Cycloalkyl containing lipids for use in lipid nanoparticles

Abstract

Compounds are provided having the following Formula (I): or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein R1, R2, R3a, R3b, L1, L2, L3, L4, and ring A are as defined herein. Use of the compounds as a component of lipid nanoparticle formulations for delivery of a therapeutic agent, compositions comprising the compounds and methods for their use and preparation are also provided.

Description

[0001] CYCLO ALKYL CONTAINING LIPIDS FOR USE IN LIPID NANOPARTICLES

[0002] BACKGROUND

[0003] Technical Field

[0004] The present disclosure generally relates to novel cationic lipids that can be used in combination with other lipid components, such as neutral lipids, cholesterol, and polymer conjugated lipids, to form lipid nanoparticles with oligonucleotides, to facilitate the intracellular delivery of therapeutic nucleic acids (e.g., oligonucleotides, messenger RNA) both in vitro and in vivo.

[0005] Description of the Related Art

[0006] There are many challenges associated with the delivery of nucleic acids to affect a desired response in a biological system. Nucleic acid-based therapeutics have enormous potential but there remains a need for more effective delivery of nucleic acids to appropriate sites within a cell or organism to realize this potential. Therapeutic nucleic acids include, e.g., messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, immune stimulating nucleic acids, antagomir, antimir, mimic, supermir, and aptamers. Some nucleic acids, such as mRNA or plasmids, can be used to effect expression of specific cellular products as would be useful in the treatment of, for example, diseases related to a deficiency of a protein or enzyme. The therapeutic applications of translatable nucleotide delivery are extremely broad as constructs can be synthesized to produce any chosen protein sequence, whether indigenous to the system. The expression products of the nucleic acid can augment existing levels of protein, replace missing or non-functional versions of a protein, or introduce a new protein and associated functionality in a cell or organism.

[0007] Some nucleic acids, such as miRNA inhibitors, can be used to effect expression of specific cellular products that are regulated by miRNA as would be useful in the treatment of, for example, diseases related to deficiency of protein or enzyme. The therapeutic applications of miRNA inhibition are extremely broad as constructs can be synthesized to inhibit one or more miRNA that would in turn regulate the expression of mRNA products. The inhibition of endogenous miRNA can augment its downstream target endogenous protein expression and restore proper function in a cell or organism as a means to treat disease associated to a specific miRNA or a group of miRNA.

[0008] Other nucleic acids can down-regulate intracellular levels of specific mRNA and, as a result, down-regulate the synthesis of the corresponding proteins through processes such as RNA interference (RNAi) or complementary binding of antisense RNA. The therapeutic applications of antisense oligonucleotide and RNAi are also extremely broad, since oligonucleotide constructs can be synthesized with any nucleotide sequence directed against a target mRNA. Targets may 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 shown the ability to specifically down-regulate target proteins through degradation of the cognate mRNA in both in vitro and in vivo models. In addition, antisense oligonucleotide constructs are currently being evaluated in clinical studies.

[0009] However, two problems currently face using oligonucleotides in therapeutic contexts. First, free RNAs are susceptible to nuclease digestion in plasma. Second, free RNAs have limited ability to gain access to the intracellular compartment where the relevant translation machinery resides. Lipid nanoparticles formed from cationic lipids with other lipid components, such as neutral lipids, cholesterol, PEG, PEGylated lipids, and oligonucleotides have been used to block degradation of the RNAs in plasma and facilitate the cellular uptake of the oligonucleotides.

[0010] There remains a need for improved cationic lipids and lipid nanoparticles for the delivery of oligonucleotides. Preferably, these lipid nanoparticles would provide optimal drug to lipid ratios, protect the nucleic acid from degradation and clearance in serum, be suitable for systemic delivery, and provide intracellular delivery of the nucleic acid. In addition, these lipid-nucleic acid particles should be well-tolerated and provide an adequate therapeutic index, such that patient treatment at an effective dose of the nucleic acid is not associated with unacceptable toxicity and / or risk to the patient. The present disclosure provides these and related advantages. BRIEF SUMMARY

[0011] In brief, the present disclosure provides lipid compounds, including stereoisomers, pharmaceutically acceptable salts, or tautomers thereof, which can be used alone or in combination with other lipid components such as neutral lipids, charged lipids, steroids (including for example, all sterols) and / or their analogs, and / or polymer conjugated lipids to form lipid nanoparticles for the delivery of therapeutic agents. In some instances, the lipid nanoparticles are used to deliver nucleic acids such as antisense and / or messenger RNA. Methods for use of such lipid nanoparticles for treatment of various diseases or conditions, such as those caused by infectious entities and / or insufficiency of a protein, are also provided.

[0012] In one embodiment, the present disclosure provides compounds having the following structure of Formula (I): or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein R1, R2, R3a, R3b, L1, L2, L3, L4, and ring A are as defined herein.

[0013] Pharmaceutical compositions comprising one or more of the foregoing compounds of Formula (I) and a therapeutic agent are also provided. In some embodiments, the pharmaceutical compositions further comprise one or more components selected from neutral lipids, charged lipids, steroids, and polymer conjugated lipids. In some embodiments, the compounds of Formula (I) combine with other components, such as neutral lipids, charged lipids, steroids, and / or polymer conjugated lipids to form lipid nanoparticles (LNPs). Such LNPs may encapsulate therapeutic agents, such as nucleic acids. In some embodiments, the LNPs are useful for delivery of the therapeutic agent.

[0014] In other embodiments, the present disclosure provides a method for administering a therapeutic agent to a patient in need thereof, the method comprising preparing a composition of lipid nanoparticles comprising the compound of Formula (I) and a therapeutic agent and delivering the composition to the patient. Such methods are useful for inducing expression of a protein in a subject, for example for expressing an antigen for purposes of vaccination or a gene editing protein.

[0015] These and other aspects of the disclosure will be apparent upon reference to the following detailed description.

[0016] DETAILED DESCRIPTION

[0017] In the following description, certain specific details are set forth to provide a thorough understanding of various embodiments of the disclosure. However, one skilled in the art will understand that the embodiments of the disclosure may be practiced without these details.

[0018] The present disclosure is based, in part, upon the discovery of novel cationic lipids that provide advantages when used in lipid nanoparticles for the in vivo delivery of an active or therapeutic agent such as a nucleic acid into a cell of a mammal. Embodiments of the present disclosure provide nucleic acid-lipid nanoparticle compositions comprising one or more of the novel cationic lipids described herein that provide increased activity of the nucleic acid and improved tolerability of the compositions in vivo, resulting in a significant increase in the therapeutic index as compared to nucleic acid-lipid nanoparticle compositions previously described.

[0019] In certain embodiments, the present disclosure provides novel cationic lipids that enable the formulation of improved compositions for the in vitro and in vivo delivery of mRNA and / or other oligonucleotides. In some embodiments, these improved lipid nanoparticle compositions are useful for expression of protein encoded by mRNA. In other embodiments, these improved lipid nanoparticles compositions are useful for upregulation of endogenous protein expression by delivering miRNA inhibitors targeting one specific miRNA or a group of miRNA regulating one target mRNA or several mRNA. In other embodiments, these improved lipid nanoparticle compositions are useful for down-regulating (e.g., silencing) the protein levels and / or mRNA levels of target genes. In some other embodiments, the lipid nanoparticles are also useful for delivery of mRNA and plasmids for expression of transgenes. In yet other embodiments, the lipid nanoparticle compositions are useful for inducing a pharmacological effect resulting from expression of a protein, e.g., increased production of red blood cells through the delivery of a suitable erythropoietin mRNA, or protection against infection through delivery of mRNA encoding for a suitable antibody.

[0020] The lipid nanoparticles and compositions of the present disclosure may be used for a variety of purposes, including the delivery of encapsulated or associated (e.g., complexed) therapeutic agents such as nucleic acids to cells, both in vitro and in vivo. Accordingly, embodiments of the present disclosure provide methods of treating or preventing diseases or disorders in a subject in need thereof by contacting the subject with a lipid nanoparticle that encapsulates or is associated with a suitable therapeutic agent, wherein the lipid nanoparticle comprises one or more of the novel cationic lipids described herein.

[0021] As described herein, embodiments of the lipid nanoparticles of the present disclosure are particularly useful for the delivery of nucleic acids, including, e.g., mRNA, antisense oligonucleotide, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomirs / antimirs), messenger-RNA-interfering complementary RNA (micRNA), DNA, multivalent RNA, dicer substrate RNA, complementary DNA (cDNA), etc. Therefore, the lipid nanoparticles and compositions of the present disclosure may be used to induce expression of a desired protein both in vitro and in vivo by contacting cells with a lipid nanoparticle comprising one or more novel cationic lipids described herein, wherein the lipid nanoparticle encapsulates or is associated with a nucleic acid that is expressed to produce the desired protein (e.g., a messenger RNA or plasmid encoding the desired protein). Alternatively, the lipid nanoparticles and compositions of the present disclosure may be used to decrease the expression of target genes and proteins both in vitro and in vivo by contacting cells with a lipid nanoparticle comprising one or more novel cationic lipids described herein, wherein the lipid nanoparticle encapsulates or is associated with a nucleic acid that reduces target gene expression (e.g., an antisense oligonucleotide or small interfering RNA (siRNA)). The lipid nanoparticles and compositions of the present disclosure may also be used for co-delivery of different nucleic acids (e.g., mRNA and plasmid DNA) separately or in combination, such as may be useful to provide an effect requiring co-localization of different nucleic acids (e.g., mRNA encoding for a suitable gene modifying enzyme and DNA segment(s) for incorporation into the host genome).

[0022] Nucleic acids for use with this disclosure may be prepared according to any available technique. For mRNA, the primary methodology of preparation is, but not limited to, enzymatic synthesis (also termed in vitro transcription) which currently represents the most efficient method to produce long sequence-specific mRNA. In vitro transcription describes a process of template-directed synthesis of RNA molecules from an engineered DNA template comprised of an upstream bacteriophage promoter sequence (e.g., including but not limited to that from the T7, T3, and SP6 coliphage) linked to a downstream sequence encoding the gene of interest. Template DNA can be prepared for in vitro transcription from a number of sources with appropriate techniques which are well known in the art including, but not limited to, plasmid DNA and polymerase chain reaction amplification (see Linpinsel, J.L and Conn, G.L., General protocols for preparation of plasmid DNA template and Bowman, J.C., Azizi, B., Lenz, T.K., Ray, P., and Williams, L.D. in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941 Conn G.L. (ed), New York, N.Y. Humana Press, 2012).

[0023] Transcription of the RNA occurs in vitro using the linearized DNA template in the presence of the corresponding RNA polymerase and adenosine, guanosine, uridine, and cytidine ribonucleoside triphosphates (rNTPs) under conditions that support polymerase activity while minimizing potential degradation of the resultant 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 kits (Life Technologies) as well as with commercially available reagents including RNA polymerases and rNTPs. The methodology for in vitro transcription of mRNA is well known in the art. (see, e.g., Losick, R., 1972, In vitro transcription, Ann Rev Biochem v.41 409-46; Kamakaka, R. T. and Kraus, W. L. 2001. In Vitro Transcription. Current Protocols in Cell Biology. 2:11.6:11.6.1- 11.6.17; Beckert, B. And Masquida, B., (2010) Synthesis of RNA by In Vitro Transcription in RNA in Methods in Molecular Biology v. 703 (Neilson, H. Ed), New York, N.Y. Humana Press, 2010; Brunelle, J.L. and Green, R., 2013, Chapter Five - In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology v. 530, 101-114; all of which are incorporated herein by reference).

[0024] The desired in vitro transcribed mRNA is then purified from the undesired components of the transcription or associated reactions (including unincorporated rNTPs, protein enzyme, salts, short RNA oligos efc.). Techniques for the isolation of the mRNA transcripts are well known in the art. Well known procedures include phenol / chloroform extraction or precipitation with either alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride. Additional, non-limiting examples of purification procedures which can be used include size exclusion chromatography (Lukavsky, P.J. and Puglisi, J.D., 2004, Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides, RNA v.10, 889-893), silica-based affinity chromatography and polyacrylamide gel electrophoresis (Bowman, J.C., Azizi, B., Lenz, T.K., Ray, P., and Williams, L.D. in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941 Conn G.L. (ed), New York, N.Y. 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).

[0025] Furthermore, while reverse transcription can yield large quantities of mRNA, the products can contain several aberrant RNA impurities associated with undesired polymerase activity which may need to be removed from the full-length mRNA preparation. These include short RNAs that result from abortive transcription initiation as well as double-stranded RNA (dsRNA) generated by RNA-dependent RNA polymerase activity, RNA-primed transcription from RNA templates and self-complementary 3' extension. It has been demonstrated that these contaminants with dsRNA structures can lead to undesired immunostimulatory activity through interaction with various innate immune sensors in eukaryotic cells that function to recognize specific nucleic acid structures and induce potent immune responses. This in turn, can dramatically reduce mRNA translation since protein synthesis is reduced during the innate cellular immune response. Therefore, additional techniques to remove these dsRNA contaminants have been developed and are known in the art including but not limited to scalable HPLC purification (see, e.g., Kariko, K., Muramatsu, H., Ludwig, J. And Weissman, D., 2011, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA, Nucl Acid Res, v. 39 el42; Weissman, D., Pardi, N., Muramatsu, H., and Kariko, K., HPLC Purification of in vitro transcribed long RNA in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, P.H. Ed), 2013). HPLC purified mRNA has been reported to be translated at much greater levels, particularly in primary cells and in vivo. A significant variety of modifications have been described in the art which are used to alter specific properties of in vitro transcribed mRNA and improve its utility. These include but are not limited to modifications to the 5' and 3' termini of the mRNA. Endogenous eukaryotic mRNA typically contains a cap structure on the 5 '-end of a mature molecule which plays an important role in mediating binding of the mRNA Cap Binding Protein (CBP), which is in turn responsible for enhancing mRNA stability in the cell and efficiency of mRNA translation. Therefore, highest levels of protein expression are achieved with capped mRNA transcripts. The 5 '-cap contains a 5 '-5 '-triphosphate linkage between the 5 '-most nucleotide and guanine nucleotide. The conjugated guanine nucleotide is methylated at the N7 position. Additional modifications include methylation of the ultimate and penultimate most 5 '-nucleotides on the 2'- hydroxyl group.

[0026] Multiple distinct cap structures can be used to generate the 5'-cap of in vitro transcribed synthetic mRNA. 5'-capping of synthetic mRNA can be performed co-transcriptionally with chemical cap analogs (i.e., capping during in vitro transcription). For example, the Anti -Reverse Cap Analog (ARC A) cap contains a 5 '-5 '-triphosphate guanine-guanine linkage where one guanine contains an N7 methyl group as well as a 3'-O-methyl group. However, up to 20% of transcripts remain uncapped during this co-transcriptional process and the synthetic cap analog is not identical to the 5 '-cap structure of an authentic cellular mRNA, potentially reducing translatability and cellular stability. Alternatively, synthetic mRNA molecules may also be enzymatically capped post-transcriptionally. These may generate a more authentic 5 '-cap structure that more closely mimics, either structurally or functionally, the endogenous 5'-cap which have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5' endonucleases and / or reduced 5' decapping. Numerous synthetic 5'-cap analogs have been developed and are known in the art to enhance mRNA stability and translatability (see, e.g., Grudzien-Nogalska, E., Kowalska, J., Su, W., Kuhn, A.N., Slepenkov, S.V., Darynkiewicz, E., Sahin, U., Jemielity, J., and Rhoads, R.E., Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, P.H. Ed), 2013).

[0027] On the 3'-terminus, a long chain of adenine nucleotides (poly-A tail) is normally added to mRNA molecules during RNA processing. Immediately after transcription, the 3' end of the transcript is cleaved to free a 3' hydroxyl to which poly-A polymerase adds a chain of adenine nucleotides to the RNA in a process called polyadenylation. The poly-A tail has been extensively shown to enhance both translational efficiency and stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly (A), poly (A) binding protein and the regulation of mRNA stability, Trends Bio Sci v. 14 373-377; Guhaniyogi, J. And Brewer, G., 2001, Regulation of mRNA stability in mammalian cells, Gene, v. 265, 11-23; Dreyfus, M. And Regnier, P., 2002, The poly (A) tail of mRNAs: Bodyguard in eukaryotes, scavenger in bacteria, Cell, v.111, 611-613).

[0028] Poly (A) tailing of in vitro transcribed mRNA can be achieved using various approaches including, but not limited to, cloning of a poly (T) tract into the DNA template or by post- transcriptional addition using Poly (A) polymerase. The first case allows in vitro transcription of mRNA 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 the enzymatic addition of a poly (A) tail to in vitro transcribed mRNA using poly (A) polymerase which catalyzes the incorporation of adenine residues onto the 3 'termini of RNA, requiring no additional manipulation of the DNA template, but results in mRNA with poly(A) tails of heterogenous 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 with commercially available reagents, various ARCA caps, Poly (A) polymerase, etc.

[0029] In addition to 5' cap and 3' poly adenylation, other modifications of the in vitro transcripts have been reported to provide benefits as related to efficiency of translation and stability. It is well known in the art that pathogenic DNA and RNA can be recognized by a variety of sensors within eukaryotes and trigger potent innate immune responses. The ability to discriminate between pathogenic and self-DNA and RNA has been shown to be based, at least in part, on structure and nucleoside modifications since most nucleic acids from natural sources contain modified nucleosides. In contrast, in vitro synthesized RNA lacks these modifications, thus rendering it immunostimulatory which in turn can inhibit effective mRNA translation as outlined above. The introduction of modified nucleosides into in vitro transcribed mRNA can be used to prevent recognition and activation of RNA sensors, thus mitigating this undesired immunostimulatory activity and enhancing translation capacity (see, e.g., Kariko, K. And Weissman, D. 2007, Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: implication for therapeutic RNA development, Curr Opin Drug Discov Devel, v.10 523-532; Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, P.H. Ed), 2013); Kariko, K., Muramatsu, H., Welsh, F.A., Ludwig, J., Kato, H., Akira, S., Weissman, D., 2008, Incorporation of Pseudouridine Into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability, Mol Ther v.16, 1833-1840. The modified nucleosides and nucleotides used in the synthesis of modified RNAs can be prepared monitored and utilized using general methods and procedures known in the art. A large variety of nucleoside modifications are available that may be incorporated alone or in combination with other modified nucleosides to some extent into the in vitro transcribed mRNA (see, e.g., US Publication No. 2012 / 0251618). In vitro synthesis of nucleoside-modified mRNA has been reported to have reduced ability to activate immune sensors with a concomitant enhanced translational capacity.

[0030] Other components of mRNA which can be modified to provide benefit in terms of translatability and stability include the 5' and 3' untranslated regions (UTR). Optimization of the UTRs (favorable 5' and 3' UTRs can be obtained from cellular or viral RNAs), either both or independently, have been shown to increase mRNA stability and translational efficiency of in vitro transcribed mRNA (see, e.g., Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, P.H. Ed), 2013).

[0031] In addition to mRNA, other nucleic acid payloads may be used for this disclosure. For oligonucleotides, methods of preparation include but are not limited to chemical synthesis and enzymatic, chemical cleavage of a longer precursor, in vitro transcription as described above, etc. Methods of synthesizing DNA and RNA nucleotides are widely used and well known in the art (see, e.g., Gait, M. J. (ed.)Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, D.C.: IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana Press, 2005; both of which are incorporated herein by reference).

[0032] For plasmid DNA, preparation for use with this disclosure commonly utilizes but is not limited to expansion and isolation of the plasmid DNA in vitro in a liquid culture of bacteria containing the plasmid of interest. The presence of a gene in the plasmid of interest that encodes resistance to a particular antibiotic (penicillin, kanamycin, etc.) allows those bacteria containing the plasmid of interest to selective grow in antibiotic-containing cultures. Methods of isolating plasmid DNA are widely used and well known in the art (see, e.g., Heilig, J., Elbing, K. L. and Brent, R (2001) Large-Scale Preparation of Plasmid DNA. Current Protocols in Molecular Biology. 41 :11: 1.7: 1.7.1-1.7.16; Rozkov, A., Larsson, B., Gillstrbm, S., Bjomestedt, R. and Schmidt, S. R. (2008), Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture. Biotechnol. Bioeng., 99: 557-566; and US6197553B1). Plasmid isolation can be performed using a variety of commercially available kits including, but not limited to Plasmid Plus (Qiagen), GenJET plasmid MaxiPrep (Thermo) and Pure Yield MaxiPrep (Promega) kits as well as with commercially available reagents. Various exemplary embodiments of Formula (I), lipid nanoparticles and compositions comprising the same, and their use to deliver active or therapeutic agents such as nucleic acids to modulate gene and protein expression, are described in further detail below.

[0033] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0034] Unless the context requires otherwise, throughout the present specification and claims, the word "comprise" and variations thereof, such as, "comprises" and "comprising" are to be construed in an open and inclusive sense, that is, as "including, but not limited to".

[0035] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. 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 features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this disclosure belongs. As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

[0037] The phrase "induce expression of a desired protein" refers to the ability of a nucleic acid to increase expression of the desired protein. To examine the extent of protein expression, a test sample (e.g, a sample of cells in culture expressing the desired protein) or a test mammal (e.g, a mammal such as a human or an animal model such as a rodent (e.g., mouse) or a non-human primate (e.g., monkey) model) is contacted with a nucleic acid (e.g., nucleic acid in combination with a lipid of the present disclosure). Expression of the desired protein in the test sample or test animal is compared to expression of the desired protein in a control sample (e.g., a sample of cells in culture expressing the desired protein) or a control mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g. , mouse) or non-human primate (e.g. , monkey) model) that is not contacted with or administered the nucleic acid. When the desired protein is present in a control sample or a control mammal, the expression of a desired protein in a control sample or a control mammal may be assigned a value of 1.0. In some embodiments, inducing expression of a desired protein is achieved when the ratio of desired protein expression in the test sample or the test mammal to the level of desired protein expression in the control sample or the control mammal is greater than 1, for example, about 1.1, 1.5, 2.0. 5.0 or 10.0. When a desired protein is not present in a control sample or a control mammal, inducing expression of a desired protein is achieved when any measurable level of the desired protein in the test sample or the test mammal is detected. One of ordinary skill in the art will understand appropriate assays to determine the level of protein expression in a sample, for example dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays, or assays based on reporter proteins that can produce fluorescence or luminescence under appropriate conditions.

[0038] The phrase "inhibiting expression of a target gene" refers to the ability of a nucleic acid to silence, reduce, or inhibit the expression of a target gene. To examine the extent of gene silencing, a test sample (e.g., a sample of cells in culture expressing the target gene) or a test mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g., mouse) or a non-human primate (e.g., monkey) model) is contacted with a nucleic acid that silences, reduces, or inhibits expression of the target gene. Expression of the target gene in the test sample or test animal is compared to expression of the target gene in a control sample (e.g., a sample of cells in culture expressing the target gene) or a control mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g., mouse) or non-human primate (e.g., monkey) model) that is not contacted with or administered the nucleic acid. The expression of the target gene in a control sample or a control mammal may be assigned a value of 100%. In particular embodiments, silencing, inhibition, or reduction of expression of a target gene is achieved when the level of target gene expression in the test sample or the test mammal relative to the level of target gene expression in the control sample or the control mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. In other words, the nucleic acids are capable of silencing, reducing, or inhibiting the expression of a target gene by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in a test sample or a test mammal relative to the level of target gene expression in a control sample or a control mammal not contacted with or administered the nucleic acid. Suitable assays for determining the level of target gene expression include, without limitation, examination of protein or mRNA levels using techniques known to those of skill in the art, such as, e.g., dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.

[0039] An "effective amount" or "therapeutically effective amount" of an active agent or therapeutic agent such as a therapeutic nucleic acid is an amount sufficient to produce the desired effect, e.g., an increase or inhibition of expression of a target sequence in comparison to the normal expression level detected in the absence of the nucleic acid. An increase in expression of a target sequence is achieved when any measurable level is detected in the case of an expression product that is not present in the absence of the nucleic acid. In the case where the expression product is present at some level prior to contact with the nucleic acid, an in increase in expression is achieved when the fold increase in value obtained with a nucleic acid such as mRNA relative to control is about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000 or greater. Inhibition of expression of a target gene or target sequence is achieved when the value obtained with a nucleic acid such as antisense oligonucleotide relative to the control is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring expression of a target gene or target sequence include, e.g., examination of protein or RNA levels using techniques known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of suitable reporter proteins, as well as phenotypic assays known to those of skill in the art.

[0040] The term "nucleic acid" as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single- or double-stranded form and includes DNA, RNA, and hybrids thereof. DNA may be in the form of antisense molecules, plasmid DNA, cDNA, PCR products, or vectors. RNA may be in the form of small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA, dicer substrate RNA or viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O- methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). "Nucleotides" contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups. "Bases" include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides.

[0041] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises partial length or entire length coding sequences necessary to produce a polypeptide or precursor polypeptide.

[0042] "Gene product," as used herein, refers to a product of a gene such as an RNA transcript or a polypeptide.

[0043] The term "lipid" refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are generally characterized by being poorly soluble in water, but soluble in many organic solvents. They are usually divided into at least three classes: (1) "simple lipids," which include fats and oils as well as waxes; (2) "compound lipids," which include phospholipids and glycolipids; and (3) "derived lipids" such as steroids.

[0044] A "steroid" is a compound comprising the following carbon skeleton:

[0045] Non-limiting examples of steroids include cholesterol, and the like.

[0046] A "cationic lipid" refers to a lipid capable of being positively charged. Exemplary cationic lipids include one or more amine group(s) which bear the positive charge. Preferred cationic lipids are ionizable such that they can exist in a positively charged or neutral form depending on pH. The ionization of the cationic lipid affects the surface charge of the lipid nanoparticle under different pH conditions. This charge state can influence plasma protein absorption, blood clearance and tissue distribution (Semple, S.C., et al., Adv. Drug Deliv Rev 32:3-17 (1998)) as well as the ability to form endosomolytic non-bilayer structures (Hafez, I.M., et al., Gene Ther 8: 1188-1196 (2001)) critical to the intracellular delivery of nucleic acids.

[0047] The term "lipid nanoparticle" refers to particles having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which include one or more of the compounds of Formula (I) or other specified components. In some embodiments, lipid nanoparticles are included in a formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, tumor, and the like). In some embodiments, the lipid nanoparticles of the disclosure comprise a nucleic acid. Such lipid nanoparticles typically comprise a compound of Formula (I) and one or more excipient selected from neutral lipids, charged lipids, steroids, and polymer conjugated lipids. In some embodiments, the active agent or therapeutic agent, such as a nucleic acid, may be encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells, e.g., an adverse immune response.

[0048] In various embodiments, the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 40 nm to about 100 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. In certain embodiments, nucleic acids, when present in the lipid nanoparticles, are resistant in aqueous solution to degradation with a nuclease. Lipid nanoparticles comprising nucleic acids and their method of preparation are disclosed in, e.g., U.S. Patent Publication Nos. 2004 / 0142025, 2007 / 0042031 and PCT Pub. Nos. WO 2013 / 016058 and WO 2013 / 086373, the full disclosures of which are herein incorporated by reference in their entirety for all purposes.

[0049] As used herein, "lipid encapsulated" refers to a lipid nanoparticle that provides an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), with full encapsulation, partial encapsulation, or both. In an embodiment, the nucleic acid (e.g., mRNA) is fully encapsulated in the lipid nanoparticle.

[0050] The term "polymer conjugated lipid" refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid. The term "pegylated lipid" refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG) and the like.

[0051] The term "neutral lipid" refers to any of several lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, but are not limited to, phosphotidylcholines such as l,2-Distearoyl- w-glycero-3-phosphocholine (DSPC), l,2-Dipalmitoyl- w-glycero-3 -phosphocholine (DPPC), l,2-Dimyristoyl- w-glycero-3- phosphocholine (DMPC), l-Palmitoyl-2-oleoyl-sw-glycero-3 -phosphocholine (POPC), 1,2- dioleoyl-sn-glycero-3 -phosphocholine (DOPC), phophatidyl ethanolamines such as 1,2-Dioleoyl- .s / / -glycero-3 -phosphoethanol amine (DOPE), sphingomyelins (SM), ceramides, steroids such as sterols and their derivatives. Neutral lipids may be synthetic or naturally derived.

[0052] The term "charged lipid" refers to any of several lipid species that exist in either a positively charged or negatively charged form independent of the pH within a useful physiological range, e.g., pH ~3 to pH ~9. Charged lipids may be synthetic or naturally derived. Examples of charged lipids include phosphatidylserines, phosphatidic acids, phosphatidylglycerols, phosphatidylinositols, sterol hemi succinates, dialkyl trimethylammonium-propanes, (e.g., DOTAP, DOTMA), dialkyl dimethylaminopropanes, ethyl phosphocholines, dimethylaminoethane carbamoyl sterols (e.g., DC-Chol).

[0053] As used herein, the term "aqueous solution" refers to a composition comprising water.

[0054] "Serum-stable" in relation to nucleic acid-lipid nanoparticles means that the nucleotide is not significantly degraded after exposure to a serum or nuclease assay that would significantly degrade free DNA or RNA. Suitable assays include, for example, a standard serum assay, a DNAse assay, or an RNAse assay.

[0055] "Systemic delivery," as used herein, refers to delivery of a therapeutic product that can result in a broad exposure of an active agent within an organism. Some techniques of administration can lead to the systemic delivery of certain agents, but not others. Systemic delivery means that a useful, preferably therapeutic, amount of an agent is exposed to most parts of the body. Systemic delivery of lipid nanoparticles can be by any means known in the art including, for example, intravenous, intraarterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery.

[0056] "Local delivery," as used herein, refers to delivery of an active agent directly to a target site within an organism. For example, an agent can be locally delivered by direct injection into a disease site such as a tumor, other target site such as a site of inflammation, or a target organ such as the liver, heart, pancreas, kidney, and the like. Local delivery can also include topical applications or localized injection techniques such as intramuscular, subcutaneous, or intradermal injection. Local delivery does not preclude a systemic pharmacological effect.

[0057] "Alkyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms that is saturated (i.e., contains no double and / or triple bonds), having from one to twenty-four carbon atoms (C1-C24 alkyl), one to sixteen carbon atoms (C1-C16 alkyl), one to twelve carbon atoms (C1-C12 alkyl), six to twenty-four carbon atoms (C6-C24 alkyl), one to eight carbon atoms (Ci-Cs alkyl) or one to six carbon atoms (Ci-Ce alkyl) and which is attached to the rest of the molecule by a single bond, e.g., methyl, ethyl, n-propyl, 1 -methylethyl (iso propyl), n-butyl, n-pentyl, 1,1 -dimethylethyl (t-butyl), 3 -methylhexyl, 2-methylhexyl, and the like. Unless stated otherwise specifically in the specification, an alkyl group is optionally substituted.

[0058] "Alkoxy" refers to a radical with a formula -ORawhere Rais an alkyl radical as defined above. Unless stated otherwise specifically in the specification, an alkoxy group is optionally substituted.

[0059] "Alkenyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms that contains at least one carbon-carbon double, having from one to twenty-four carbon atoms (C2-C24 alkenyl), one to twelve carbon atoms (C2-C12 alkenyl), six to twenty-four carbon atoms (C6-C24 alkenyl), two to sixteen carbon atoms (C2-C16 alkenyl), four to twelve carbon atoms (C4-C12 alkenyl), one to eight carbon atoms (C2-C8 alkenyl) or one to six carbon atoms (C2-C6 alkenyl) and which is attached to the rest of the molecule by a single bond, e.g., ethenyl, n-propenyl, 1 -methylethenyl, n-butenyl, n-pentenyl, 1,1 -dimethylethenyl, 3- methylhexenyl, 2-methylhexenyl, and the like. Unless stated otherwise specifically in the specification, an alkenyl group is optionally substituted.

[0060] " Alkynyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms that contains at least one carbon-carbon triple bond, having from one to twenty-four carbon atoms (C2-C24 alkynyl), one to twelve carbon atoms (C2-C12 alkynyl), one to eight carbon atoms (C2-C8 alkynyl) or one to six carbon atoms (C2-C6 alkynyl) and which is attached to the rest of the molecule by a single bond, e.g., ethynyl, n-propynyl, 1-methylethynyl, n-butynyl, n-pentynyl, 1,1-dimethylethynyl, 3 -methylhexynyl, 2-methylhexynyl, and the like. Unless stated otherwise specifically in the specification, an alkynyl group is optionally substituted.

[0061] "Alkylene" or "alkylene chain" refers to a straight or branched divalent saturated hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen. In some embodiments, an alkylene chain has from one to twenty -four carbon atoms (C1-C24 alkylene), one to fifteen carbon atoms (C1-C15 alkylene), one to twelve carbon atoms (C1-C12 alkylene), one to eight carbon atoms (Ci-Cs alkylene), one to six carbon atoms (Ci-Ce alkylene), four to six carbon atoms (C4-C6 alkylene), two to four carbon atoms (C2-C4 alkylene), one to two carbon atoms (C1-C2 alkylene), e.g., methylene, ethylene, propylene, w-butylene, and the like. 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 carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, an alkylene chain is optionally substituted. "Alkenylene" or "alkenylene chain" refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen and which comprises at least one carbon-carbon double bond. In some embodiments, an alkenylene chain has from two to twenty-four carbon atoms (C2-C24 alkenylene), two to fifteen carbon atoms (C2-C15 alkenylene), two to twelve carbon atoms (C2-C12 alkenylene), two to eight carbon atoms (C2-C8 alkenylene), two to six carbon atoms (C2-C6 alkenylene), four to six carbon atoms (C4-C6 alkenylene), two to four carbon atoms (C2-C4 alkenylene), e.g., ethenylene, propenylene, w-butenylene, and the like. The alkenylene 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 alkenylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, an alkenylene chain is optionally substituted.

[0062] "Cycloalkyl" or "carbocyclic ring" refers to a stable non aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems, having from three to fifteen ring carbon atoms (C3-C15), from three to ten ring carbon atoms (C3-C10) or from three to eight ring carbon atoms (Cs-Cs), and which is saturated or unsaturated and attached to the rest of the molecule by a single bond. Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethyl bicyclo[2.2.1]heptanyl, and the like. Unless otherwise stated specifically in the specification, a cycloalkyl group is optionally substituted.

[0063] "Aryl" refers to a carbocyclic ring system radical comprising hydrogen, 6 to 18 carbon atoms and at least one aromatic ring. For purposes of this disclosure, the aryl radical is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused or bridged ring systems. Aryl radicals include, but are not limited to, aryl radicals derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, -indacene, indane, indene, naphthalene, phenal ene, phenanthrene, pleiadene, pyrene, and triphenylene.

[0064] "Arylalkyl" refers to a radical of the formula -Rb-Rc where Rb is an alkylene or alkenylene as defined above and Rcis one or more aryl radicals as defined above, for example, benzyl, diphenylmethyl and the like. Unless stated otherwise specifically in the specification, an arylalkyl group is optionally substituted.

[0065] "Heterocyclyl" or "heterocyclic ring" refers to a stable 3- to 18-membered non-aromatic ring radical having one to twelve ring carbon atoms (e.g., two to twelve) and from one to six ring heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Unless stated otherwise specifically in the specification, the heterocyclyl radical is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused, spirocyclic ("spiro-heterocyclyl") and / or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclyl radical is optionally oxidized; the nitrogen atom is optionally quaternized; and the heterocyclyl radical is partially or fully saturated. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[l,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 specifically in the specification, a heterocyclyl group is optionally substituted.

[0066] "Heteroaryl" refers to a 5- to 18-membered, for example 5- to 6-membered, ring system radical comprising one to thirteen ring carbon atoms, one to six ring heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, and at least one aromatic ring. Heteroaryl radicals may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon, or sulfur atoms in the heteroaryl radical may be optionally oxidized; the nitrogen atom may be optionally quaternized. Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodi oxolyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[Z»][l,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodi oxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotri azolyl, benzo[4,6]imidazo[l,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1- oxidopyrazinyl, 1-oxidopyridazinyl, 1 -phenyl- 1 / / -pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl ( / .< ., thienyl). Unless stated otherwise specifically in the specification, heteroaryl groups are optionally substituted.

[0067] The term "substituted" used herein means any of the above groups (e.g., alkyl, alkylhydroxyl, alkenyl, alkynyl, alkylene, cycloalkyl, aryl, aralkyl or heterocyclyl) wherein at least one hydrogen atom is replaced by a bond to a non-hydrogen atoms such as, but not limited to: a halogen atom such as F, Cl, Br, and I; oxo groups (=0); hydroxyl groups (-0H); alkoxy groups (-0Ra, where Rais C1-C12 alkyl or cycloalkyl); carboxyl groups (-0C(=0)Raor - C(=0)0Ra, where Rais H, C1-C12 alkyl or cycloalkyl); amine groups (-NRaRb, where Raand Rbare each independently H, C1-C12 alkyl or cycloalkyl); C1-C12 alkyl groups; and cycloalkyl groups. In some embodiments the substituent is a C1-C12 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 a oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group. In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group.

[0068] "Optional" or "optionally" (e.g., optionally substituted) means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. For example, "optionally substituted alkyl" means that the alkyl radical may or may not be substituted and that the description includes both substituted alkyl radicals and alkyl radicals having no substitution. In some embodiments, "optionally substituted" means a particular radical is substituted with one or more substituents selected from the group consisting of halo (e.g., F, Cl, Br, and I), oxo (=0), hydroxyl (-0H), alkoxy (-0Ra, where Rais C1-C12 alkyl), cycloalkoxy (-0Ra, where Rais Cs-Cs cycloalkyl), carboxyl (-0C(=0)Raor -C(=0)0Ra, where Rais H, Ci- C12 alkyl, or Cs-Cs cycloalkyl), amine (-NRaRb, where Raand Rbare each independently H, Ci- C12 alkyl, or Cs-Cs cycloalkyl), C1-C12 alkyl, and Cs-Cs cycloalkyl.

[0069] In some embodiments, "optionally substituted" means substituted with one or more halo substituents. In some embodiments, "optionally substituted" means substituted with one or more oxo substituents. In some embodiments, "optionally substituted" means substituted with one or more hydroxyl substituents. In certain embodiments, "optionally substituted" means substituted with one or more alkoxy substituents. In some embodiments, "optionally substituted" means substituted with one or more cycloalkoxy substituents. In certain embodiments, "optionally substituted" means substituted with one or more carboxy substituents. In some embodiments, "optionally substituted" means substituted with one or more amine substituents. In certain embodiments, "optionally substituted" means substituted with one or more C1-C12 alkyl substituents. In some embodiments, "optionally substituted" means substituted with one or more C3-C8 cycloalkyl substituents.

[0070] When a functional group is described as "optionally substituted," and in turn, substituents on the functional group are also "optionally substituted" and so on, for the purposes of this disclosure, such iterations are limited to five, preferably such iterations are limited to two. In some embodiments, such iterations are limited to one. In some embodiments, such iterations are limited to zero.

[0071] This disclosure is also meant to encompass all pharmaceutically acceptable compounds of the compound of Formula (I) being isotopically labelled by having one or more atoms replaced by an atom having a different atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, and iodine, such as2H,3H,nC,13C,14C,13N,15N,15O,17O,18O,31P,32P,35S,18F,36C1,123I, and125I, respectively. These radiolabeled compounds could be useful to help determine or measure the effectiveness of the compounds, by characterizing, for example, the site or mode of action, or binding affinity to pharmacologically important site of action. Certain isotopically labelled compounds of Formula (I), for example, those incorporating a radioactive isotope, are useful in drug and / or substrate tissue distribution studies. The radioactive isotopes tritium, e.g.,3H, and carbon- 14, z.e.,14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

[0072] Substitution with heavier isotopes such as deuterium, z.e.,2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.

[0073] Substitution with positron emitting isotopes, such asnC,18F,15O and13N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically labeled compounds of Formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the Preparations and Examples as set out below using an appropriate isotopically labeled reagent in place of the non-labeled reagent previously employed.

[0074] This disclosure is also meant to encompass the in vivo metabolic products of the disclosed compounds. Such products may result from, for example, the oxidation, reduction, hydrolysis, amidation, esterification, and the like of the administered compound, primarily due to enzymatic processes. Accordingly, the disclosure includes compounds produced by a process comprising administering a compound of this disclosure to a mammal for a period sufficient to yield a metabolic product thereof. Such products are typically identified by administering a radiolabeled compound of the disclosure in a detectable dose to an animal, such as rat, mouse, guinea pig, monkey, or to human, allowing sufficient time for metabolism to occur, and isolating its conversion products from the urine, blood, or other biological samples.

[0075] "Stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. "Mammal" includes humans and both domestic animals such as laboratory animals and household pets (e.g., cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and non-domestic animals such as wildlife and the like.

[0076] "Pharmaceutically acceptable carrier, diluent or excipient" includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.

[0077] "Pharmaceutically acceptable salt" includes both acid and base addition salts.

[0078] "Pharmaceutically acceptable acid addition salt" refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor- 10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfuric acid, ethane- 1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2- oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene- 1,5-disulfonic acid, naphthal ene-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, -toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like.

[0079] "Pharmaceutically acceptable base addition salt" refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, A-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

[0080] Often crystallizations produce a solvate of the compound of the disclosure. As used herein, the term "solvate" refers to an aggregate that comprises one or more molecules of a compound of the disclosure with one or more molecules of solvent. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of the present disclosure may exist as a hydrate, including a monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate and the like, as well as the corresponding solvated forms. The compound of the disclosure may be true solvates, while in other cases, the compound of the disclosure may merely retain adventitious water or be a mixture of water plus some adventitious solvent.

[0081] A "pharmaceutical composition" refers to a formulation of a compound of the disclosure and a medium generally accepted in the art for the delivery of the biologically active compound to mammals, e.g., humans. Such a medium includes all pharmaceutically acceptable carriers, diluents, or excipients therefor.

[0082] "Effective amount" or "therapeutically effective amount" refers to that amount of a compound of the disclosure which, when administered to a mammal, preferably a human, is sufficient to effect treatment in the mammal, preferably a human. The amount of a lipid nanoparticle of the disclosure which constitutes a "therapeutically effective amount" will vary depending on the compound, the condition and its severity, the manner of administration, and the age of the mammal to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.

[0083] "Treating" or "treatment" as used herein covers the treatment of the disease or condition of interest in a mammal, preferably a human, having the disease or condition of interest, and includes:

[0084] (i) preventing the disease or condition from occurring in a mammal, in particular, when such mammal is predisposed to the condition but has not yet been diagnosed as having it;

[0085] (ii) inhibiting the disease or condition, z.e., arresting its development;

[0086] (iii) relieving the disease or condition, z.e., causing regression of the disease or condition; or (iv) relieving the symptoms resulting from the disease or condition, i.e., relieving pain without addressing the underlying disease or condition. As used herein, the terms "disease" and "condition" may be used interchangeably or may be different in that the malady or condition may not have a known causative agent (so that etiology has not yet been worked out) and it is therefore not yet recognized as a disease but only as an undesirable condition or syndrome, wherein a more or less specific set of symptoms have been identified by clinicians.

[0087] The compounds of the disclosure, or their pharmaceutically acceptable salts may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (5)- or, as (D)- or (L)- for amino acids. The present disclosure is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (5)-, or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high-pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

[0088] A "stereoisomer" refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable. The present disclosure contemplates various stereoisomers and mixtures thereof and includes "enantiomers", which refers to two stereoisomers whose molecules are non-superimposable mirror images of one another.

[0089] A "tautomer" refers to a proton shift from one atom of a molecule to another atom of the same molecule. The present disclosure includes tautomers of any said compounds. Compounds

[0090] In an aspect, the disclosure provides novel lipid compounds which can combine with other lipid components such as neutral lipids, charged lipids, steroids, and / or polymer conjugated lipids to form lipid nanoparticles with oligonucleotides. Without wishing to be bound by theory, it is thought that these lipid nanoparticles shield oligonucleotides from degradation in the serum and provide for effective delivery of oligonucleotides to cells in vitro and in vivo.

[0091] Accordingly, one embodiment provides a compound having the following Formula (I): or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein: ring A is an optionally substituted Cs-Cs cycloalkyl;

[0092] L1and L2are each independently optionally substituted C4-C12 alkylene; indicates a covalent bond to ring A;

[0093] L3ais a direct bond or optionally substituted C1-C4 alkylene;

[0094] L4is a direct bond or optionally substituted C1-C4 alkylene;

[0095] R1is -NRla-C(=O)-Rlb, -C(=O)-N(Rla)Rlb, -O-C(=O)-Rla, -C(=O)-O-Rla, or -O-C(=O)- O-Rla;

[0096] Rlaand Rlbare each independently optionally substituted C1-C24 alkyl, optionally substituted C2-C24 alkenyl, or optionally substituted C2-C24 alkynyl;

[0097] R2is -NR2a-C(=O)-R2b, -C(=O)-N(R2a)R2b, -O-C(=O)-R2a, -C(=O)-O-R2a, or -O-C(=O)- O-R2a;

[0098] R2aand R2bare each independently optionally substituted C1-C24 alkyl, optionally substituted C2-C24 alkenyl, or optionally substituted C2-C24 alkynyl;

[0099] R3aand R3bare each independently hydrogen or optionally substituted C1-C4 alkyl, or R3aand R3b, together with the nitrogen to which they are attached, join to form an optionally substituted 3-8 membered heterocyclyl or an optionally substituted 5-6 membered heteroaryl; and

[0100] R4is optionally substituted C1-C12 alkyl, optionally substituted C2-C12 alkenyl, or optionally substituted C2-C12 alkynyl.

[0101] In some embodiments, ring A is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In certain embodiments, ring A is cyclobutyl or cyclohexyl. In some embodiments, ring A is unsubstituted.

[0102] In certain embodiments, L1is C4-C10 alkylene. In some embodiments, L1is C4 alkylene, C5 alkylene, C7 alkylene, Cs alkylene, or C9 alkylene.

[0103] In some embodiments, L2is C4-C10 alkylene. In certain embodiments, L2is C4 alkylene, Cs alkylene, C7 alkylene, Cs alkylene, or C9 alkylene. In certain embodiments, L1and L2are unbranched and unsubstituted. In some embodiments, L1, L2, or both are substituted with one or more fluoro substituents. In certain embodiments, L1and L2are the same. In some embodiments, L1and L2are different.

[0104] In some embodiments, L3is -NR4-C(=O)-* and R4is optionally substituted C1-C10 alkyl. In certain embodiments, L3is -NR4-C(=O)-* and R4is unsubstituted methyl, unsubstituted C3 alkyl, unsubstituted Cs alkyl, or unsubstituted C10 alkyl. In some embodiments, L3has one of the following structures:

[0105] I , with one or more oxo substituents. In certain embodiments, L3has the following structure:

[0106] In certain embodiments, L3is -C(=O)-NR4-* and R4is optionally substituted C1-C10 alkyl. In some embodiments, L3is -C(=O)-O-L3a-* and L3ais a direct bond, methylene, or ethylene. In certain embodiments, L3is -O-C(=O)-L3a-* and L3ais a direct bond or methylene. In some embodiments, L4is a direct bond or methylene.

[0107] In certain embodiments, R1is -NRla-C(=O)-Rlbor -C(=O)-N(Rla)Rlb. In some embodiments, R1-O-C(=O)-Rlaor -C(=O)-O-Rla. In certain embodiments, Rlais C4-C18 alkyl. In some embodiments, Rlais C4 alkyl, Ce alkyl, Cs alkyl, C9 alkyl, C10 alkyl, C12 alkyl, C14 alkyl, Ci6 alkyl, C17 alkyl, or Cis alkyl. In certain embodiments, Rlbis C4-C16 alkyl. In some embodiments, Rlbis C4 alkyl, Ce alkyl, Cs alkyl, C9 alkyl, C10 alkyl, C12 alkyl, C14 alkyl, Ci6 alkyl, C17 alkyl, or Cis alkyl. In some embodiments, Rlais unbranched and unsubstituted. In certain embodiments, Rlais branched. In some embodiments, Rlais optionally substituted with one or more fluoro substituents. In some embodiments, Rlbis unbranched and unsubstituted. In certain embodiments, Rlbis branched. In some embodiments, Rlbis optionally substituted with one or more fluoro substituents.

[0108] In some embodiments, R1has one of the following structures: In certain embodiments, R1has one of the following structures: In some embodiments, R2is -NR2a-C(=O)-R2bor -C(=O)-N(R2a)R2b. In certain embodiments, R2-O-C(=O)-R2aor -C(=O)-O-R2a. In some embodiments, R2ais C4-C18 alkyl. In certain embodiments, R2ais C4 alkyl, Ce alkyl, Cs alkyl, C9 alkyl, C10 alkyl, C11 alkyl, C12 alkyl, C14 alkyl, Ci6 alkyl, C17 alkyl, or Cis alkyl. In some embodiments, R2bis C4-C16 alkyl. In some embodiments, R2bis C4 alkyl, Ce alkyl, Cs alkyl, C9 alkyl, C10 alkyl, C12 alkyl, C14 alkyl, Ci6 alkyl, C17 alkyl, or Cis alkyl. In certain embodiments, R2ais unbranched and unsubstituted. In some embodiments, R2ais branched. In certain embodiments, R2ais optionally substituted with one or more fluoro substituents. In some embodiments, R2bis unbranched and unsubstituted. In certain embodiments, R2bis branched. In some embodiments, R2bis optionally substituted with one or more fluoro substituents. In certain embodiments, R2has one of the following structures:

[0109] In some embodiments, R2has one of the following structures:

[0110]

[0111] In some embodiments, R3ais unsubstituted C1-C4 alkyl. In certain embodiments, R3bis unsubstituted C1-C4 alkyl. In some embodiments, R3aand R3bare the same. In certain embodiments, R3aand R3bare both methyl.

[0112] In some embodiments, R3aand R3b, together with the nitrogen to which they are attached, join to form an optionally substituted 3-8 membered heterocyclyl. In certain embodiments, R3aand R3b, together with the nitrogen to which they are attached, join to form an optionally substituted 5-6 membered heterocyclyl. In some embodiments, R3aand R3b, together with the nitrogen to which they are attached, join to form an optionally substituted 5 membered heterocyclyl. In some more specific embodiments, R3aand R3b, together with the nitrogen to which they are attached, join to form an unsubstituted pyrrolidinyl.

[0113] In various embodiments, the compound has one of the structures set forth in Table 1 below. It is understood that any embodiment of the compounds of Formula (I), as set forth above, and any specific substituent and / or variable in the compound Formula (I), as set forth above, may be independently combined with other embodiments and / or substituents and / or variables of compounds of Formula (I) to form embodiments of the disclosure not specifically set forth above. In addition, in the event that a list of substituents and / or variables is listed for any particular G group, R group, or L group 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 that the remaining list of substituents and / or variables will be considered to be within the scope of embodiments of the disclosure.

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

[0115] For the purposes of administration, the compounds of the present disclosure (typically in the form of lipid nanoparticles in combination with a therapeutic agent) may be administered as a raw chemical or may be formulated as pharmaceutical compositions. Pharmaceutical compositions (e.g., lipid nanoparticles) of the present disclosure comprise a compound of Formula (I) and one or more pharmaceutically acceptable carrier, diluent, or excipient. The compound of Formula (I) is present in the composition in an amount which is effective to form a lipid nanoparticle and deliver the therapeutic agent, e.g., for treating a particular disease or condition of interest. Appropriate concentrations and dosages can be readily determined by one skilled in the art.

[0116] An embodiment provides a composition (e.g., lipid nanoparticles) comprising a compound of Formula (I) and a therapeutic agent. In some embodiments, the composition (e.g., lipid nanoparticles) further comprises one or more excipient selected from neutral lipids, steroids, and polymer conjugated lipids.

[0117] In some embodiments, the therapeutic agent comprises a nucleic acid. In certain embodiments, the nucleic acid is selected from antisense and messenger RNA.

[0118] In certain embodiments, the composition comprises one or more neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC.

[0119] In some embodiments, the molar ratio of the compound to the neutral lipid ranges from about 2: 1 to about 8: 1. In certain embodiments, the steroid is cholesterol. In some embodiments, the molar ratio of the compound to cholesterol ranges from about 2: 1 to 1 : 1. In some embodiments, the ratio is from 5 : 1 to 1 : 1 or from 2: 1 to 1 : 1. In certain embodiments, the polymer conjugated lipid is a pegylated lipid. In various embodiments, the polymer conjugated lipid is a pegylated lipid. For example, some embodiments include a pegylated diacylglycerol (PEG-DAG) such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4- 0-(2',3'-di(tetradecanoyloxy)propyl-l-0-(co-methoxy(polyethoxy)ethyl)butanedioate (PEG-S- DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co- methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3- di(tetradecanoxy)propyl-N-(co-methoxy(polyethoxy)ethyl)carbamate.

[0120] In some embodiments, the molar ratio of the compound to the pegylated lipid ranges from about 100: 1 to about 10: 1 or from about 100: 1 to about 25: 1. In some embodiments, the molar ratio of the compound to pegylated lipid ranges from about 100: 1 to about 20: 1 or from about 100: 1 to about 10: 1.

[0121] In some embodiments, the pegylated lipid is PEG-DMG. In certain embodiments, the pegylated lipid has the following Formula (II): or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:

[0122] R5and R6are each independently a straight or branched, alkyl, alkenyl or alkynyl from 10 to 30 carbon atoms, wherein the alkyl, alkenyl or alkynyl is optionally interrupted by one or more ester bonds; and w has a value ranging from 30 to 60.

[0123] In certain embodiments, R5and R6are each independently straight alkyl chain containing from 12 to 16 carbon atoms. In some embodiments, w is 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55. In certain embodiments, w has a value ranging from 40 to 50. In some embodiments, w ranges from 42 to 48. In some embodiments, w is 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0124] In some embodiments, the lipid nanoparticle or composition comprises a plurality of pegylated lipids of Formula (II) and the average w for the plurality ranges from 40 to 50. In some embodiments, the average w is 43, 44, 45, 46, 47, or 48.

[0125] Synthesis of pegylated lipids can be found in US Patent No. 9,738,593, the disclosure of which is hereby incorporated by reference. In some embodiments, the lipid nanoparticle or composition comprises 45-50 mol % of a compound of Formula (I), 5-15 mol% DSPC, 35-45 mol% cholesterol, and 1.5-2.5 mol% of a pegylated lipid based on the total moles of each of the components.

[0126] Administration of the compositions of the disclosure can be carried out via any of the accepted modes of administration of agents for serving similar utilities. For example, the pharmaceutical compositions of the disclosure may be formulated into preparations into a liquid solution such as solutions, suspensions, injections, etc. A typical route of administering pharmaceutical compositions of the present disclosure includes parenteral injection. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intradermal, intrastemal injection or infusion techniques. In some embodiments, the administering is performed via an intravenous or intramuscular injection. Pharmaceutical compositions of the disclosure are formulated to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a subject or patient take the form of one or more dosage units, where for example, a single injection may be a single dosage unit, and a container (e.g., a vial) of LNPs of the disclosure may hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition or LNPs to be administered will, in any event, contain a therapeutically effective amount of a therapeutic agent of the disclosure (e.g., antisense RNA, mRNA, etc ), or a pharmaceutically acceptable salt thereof, for treatment of a disease or condition of interest in accordance with the teachings of this disclosure.

[0127] A pharmaceutical composition of the disclosure may be in the form of a liquid solution. In some embodiments, the pharmaceutical composition comprises carrier(s) that are liquid, with the compositions being, for example, an injectable liquid.

[0128] The pharmaceutical composition may be in the form of a liquid, for example, a solution, emulsion, or suspension. The liquid may be formulated as a liquid for delivery by injection, as an example. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer, and isotonic agent may be included.

[0129] The liquid pharmaceutical compositions of the disclosure, whether they be solutions, suspensions or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose; agents to act as cryoprotectants such as sucrose or trehalose. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile.

[0130] A liquid pharmaceutical composition of the disclosure intended for parenteral should contain an amount of a LNP of the disclosure such that a suitable dosage will be obtained.

[0131] The pharmaceutical compositions of the disclosure may be prepared by methodology well known in the pharmaceutical art. For example, a pharmaceutical composition intended to be administered by injection can be prepared by combining the lipid nanoparticles of the disclosure with sterile, distilled water or other carrier to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the compound of the disclosure to facilitate dissolution or homogeneous suspension of the compound in the aqueous delivery system.

[0132] The compositions of the disclosure are administered in a therapeutically effective amount, which will vary depending upon a variety of factors including (1) the activity of the specific therapeutic agent employed; (2) the metabolic stability and length of action of the therapeutic agent; (3) the age, body weight, general health, sex, and diet of the patient; (4) the mode and time of administration; (5) the rate of excretion; (6) the drug combination; (7) the severity of the particular disorder or condition; and (8) the subject undergoing therapy.

[0133] Compositions of the disclosure may also be administered simultaneously with, prior to, or after administration of one or more other therapeutic agents. Such combination therapy includes administration of a single pharmaceutical dosage formulation of a composition of the disclosure and one or more additional active agents, as well as administration of the composition of the disclosure and each active agent in its own separate pharmaceutical dosage formulation. For example, a composition of the disclosure and the other active agent can be administered to the patient together in a single liquid dosage composition such as an injection, or each agent administered in separate dosage formulations. Where separate dosage formulations are used, the compositions of the disclosure and one or more additional active agents can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially; combination therapy is understood to include all these regimens.

[0134] Preparation methods for the above compounds and compositions are described herein below and / or known in the art. It will be appreciated by those skilled in the art that in the process described herein the functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxy, amino, mercapto and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (for example, t- butyldimethylsilyl, / -butyldiphenylsilyl or trimethyl silyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino and guanidino include / -butoxy carbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include -C(O)-R" (where R" is alkyl, aryl or arylalkyl), / ?-methoxybenzyl, trityl and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl or arylalkyl esters. Protecting groups may be added or removed in accordance with standard techniques, which are known to one skilled in the art and as described herein. The use of protecting groups is described in detail in Green, T.W. and P.G.M. Wutz, Protective Groups in Organic Synthesis (1999), 3rdEd., Wiley. As one of skill in the art would appreciate, the protecting group may also be a polymer resin such as a Wang resin, Rink resin or a 2-chlorotrityl-chloride resin.

[0135] Furthermore, all compounds of the disclosure which exist in free base or acid form can be converted to their pharmaceutically acceptable salts by treatment with the appropriate inorganic or organic base or acid by methods known to one skilled in the art. Salts of the compounds of the disclosure can be converted to their free base or acid form by standard techniques (e.g., pH adjustment).

[0136] The following examples show methods of preparing compounds of this disclosure, / .<?., compounds of Formula (I): or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein R1, R2, R3a, R3b, L1, L2, L3, L4, and ring A are as defined herein. GENERAL REACTION SCHEME 1

[0137] Embodiments of the compound of Formula (I) can be prepared according to General Reaction Scheme 1. The variables shown in the example above are selected such that the final compound is a compound of Formula (I). Namely, the variables R1, R2, R3a, R3b, L1, L2, L3, L4, and A are as defined by embodiments of the disclosure. The variables Glaand Glb, G2aand G2b, and G3aand G3bare respective complimentary reactive groups that undergo a chemical reaction to form a linkage between the synthons as represented by General Reaction Scheme 1 (e.g., a primary or secondary amine and a halo, a carboxylic acid or activated ester and an amine or hydroxy). Additionally, variables R1', R2, and L3' are selected such that the variables R1, R2, and L3(respectively) are formed because of the chemical reactions as indicated. Appropriate reaction conditions (e.g., acidic, basic, aprotic, heating, time, efc.) are selected to facilitate the appropriate reaction steps as shown. Work-up and purification steps are not necessarily shown but are performed as necessary. GENERAL REACTION SCHEME 2

[0138] Embodiments of the compound of Formula (I) can also be prepared according to General Reaction Scheme 2. The variables shown in the example above are selected such that the final compound is a compound of Formula (I). Namely, the variables R1, R2, R3a, R3b, R4, L1, L2, L4, and A are as defined by embodiments of the disclosure. The primary amine starting material shown undergoes a reductive amination with the ketone containing species shown. After completion, the resultant secondary amine is coupled with an appropriate reagent (e.g., an acid chloride) to connect R4to the nitrogen of the secondary amine and yield a product compound of Formula (I). Appropriate reaction conditions (e.g., acidic, basic, aprotic, heating, time, efc.) are selected to facilitate the appropriate reaction steps as shown. Work-up and purification steps are not necessarily shown but are performed as necessary. GENERAL REACTION SCHEME 3

[0139] Embodiments of the compound of Formula (I) can also be prepared according to General Reaction Scheme 3. The variables shown in the example above are selected such that the final compound is a compound of Formula (I). Namely, the variables R1, R2, R3a, R3b, R4, L1, L2, L4, and A are as defined by embodiments of the disclosure. The primary amine starting material shown undergoes a reductive amination with the ketone containing species shown. After completion, the resultant secondary amine is coupled with an appropriate reagent (e.g., a carboxylic acid) to connect A to the nitrogen of the secondary amine and yield a product compound of Formula (I). Appropriate reaction conditions (e.g., acidic, basic, aprotic, heating, time, efc.) are selected to facilitate the appropriate reaction steps as shown. Work-up and purification steps are not necessarily shown but are performed as necessary.

[0140] It should also be noted that various alternative strategies for preparation of compounds of Formula (I) are available to those of ordinary skill in the art. For example, appropriate protecting groups may be required to mask desired substituents (e.g., a substituent on A). The use of protecting groups as needed and other modification to the above General Reaction Scheme 1 will be readily apparent to one of ordinary skill in the art.

[0141] It is understood that one skilled in the art may be able to make these compounds by similar methods or by combining other methods known to one skilled in the art. It is also understood that one skilled in the art would be able to make, in a similar manner as described below, other compounds of Formula (I) not specifically illustrated below by using the appropriate starting components and modifying the parameters of the synthesis as needed. In general, starting components may be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, etc. or synthesized according to sources known to those skilled in the art (see, e.g., Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)) or prepared as described in this disclosure. Purification (e.g., silica gel chromatography, filtration, extraction, efc.) after each described reaction step is performed as needed.

[0142] EXAMPLE 1

[0143] IMMUNOGLOBULIN G (IGG) MRNA IN VIVO EVALUATION USING LIPID NANOPARTICLE COMPOSITIONS

[0144] A lipid of Formula (I), DSPC, cholesterol and PEG-lipid of Formula (II) are solubilized in ethanol at a molar ratio of 47.5: 10:40.7: 1.8. Lipid nanoparticles (LNP) are prepared at a total lipid to mRNA weight ratio of approximately 10: 1 to 40: 1. Briefly, the mRNA is diluted to 0.2 mg / mL in 10 to 50 mM citrate buffer, pH 4 to 6 or 10 to 25 mM acetate buffer, pH 4 to 6. Syringe pumps are used to mix the ethanolic lipid solution with the mRNA aqueous solution at a ratio of about 1 :5 to 1 :3 (vol / vol) with total flow rates above 15 mL / min. The ethanol is then removed, and the external buffer replaced with PBS by dialysis. Finally, the lipid nanoparticles are filtered through a 0.2 pm pore sterile filter.

[0145] Studies are performed in 6-8-week-old CD-l / ICR mice (Charles River or Inotiv) according to guidelines established by an institutional animal care committee (ACC) and the Canadian Council on Animal Care (CCAC). Varying doses of mRNA-lipid nanoparticle are systemically administered by tail vein injection and animals euthanized at a specific time point (e.g., 24 hours) post-administration. The whole blood is collected, and the serum subsequentially separated by centrifuging the tubes of the whole blood at 2000 * g for 10 minutes at 4 °C and stored at -80 °C until use for analysis.

[0146] For immunoglobulin G (IgG) ELISA (Life Diagnostics Human IgG ELISA kit), the serum samples are diluted at 100 to 20,000 folds with 1 x diluent solution. 100 pL of diluted serum is dispensed into anti-human IgG coated 96-well plate in duplicate alongside human IgG standards and incubated in a plate shaker at 150 rpm at 25 °C for 45 minutes. The wells are washed 5 times with lx wash solution using a plate washer (400 pL / well). 100 pL of HRP conjugate is added into each well and incubated in a plate shaker at the same condition above. The wells are washed 5 times again with 1 x wash solution using a plate washer (400 pL / well). 100 pL of TMB reagent is added into each well and incubated in a plate shaker at the same condition above. The reaction is stopped by adding 100 pL of Stop solution to each well. The absorbance is read at 450 nm (A450) with a microplate reader. The amount of human IgG in mouse serum is determined by plotting A450 values for the assay standard against human IgG concentration.

[0147] EXAMPLE 2

[0148] DETERMINATION OF PKA OF FORMULATED LIPIDS

[0149] As described elsewhere, the pKa of formulated lipids is correlated with the effectiveness of LNPs for delivery of nucleic acids (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176 (2010)). In some embodiments, the preferred range of pKa is ~5 to ~7. The pKaof each lipid may be determined in lipid nanoparticles using an assay based on fluorescence of 2-(p-toluidino)-6- napthalene sulfonic acid (TNS). Lipid nanoparticles comprising compound of Formula (I) / DSPC / cholesterol / PEG-lipid (47.5:10:40.7: 1.8 mol%) in PBS at a concentration of 0.4 mM total lipid are prepared using the in-line process as described in Example 1. TNS is prepared as a 100 pM stock solution in distilled water. Vesicles are diluted to 24 pM lipid in 2 mL of buffered solutions containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl, where the pH ranged from 2.5 to 11. An aliquot of the TNS solution is added to give a final concentration of 1 pM and following vortex mixing fluorescence intensity is measured at room temperature in a SLM Aminco Series 2 Luminescence Spectrophotometer using excitation and emission wavelengths of 321 nm and 445 nm. A sigmoidal best fit analysis is applied to the fluorescence data and the pKais measured as the pH giving rise to half-maximal fluorescence intensity.

[0150] EXAMPLE 3

[0151] DETERMINATION OF EFFICACY OF LIPID NANOPARTICLE FORMULATIONS CONTAINING VARIOUS CATIONIC LIPIDS USING IGG MRNA EXPRESSION RODENT MODEL

[0152] Representative compounds of the disclosure shown in Table 2 were formulated using the following molar ratio: 47.5% cationic lipid / 10% DSPC / 40.7% Cholesterol / 1.8% PEG lipid. The activity was determined by measuring the amount of human IgG in mouse serum as described in Example 1. The activity was compared at a dose of 1.0 or 0.3 mg mRNA / kg and expressed as pg IgG / mL serum measured 24 hours after administration. Compound numbers in Table 2 refer to the compound numbers of Table 1.

[0153] Table 2. Novel Cationic Lipids and Associated Activity SYNTHETIC EXAMPLE 1

[0154] N 1-BUTYL-N19,N19-DIDECYL-10-((1S,3S)-3-(DIMETHYLAMINO)-N-METHYLCYCLOBUTANE-1- CARBOXAMIDO)-N1-(HEPTADECAN-9-YL)NONADECANEDIAMIDE (COMPOUND 1-1)

[0155] Synthesis of Compound 1-1

[0156] JH NMR (400 MHz, CDCh) 6 4.62 - 4.50 (m, 1H), 3.69 - 3.45 (m, 1H), 3.32 - 3.23 (m, 2H), 3.23 - 3.15 (m, 2H), 3.11 - 2.97 (m, 2H), 2.87 - 2.74 (m, 1H), 2.67 (d, J= 5.5 Hz, 4H), 2.33 - 2.11 (m, 14H), 1.75 - 1.03 (m, 94H), 1.00 - 0.83 (m, 15H). m / z calculated for Chemical Formula: C68H134N4O3 = 1055.1. Found [M+H]+= 1056.4.

[0157] SYNTHETIC EXAMPLE 2

[0158] 1 , 19-BIS(DIDECYL AMINO)- 1 , 19-DIOXONONADEC AN- 10- YL ( 1 S,3s)-3- ((DIMETHYLAMINO)METHYL)CYCLOBUTANE-1-CARBOXYLATE (COMPOUND 1-2)

[0159] Synthesis of Compound 1-2

[0160] 'HNMR (400 MHz, CDCh) 6 3.32 - 3.23 (m, 4H), 3.23 - 3.15 (m, 4H), 3.04 - 2.93 (m, 1H), 2.46 - 2.14 (m, 15H), 1.99 - 1.86 (m, 2H), 1.73 - 1.41 (m, 19H), 1.36 - 1.23 (m, 78H), 0.92 - 0.83 (m, 12H). m / z calculated For Chemical Formula: C67H131N3O4 = 1042.0. Found [M+H]+= 1043.5. SYNTHETIC EXAMPLE 3

[0161] 1,13-BIS(DIDECYLAMINO)-1,13-DIOXOTRIDECAN-7-YL (1R,3R)-3-

[0162] ((DIMETHYLAMINO)METHYL)CYCLOBUTANE-1-CARBOXYLATE (COMPOUND 1-3) Synthesis of Compound 1-3

[0163] 'HNMR (400 MHz, CDCh) 6 4.91 - 4.78 (m, 1H), 3.31 - 3.14 (m, 8H), 3.09 - 2.90 (m, 1H), 2.65 - 2.10 (m, 15H), 2.04 - 1.84 (m, 2H), 1.77 - 1.04 (m, 87H), 0.92 - 0.83 (m, 12H). m / z calculated For Chemical Formula: C61H119N3O4 = 957.9. Found [M+H]+= 959.3.

[0164] SYNTHETIC EXAMPLE 4 N1 ,N1 ,N19,N 19-TETRAKIS(DECYL)- 10-((l S,3S)-3-(DIMETHYLAMINO)-N-

[0165] METHYLCYCLOBUTANE-1-CARBOXAMIDO)NONADECANEDIAMIDE (COMPOUND 1-4)

[0166] Synthesis of Compound 1-4

[0167] 1H NMR (400 MHz, CDCh) 6 4.61 - 4.48 (m, 1H), 3.53 - 3.44 (m, 1H), 3.31 - 3.23 (m, 4H), 3.23 - 3.14 (m, 4H), 2.88 - 2.75 (m, 1H), 2.73 - 2.61 (m, 4H), 2.32 - 2.14 (m, 14H), 1.83 -

[0168] 0.98 (m, 98H), 0.92 - 0.83 (m, 12H). m / z calculated for Chemical Formula: C67H132N4O3 = 1041.0. Found [M+H]+= 1042.4. SYNTHETIC EXAMPLE 5

[0169] N 1 ,N 19-DIBUTYL- 10-(( 1 s,3s)-N -DEC YL-3-(DIMETHYLAMINO)CYCLOBUT ANE- 1 -

[0170] CARBOXAMIDO)-N1,N19-DIOCTYLNONADECANEDIAMIDE (COMPOUND 1-5)

[0171] Synthesis of Compound 1-5

[0172] 'HNMR (400 MHz, CDCh) 6 3.50 - 3.39 (m, 1H), 3.34 - 3.27 (m, 4H), 3.25 - 3.16 (m, 4H), 3.09 - 2.93 (m, 2H), 2.83 - 2.57 (m, 2H), 2.31 - 2.13 (m, 14H), 1.73 - 1.04 (m, 84H), 0.99 - 0.83 (m, 15H). m / z calculated for Chemical Formula: C60H118N4O3 = 942.9. Found [M+H]+= 944.3.

[0173] SYNTHETIC EXAMPLE 6

[0174] 10-((l S,3S)-N-DECYL-3-(DIMETHYLAMINO)CYCLOBUTANE- 1-CARBOXAMIDO)-N 1 ,N1 ,N19,N19-

[0175] TETRAOCTYLNONADECANEDIAMIDE (COMPOUND 1-6)

[0176] Synthesis of 10-oxononadecanedioyl di chloride

[0177] To a mixture of commercially available 10-oxononadecanedioic acid (3.0 g, 8.8 mmol) and DMF (catalytic) in dichloromethane (180 mL) was added oxalyl chloride (2.59 mL, 30.7 mmol). The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated to dryness to give compound 10-oxononadecanedioyl dichloride which was used in the next step without further purification.

[0178] Synthesi s of N 1 ,N1 ,N 19,N 19-tetraoctyl- 10-oxononadecanediamide

[0179] To a mixture of triethylamine (0.58 mL, 4.2 mmol) and dioctylamine (636 mg, 2.6 mmol) in dichloromethane (5.3 mL) was added compound 10-oxononadecanedioyl di chloride (0.40 g,

[0180] 1.1 mmol) in dichloromethane (1 mL). The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated, and the crude material was suspended in hexanes then filtered. The filtrate was purified via flash chromatography (0% to 20% EtOAc in hexanes) to give compound N1,N1,N19, N19-tetraoctyl-10-oxononadecanediamide (646 mg, 78%).

[0181] Synthesis of 10-(decylamino)-N 1 ,N1 ,N19,N 19-tetraoctylnonadecanediamide

[0182]

[0183] Compound 10-(decylamino)-Nl,Nl,N19,N19-tetraoctylnonadecanediamide was prepared from compound Nl, N1,N19, N19-tetraoctyl-10-oxononadecanediamide and decylamine according to the procedure for the synthesis of Nl,Nl,N19,N19-tetrakis(decyl)-10- (decylamino)nonadecanediamide. Yield (231 mg, 65%).

[0184] Synthesis of 10-(( 1 s,3 s)-N-decyl-3 -(dimethylamino)cyclobutane- 1 -carboxamido)-

[0185] Nl,Nl,N19,N19-tetraoctylnonadecanediamide (Compound 1-6)

[0186]

[0187] A mixture of compound 10-(decylamino)-Nl,Nl,N19,N19-tetraoctylnonadecanediamide

[0188] (114 mg, 0.12 mmol), DIEA (0.085 mL, 0.49 mmol), (ls,3s)-3-(dimethylamino)cyclobutane-l- carboxylic acid hydrochloride (24 mg, 0.14 mmol), and HATU (61 mg, 0.16 mmol) in dichloromethane (5 mL) was stirred at room temperature for 1 h. The reaction mixture was concentrated, and the crude material was partitioned between EtOAc and sat. NaHCCh. Purification via flash chromatography (5% to 65% EtOAc in hexanes with 1% EtsN) gave Compound 1-6 (80 mg, 62%).

[0189] 'HNMR (400 MHz, CDCh) 6 3.51 - 3.39 (m, 1H), 3.32 - 3.24 (m, j4H), 3.20 - 3.19 (m, 4H), 3.07 - 2.92 (m, 2H), 2.84 - 2.56 (m, 2H), 2.31 - 2.09 (m, 14H), 1.72 - 1.06 (m, 98H), 0.93

[0190] - 0.83 (m, 15H). m / z calculated for Chemical Formula: C68H134N4O3 = 1055.1. Found [M+H]+=

[0191] 1056.3. SYNTHETIC EXAMPLE 7

[0192] N1,N1,N 13, N 13-TETRAKIS(DECYL)-7-((1S,3S)-N-DECYL-3-(DIMETHYLAMINO)CYCLOBUTANE- 1-CARBOXAMIDO)TRIDECANEDIAMIDE (COMPOUND 1-7) Synthesis of Compound 1-7

[0193] 'HNMR (400 MHz, CDCh) 6 3.53 - 3.38 (m, 1H), 3.31 - 3.14 (m, 8H), 3.05 - 2.91 (m, 2H), 2.83 - 2.71 (m, 1H), 2.69 - 2.56 (m, 1H), 2.30 - 2.08 (m, 13H), 1.76 - 1.03 (m, 99H), 0.94 - 0.83 (m, 15H). m / z calculated for Chemical Formula: C70H138N4O3 = 1083.1. Found [M+H]+= 1084.4. SYNTHETIC EXAMPLE 8

[0194] N1 ,N1 ,N19,N 19-TETRAKIS(DECYL)- 10-((1R,3R)-N-DECYL-3-

[0195] (DIMETHYLAMINO)CYCLOBUTANE-1-CARBOXAMIDO)NONADECANEDIAMIDE (COMPOUND 1-8)

[0196] Synthesis of Compound 1-8 'HNMR (400 MHz, CDCh) 6 3.60 - 3.52 (m, 1H), 3.49 - 3.42 (m, 4H), 3.41 - 3.28 (m,

[0197] 5H), 3.29 - 3.17 (m, 1H), 3.15 - 2.90 (m, 2H), 2.60 - 2.48 (m, 2H), 2.48 - 2.39 (m, 4H), 2.33 - 2.22 (m, 8H), 1.89 - 1.21 (m, 115H), 1.10 - 1.01 (m, 15H). m / z calculated for Chemical Formula: C76H150N4O3 = 1167.2. Found [M+H]+= 1168.4. SYNTHETIC EXAMPLE 9

[0198] N1,N1,N19,N19-TETRAKIS(DECYL)-10-((1S,3S)-N-DECYL-3-(DIMETHYLAMINO)CYCLOBUTANE-

[0199] 1-CARBOXAMIDO)NONADECANEDIAMIDE (COMPOUND 1-9) Synthesis of N1 ,N1 ,N19,N 19-tetrakis(decyl)- 10-oxononadecanediamide

[0200] A mixture of commercially available 10-oxononadecanedioic acid (2.0 g, 5.8 mmol), didecylamine (3.82 g, 12.8 mmol), DIEA (6.1 mL, 35 mmol), and HATU (5.77 g, 15.2 mmol) in dichloromethane (29 mL) was stirred at room temperature for 20 h. The reaction mixture was concentrated and partitioned between EtOAc and sat. NaHCCh. The organic layer was separated and concentrated. Purification via flash chromatography (5% to 50% EtOAc in hexanes) gave compound N1,N1,N19, N19-tetrakis(decyl)-l O-oxononadecanediamide (4.29 g, 81%).

[0201] Synthesis of N1 ,N1 ,N19,N 19-tetrakis(decyl)- 10-(decylamino)nonadecanediamide

[0202]

[0203] A mixture of compound N1,N1,N19, N19-tetrakis(decyl)-10-oxononadecanediamide (1.5 g, 1.66 mmol), decylamine (393 mg, 2.5 mmol), acetic acid (0.14 mL, 2.5 mmol), and sodium triacetoxyborohydride (705 mg, 3.3 mmol) in di chloroethane (10 mL) was stirred at room temperature for 20 h. The reaction mixture was concentrated, and the crude was partitioned between EtOAc and sat. NaHCCh. The organic layer was separated and concentrated. Purification via flash chromatography (10% to 100% EtOAc in hexanes) gave compound Nl,Nl,N19,N19-tetrakis(decyl)-10-(decylamino)nonadecanediamide (1.17 g, 67%). Synthesis of tert-butyl ((ls,3s)-3-((l,19-bis(didecylamino)-l,19-dioxononadecan-10- yl)(decyl)carbamoyl)cyclobutyl)carbamate

[0204]

[0205] A mixture of compound N1 ,N1 ,N19,N 19-tetrakis(decyl)- 10- (decylamino)nonadecanediamide (200mg, 0.19 mmol), (ls,3s)-3-((tert- butoxycarbonyl)amino)cyclobutane-l -carboxylic acid (43 mg, 0.2 mmol), DIEA (0.1 mL, 0.58 mmol), and HATU (95 mg, 0.25 mmol) in dichloromethane (1 mL) was stirred at room temperature for 40 min. The reaction mixture was concentrated, and the crude was partitioned between EtOAc and sat. NaHCCh. The organic layer was separated and concentrated. Purification via flash chromatography (5% to 100% EtOAc in hexanes) gave compound tertbutyl (( 1 s,3 s)-3 -((1,19-bis(didecylamino)- 1 , 19-di oxononadecan- 10- yl)(decyl)carbamoyl)cyclobutyl)carbamate (180mg, 76%).

[0206] Synthesis of 10-(( 1 s,3 s)-3 -amino-N-decyl cyclobutane- 1 -carboxamido)-N 1 ,N1 ,N19,N 19- tetrakis(decyl)nonadecanediamide

[0207] A mixture of compound tert-butyl ((ls,3s)-3-((l,19-bis(didecylamino)-l,19- di oxononadecan- 10-yl)(decyl)carbamoyl)cy cl obutyl)carbamate (180 mg, 0.15 mmol) and trifluoroacetic acid (1 mL) in dichloromethane (1.5 mL) was stirred at room temperature for 1 h. The reaction mixture was concentrated, and the crude was partitioned between EtOAc and sat. NaHCCh. The organic layer was separated and concentrated to give 10-((ls,3s)-3-amino-N- decylcyclobutane- 1 -carboxamido)-N 1 ,N1 ,N19,N 19-tetrakis(decyl)nonadecanediamide (183 mg, quantitative). Synthesis of N1 ,N1 ,N19,N 19-tetrakis(decyl)- 10-(( 1 s,3 s)-N-decyl-3 -

[0208] (dimethylamino)cyclobutane- 1 -carboxamido)nonadecanediamide (Compound 1-9)

[0209]

[0210] A mixture of compound 10-((ls,3s)-3-amino-N-decylcyclobutane-l-carboxamido)- Nl,Nl,N19,N19-tetrakis(decyl)nonadecanediamide (120 mg, 0.11 mmol), 37% formaldehyde in water (2 mL), and sodium triacetoxyborohydride (94 mg, 0.44 mmol) in methanol (4 mL) was stirred at room temperature for 20 h. Additional 37% formaldehyde in water (2 mL) and sodium triacetoxyborohydride (94 mg, 0.44 mmol) was added and the reaction mixture was stirred for a further 72 h. The reaction mixture was concentrated, and the crude was partitioned between EtOAc and sat. NaHCCh. The organic layer was separated and concentrated. Purification via flash chromatography (5% to 65% EtOAc in hexanes with 1% EtsN) gave Compound 1-9 (80 mg, 65%).

[0211] 'HNMR (400 MHz, CDCh) 8 3.50 - 3.40 (m, 1H), 3.32 - 3.22 (m, 4H), 3.22 - 3.13 (m,

[0212] 4H), 3.05 - 2.93 (m, 2H), 2.82 - 2.57 (m, 2H), 2.32 - 2.09 (m, 14H), 1.77 - 1.06 (m, 116H), 0.92

[0213] - 0.83 (m, 15H). m / z calculated for Chemical Formula: C76H150N4O3 = 1167.2. Found [M+H]+=

[0214] 1168.4. SYNTHETIC EXAMPLE 10

[0215] 1 , 13-BIS(DIDEC YLAMINO)- 1 , 13-DIOXOTRIDE CAN-7- YL ( 1 S,3s)-3- (DIMETHYLAMINO)CYCLOBUTANE-l-CARBOXYLATE (COMPOUND 1-10) Synthesis of Compound I- 10

[0216] 'HNMR (400 MHz, CDCh) 6 4.92 - 4.81 (m, 1H), 3.33 - 3.25 (m, 4H), 3.25 - 3.16 (m, 4H), 2.78 - 2.54 (m, 2H), 2.38 - 2.23 (m, 6H), 2.21 - 2.03 (m, 8H), 1.75 - 1.05 (m, 85H), 0.94 - 0.86 (m, 12H). m / z calculated For Chemical Formula: C60H117N3O4 = 943.9. Found [M+H]+= 945.2..

[0217] SYNTHETIC EXAMPLE 11

[0218] 1 , 19-BIS(DIDECYL AMINO)- 1 , 19-DIOXONONADEC AN- 10- YL ( 1 S,4s)-4-

[0219] (DIMETHYLAMINO)CYCLOHEXANE-l-CARBOXYLATE (COMPOUND 1-11)

[0220] Synthesis of N1 ,N1 ,N19,N 19-tetrakis(decyl)- 10-hydroxynonadecanediamide

[0221]

[0222] A mixture of compound N1,N1,N19, N19-tetrakis(decyl)-10-oxononadecanediamide (500 mg, 0.56 mmol) and sodium borohydride (21 mg, 0.56 mmol) in MeOH (10 mL) was stirred at room temperature for 20 h. The reaction mixture was concentrated, and the crude material was partitioned between EtOAc and sat. NaHCCh to give compound N1 ,N1 ,N19,N 19-tetrakis(decyl)-

[0223] 10-hydroxynonadecanediamide (470 mg, 94%).

[0224] Synthesis of l,19-bis(didecylamino)-l,19-di oxononadecan- 10-yl (ls,4s)-4- (dimethylamino)cyclohexane-l -carboxylate (Compound 1-11)

[0225]

[0226] A mixture of compound N1,N1, N 19, N19-tetrakis(decyl)-10-hydroxynonadecanediamide (157 mg, 0.17 mmol), (ls,4s)-4-(dimethylamino)cyclohexane-l -carboxylic acid (33 mg, 0.19 mmol), DMAP (32 mg, 0.26 mmol), and DCC (72 mg, 0.35 mmol) in di chloromethane (2 mL) was stirred at room temperature for 20 h. The reaction mixture was concentrated, and the crude material was suspended in hexanes then filtered. The filtrate was purified via flash chromatography (5% to 100% EtOAc in hexanes with 1% EtsN) followed by a second purification via flash chromatography (1% to 10% MeOH in CHCh) to give Compound 1-11 (81 mg, 44%). 'HNMR (400 MHz, CDCh) 6 4.97 - 4.83 (m, 1H), 3.34 - 3.26 (m, 4H), 3.25 - 3.17 (m,

[0227] 4H), 2.59 - 2.49 (m, 1H), 2.35 - 2.08 (m, 13H), 1.86 - 1.03 (m, 102H), 0.94 - 0.85 (m, 12H). m / z calculated For Chemical Formula: C68H133N3O4 = 1056.0. Found [M+H]+= 1057.3. SYNTHETIC EXAMPLE 12

[0228] 6-((1S,3S)-3-(DIMETHYLAMINO)-N-OCTYLCYCLOBUTANE-1-CARBOXAMIDO)-N1,N1,N16,N16-

[0229] TETRAOCTYLHEXADECANEDIAMIDE (COMPOUND 1-12)

[0230] Synthesis of Compound 1-12

[0231] 1H NMR (400 MHz, CDCh) 6 3.53 - 3.41 (m, 1H), 3.35 - 3.12 (m, 8H), 3.10 - 2.93 (m, 2H), 2.84 - 2.57 (m, 2H), 2.31 - 2.11 (m, 14H), 1.77 - 1.03 (m, 86H), 0.93 - 0.83 (m, 15H). m / z calculated for Chemical Formula: C63H124N4O3 = 985.0. Found [M+H]+= 986.3.

[0232] SYNTHETIC EXAMPLE 13

[0233] 6-((1S,3S)-N-DECYL-3-(DIMETHYLAMINO)CYCLOBUTANE-1-CARBOXAMIDO)-N1,N1,N16,N16-

[0234] TETRAOCTYLHEXADECANEDIAMIDE (COMPOUND 1-13)

[0235] Synthesis of Compound 1-13

[0236] 1H NMR (400 MHz, CDCh) 6 3.50 - 3.36 (m, 1H), 3.32 - 3.13 (m, 8H), 3.11 - 2.78 (m, 3H), 2.66 - 2.14 (m, 13H), 1.79 - 1.04 (m, 89H), 0.93 - 0.83 (m, 15H). m / z calculated for Chemical Formula: C60H118N4O3 = 1013.0. Found [M+H]+= 1014.3.

[0237] SYNTHETIC EXAMPLE 14

[0238] 10-(N-((1S,3S)-3-(DIMETHYLAMINO)CYCLOBUTYL)NONANAMIDO)-N1,N1,N19,N19-

[0239] TETRAOCTYLNONADECANEDIAMIDE (COMPOUND 1-14) Synthesis of Compound 1-14

[0240] 'H NMR (400 MHz, CDCh) 6 3.99 - 3.73 (m, 0.5H), 3.65 - 3.53 (m, 0.5H), 3.32 - 3.12 (m, 8H), 3.11 - 2.88 (m, 1H), 2.60 - 2.01 (m, 15H), 1.98 - 1.03 (m, 94H), 0.93 - 0.83 (m, 15H). m / z calculated for Chemical Formula: C66H130N4O3 = 1027.0. Found [M+H]+= 1028.3.

[0241] SYNTHETIC EXAMPLE 15

[0242] N1 ,N1 ,N19,N 19-TETRAKIS(DECYL)- 10-((l S,3S)-3-(DIMETHYLAMINO)-N-

[0243] PROPYLCYCLOBUTANE-1-CARBOXAMIDO)NONADECANEDIAMIDE (COMPOUND 1-15)

[0244] Synthesis of Compound 1-15

[0245] 'HNMR (400 MHz, CDCh) 6 3.52 - 3.39 (m, 1H), 3.32 - 3.23 (m, 4H), 3.23 - 3.14 (m, 4H), 3.06 - 2.88 (m, 2H), 2.85 - 2.53 (m, 2H), 2.31 - 2.04 (m, 14H), 1.79 - 1.03 (m, 97H), 0.95 - 0.83 (m, 15H). m / z calculated for Chemical Formula: C69Hi36N4Ch = 1069.1. Found [M+H]+= 1070.3.

[0246] SYNTHETIC EXAMPLE 16

[0247] 10-((l S,3S)-N-DECYL-3-(DIMETHYLAMINO)CYCLOBUTANE- 1-CARBOXAMIDO)-N 1 ,N1 ,N19,N19-

[0248] TETRAHEXYLNONADECANEDIAMIDE (COMPOUND 1-16)

[0249] Synthesis of Compound 1-16

[0250] 'HNMR (400 MHz, CDCh) 6 3.50 - 3.39 (m, 1H), 3.32 - 3.24 (m, 4H), 3.23 - 3.15 (m, 4H), 3.09 - 2.91 (m, 2H), 2.83 - 2.54 (m, 2H), 2.32 - 2.05 (m, 14H), 1.80 - 1.04 (m, 79H), 0.94 - 0.83 (m, 15H). m / z calculated for Chemical Formula: C60H118N4O3 = 942.9. Found [M+H]+= 944.2. SYNTHETIC EXAMPLE 17

[0251] 1 , 19-BIS(DIOCTYLAMINO)- 1 , 19-DIOXONONADECAN- 10- YL ( 1 s,3s)-3- ((DIMETHYLAMINO)METHYL)CYCLOBUTANE-1-CARBOXYLATE (COMPOUND 1-17)

[0252] Synthesis of Compound 1-17

[0253] 'HNMR (400 MHz, CDCh) 6 4.89 - 4.77 (m, 1H), 3.32 - 3.15 (m, 8H), 3.06 - 2.91 (m, 1H), 2.49 - 2.11 (m, 15H), 1.98 - 1.86 (m, 2H), 1.76 - 1.04 (m, 81H), 0.93 - 0.83 (m, 12H). m / z calculated For Chemical Formula: C59H115N3O4 = 929.9. Found [M+H]+= 931.1.

[0254] SYNTHETIC EXAMPLE 18

[0255] 1 , 19-BIS(DIOCTYLAMINO)- 1 ,19-DIOXONONADECAN- 10-YL (1 s,4s)-4- (DIMETHYLAMINO)CYCLOHEXANE-l-CARBOXYLATE (COMPOUND 1-18)

[0256] Synthesis of Compound 1-18

[0257] 'HNMR (400 MHz, CDCh) 6 3.34 - 3.26 (m, 4H), 3.25 - 3.17 (m, 4H), 2.58 - 2.49 (m, 1H), 2.33 - 2.24 (m, 10H), 2.23 - 2.09 (m, 3H), 1.77 - 1.44 (m, 25H), 1.39 - 1.20 (m, 60H), 0.95 - 0.85 (m, 12H). m / z calculated For Chemical Formula: C60H117N3O4 = 943.9. Found [M+H]+= 945.1.

[0258] SYNTHETIC EXAMPLE 19

[0259] 1,13-BIS(DIDECYLAMINO)-1,13-DIOXOTRIDECAN-7-YL 2-((1S,3S)-3-

[0260] (DIMETHYLAMINO)CYCLOBUTYL)ACETATE (COMPOUND 1-19) Synthesis of Compound 1-19

[0261] 'HNMR (400 MHz, CDCh) 6 4.89 - 4.77 (m, 1H), 3.31 - 3.23 (m, 4H), 3.22 - 3.14 (m, 4H), 2.53 - 2.44 (m, 1H), 2.42 - 2.36 (m, 2H), 2.34 - 2.20 (m, 7H), 2.10 (s, 6H), 1.70 - 1.02 (m, 88H), 0.92 - 0.83 (m, 12H). m / z calculated For Chemical Formula: C61H119N3O4 = 957.9. Found [M+H]+= 945.1. SYNTHETIC EXAMPLE 20

[0262] 1 , 13-BIS(DIDEC YLAMINO)- 1 , 13-DIOXOTRIDE CAN-7- YL ( 1 S,4s)-4-

[0263] (DIMETHYLAMINO)CYCLOHEXANE-l-CARBOXYLATE (COMPOUND 1-20)

[0264] Synthesis of Compound 1-20 1H NMR (400 MHz, CDCh) 6 4.91 - 4.81 (m, 1H), 3.31 - 3.23 (m, 4H), 3.22 - 3.14 (m,

[0265] 4H), 2.53 - 2.45 (m, 1H), 2.30 - 2.23 (m, 10H), 2.19 - 2.01 (m, 3H), 1.72 - 1.02 (m, 91H), 0.92 - 0.83 (m, 12H). m / z calculated For Chemical Formula: C62H121N3O4 = 971.9. Found [M+H]+= 973.3. SYNTHETIC EXAMPLE 21

[0266] N1,N17-DIDECYL-9-(N-((1S,3S)-3-(DIMETHYLAMINO)CYCLOBUTYL)NONANAMIDO)-N1,N17-

[0267] DIOCTYLHEPTADECANEDIAMIDE (COMPOUND 1-21) Synthesis of Compound 1-21

[0268] 'HNMR (400 MHz, CDCh) 6 3.99 - 3.80 (m, 0.5H), 3.68 - 3.52 (m, 0.5H), 3.35 - 3.27 (m, 4H), 3.26 - 3.18 (m, 4H), 3.14 - 2.95 (m, 1H), 2.54 - 2.06 (m, 15H), 2.06 - 1.03 (m, 97H), 0.96 - 0.87 (m, 15H). m / z calculated for Chemical Formula: C68H134N4O3 = 1055.1. Found [M+H]+= 1056.4. SYNTHETIC EXAMPLE 22

[0269] N1,N 1-DIDECYL-10-(N-((1S,3S)-3-(DIMETHYLAMINO)CYCLOBUTYL)NONANAMIDO)-N19,N19-

[0270] DIOCTYLNONADECANEDIAMIDE (COMPOUND 1-22)

[0271] Synthesis of Compound 1-22 'HNMR (400 MHz, CDCh) 6 4.01 - 3.78 (m, 0.5H), 3.68 - 3.53 (m, 0.5H), 3.35 - 3.27

[0272] (m, 4H), 3.27 - 3.18 (m, 4H), 3.19 - 2.94 (m, 2H), 2.57 - 2.05 (m, 16H), 2.04 - 1.05 (m, 104H), 0.96 - 0.87 (m, 15H). m / z calculated for Chemical Formula: C70H138N4O3 = 1083.1. Found [M+H]+= 1084.4. SYNTHETIC EXAMPLE 23

[0273] 3-ETHYLNONYL 10-(N-((1S,3S)-3-(DIMETHYLAMINO)CYCLOBUTYL)NONANAMIDO)-19- (DIOCTYLAMINO)-19-OXONONADECANOATE (COMPOUND 1-23)

[0274] Synthesis of Compound 1-23

[0275] 'HNMR (400 MHz, CDCh) 6 4.08 (t, J= 7.1 Hz, 2H), 3.95 - 3.77 (m, 0.5H), 3.64 - 3.51 (m, 0.5H), 3.32 - 3.23 (m, 2H), 3.23 - 3.15 (m, 2H), 3.14 - 2.91 (m, 1H), 2.52 - 2.01 (m, 15H), 2.00 - 1.06 (m, 85H), 0.93 - 0.81 (m, 15H). m / z calculated For Chemical Formula: C61H119N3O4 = 957.9. Found [M+H]+= 959.3.

[0276] SYNTHETIC EXAMPLE 24

[0277] 3-ETHYLNONYL 10-((1S,3S)-N-DECYL-3-(DIMETHYLAMINO)CYCLOBUTANE-1-CARBOXAMIDO)-

[0278] 19-(DIOCTYLAMINO)-19-OXONONADECANOATE (COMPOUND 1-24)

[0279] Synthesis of Compound 1-24

[0280] 'HNMR (400 MHz, CDCh) 6 4.12 - 4.04 (m, 2H), 3.49 - 3.39 (m, 1H), 3.32 - 3.15 (m, 4H), 3.07 - 2.91 (m, 2H), 2.83 - 2.56 (m, 2H), 2.34 - 2.08 (m, 14H), 1.73 - 1.03 (m, 89H), 0.93 - 0.81 (m, 15H). m / z calculated For Chemical Formula: C63H123N3O4 = 986.0. Found [M+H]+= 987.4.

[0281] SYNTHETIC EXAMPLE 25

[0282] NONYL 10-(N-((1S,3S)-3-(DIMETHYLAMINO)CYCLOBUTYL)NONANAMIDO)-19- (DIOCTYLAMINO)-19-OXONONADECANOATE (COMPOUND 1-25) Synthesis of Compound 1-25

[0283] 'HNMR (400 MHz, CDCh) 6 4.05 (t, J= 6.7 Hz, 2H), 3.96 - 3.78 (m, 0.5H), 3.64 - 3.48 (m, 0.5H), 3.32 - 3.23 (m, 3H), 3.23 - 3.15 (m, 2H), 3.12 - 2.92 (m, 1H), 2.54 - 2.04 (m, 15H), 2.00 - 1.00 (m, 86H), 0.88 (qd, J= 6.4, 2.9 Hz, 12H). m / z calculated For Chemical Formula: C59H115N3O4 = 929.9. Found [M+H]+= 931.2.

[0284] SYNTHETIC EXAMPLE 26

[0285] NONYL 10-((l S,3S)-N-DECYL-3-(DIMETHYLAMINO)CYCLOBUTANE- 1-CARBOXAMIDO)- 19- (DIOCTYLAMINO)-19-OXONONADECANOATE (COMPOUND 1-26) Synthesis of Compound 1-26

[0286] 'HNMR (400 MHz, CDCh) 6 4.09 - 4.01 (m, 2H), 3.50 - 3.40 (m, 0.5H), 3.32 - 3.21 (m, 2H), 3.23 - 3.14 (m, 2H), 3.06 - 2.92 (m, 2H), 2.82 - 2.56 (m, 2H), 2.33 - 2.06 (m, 14H), 1.74 - 1.03 (m, 87H), 0.93 - 0.83 (m, 12H). m / z calculated For Chemical Formula: C61H119N3O4 = 957.9. Found [M+H]+= 959.2. SYNTHETIC EXAMPLE 27

[0287] BIS(2-BUT YLOCT YL) 6-(( 1 s,3 S)-N-DEC YL-3 -(DIMETH YL AMINO)C YCLOBUTANE- 1 - CARBOXAMIDO)HEXADECANEDIOATE (COMPOUND 1-27)

[0288] Synthesis of bis(2 -butyloctyl) 6-oxohexadecanedioate

[0289] bis(2 -butyloctyl) 6-oxohexadecanedioate was prepared from 6-oxohexadecanedioic acid and commercially available 2-butyloctan-l-ol according to the procedure for bis(2 -butyloctyl) 10-oxononadecanedioate. Purification was performed via flash chromatography (0% to 20% EtOAc in hexanes). Yield (386 mg, 40%).

[0290] Synthesis of bis(2 -butyloctyl) 6-(decylamino)hexadecanedioate bis(2 -butyloctyl) 6-(decylamino)hexadecanedioate was prepared from bis(2 -butyloctyl) 6-oxohexadecanedioate and decylamine according to the procedure for bis(2 -butyloctyl) 10- (decylamino)nonadecanedioate. Yield (409 mg, 87%). Synthesis of Compound

[0291] Compound 1-27 was prepared from bis(2 -butyloctyl) 6-(decylamino)hexadecanedioate and commercially available (ls,3s)-3-(dimethylamino)cyclobutane-l-carboxylic acid hydrochloride according to the procedure for compound 1-33. Yield (92 mg, 73%).

[0292] 'HNMR (400 MHz, CDCh) 6 3.99 - 3.92 (m, 4H), 3.52 - 3.41 (m, 1H), 3.06 - 2.92 (m, 2H), 2.81 - 2.56 (m, 2H), 2.34 - 2.06 (m, 14H), 1.70 - 1.05 (m, 76H), 0.93 - 0.84 (m, 15H). ESIMS: m / z calculated for chemical formula: C57H110N2O5 = 902.8. Found [M+H]+= 904.1.

[0293] SYNTHETIC EXAMPLE 28

[0294] 2-BUT YLOCT YL 10-(( 1 S,3 S)-N-DECYL-3 -((DIMETHYLAMINO)METHYL)CYCLOBUTANE- 1 - CARBOXAMIDO)-19-(DIDECYLAMINO)-19-OXONONADEC ANOATE (COMPOUND 1-28)

[0295] Synthesis of 2-butyloctyl 10-((ls,3s)-3-(((tert-butoxycarbonyl)amino)methyl)-N- decylcyclobutane- 1 -carboxamido)- 19-(didecylamino)- 19-oxononadecanoate

[0296]

[0297] 2-butyloctyl 10-((ls,3s)-3-(((tert-butoxycarbonyl)amino)methyl)-N-decylcyclobutane-l- carboxamido)-19-(didecylamino)-l 9-oxononadecanoate was prepared from 2-butyloctyl 10-

[0298] (decylamino)-19-(didecylamino)-19-oxononadecanoate and commercially available 2-((ls,3s)-3- ((tert-butoxycarbonyl)amino)cyclobutyl)acetic acid according to the procedure for bis(2- butyloctyl) 10-(( 1 s,3 s)-3 -((tert-butoxycarbonyl)amino)-N-decylcyclobutane- 1 - carboxamido)nonadecanedioate. (275 mg, 90%).

[0299] Synthesis of 2-butyloctyl 10-((ls,3s)-3-(aminomethyl)-N-decylcyclobutane-l-carboxamido)-19- (didecylamino)- 19-oxononadecanoate

[0300]

[0301] 2-butyloctyl 10-(( 1 s,3 s)-3 -(aminomethyl)-N-decylcyclobutane- 1 -carboxamido)- 19-

[0302] (didecylamino)- 19-oxononadecanoate was prepared from 2-butyloctyl 10-((ls,3s)-3-(((tert- butoxycarbonyl)amino)methyl)-N-decylcyclobutane- 1 -carboxamido)- 19-(didecylamino)- 19- oxononadecanoate according to the procedure for bis(2 -butyloctyl) 10-((ls,3s)-3-amino-N- decylcyclobutane- l-carboxamido)nonadecanedioate. (278 mg, quantitative).

[0303] Synthesis of Compound 1-28 Compound 1-28 was prepared from 2-butyloctyl 10-((ls,3s)-3-(aminomethyl)-N- decylcyclobutane-l-carboxamido)-19-(didecylamino)-19-oxononadecanoate according to the procedure for compound 1-40. Yield (126 mg, 49%).

[0304] 'HNMR (400 MHz, CDCh) 6 4.00 - 3.93 (m, 2H), 3.45 (s, 1H), 3.32 - 3.16 (m, 4H), 3.14 - 2.91 (m, 3H), 2.42 - 2.13 (m, 15H), 2.06 - 1.97 (m, 2H), 1.70 - 1.01 (m, 96H), 0.93 - 0.84 (m,

[0305] 15H). ESI-MS: m / z calculated for chemical formula: C69Hi3sN3O4 = 1070.1. Found [M+H]+= 1071.4.

[0306] SYNTHETIC EXAMPLE 29

[0307] BIS(2-BUTYLOCTYL) 10-(N-DECYL-2-((1S,3S)-3- (DIMETHYLAMINO)CYCLOBUT YL)ACETAMIDO)NONADECANEDIOATE (COMPOUND 1-29)

[0308] Synthesis of bis(2 -butyloctyl) 10-(2-((ls,3s)-3-((tert-butoxycarbonyl)amino)cyclobutyl)-N- decy 1 acetami do)nonadecanedi oate bis(2 -butyloctyl) 10-(2-((ls,3s)-3-((tert-butoxycarbonyl)amino)cyclobutyl)-N- decyl acetami do)nonadecanedi oate was prepared from bis(2 -butyloctyl) 10- (decylamino)nonadecanedioate and commercially available 2-((ls,3s)-3-((tert- butoxy carbonyl)amino)cyclobutyl)acetic acid according to the procedure for bis(2 -butyloctyl) 10-(( 1 s,3 s)-3 -((tert-butoxycarbonyl)amino)-N-decylcyclobutane- 1 - carboxamido)nonadecanedioate. Yield (245 mg, 76%). Synthesis of bis(2 -butyloctyl) 10-(2-((ls,3s)-3-aminocyclobutyl)-N- decy 1 acetami do)nonadecanedi oate bis(2 -butyloctyl) 10-(2-((ls,3s)-3-aminocyclobutyl)-N-decylacetamido)nonadecanedioate was prepared from bis(2-butyloctyl) 10-(2-((ls,3s)-3-((tert-butoxycarbonyl)amino)cyclobutyl)- N-decylacetamido)nonadecanedioate according to the procedure for bis(2 -butyloctyl) 10- ((ls,3s)-3-amino-N-decylcyclobutane-l-carboxamido)nonadecanedioate. Yield (200 mg, 90%).

[0309] Synthesis of Compound 1-29

[0310] formaldehyde in water NaBH(OAc)3MeOH

[0311] Compound 1-29 was prepared from bis(2-butyloctyl) 10-(2-((ls,3s)-3-aminocyclobutyl)- N-decylacetamido)nonadecanedioate according to the procedure for compound 1-40. Yield (132 mg, 64%). 'HNMR (400 MHz, CDCh) 6 4.00 - 3.93 (m, 4H), 3.59 - 3.50 (m, 1H), 3.06 - 2.93 (m,

[0312] 2H), 2.49 - 2.24 (m, 10H), 2.10 (s, 6H), 1.68 - 1.05 (m, 83H), 0.93 - 0.83 (m, 15H). ESI-MS: m / z calculated for chemical formula: C61H118N2O5 = 958.9. Found [M+H]+= 960.2.

[0313] SYNTHETIC EXAMPLE 30

[0314] BIS(2-HEXYLDECYL) 7-((1R,3R)-N-DECYL-3-(DIMETHYLAMINO)CYCLOBUTANE-1- CARBOXAMIDO)TRIDECANEDIOATE (COMPOUND 1-30)

[0315] Synthesis of Compound 1-30

[0316] Compound 1-30 was prepared from bis(2-hexyldecyl) 7-(decylamino)tridecanedioate and commercially available (lr,3r)-3-(dimethylamino)cyclobutane-l-carboxylic acid hydrochloride according to the procedure for compound 1-33. Yield (87 mg, 76%). 'HNMR (400 MHz, CDCh) 6 4.03 - 3.96 (m, 4H), 3.49 - 3.37 (m, 1H), 3.26 - 3.14 (m,

[0317] 1H), 3.11 - 3.04 (m, 1H), 3.00 - 2.74 (m, 2H), 2.46 - 2.25 (m, 6H), 2.23 - 2.06 (m, 8H), 1.72 - 1.07 (m, 88H), 0.96 - 0.87 (m, 15H). ESI-MS: m / z calculated for chemical formula: C62H120N2O5 = 972.9. Found [M+H]+= 974.2.

[0318] SYNTHETIC EXAMPLE 31 BIS(2-HEX YLDEC YL) 7-(( 1 s,3 S)-N-DEC YL-3 -(DIMETH YL AMINO)C YCLOBUTANE- 1 -

[0319] CARBOXAMIDO)TRIDECANEDIOATE (COMPOUND 1-31)

[0320] Synthesis of bis(2 -hexyldecyl) 7-oxotridecanedioate

[0321]

[0322] To a mixture of commercially available 7 -oxotri decanedioic acid (7.74 mmol, 2.0g), commercially available 2-hexyl-l -decanol (20.4 mmol, 4.96 g), DMAP (17.5 mmol, 2.14 g) in DCM (70 mL) was added DCC (23.4 mmol, 4.82 g). The reaction mixture was stirred at room temperature for 24h. The reaction mixture was filtered and the filtrate was concentrated. The resultant crude material was washed with hexanes and filtered. The filtrate was purified via flash chromatography (1% to 10% EtOAc in hexanes) to give compound bis(2-hexyldecyl) 7- oxotri decanedioate (5.1 g, 93%). Synthesis of bis(2 -hexyldecyl) 7-(decylamino)tridecanedioate A mixture of compound bis(2 -hexyldecyl) 7-oxotridecanedioate (2.55 mmol, 1.8 g), decan-l-amine (3.82 mmol, 600 mg), acetic acid (3.82 mmol, 0.22 mL), and sodium triacetoxyborohydride (5.1 mmol, 1.1 g) in di chloroethane (15 mL) was stirred at room temperature for 20 h. An additional amount of sodium triacetoxyborohydride (500 mg) was added to promote reaction completion. The reaction mixture was concentrated, quenched with sat. NaHCCh and extracted with EtOAc. The organic layer was separated, dried over Na2SC>4 and concentrated. Purification via flash chromatography (5% to 100% EtOAc in hexanes) gave compound bis(2-hexyldecyl) 7-(decylamino)tridecanedioate (1.6 g, 75%). Synthesis of Compound

[0323] Compound 1-31 was prepared from bis(2-hexyldecyl) 7-(decylamino)tridecanedioate and commercially available (ls,3s)-3-(dimethylamino)cyclobutane-l-carboxylic acid hydrochloride according to the procedure for compound 1-33. Yield (83 mg, 72%). JHNMR (400 MHz, CDCh) 54.02 - 3.95 (m, 4H), 3.53 - 3.44 (m, 1H), 3.09 - 2.95 (m,

[0324] 2H), 2.86 - 2.59 (m, 2H), 2.38 - 2.11 (m, 14H), 1.77 - 1.09 (m, 88H), 0.95 - 0.86 (m, 15H). ESI-MS: m / z calculated for chemical formula: C62H120N2O5 = 972.9. Found [M+H]+= 974.2. SYNTHETIC EXAMPLE 32

[0325] 2-BUTYLOCTYL 10-((1R,4R)-N-DECYL-4-(DIMETHYLAMINO)CYCLOHEXANE- 1 -CARBOXAMIDO)- 19- (DIDECYLAMINO)-19-OXONONADECANOATE (COMPOUND 1-32) Synthesis of Compound 1-32

[0326] Compound 1-32 was prepared from 2-butyloctyl 10-(decylamino)-19-(didecylamino)-19- oxononadecanoate and commercially available(lr,4r)-4-(dimethylamino)cyclohexane-l- carboxylic acid according to the procedure for compound 1-33. Yield (67 mg, 58%).

[0327] 'HNMR (400 MHz, CDCh) 6 4.00 - 3.93 (m, 2H), 3.71 - 3.59 (m, 1H), 3.32 - 3.12 (m, 4H), 3.07 - 2.94 (m, 2H), 2.49 - 2.38 (m, 1H), 2.35 - 2.15 (m, 12H), 2.07 - 1.93 (m, 2H), 1.83 -

[0328] 1.02 (m, 106H), 0.95 - 0.83 (m, 15H). ESI-MS: m / z calculated for chemical formula: C7oHi37N304= 1084.1. Found [M+H]+= 1085.3.

[0329] SYNTHETIC EXAMPLE 33

[0330] 2-BUTYLOCTYL 10-((l S,4S)-N-DECYL-4-(DIMETHYLAMINO)CYCLOHEXANE- 1 -CARBOXAMIDO)- 19- (DIDEC YLAMINO)- 19 -OXONONADECANOATE (COMPOUND 1-33 ) Synthesis of 19-(didecylamino)-10,19-di oxononadecanoic acid

[0331] A mixture of commercially available 10-oxononadecanedioic acid (29.2 mmol, 10 g), didecylamine (29.2 mmol, 8.69 g), DIEA (87.6 mmol, 15.3 mL), and HATU (38 mmol, 14.4 g) in dichloromethane (146 mL) was stirred at room temperature for 48 h. The reaction mixture was concentrated and the resultant crude material was partitioned between EtOAc and IM HC1. Purification via flash chromatography (10% to 70% EtOAc in hexanes) to give 19- (didecylamino)-10,19-di oxononadecanoic acid (3.27 g, 18%). Synthesis of 2-butyloctyl 19-(didecylamino)-10,19-di oxononadecanoate

[0332] A mixture of 19-(didecylamino)-10,19-di oxononadecanoic acid (1.93 mmol, 1.2 g), 2- butyloctan-l-ol (3.38 mmol, 0.755 mL), DMAP (2.89 mmol, 354 mg), and DCC (3.86 mmol, 796 mg) in dichloromethane (10 mL) was stirred at room temperature for 48 h. The reaction mixture was concentrated and the crude material was suspended in hexanes and filtered. Purification via flash chromatography (1% to 15% EtOAc in hexanes) gave 2-butyloctyl 19- (didecylamino)-10,19-di oxononadecanoate (1.4 g, 92%).

[0333] Synthesis of 2-butyloctyl 10-(decylamino)-19-(didecylamino)-19-oxononadecanoate

[0334] 2-butyloctyl 10-(decylamino)-19-(didecylamino)-19-oxononadecanoate was prepared from 2-butyloctyl 19-(didecylamino)-10,19-di oxononadecanoate according to the procedure for bis(2 -butyloctyl) 10-(decylamino)nonadecanedioate. Yield (1.4 g, 85%).

[0335] Synthesis of Compound 1-33

[0336]

[0337] Compound 1-33 was prepared from 2-butyloctyl 10-(decylamino)-19-(didecylamino)-19- oxononadecanoate and commercially available (ls,4s)-4-(dimethylamino)cyclohexane-l- carboxylic acid according to the procedure for bis(2 -butyloctyl) 10-((ls,3s)-3-((tert- butoxycarbonyl)amino)-N-decylcyclobutane-l-carboxamido)nonadecanedioate. Four equivalents of DIEA was used. Purification was performed via flash chromatography (5% to 65% EtOAc in hexanes with 1% triethylamine) Yield (80 mg, 69%).

[0338] 'HNMR (400 MHz, CDCh) 6 4.00 - 3.93 (m, 2H), 3.68 - 3.57 (m, 1H), 3.32 - 3.14 (m, 4H), 3.06 - 2.96 (m, 2H), 2.69 - 2.61 (m, 0.5H), 2.58 - 2.49 (m, 0.5H), 2.33 - 2.22 (m, 10H), 2.16 - 2.09 (m, 1H), 2.04 - 1.91 (m, 4H), 1.74 (s, 5H), 1.66 - 1.02 (m, 100H), 0.95 - 0.83 (m, 15H).

[0339] ESI-MS: m / z calculated for chemical formula: C?oHi37N304 = 1084.1. Found [M+H]+= 1085.3.

[0340] SYNTHETIC EXAMPLE 34

[0341] BIS(2-BUTYLOCTYL) 10-((1S,4S)-N-DECYL-4-(DIMETHYLAMINO)CYCLOHEXANE-1-

[0342] CARBOXAMIDO)NONADECANEDIOATE (COMPOUND 1-34)

[0343] Synthesis of bis(2 -butyloctyl) 10-((ls,4s)-4-((tert-butoxycarbonyl)amino)-N-decylcyclohexane- 1 -carboxamido)nonadecanedioate

[0344] bis(2 -butyloctyl) 10-((ls,4s)-4-((tert-butoxycarbonyl)amino)-N-decylcy cl ohexane-1- carboxamido)nonadecanedioate was prepared from bis(2 -butyloctyl) 10- (decylamino)nonadecanedioate and commercially available (ls,4s)-4-((tert- butoxy carbonyl)amino)cy cl ohexane-1 -carboxylic acid according to the procedure for bis(2- butyloctyl) 10-(( 1 s,3 s)-3 -((tert-butoxycarbonyl)amino)-N-decylcyclobutane- 1 - carboxamido)nonadecanedioate. Yield (510 mg, quantitative).

[0345] Synthesis of bis(2 -butyloctyl) 10-(( Is, 4s)-4-amino-N-decylcy cl ohexane-1 - carboxamido)nonadecanedioate

[0346] bis(2 -butyloctyl) 10-((ls,4s)-4-amino-N-decylcyclohexane-l- carboxamido)nonadecanedioate was prepared from bis(2 -butyloctyl) 10-((ls,4s)-4-((tert- butoxycarbonyl)amino)-N-decylcyclohexane-l-carboxamido)nonadecanedioate according to the procedure for bis(2 -butyloctyl) 10-((ls,3s)-3-amino-N-decylcyclobutane-l- carboxamido)nonadecanedioate. Yield (470 mg, quantitative).

[0347] Synthesis of Compound 1-34 formaldehyde in water NaBH(OAc)3MeOH Compound 1-34 was prepared from bis(2-butyloctyl) 10-((ls,4s)-4-amino-N- decylcyclohexane-l-carboxamido)nonadecanedioate according to the procedure for compound I- 40. Yield (75 mg, 52%).

[0348] 'HNMR (400 MHz, CDCh) 6 4.00 - 3.92 (m, 4H), 3.69 - 3.56 (m, 1H), 3.08 - 2.95 (m, 2H), 2.72 - 2.49 (m, 1H), 2.34 - 2.19 (m, 10H), 2.16 - 2.09 (m, 1H), 2.07 - 1.91 (m, 4H), 1.79

[0349] (s, 4H), 1.67 - 1.02 (m, 82H), 0.95 - 0.83 (m, 15H). ESI-MS: m / z calculated for chemical formula: C62H120N2O5 = 972.9. Found [M+H]+= 974.2.

[0350] SYNTHETIC EXAMPLE 35

[0351] BIS(2-BUTYLOCTYL) 10-((1R,3R)-N-DECYL-3-((DIMETHYLAMINO)METHYL)CYCLOBUTANE-1- CARBOXAMIDO)NONADECANEDIOATE (COMPOUND 1-35)

[0352] Synthesis of bis(2 -butyloctyl) 10-((lr,3r)-3-(((tert-butoxycarbonyl)amino)methyl)-N- decylcyclobutane- 1 -carboxamido)nonadecanedioate bis(2 -butyloctyl) 10-((lr,3r)-3-(((tert-butoxycarbonyl)amino)methyl)-N- decylcyclobutane-l-carboxamido)nonadecanedioate was prepared from bis(2 -butyloctyl) 10- (decylamino)nonadecanedioate and commercially available (Ir, 3 r)-3 -(((tertbutoxy carbonyl)amino)methyl)cy cl obutane-1 -carboxylic acid according to the procedure for bis(2 -butyloctyl) 10-(( 1 s,3 s)-3 -((tert-butoxycarbonyl)amino)-N-decylcyclobutane- 1 - carboxamido)nonadecanedioate. Yield (388 mg, 88%).

[0353] Synthesis of bis(2 -butyloctyl) 10-((lr,3r)-3-(aminomethyl)-N-decylcyclobutane-l- carboxamido)nonadecanedioate bis(2 -butyloctyl) 10-((lr,3r)-3-(aminomethyl)-N-decylcyclobutane-l- carboxamido)nonadecanedioate was prepared from bis(2 -butyloctyl) 10-((l r, 3 r)-3 -(((tertbutoxy carbonyl)amino)methyl)-N-decylcyclobutane-l-carboxamido)nonadecanedioate according to the procedure for bis(2 -butyloctyl) 10-((ls,3s)-3-amino-N-decylcyclobutane-l- carboxamido)nonadecanedioate. Yield (340 mg, 86%).

[0354] Synthesis of Compound 1-35

[0355] formaldehyde in water NaBH(OAc)3MeOH

[0356] Compound 1-35 was prepared from bis(2-butyloctyl) 10-((lr,3r)-3-(aminomethyl)-N- decylcyclobutane-l-carboxamido)nonadecanedioate according to the procedure for compound I- 40. Yield (90 mg, 73%). 'HNMR (400 MHz, CDCh) 6 4.00 - 3.93 (m, 4H), 3.50 - 2.84 (m, 4H), 2.62 - 2.13 (m,

[0357] 15H), 2.09 - 1.82 (m, 2H), 1.72 - 1.04 (m, 82H), 0.93 - 0.83 (m, 15H). ESI-MS: m / z calculated for chemical formula: C61H118N2O5 = 958.9. Found [M+H]+= 960.2.

[0358] SYNTHETIC EXAMPLE 36

[0359] BIS(2-BUTYLOCTYL) 10-((l s,3 S)-N-DECYL-3-((DIMETHYLAMINO)METHYL)CYCLOBUTANE- 1 -

[0360] CARBOXAMIDO)NONADECANEDIOATE (COMPOUND 1-36)

[0361] Synthesis of bis(2 -butyloctyl) 10-((ls,3s)-3-(((tert-butoxycarbonyl)amino)methyl)-N- decylcyclobutane- 1 -carboxamido)nonadecanedioate

[0362] bis(2 -butyloctyl) 10-((ls,3s)-3-(((tert-butoxycarbonyl)amino)methyl)-N- decylcyclobutane- l-carboxamido)nonadecanedioate was prepared from bis(2 -butyloctyl) 10- (decylamino)nonadecanedioate and commercially available (ls,3s)-3-(((tert- butoxy carbonyl)amino)methyl)cy cl obutane-1 -carboxylic acid according to the procedure for bis(2 -butyloctyl) 10-(( 1 s,3 s)-3 -((tert-butoxycarbonyl)amino)-N-decylcyclobutane- 1 - carboxamido)nonadecanedioate. Yield (300 mg, 70%).

[0363] Synthesis of bis(2 -butyloctyl) 10-((ls,3s)-3-(aminomethyl)-N-decylcyclobutane-l- carboxamido)nonadecanedioate

[0364] bis(2 -butyloctyl) 10-((ls,3s)-3-(aminomethyl)-N-decylcyclobutane-l- carboxamido)nonadecanedioate was prepared from bis(2-butyloctyl) 10-((ls,3s)-3-(((tert- butoxycarbonyl)amino)methyl)-N-decylcyclobutane-l-carboxamido)nonadecanedioate according to the procedure for bis(2 -butyloctyl) 10-((ls,3s)-3-amino-N-decylcyclobutane-l- carboxamido)nonadecanedioate. Yield (298 mg, quantitative). formaldehyde in water NaBH(OAc)3MeOH Compound 1-36 was prepared from bis(2-butyloctyl) 10-((ls,3s)-3-(aminomethyl)-N- decylcyclobutane-l-carboxamido)nonadecanedioate according to the procedure for compound I- 40. A second purification via flash chromatography (2% to 12% MeOH in chloroform) was required. Yield (58 mg, 47%). 'HNMR (400 MHz, CDCh) 6 4.00 - 3.93 (m, 4H), 3.50 - 3.40 (m, 1H), 3.15 - 2.90 (m,

[0365] 3H), 2.44 - 2.14 (m, 15H), 2.07 - 1.95 (m, 2H), 1.69 - 1.07 (m, 84H), 0.93 - 0.84 (m, 15H). ESIMS: m / z calculated for chemical formula: C61H118N2O5 = 958.9. Found [M+H]+= 960.2.

[0366] SYNTHETIC EXAMPLE 37

[0367] BIS(2-BUT YLOCTYL) 10-((1R,3R)-N-DECYL-3-(PYRROLIDIN- 1 -YL)CYCLOBUTANE- 1 - CARBOXAMIDO)NONADECANEDIOATE (COMPOUND 1-37)

[0368] Compound 1-37 was prepared from bis(2-butyloctyl) 10-((lr,3r)-3-amino-N- decylcyclobutane- l-carboxamido)nonadecanedioate according to the procedure for compound I- 40. Yield (62 mg, 39%). 'H NMR (400 MHz, CDCh) 6 4.00 - 3.93 (m, 4H), 3.45 - 3.19 (m, 2H), 3.12 - 2.85 (m, 3H), 2.49 - 2.36 (m, 5H), 2.33 - 2.08 (m, 6H), 1.83 - 1.75 (m, 4H), 1.68 - 1.04 (m, 81H), 0.93 - 0.84 (m, 15H). ESI-MS: m / z calculated for chemical formula: C62H118N2O5 = 970.9. Found [M+H]+= 972.1.

[0369] SYNTHETIC EXAMPLE 38

[0370] BIS(2-BUTYLOCTYL) 10-((l s,3 S)-N-DECYL-3-(PYRROLIDIN- 1 -YL)CYCLOBUTANE-1 - CARBOXAMIDO)NONADECANEDIOATE (COMPOUND 1-38)

[0371] Synthesis of Compound 1-38

[0372] A mixture of bis(2 -butyloctyl) 10-((ls,3s)-3-amino-N-decylcyclobutane-l- carboxamido)nonadecanedioate (0.16 mmol, 150 mg), 1,4-dibromobutane (0.18 mmol, 39 mg), and DIEA (0.49 mmol, 0.085 mL) in acetonitrile (15 mL) was heated at 75 °C for 48 h.

[0373] Purification via flash chromatography (5% to 65% EtOAc in hexanes with 1% tri ethylamine) gave compound 1-38 (43 mg, 27%). 'H NMR (400 MHz, CDCh) 6 4.00 - 3.93 (m, 4H), 3.49 - 3.41 (m, 1H), 3.07 - 2.92 (m, 3H), 2.89 - 2.74 (m, 1H), 2.54 - 2.45 (m, 4H), 2.34 - 2.15 (m, 8H), 1.82 - 1.72 (m, 4H), 1.70 - 1.04 (m, 81H), 0.93 - 0.83 (m, 15H). ESI-MS: m / z calculated for chemical formula: C62H118N2O5 = 970.9. Found [M+H]+= 972.1.

[0374] SYNTHETIC EXAMPLE 39

[0375] BIS(2-BUTYLOCTYL) 10-((1R,3R)-N-DECYL-3-(DIMETHYLAMINO)CYCLOBUTANE-1- CARBOXAMIDO)NONADECANEDIOATE (COMPOUND 1-39)

[0376] Synthesis of bis(2 -butyloctyl) 10-((lr,3r)-3-((tert-butoxycarbonyl)amino)-N-decylcyclobutane-l- carb oxami do)nonadecanedi oate bis(2 -butyloctyl) 10-((lr,3r)-3-((tert-butoxycarbonyl)amino)-N-decylcyclobutane-l- carboxamido)nonadecanedioate was prepared from bis(2 -butyloctyl) 10- (decylamino)nonadecanedioate according to the procedure for bis(2 -butyloctyl) 10-((ls,3s)-3- ((tert-butoxycarbonyl)amino)-N-decylcyclobutane- 1 -carboxamido)nonadecanedioate. Yield (494 mg, 95%). Synthesis of bis(2 -butyloctyl) 10-((lr,3r)-3-amino-N-decylcyclobutane-l- carb oxami do)nonadecanedi oate bis(2 -butyloctyl) 10-((lr,3r)-3-amino-N-decylcyclobutane-l- carboxamido)nonadecanedioate was prepared from bis(2 -butyloctyl) 10-((l r, 3 r)-3 -((tertbutoxy carbonyl)amino)-N-decylcy cl obutane-l-carboxamido)nonadecanedioate according to the procedure for bis(2 -butyloctyl) 10-((ls,3s)-3-amino-N-decylcyclobutane-l- carboxamido)nonadecanedioate. Yield (346 mg, 78%). Synthesis of Compound 1-39

[0377] formaldehyde in water NaBH(OAc)3MeOH

[0378] Compound 1-39 was prepared from bis(2-butyloctyl) 10-((lr,3r)-3-amino-N- decylcyclobutane- l-carboxamido)nonadecanedioate according to the procedure for compound I-

[0379] 40. Yield (67 mg, 54%). 'HNMR (400 MHz, CDCh) 8 3.99 - 3.93 (m, 4H), 3.49 - 3.16 (m, 2H), 3.11 - 2.87 (m,

[0380] 2H), 2.78 - 2.12 (m, 14H), 1.74 - 1.03 (m, 81H), 0.93 - 0.83 (m, 15H). ESI-MS: m / z calculated for chemical formula: C60H116N2O5 = 944.9. Found [M+H]+= 946.1.

[0381] SYNTHETIC EXAMPLE 40

[0382] BIS(2-BUTYLOCTYL) 10-((1S,3S)-N-DECYL-3-(DIMETHYLAMINO)CYCLOBUTANE-1-

[0383] CARBOXAMIDO)NONADECANEDIOATE (COMPOUND 1-40)

[0384] Synthesis of bis(2 -butyloctyl) 10-oxononadecanedioate

[0385] To a mixture of commercially available 10-oxononadecanedioic acid (17.5 mmol, 6.0 g), 2-butyloctan-l-ol (61.2 mmol, 11.4 g), and DMAP (52.6 mmol, 6.42 g) in di chloromethane (500 mL) was added DCC (70.1 mmol, 14.4 g). The reaction was stirred at room temperature for 20 h. The precipitate was filtered and the filtrate was purified via flash chromatography (0% to 5% EtOAc in hexanes) to give bi s(2 -butyloctyl) 10-oxononadecanedioate (9.7 g, 81%).

[0386] Synthesis of bis(2 -butyloctyl) 10-(decylamino)nonadecanedioate A mixture of bis(2 -butyloctyl) 10-oxononadecanedioate (4.42 mmol, 3.0 g), decan- 1- amine (6.63 mmol, 1.04 g), acetic acid (6.18 mmol, 0.35 mL), and sodium triacetoxyborohydride (17.7 mmol, 3.75 g) in di chloroethane (26 mL) was stirred at room temperature for 20 h. The reaction mixture was concentrated, quenched with sat. NaHCCh and extracted with EtOAc. The organic layer was separated, dried over Na2SC>4 and concentrated. Purification via flash chromatography (5% to 100% EtOAc in hexanes) gave bis(2 -butyloctyl) 10- (decylamino)nonadecanedioate (2.67 g, 74%).

[0387] Synthesis of bis(2 -butyloctyl) 10-((l s,3 s)-3-((tert-butoxycarbonyl)amino)-N-decylcyclobutane- 1 - carb oxami do)nonadecanedi oate

[0388] A mixture of bis(2 -butyloctyl) 10-(decylamino)nonadecanedioate (0.46 mmol, 398mg), commercially available (ls,3s)-3-((tert-butoxycarbonyl)amino)cyclobutane-l-carboxylic acid (0.51 mmol, 110 mg), DIEA (1.53 mmol, 0.27 mL), and HATU (0.664 mmol, 253 mg) in dichloromethane (6 mL) was stirred at room temperature for 20 h. The reaction mixture was concentrated and the resultant crude material was partitioned between EtOAc and saturated sodium bicarbonate. The organic layer was separated, dried over Na2SO4 and concentrated. Purification via flash chromatography (5% to 100% EtOAc in hexanes) gave bis(2 -butyloctyl) 10-(( 1 s,3 s)-3 -((tert-butoxycarbonyl)amino)-N-decylcyclobutane- 1 - carboxamido)nonadecanedioate (494 mg, 95%). Synthesis of bis(2 -butyloctyl) 10-(( 1 s,3 s)-3 -amino-N-decylcyclobutane- 1 - carb oxami do)nonadecanedi oate

[0389] A mixture of bis(2 -butyloctyl) 10-((ls,3s)-3-((tert-butoxycarbonyl)amino)-N- decylcyclobutane- l-carboxamido)nonadecanedioate (0.485 mmol, 495 mg) and trifluoroacetic acid (2 mL) in dichloromethane (4 mL) was stirred at room temperature for 1 h. The reaction mixture was concentrated and the resultant crude material was partitioned between EtOAc and saturated sodium bicarbonate. The organic layer was separated, dried over Na2SC>4 and concentrated to give bis(2-butyloctyl) 10-((ls,3s)-3-amino-N-decylcyclobutane-l- carboxamido)nonadecanedioate (350 mg, 79%).

[0390] Synthesis of Compound 1-40

[0391] formaldehyde in water NaBH(OAc)3MeOH

[0392] To a solution of bis(2 -butyloctyl) 10-((ls,3s)-3-amino-N-decylcyclobutane-l- carboxamido)nonadecanedioate (0.13 mmol, 120 mg) in methanol (4 mL) was added 37% formaldehyde in water (1 mL) and sodium triacetoxyborohydride (0.55 mmol, 116 mg) in 2 portions 10 minutes apart. After the last addition, the reaction mixture was allowed to stir at room temperature for 20 min. The reaction mixture was concentrated and the crude material was partitioned between EtOAc and saturated sodium bicarbonate. The organic layer was separated, dried over Na2SC>4 and concentrated. Purification via flash chromatography (5% to 65% EtOAc in hexanes with 1% tri ethylamine) gave compound 1-40 (82 mg, 66%). 'HNMR (400 MHz, CDCh) 6 4.00 - 3.93 (m, 4H), 3.50 - 3.41 (m, 1H), 3.05 - 2.90 (m,

[0393] 2H), 2.82 - 2.57 (m, 2H), 2.33 - 2.24 (m, 4H), 2.24 - 2.10 (m, 10H), 1.65 - 1.54 (m, 8H), 1.53 - 1.36 (m, 6H), 1.35 - 1.07 (m, 66H), 0.93 - 0.84 (m, 15H). ESLMS: m / z calculated for chemical formula: C60H116N2O5 = 944.9. Found [M+H]+= 946.1.

[0394] SYNTHETIC EXAMPLE 41 9-(N-(( 1 s,3 s)-3 -(DIMETHYLAMINO)C YCLOBUT YL)NONANAMIDO)-N 1 ,N1 ,N17,N 17-

[0395] TETRANONYLHEPTADECANEDIAMIDE (COMPOUND 1-41) Synthesis of N1 ,N1 ,N17,N 17-tetranonyl-9-oxoheptadecanediamide

[0396] Nl,Nl,N17,N17-tetranonyl-9-oxoheptadecanediamide was prepared from 9- oxoheptadecanedioic acid and dinonylaine according to the procedure for N1,N1,N19,N19- tetrakis(decyl)-10-oxononadecanediamide. Yield (189 mg, 73%).

[0397] Synthesis of tert-butyl ((ls,3s)-3-((l,17-bis(dinonylamino)-l,17-dioxoheptadecan-9- yl)amino)cyclobutyl)carbamate

[0398] tert-butyl (( 1 s, 3 s)-3 -(( 1 , 17 -bi s(dinonylamino)- 1 , 17-dioxoheptadecan-9- yl)amino)cyclobutyl)carbamate was prepared from Nl,Nl,N17,N17-tetranonyl-9- oxoheptadecanediamide and commercially available tert-butyl ((ls,3s)-3- aminocyclobutyl)carbamate according to the procedure for bis(2 -butyloctyl) 10- (decylamino)nonadecanedioate. Yield (220 mg, 96%).

[0399] Synthesis of tert-butyl ((ls,3s)-3-(N-(l,17-bis(dinonylamino)-l,17-dioxoheptadecan-9- yl)nonanamido)cyclobutyl)carbamate

[0400] tert-butyl (( 1 s,3 s)-3 -(N-(l , 17-bis(dinonylamino)- 1 , 17-dioxoheptadecan-9- yl)nonanamido)cyclobutyl)carbamate was prepared from tert-butyl ((ls,3s)-3-((l,17- bis(dinonylamino)-l, 17-dioxoheptadecan-9-yl)amino)cyclobutyl)carbamate and nonanoyl chloride according to the procedure for tert-butyl ((ls,3s)-3-(N-(l,19-bis(dioctylamino)-l,19- di oxononadecan- 10-yl)nonanamido)cy cl obutyl)carbamate. Yield (251 mg, quantitative).

[0401] Synthesis of 9-(N-(( 1 s,3 s)-3 -aminocyclobutyl)nonanamido)-N 1 ,N1 ,N17,N 17- tetranonylheptadecanediamide

[0402]

[0403] 9-(N-(( 1 s,3 s)-3 -aminocyclobutyl)nonanamido)-N 1 ,N1 ,N17,N 17- tetranonylheptadecanediamide was prepared from tert-butyl ((ls,3s)-3-(N-(l,17- bis(dinonylamino)-!, 17-dioxoheptadecan-9-yl)nonanamido)cyclobutyl)carbamate according to the procedure for bis(2 -butyloctyl) 10-((ls,3s)-3-amino-N-decylcyclobutane-l- carboxamido)nonadecanedioate. Yield (178 mg, 78%).

[0404] Synthesis of Compound 1-41

[0405] formaldehyde in water NaBH(OAc)3MeOH

[0406] Compound 1-41 was prepared from 9-(N-((ls,3s)-3-aminocyclobutyl)nonanamido)- Nl,Nl,N17,N17-tetranonylheptadecanediamide according to the procedure for compound 1-40. Yield (125 mg, 53%). JH NMR (400 MHz, CDCh) 6 3.96 - 3.82 (m, 0.5H), 3.67 - 3.54 (m, 0.5H), 3.36 - 3.19

[0407] (m, 8H), 3.14 - 2.91 (m, 1H), 2.57 - 2.05 (m, 16H), 2.03 - 1.04 (m, 102H), 0.95 - 0.85 (m, 15H). ESI-MS: m / z calculated for chemical formula: C68H134N4O3 = 1055.1. Found [M+H]+= 1056.4.

[0408] SYNTHETIC EXAMPLE 42

[0409] BIS(2-BUTYLOCTYL) 10-(N-((1R,3R)-3- ((DIMETHYLAMINO)METHYL)CYCLOBUT YL)NONANAMIDO)NONADECANEDIOATE (COMPOUND 1-42)

[0410] Synthesis of bis(2 -butyloctyl) 10-(((lr,3r)-3- ((dimethylamino)methyl)cyclobutyl)amino)nonadecanedioate

[0411] bis(2 -butyloctyl) 10-(((lr,3r)-3- ((dimethylamino)methyl)cyclobutyl)amino)nonadecanedioate was prepared from bis(2- butyloctyl) 10-oxononadecanedioate and commercially available (lr,3r)-3- ((dimethylamino)methyl)cyclobutan-l -amine dihydrochloride according to the procedure for bis(2 -butyloctyl) 10-(decylamino)nonadecanedioate. Purification was performed via flash chromatography (2% to 15% MeOH in chloroform). Yield (300 mg, 51%).

[0412] Synthesis of Compound 1-42)

[0413]

[0414] A mixture of bis(2 -butyloctyl) 10-(((lr,3r)-3- ((dimethylamino)methyl)cyclobutyl)amino)nonadecanedioate (0.126 mmol, 100 mg), nonanoyl chloride (0.139 mmol, 0.025 mL), and triethylamine (0.25 mmol, 0.035 mL) in dichloromethane (0.63 mL) was stirred at room temperature for 20 h. The reaction mixture was quenched with methanol then concentrated. Purification via flash chromatography (5% to 65% EtOAc in hexanes with 1% triethylamine) to give compound 1-42 (77 mg, 65%).

[0415] 'HNMR (400 MHz, CDCh) 6 4.00 - 3.94 (m, 4H), 3.73 - 3.51 (m, 2H), 3.17 - 3.06 (m, 2H), 2.74 - 2.60 (m, 1H), 2.45 - 2.13 (m, 14H), 2.09 - 1.96 (m, 0.5H), 1.84 - 1.71 (m, 2H), 1.67 - 1.55 (m, 11H), 1.43 - 1.04 (m, 66H), 0.93 - 0.83 (m, 15H). ESI-MS: m / z calculated for chemical formula: C59H114N2O5 = 930.9. Found [M+H]+= 932.2.

[0416] SYNTHETIC EXAMPLE 43

[0417] BIS(2-BUTYLOCTYL) 10-(N-((1R,3R)-3-((DIMETHYLAMINO)METHYL)

[0418] CYCLOBUTYL)NONANAMIDO)NONADECANEDIOATE (COMPOUND 1-43) Synthesis of bis(2 -butyloctyl) 10-((lr,4r)-4-((tert-butoxycarbonyl)amino)-N-decylcyclohexane- 1 -carboxamido)nonadecanedioate bis(2 -butyloctyl) 10-((lr,4r)-4-((tert-butoxycarbonyl)amino)-N-decylcyclohexane-l- carboxamido)nonadecanedioate was prepared from bis(2 -butyloctyl) 10- (decylamino)nonadecanedioate and commercially available (lr,4r)-4-((tert- butoxycarbonyl)amino)cyclohexane-l -carboxylic acid according to the procedure for bis(2- butyloctyl) 10-(( 1 s,3 s)-3 -((tert-butoxycarbonyl)amino)-N-decylcyclobutane- 1 - carboxamido)nonadecanedioate. Yield (510 mg, quantitative).

[0419] Synthesis of bis(2 -butyloctyl) 10-(( 1 r,4r)-4-amino-N-decylcyclohexane- 1 - carb oxami do)nonadecanedi oate

[0420] bis(2 -butyloctyl) 10-((lr,4r)-4-amino-N-decylcyclohexane-l- carboxamido)nonadecanedioate was prepared from bis(2 -butyloctyl) 10-((lr,4r)-4-((tert- butoxycarbonyl)amino)-N-decylcyclohexane-l-carboxamido)nonadecanedioate according to the procedure for bis(2 -butyloctyl) 10-((ls,3s)-3-amino-N-decylcyclobutane-l- carboxamido)nonadecanedioate. Yield (410 mg, 89%).

[0421] Synthesis of Compound 1-43 formaldehyde in water NaBH(OAc)3MeOH Compound 1-43 was prepared from bis(2-butyloctyl) 10-((lr,4r)-4-amino-N- decylcyclohexane-l-carboxamido)nonadecanedioate according to the procedure for compound I- 40. Yield (75 mg, 52%).

[0422] 'HNMR (400 MHz, CDCh) 6 4.00 - 3.93 (m, 4H), 3.71 - 3.60 (m, 0.5H), 3.07 - 2.94 (m, 2H), 2.50 - 2.37 (m, 1H), 2.36 - 2.16 (m, 11H), 2.06 - 1.91 (m, 2H), 1.86 - 1.07 (m, 88H), 0.93 - 0.81 (m, 15H). ESI-MS: m / z calculated for chemical formula: C62H120N2O5 = 972.9. Found [M+H]+= 974.3.

[0423] The various embodiments described above can be combined to provide further embodiments. All the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification, including U.S. Provisional Patent Application No. 63 / 734,609, filed on December 16, 2024, are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications, and publications to provide yet further embodiments.

[0424] These and other changes can be made to the embodiments considering the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims

CLAIMS1. A compound having the following Formula (I):or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein: ring A is an optionally substituted Cs-Cs cycloalkyl;L1and L2are each independently optionally substituted C4-C12 alkylene;indicates a covalent bond to ring A;L3ais a direct bond or optionally substituted C1-C4 alkylene;L4is a direct bond or optionally substituted C1-C4 alkylene;R1is -NRla-C(=O)-Rlb, -C(=O)-N(Rla)Rlb, -O-C(=O)-Rla, -C(=O)-O-Rla, or -O-C(=O)- O-Rla;Rlaand Rlbare each independently optionally substituted C1-C24 alkyl, optionally substituted C2-C24 alkenyl, or optionally substituted C2-C24 alkynyl;R2is -NR2a-C(=O)-R2b, -C(=O)-N(R2a)R2b, -O-C(=O)-R2a, -C(=O)-O-R2a, or -O-C(=O)- O-R2a;R2aand R2bare each independently optionally substituted C1-C24 alkyl, optionally substituted C2-C24 alkenyl, or optionally substituted C2-C24 alkynyl;R3aand R3bare each independently hydrogen or optionally substituted C1-C4 alkyl, or R3aand R3b, together with the nitrogen to which they are attached, join to form an optionally substituted 3-8 membered heterocyclyl or an optionally substituted 5-6 membered heteroaryl; andR4is optionally substituted C1-C12 alkyl, optionally substituted C2-C12 alkenyl, or optionally substituted C2-C12 alkynyl.

2. The compound of claim 1, wherein ring A is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

3. The compound of any one of claims 1-2, wherein ring A is cyclobutyl or cyclohexyl.

4. The compound of any one of claims 1-3, wherein ring A is unsubstituted.

5. The compound of any one of claims 1-4, wherein L1is C4-C10 alkylene.

6. The compound of any one of claims 1-5, wherein L1is C4 alkylene, C5 alkylene, C7 alkylene, Cs alkylene, or C9 alkylene.

7. The compound of any one of claims 1-6, wherein L2is C4-C10 alkylene.

8. The compound of any one of claims 1-7, wherein L2is C4 alkylene, C5 alkylene, C7 alkylene, Cs alkylene, or C9 alkylene.

9. The compound of any one of claims 1-8, wherein L1and L2are unbranched and unsubstituted.

10. The compound of any one of claims 1-9, wherein L1, L2, or both are substituted with one or more fluoro substituents.

11. The compound of any one of claims 1-10, wherein L1and L2are the same.

12. The compound of any one of claims 1-10, wherein L1and L2are different.

13. The compound of any one of claims 1-12, wherein L3is -NR4-C(=O)-* and R4is optionally substituted C1-C10 alkyl.

14. The compound of any of any one of claims 1-13, wherein L3is -NR4-C(=O)-* and R4is unsubstituted methyl, unsubstituted C3 alkyl, unsubstituted Cs alkyl, or unsubstituted C10 alkyl.

15. The compound of any one of claims 1-14, wherein L3has one of the following structures:

16. The compound of any one of claims 1-12, wherein L3is -NR4-* and R4is C7-C11 alkyl optionally substituted with one or more oxo substituents.

17. The compound of any one of claims 1-12, wherein L3has the following structure:

18. The compound of any one of claims 1-12, wherein L3is -C(=0)-NR4-* and R4is optionally substituted C1-C10 alkyl.

19. The compound of any one of claims 1-12, wherein L3is -C(=O)-O-L3a-* and L3ais a direct bond, methylene, or ethylene.

20. The compound of any one of claims 1-12, wherein L3is -O-C(=O)-L3a-* and L3ais a direct bond or methylene.

21. The compound of any one of claims 1-12, wherein L4is a direct bond or methylene.

22. The compound of any one of claims 1-21, wherein R1is -NRla-C(=O)-Rlbor -C(=O)-N(Rla)Rlb.

23. The compound of any one of claims 1-21, wherein R1-O-C(=O)-Rlaor -C(=O)-O-Rla.

24. The compound of any one of claims 1-23, wherein Rlais C4-C18 alkyl.

25. The compound of any one of claims 1-24, wherein Rlais C4 alkyl, Ce alkyl, Cs alkyl, C9 alkyl, C10 alkyl, C12 alkyl, C14 alkyl, Ci6 alkyl, C17 alkyl, or Cis alkyl.

26. The compound of any one of claims 1-25, wherein Rlbis C4-C16 alkyl.

27. The compound of any one of claims 1-26, wherein Rlbis C4 alkyl, Ce alkyl, Cs alkyl, C9 alkyl, C10 alkyl, C12 alkyl, C14 alkyl, Ci6 alkyl, C17 alkyl, or Cis alkyl.

28. The compound of any one of claims 1-27, wherein Rlais unbranched and unsubstituted.

29. The compound of any one of claims 1-27, wherein Rlais branched.

30. The compound of any one of claims 1-27, wherein Rlais optionally substituted with one or more fluoro substituents.

31. The compound of any one of claims 1-30, wherein Rlbis unbranched and unsubstituted.

32. The compound of any one of claims 1-30, wherein Rlbis branched.

33. The compound of any one of claims 1-30, wherein Rlbis optionally substituted with one or more fluoro substituents.

34. The compound of any one of claims 1-21, wherein R1has one of the following structures:

35. The compound of any one of claims 1-21, wherein R1has one of the following structures:

36. The compound of any one of claims 1-35, wherein R2is -NR2a-C(=O)-R2bor - C(=O)-N(R2a)R2b.

37. The compound of any one of claims 1-35, wherein R2-O-C(=O)-R2aor -C(=O)- O-R2a.

38. The compound of any one of claims 1-37, wherein R2ais C4-C18 alkyl.

39. The compound of any one of claims 1-38, wherein R2ais C4 alkyl, Ce alkyl, Cs alkyl, C9 alkyl, C10 alkyl, C11 alkyl, C12 alkyl, C14 alkyl, Ci6 alkyl, C17 alkyl, or Cis alkyl.

40. The compound of any one of claims 1-39, wherein R2bis C4-C16 alkyl.

41. The compound of any one of claims 1-40, wherein R2bis C4 alkyl, Ce alkyl, Cs alkyl, C9 alkyl, C10 alkyl, C12 alkyl, C14 alkyl, Ci6 alkyl, C17 alkyl, or Cis alkyl.

42. The compound of any one of claims 1-41, wherein R2ais unbranched and unsubstituted.

43. The compound of any one of claims 1-41, wherein R2ais branched.

44. The compound of any one of claims 1-41, wherein R2ais optionally substituted with one or more fluoro substituents.

45. The compound of any one of claims 1-44, wherein R2bis unbranched and unsubstituted.

46. The compound of any one of claims 1-44, wherein R2bis branched.

47. The compound of any one of claims 1-44, wherein R2bis optionally substituted with one or more fluoro substituents.

48. The compound of any one of claims 1-35, wherein R2has one of the following structures:

49. The compound of any one of claims 1-35, wherein R2has one of the following structures:

50. The compound of any one of claims 1-49, wherein R3ais unsubstituted C1-C4 alkyl.

51. The compound of any one of claims 1-50, wherein R3bis unsubstituted C1-C4 alkyl.

52. The compound of any one of claims 1-51, wherein R3aand R3bare the same.

53. The compound of claim 52, wherein R3aand R3bare both methyl.

54. The compound of any one of claims 1-49, wherein R3aand R3b, together with the nitrogen to which they are attached, join to form an optionally substituted 3-8 membered heterocyclyl.

55. The compound of any one of claims 1-49, wherein R3aand R3b, together with the nitrogen to which they are attached, join to form an unsubstituted pyrrolidinyl.

56. The compound of any one of claims 1-55, wherein the compound has one of the following structures:or a pharmaceutically acceptable salt thereof.

57. A lipid nanoparticle comprising the compound of any one of claims 1-56 and a therapeutic agent.

58. A composition comprising the compound of any one of claims 1-56, or the nanoparticle of claim 57, and a therapeutic agent and optionally a pharmaceutically acceptable excipient.

59. The lipid nanoparticle or composition of any one of claims 57-58, further comprising one or more component selected from neutral lipids, steroids, and polymer conjugated lipids.

60. The lipid nanoparticle or composition of any one of claims 57-59, comprising one or more neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.

61. The lipid nanoparticle or composition of claim 60, wherein the neutral lipid is DSPC.

62. The lipid nanoparticle or composition of any one of claims 59-61, wherein the molar ratio of the compound to the neutral lipid ranges from about 2: 1 to about 8: 1.

63. The lipid nanoparticle or composition of any one of claims 59-62, wherein the steroid is cholesterol.

64. The lipid nanoparticle or composition of claim 63, wherein the molar ratio of the compound to cholesterol ranges from about 5: 1 to about 1 : 1 or from about 2: 1 to about 1 : 1.

65. The lipid nanoparticle or composition of any one of claims 59-64, wherein the polymer conjugated lipid is a pegylated lipid.

66. The lipid nanoparticle or composition of any one of claims 59-65, wherein the molar ratio of the compound to the polymer conjugated lipid ranges from about 100: 1 to about 20: 1 or from about 100: 1 to about 10: 1.

67. The lipid nanoparticle or composition of any one of claims 65-66, wherein the pegylated lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer or a PEG dialkyoxypropylcarbamate.

68. The lipid nanoparticle or composition of any one of claims 65-66, wherein the pegylated lipid has the following Formula (II):or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:R5and R6are each independently a straight or branched alkyl, alkenyl, or alkynyl containing from 10 to 30 carbon atoms, wherein each alkyl, alkenyl, or alkynyl is optionally substituted with at least one fluoro; and w is an integer ranging from 30 to 60.

69. The lipid nanoparticle or composition of claim 68, wherein R5and R6are each independently a straight alkyl chain containing from 12 to 16 carbon atoms.

70. The lipid nanoparticle or composition of claim 68, wherein w is an integer ranging from 40 to 50.

71. The lipid nanoparticle or composition of any one of claims 57-70, wherein the therapeutic agent comprises a nucleic acid.

72. The lipid nanoparticle or composition of claim 71, wherein the nucleic acid comprises an antisense RNA, a messenger RNA, or a combination thereof.

73. The lipid nanoparticle or composition of any one of claims 57-72, wherein the therapeutic agent comprises a Cas9 mRNA or a ribonucleoprotein.

74. The lipid nanoparticle or composition of claim 72, wherein the messenger RNA encodes an antigen.

75. The lipid nanoparticle or composition of claim 74, wherein the antigen is an influenza antigen or a respiratory syncytial virus (RSV) antigen.

76. The lipid nanoparticle or composition of claim 75, wherein the influenza antigen is an influenza A antigen or an influenza B antigen.

77. The lipid nanoparticle or composition of any one of claims 57-76, wherein the lipid nanoparticle has a diameter of 40 nm to 100 nm.

78. A method for administering a therapeutic agent to a patient in need thereof, the method comprising preparing or providing the lipid nanoparticle or composition of any one of claims 57-77 and administering the composition to the patient.

79. A method for inducing expression of a desired protein in a subject in need thereof, the method comprising administering a therapeutically effective amount of the lipid nanoparticle or pharmaceutical composition of any one of claims 57-77 to the subject, wherein the therapeutic agent comprises a nucleic acid.

80. The method of claim 79, wherein the nucleic acid is an antisense RNA, a mRNA or a Cas9 mRNA.

81. The method of claim 80, wherein the mRNA encodes an antigen.

82. The method of any one of claims 78-81, wherein the method is for vaccination against a viral pathogen.

83. The method of claim 80, wherein the nucleic acid is a Cas9 mRNA, and the method is for editing a gene of interest.

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