Lipids for lipid nanoparticle formulations

Abstract

Provided are compounds having the following structure (I): (I) or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein R1, R1, L1, L2, L2a, L2b, and A are as defined herein. Uses of the compounds as components of lipid nanoparticle formulations for the delivery of therapeutic agents, compositions comprising the compounds, and methods of their use and preparation are also provided.

Description

BACKGROUND TECHNICAL FIELD

[0002] The present disclosure relates generally to novel cationic lipids that can be used in combination with other lipid components (e.g., neutral lipids, cholesterol, and polymer-conjugated lipids) to form lipid nanoparticles with oligonucleotides to facilitate intracellular delivery of therapeutic nucleic acids (e.g., oligonucleotides, messenger RNA) in vitro and in vivo.

[0003] DESCRIPTION OF RELATED ART

[0004] Many challenges associated with nucleic acid delivery impact the desired response in a biological system. Nucleic acid-based therapies have tremendous potential, but there remains a need to more effectively deliver nucleic acids to the appropriate site within a cell or organism to realize this potential. Therapeutic nucleic acids include, e.g., messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, immunostimulatory nucleic acids, antagomirs, antimir, mimics, supermirs, and aptamers. Some nucleic acids, such as mRNA or plasmids, can be used to effect expression of a particular cellular product, which would be useful in treating, e.g., diseases associated with a deficiency in a protein or enzyme. Therapeutic applications of translatable nucleotide delivery are extremely broad, as constructs can be synthesized to produce any selected protein sequence, whether or not the sequence is intrinsic to the system. The expression product of the nucleic acid can increase the existing level of a protein, replace a missing or nonfunctional form of a protein, or introduce a new protein and associated function in a cell or organism.

[0005] Some nucleic acids, such as miRNA inhibitors, can be used to effect expression of a particular cellular product modulated by a miRNA, which would be useful in treating, e.g., diseases associated with a deficiency in a protein or enzyme. Therapeutic applications of miRNA inhibition are extremely broad, as constructs can be synthesized to inhibit one or more miRNAs, which in turn modulate the expression of mRNA products. Inhibition of an endogenous miRNA can increase the expression of its downstream target endogenous protein and restore proper function in a cell or organism as a means to treat a disease associated with a particular miRNA or set of miRNAs.

[0006] Other nucleic acids can down-regulate the intracellular levels of specific mRNAs, and thus the synthesis of the corresponding proteins, through processes such as RNA interference (RNAi) or complementary binding of antisense RNA. Therapeutic applications of antisense oligonucleotide and RNAi are also very broad, as oligonucleotide constructs with any nucleotide sequence directed against a target mRNA can be synthesized. Targets can include mRNAs from normal cells, mRNAs associated with disease states (e.g., cancer), and mRNAs of infectious agents (e.g., viruses). To date, antisense oligonucleotide constructs have shown the ability to specifically down-regulate target proteins through degradation of cognate mRNAs in in vitro and in vivo models. In addition, antisense oligonucleotide constructs are currently being evaluated in clinical studies.

[0007] However, two problems currently exist with the use of oligonucleotides in a therapeutic setting. First, free RNA is sensitive to nuclease digestion in the plasma. Second, free RNA has limited ability to enter the intracellular compartments where the relevant translational machinery resides. Lipid nanoparticles formed from cationic lipids along with other lipid components (e.g., neutral lipids, cholesterol, PEG, PEGylated lipids) and oligonucleotides have been used to block the degradation of RNA in the plasma and to facilitate cellular uptake of the oligonucleotides.

[0008] There remains a need for improved cationic lipids and lipid nanoparticles for the delivery of oligonucleotides. Preferably, these lipid nanoparticles will 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 a sufficient therapeutic index such that patient treatment with effective doses of the nucleic acid is not associated with unacceptable toxicity and / or risk to the patient. The present disclosure provides these and related advantages.

[0009] BRIEF SUMMARY

[0010] Briefly, the present disclosure provides lipid compounds (including stereoisomers, pharmaceutically acceptable salts, or tautomers thereof) that can be used alone or in combination with other lipid components (e.g., neutral lipids, charged lipids, steroids (including, e.g., all sterols) and / or analogs thereof, and / or polymer-conjugated lipids) to form lipid nanoparticles for the delivery of therapeutic agents. In some cases, the lipid nanoparticles are used to deliver nucleic acids such as antisense RNA and / or messenger RNA. Methods of using such lipid nanoparticles to treat various diseases or conditions (e.g., those caused by infectious entities and / or protein deficiencies) are also provided.

[0011] In one embodiment, a compound having the following structure (I) is provided:

[0012] (I)

[0013] or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein R 1 , R 2 , L 1 , L 2 , L 2a , L 2b , and A are as defined herein.

[0014] Also provided are pharmaceutical compositions comprising one or more of the above compounds of structure (I) and a therapeutic agent. In some embodiments, the pharmaceutical compositions further comprise one or more components selected from the group consisting of neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. Such compositions can be used to form lipid nanoparticles for use in delivering therapeutic agents.

[0015] In other embodiments, the present disclosure provides methods for administering a therapeutic agent to a patient in need thereof, the methods comprising preparing a composition of a lipid nanoparticle comprising a compound of structure (I) and a therapeutic agent, and delivering the composition to the patient. Such methods can be used to induce expression of a protein in an individual, for example, to express an antigen for purposes of vaccination or gene editing proteins.

[0016] These and other aspects of the present disclosure will become apparent upon reference to the following detailed description.

[0017] DETAILED DESCRIPTION

[0018] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the present disclosure. However, one skilled in the art will understand that the present disclosure can be practiced without these details.

[0019] The present disclosure is based, in part, on the discovery of novel cationic (amino) lipids that provide a variety of advantages when used in lipid nanoparticles for the in vivo delivery of active or therapeutic agents, such as nucleic acids, into cells of a mammal. In particular, 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 nucleic acid activity and improved composition tolerability in vivo compared to previously described nucleic acid-lipid nanoparticle compositions, resulting in a significant improvement in therapeutic index.

[0020] In particular embodiments, the present disclosure provides novel cationic lipids that enable improved compositions for the delivery of mRNA and / or other oligonucleotides in vitro and in vivo. In some embodiments, these improved lipid nanoparticle compositions can be used to express proteins encoded by mRNA. In other embodiments, these improved lipid nanoparticle compositions can be used to upregulate endogenous protein expression by delivering an miRNA inhibitor that targets a particular miRNA or a group of miRNAs to modulate a target mRNA or several mRNAs. In other embodiments, these improved lipid nanoparticle compositions can be used to downregulate (e.g., silence) protein levels and / or mRNA levels of a target gene. In some other embodiments, the lipid nanoparticles can also be used to deliver mRNA and plasmids to express transgenes. In other embodiments, the lipid nanoparticle compositions can be used to induce pharmacological effects resulting from protein expression, such as increasing red blood cell production by delivering an appropriate erythropoietin mRNA, or protecting against infection by delivering an mRNA encoding an appropriate antibody.

[0021] The lipid nanoparticles and compositions of the present disclosure can be used for a variety of purposes, including the delivery of encapsulated or associated (e.g., complexed) therapeutic agents, such as nucleic acids, to cells in vitro and in vivo. Accordingly, embodiments of the present disclosure provide methods of treating or preventing a disease or disorder in an individual in need thereof by contacting the individual with a lipid nanoparticle encapsulating an appropriate therapeutic agent or a lipid nanoparticle associated with an appropriate therapeutic agent, wherein the lipid nanoparticle comprises one or more novel cationic lipids described herein.

[0022] 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 oligonucleotides, plasmid DNA, microRNAs (miRNAs), miRNA inhibitors (antagomirs / antimirs), complementary RNAs that interfere with messenger RNAs (micRNAs), DNA, multivalent RNA, Dicer substrate RNA, complementary DNA (cDNA), and the like. Thus, the lipid nanoparticles and compositions of the present disclosure can be used to induce the expression of a desired protein in vitro and in vivo by contacting a cell with a lipid nanoparticle comprising one or more of the 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 can be used to reduce the expression of a target gene and protein in vitro and in vivo by contacting a cell with a lipid nanoparticle comprising one or more of the novel cationic lipids described herein, wherein the lipid nanoparticle encapsulates or is associated with a nucleic acid that reduces the expression of the target gene (e.g., an antisense oligonucleotide or small interfering RNA (siRNA)). The lipid nanoparticles and compositions of the present disclosure can also be used to co-deliver different nucleic acids (e.g., mRNA and plasmid DNA) separately or in combination, as can be useful for providing the functions of both nucleic acids (e.g., mRNA encoding a suitable gene modifying enzyme and a segment of DNA for incorporation into the host genome) that are required for the co-localization

[0023] ​Nucleic acids for use with the present disclosure can be prepared according to any available technique. For mRNA, the primary method of preparation is, but is not limited to, enzymatic synthesis (also known as in vitro transcription), which represents the most efficient method for producing long sequence-specific mRNA currently available. In vitro transcription describes the process of template-directed synthesis of RNA molecules from an engineered DNA template containing an upstream bacteriophage promoter sequence (e.g., including but not limited to promoter sequences from T7, T3, and SP6 E. coli bacteriophages) linked to the downstream sequence encoding the gene of interest. Template DNA for in vitro transcription can be prepared from a variety of sources using appropriate techniques 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 Vol. 941 Conn G.L. (Editor), New York, N.Y. Humana Press, 2012).

[0024] RNA transcription is performed in vitro using linearized DNA templates in the presence of a corresponding RNA polymerase and adenosine, guanosine, uridine, and cytidine ribonucleotide triphosphates (rNTPs) under conditions that support polymerase activity while minimizing potential degradation of the resulting mRNA transcript. In vitro transcription can be performed using a variety of commercially available kits as well as commercially available reagents including RNA polymerases and rNTPs including, but not limited to, RiboMax Large Scale RNA Production Systems (Promega), MegaScript Transcription Kit (Life Technologies). Methods for in vitro transcription of mRNA are well known in the art. (See, e.g., Losick, R., 1972, In vitro transcription, Ann Rev Biochem Vol. 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 Vol. 703 (Neilson, H. Ed.), New York, N.Y. Humana Press, 2010; Brunelle, J. L. and Green, R., 2013, Chapter 5 - In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology Vol. 530, 101-114; all incorporated herein by reference).

[0025] The desired in vitro transcribed mRNA is then purified from the undesired components of the transcription or associated reactions, including unincorporated rNTPs, proteases, salts, short RNA oligomers, etc. Techniques for isolating mRNA transcripts are well known in the art. Well known procedures include phenol / chloroform extraction or precipitation with alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride. Other non-limiting examples of purification procedures that 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 the SV Total Isolation System (Promega) and the In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).

[0026] Furthermore, while reverse transcription can produce large amounts of mRNA, the product can contain many abnormal RNA impurities associated with undesirable polymerase activity that can need to be removed from the full-length mRNA preparation. These include short RNAs produced from aborted transcription initiation, as well as double-stranded RNA (dsRNA) produced from RNA-dependent RNA polymerase activity, transcription from RNA templates initiated by RNA and self-complementary 3’ extensions. It has been demonstrated that these contaminants with dsRNA structure can lead to undesirable immune stimulatory activity by interacting with various innate immune sensors in eukaryotic cells that perform the function of recognizing specific nucleic acid structures and inducing potent immune responses. This in turn can significantly reduce mRNA translation as protein synthesis is reduced during innate cellular immune responses. Therefore, additional techniques have been developed to remove these dsRNA contaminants 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 is reported to translate at much higher levels, particularly in primary cells and in vivo.

[0027] A wide variety of modifications have been described in the art for altering the specific properties of in vitro transcribed mRNA and improving its utility. These include, but are not limited to, modifications to the 5' and 3' ends of the mRNA. Endogenous eukaryotic mRNA typically contains a cap structure at the 5'-end of the mature molecule, which plays an important role in mediating the binding of mRNA cap-binding proteins (CBPs) which in turn are responsible for enhancing the stability of the mRNA and efficiency of mRNA translation in the cell. Thus, the highest levels of protein expression are achieved with capped mRNA transcripts. The 5'-cap contains a 5'-5'- triphosphate linkage between the 5'-most nucleotide and a guanine nucleotide. The conjugated guanine nucleotide is methylated at the N7 position. Additional modifications include methylation of the penultimate and antepenultimate most 5'-nucleotides on the 2'-hydroxyl groups.

[0028] A variety of different cap structures can be used to generate 5'-caps of in vitro transcribed synthetic mRNA. 5'-capping of synthetic mRNA can be performed co-transcriptionally (i.e., capping during in vitro transcription) with chemical cap analogs. For example, the anti-reverse cap analog (ARCA) cap contains a 5'-5'- triphosphate guanine-guanine linkage, with one guanine containing a N7 methyl group and a 3'-0-methyl group. However, during this co-transcriptional process, up to 20% of transcripts remain uncapped, and the synthetic cap analog is not identical to the 5'-cap structure of authentic cellular mRNA, which can reduce translatability and cellular stability. Alternatively, synthetic mRNA molecules can also be enzymatically capped post-transcriptionally. These can generate more authentic 5'-cap structures that more closely mimic the endogenous 5'-cap in structure or function, with enhanced binding of cap-binding proteins, prolonged half-life, reduced sensitivity to 5' endonucleases, and / or reduced 5' de-capping. A number of synthetic 5'-cap analogs have been developed, and it is known in the art that the synthetic 5'-caps 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).

[0029] At the 3' end, long-chain adenine nucleotides (poly-A tails) are typically added to the mRNA molecule during RNA processing. Immediately after transcription, the 3' end of the transcript is cleaved to release the 3' hydroxyl group, and during a process called polyadenylation, poly-A polymerase adds a chain of adenine nucleotides to the 3' hydroxyl group into the RNA. Poly(A) tails have been widely shown to enhance mRNA translation efficiency and stability (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).

[0030] Poly(A) tailing of in vitro transcribed mRNA can be achieved using various methods, including but not limited to cloning poly(T) tracts into a DNA template or adding them post-transcriptionally using a poly(A) polymerase. The first approach allows for the in vitro transcription of mRNA with a defined poly(A) tail, depending on the size of the poly(T) tract, but requires additional manipulation of the template. The latter approach involves enzymatically adding the poly(A) tail to in vitro transcribed mRNA using a poly(A) polymerase that catalyzes the incorporation of adenine residues into the 3' end of the RNA. This does not require additional manipulation of the DNA template but produces mRNA with poly(A) tails of uneven length. A variety of commercially available kits and reagents, various ARCA caps, poly(A) polymerases, etc., can be used for 5'-capping and 3'-poly(A) tailing. The kits include, but are not limited to, the Poly(A) Polymerase Tailing kit (EpiCenter), the mMESSAGE mMACHINE T7 Ultra kit, and the Poly(A) Tailing kit (Life Technologies).

[0031] It has been reported that in addition to 5' cap and 3' polyadenylation, other modifications of in vitro transcripts provide benefits related to translational efficiency and stability. It is well known in the art that pathogenic DNA and RNA can be recognized by various sensors within eukaryotes and trigger potent innate immune responses. The ability to distinguish pathogenic from self DNA and RNA has been shown to be based at least in part on structure and nucleoside modifications, as most nucleic acids of natural origin contain modified nucleosides. In vitro synthesized RNA, in contrast, lacks these modifications, thus rendering it immunostimulatory, which in turn can inhibit efficient mRNA translation as outlined above. Incorporation of modified nucleosides into in vitro transcribed mRNA can be used to prevent recognition and activation of RNA sensors, thus mitigating this undesirable immunostimulatory activity and enhancing translational 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).Modified nucleosides and nucleotides used in the synthesis of modified RNA can be prepared, monitored, and utilized using general methods and procedures known in the art. A wide variety of nucleoside modifications are available that can be incorporated into in vitro transcribed mRNA to some extent, either alone or in combination with other modified nucleosides (see, e.g., U.S. Pub. No. 2012 / 0251618). In vitro synthesis of nucleoside-modified mRNA has been reported to reduce the ability to activate immune sensors, while concomitantly enhancing the ability to translate.

[0032] Other components of mRNA that can be modified to provide benefits in terms of translatability and stability include 5' and 3' untranslated regions (UTRs). Optimization of UTRs, which can be derived from cellular or viral RNAs to advantageously 5' and 3' UTRs, either simultaneously or separately, have been shown to improve mRNA stability and translation 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).

[0033] In addition to mRNA, other nucleic acid payloads can be used in the present disclosure. For oligonucleotides, methods of preparation include, but are not limited to, chemical synthesis and enzymatic, chemical cleavage of longer precursors, in vitro transcription as described above, and the like. 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).

[0034] For plasmid DNA, preparations for use with the present disclosure generally utilize, but are not limited to, in vitro amplification and isolation of plasmid DNA from liquid cultures of bacteria containing the plasmid of interest. The presence of genes encoding resistance to specific antibiotics (penicillin, kanamycin, etc.) in the plasmid of interest allows those bacteria containing the plasmid of interest to be selectively grown in cultures containing the antibiotic. Methods for isolating plasmid DNA are widely used and well known in the art (see, e.g., Heilig, J., Elbing, K. L., and Brent, R (2001) Large-Scale Preparation of Plasmid DNA. Current Protocols in Molecular Biology. 41:II:1.7:1.7.1-1.7.16; Rozkov, A., Larsson, B., Gillström, S., Björnestedt, 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 U.S. Patent No. 6,197,553). Plasmid isolation can be performed using a variety of commercially available kits as well as commercially available reagents, including but not limited to, Plasmid Plus (Qiagen), GenJET Plasmid MaxiPrep (Thermo), and PureYield MaxiPrep (Promega) kits.

[0035] Various exemplary embodiments of the cationic lipids, lipid nanoparticles, and compositions comprising the same of the present disclosure, 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.

[0036] As used herein, the following terms have the meanings ascribed to them unless otherwise specified.

[0037] Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof (e.g., “comprises” and “comprising”) are to be construed in an open, inclusive sense, that is, as “including, but not limited to.”

[0038] Throughout the entire specification, reference 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 disclosure. The appearances of the phrase “in one embodiment” or “in an embodiment” in various places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0040] The phrase “inducing expression of a desired protein” means that the nucleic acid is capable of increasing expression of the desired protein. To examine the extent of protein expression, a test sample (e.g., a sample of cultured cells expressing the desired protein) or a test mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model) can be contacted with the nucleic acid (e.g., a nucleic acid in combination with a lipid of the 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 cultured cells expressing the desired protein) or a control mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model) that is not contacted with the nucleic acid or that is not administered the nucleic acid. When the desired protein is present in the control sample or control mammal, expression of the desired protein in the control sample or control mammal can be assigned a value of 1.0. In particular embodiments, expression of the desired protein in the test sample or test mammal is induced when the ratio of the level of expression of the desired protein in the test sample or test mammal to the level of expression of the desired protein in the control sample or control mammal is greater than 1, e.g., about 1.1, 1.5, 2.0, 5.0, or 10.0. When the desired protein is not present in the control sample or control mammal, expression of the desired protein is induced when any measurable level of the desired protein is detected in the test sample or test mammal. One of ordinary skill in the art will appreciate the appropriate assay to determine the level of protein expression in a sample, e.g., dot blot, Northern blot, in situ hybridization, ELISA, immunoprecipitation, enzyme function and phenotype assays, or assays based on reporter proteins that produce fluorescence or luminescence under appropriate conditions.

[0041] The phrase "inhibiting expression of a target gene" means that the nucleic acid is capable of silencing, reducing, or inhibiting expression of the target gene. To check the degree of gene silencing, a test sample (e.g., a sample of cultured cells 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., a mouse) or a non-human primate (e.g., a monkey) model) is contacted with a nucleic acid that silences, reduces, or inhibits expression of the target gene. The expression of the target gene in the test sample or test animal is compared to the expression of the target gene in a control sample (e.g., a sample of cultured cells 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., a mouse) or a non-human primate (e.g., a monkey) model) that has not been contacted or administered the nucleic acid. The expression of the target gene in the control sample or control mammal can be assigned a value of 100%. In particular embodiments, silencing, inhibiting, or reducing expression of the target gene is achieved when the level of target gene expression in the test sample or test mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% relative to the level of target gene expression in the control sample or control mammal. In other words, the nucleic acid is capable of silencing, reducing, or inhibiting target gene expression in the test sample or test mammal by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to the level of target gene expression in the control sample or control mammal that has not been contacted or administered the nucleic acid. Suitable assays for determining the level of target gene expression include, but are not limited to, checking protein or mRNA levels using techniques known to those of skill in the art, such as dot blots, Northern blots, in situ hybridizations, ELISAs, immunoprecipitations, enzyme function, and phenotypic assays known to those of skill in the art.

[0042] An "effective amount" or "therapeutically effective amount" of an active agent or therapeutic agent, such as a therapeutic nucleic acid, is an amount that is sufficient to effect a desired effect, such as an increase or inhibition of expression of a target sequence, as compared to normal expression levels detected in the absence of the nucleic acid. An increase in expression of a target sequence is achieved when any measurable level of the expression product is detected in the absence of the nucleic acid. An increase in expression is achieved when the fold increase in the value obtained with the nucleic acid, such as an mRNA, relative to a control, is about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000 or greater, in the presence of the nucleic acid prior to contact. Inhibition of expression of a target gene or target sequence is achieved when the value obtained with the nucleic acid, such as an antisense oligonucleotide, is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 0% relative to a control. Suitable assays for measuring expression of a target gene or target sequence include examination of protein or RNA levels, such as by spot blot, Northern blot, in situ hybridization, ELISA, immunoprecipitation, enzymatic function, fluorescence or luminescence of suitable reporter proteins, and phenotypic assays known to those of skill in the art.

[0043] 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 can be in the form of an antisense molecule, plasmid DNA, cDNA, PCR product, or vector. RNA can be in the form of a 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 those that contain known analogues of natural nucleotides so long as the analogue contains a functional group that is capable of hydrogen bonding to a natural nucleotide base. The term encompasses nucleic acids containing known nucleotide analogues or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties. Examples of such analogues include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless specifically otherwise limited, the term encompasses nucleic acids containing known enantiomeric, diastereomeric, and tautomeric forms of these bases, as well as the particular geometric and positional isomers. Unless specifically 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 can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of nucleotides and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). A "nucleotide" contains a sugar, either deoxyribose (DNA) or ribose (RNA); a base; and a phosphate group. Nucleotides are linked together by the phosphate groups. "Bases" include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, including but not limited to modifications that place new reactive groups (such as, but not limited to, amines, alcohols, thiols, carboxylates, and hydrocarbyl halides).

[0044] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises all or a portion of the coding sequence necessary for the production of a polypeptide or precursor polypeptide.

[0045] "Gene product" as used herein refers to a product of a gene, such as an RNA transcript or a polypeptide.

[0046] The term "lipid" refers to a group of organic compounds that includes, but is not limited to, esters of fatty acids, and are typically characterized by being insoluble in water but soluble in many organic solvents. They are generally classified into at least three groups: (1) "simple lipids" including fats and oils and waxes; (2) "compound lipids" including phospholipids and glycolipids; and (3) "derived lipids" such as steroids.

[0047] A "steroid" is a compound comprising the following carbon skeleton: .

[0048] Non-limiting examples of steroids include cholesterol and the like.

[0049] A "cationic lipid" refers to a lipid that is capable of carrying a positive charge. Exemplary cationic lipids include one or more amine groups that carry a positive charge. Preferred cationic lipids are ionizable such that they can exist in a positively charged or neutral form depending on the pH. Ionization of the cationic lipid under different pH conditions affects the surface charge of the lipid nanoparticle. This charge state can affect plasma protein uptake, blood clearance, and tissue distribution critical for intracellular delivery of nucleic acids (Semple, S.C., et al., Adv. Drug Deliv Rev 32:3-17 (1998)) as well as the ability to form endosomolytic nonbilayer structures (Hafez, I.M., et al., Gene Ther 8:1188-1196 (2001)).

[0050] The term "lipid nanoparticle" refers to a particle having at least one dimension on the order of a nanometer (e.g., 1-1,000 nm) that comprises one or more of the compounds of Formula (I) or other specified cationic lipids. In some embodiments, the lipid nanoparticle is contained in a formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), to a target site of interest (e.g., a cell, tissue, organ, tumor, etc.). In some embodiments, the lipid nanoparticle of the present disclosure comprises a nucleic acid. Such lipid nanoparticles typically comprise a compound of structure (I) and one or more excipients selected from the group consisting of neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. In some embodiments, the active agent or therapeutic agent, such as a nucleic acid, can be encapsulated in the lipid portion of the lipid nanoparticle or in an aqueous space enclosed by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesired effects induced by mechanisms of the host organism or cell, such as an adverse immune response.

[0051] In various embodiments, the average diameter of the lipid nanoparticle is about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or 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 is substantially non-toxic. In certain embodiments, the nucleic acid, when present in the lipid nanoparticle, is resistant to degradation by nucleases in an aqueous solution. Lipid nanoparticles comprising nucleic acids and methods of making the same are disclosed in, e.g., U.S. Patent Publication Nos. 2004 / 0142025, 2007 / 0042031, and PCT Publication Nos. WO 2013 / 016058 and WO 2013 / 086373, the entire disclosures of which are incorporated by reference herein in their entireties for all purposes.

[0052] As used herein, “encapsulated lipid” refers to a lipid nanoparticle that provides complete encapsulation, partial encapsulation, or both, of an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA). In embodiments, a nucleic acid (e.g., mRNA) is completely encapsulated in a lipid nanoparticle.

[0053] The term “polymer-conjugated lipid” refers to a molecule comprising a lipid moiety and a polymer moiety. An example of a polymer-conjugated lipid is a PEGylated lipid. The term “PEGylated lipid” refers to a molecule comprising a lipid moiety and a polyethylene glycol moiety. PEGylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-DMG), and the like.

[0054] The term “neutral lipid” refers to any of a variety of lipid species that exist in an uncharged or neutral zwitterionic form at a selected pH. Such lipids at physiological pH include, but are not limited to: phosphatidylcholines such as 1,2-distearyl- sn - glycerol-3-phosphocholine (DSPC), 1,2-dipalmitoyl- sn - glycerol-3-phosphocholine (DPPC), 1,2-dimyristoyl- sn- glycerol-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl- sn - glycerol-3-phosphocholine (POPC), 1,2-dioleoyl- sn - glycerol-3-phosphocholine (DOPC), phosphatidylethanolamines such as 1,2-dioleoyl- sn - glycerol-3-phosphoethanolamine (DOPE), sphingomyelin (SM), ceramide, steroids such as cholesterols and derivatives thereof. Neutral lipids can be synthetic or of natural origin.

[0055] The term "charged lipid" refers to any of a variety of lipid species that exist in a positively or negatively charged form that is independent of pH, within useful physiological ranges (e.g., pH ~3 to pH ~9). Charged lipids can be synthetic or of natural origin. Examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, cholesterol hemisuccinate, dialkyldimethylammonium propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, dimethylaminoethane carbamoyl cholesterol (e.g., DC-Chol).

[0056] As used herein, the term "aqueous solution" refers to a composition comprising water.

[0057] With respect to nucleic acid-lipid nanoparticles, "serum stable" means that the nucleotides are not significantly degraded upon exposure to serum or nuclease assays that would significantly degrade free DNA or RNA. Suitable assays include, for example, standard serum assays, DNase assays, or RNase assays.

[0058] As used herein, "systemic delivery" refers to the delivery of a therapeutic product that can result in widespread exposure of an active agent within an organism. Some administration techniques can result in systemic delivery of certain agents, but not others. Systemic delivery means that a useful, preferably therapeutic, amount of an agent is exposed to a substantial portion of the body. Systemic delivery of a lipid nanoparticle can be performed by any means known in the art, including, for example, intravenous, intraarterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of a lipid nanoparticle is performed by intravenous delivery.

[0059] As used herein, "local delivery" refers to the direct delivery of an active agent to a target site within an organism. For example, an agent can be locally delivered by direct injection into a disease site (e.g., a tumor), other target site (e.g., an inflammation site), or target organ (e.g., liver, heart, pancreas, kidney, etc.). Local delivery can also include topical application or local injection techniques, such as intramuscular, subcutaneous, or intradermal injection. Local delivery does not preclude systemic pharmacological effects.

[0060] "Alkyl" refers to a straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms. It is saturated (i.e., does not contain double and / or triple bonds) and has one to twenty-four carbon atoms (C1-C2). 24 Alkyl groups, with one to sixteen carbon atoms (C1-C6). 16 Alkyl groups, with one to twelve carbon atoms (C1-C2). 12 Alkyl groups, and six to twenty-four carbon atoms (C6-C6). 24 Alkyl groups (one to eight carbon atoms (C1-C8 alkyl) or one to six carbon atoms (C1-C6 alkyl) are attached to the rest of the molecule by single bonds, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, etc. Unless otherwise specified in this specification, alkyl groups are optionally substituted.

[0061] "alkylhydroxyl" means an alkyl group as defined herein that contains at least one hydroxyl (OH) substituent.

[0062] "Alkenyl" refers to a straight-chain or branched hydrocarbon chain group composed only of carbon and hydrogen atoms, containing at least one carbon-carbon double bond and having one to twenty-four carbon atoms (C2-C4). 24 alkenyl), one to twelve carbon atoms (C2-C) 12 alkenyl), six to twenty-four carbon atoms (C6-C) 24 alkenyl), two to sixteen carbon atoms (C2-C) 16 alkenyl), four to twelve carbon atoms (C4-C) 12 The alkenyl group (e.g., vinyl, propenyl, 1-methylvinyl, n-butenyl, n-pentenyl, 1,1-dimethylvinyl, 3-methylhexenyl, 2-methylhexenyl, etc.) has one to eight carbon atoms (C2-C8 alkenyl) or one to six carbon atoms (C2-C6 alkenyl) and is connected to the rest of the molecule by a single bond. Unless otherwise specified in this specification, the alkenyl group is optionally substituted.

[0063] "Alkyne" refers to a straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, containing at least one carbon-carbon triple bond and having one to twenty-four carbon atoms (C2-C4). 24 alkynyl group), one to twelve carbon atoms (C2-C) 12 The alkynyl group (containing one to eight carbon atoms (C2-C8 alkynyl) or one to six carbon atoms (C2-C6 alkynyl) is attached to the rest of the molecule by a single bond, for example, ethynyl, n-propynyl, 1-methylethynyl, n-butynyl, n-pentynyl, 1,1-dimethylethynyl, 3-methylhexynyl, 2-methylhexynyl, etc. Unless otherwise specified in this specification, the alkynyl group is optionally substituted.

[0064] “Alkylene” or “alkylene chain” refers to a straight or branched divalent saturated hydrocarbon chain consisting solely of carbon and hydrogen atoms, which links the rest of the molecule to a radical group. In some embodiments, the alkylene chain has one to twenty-four carbon atoms (Ci-C 24 alkylene), one to fifteen carbon atoms (Ci-C 15 alkylene), one to twelve carbon atoms (Ci-C 12 alkylene), one to eight carbon atoms (Ci-C8alkylene), one to six carbon atoms (Ci-C6alkylene), four to six carbon atoms (C4-C6alkylene), two to four carbon atoms (C2-C4alkylene), one to two carbon atoms (Ci-C2alkylene), e.g., methylene, ethylene, propylene, n-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 specifically stated otherwise in the specification, the alkylene chain is optionally substituted. “Cycloalkyl” or “carbocyclic” refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which can include a fused or bridged ring system, having three to fifteen ring carbon atoms (C3-C 15 ), three to ten ring carbon atoms (C3-C 10 ), or three to eight ring carbon atoms (C3-C8), and which is saturated or unsaturated and attached to the rest of the molecule through 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-dimethyldicyclo[2.2.1]heptyl, and the like. Unless specifically stated otherwise in the specification, the cycloalkyl radical is optionally substituted.

[0065] “Aryl” refers to a carbocyclic ring system radical comprising hydrogen, six to eighteen carbon atoms, and at least one aromatic ring. For purposes of this disclosure, aryl radicals are monocyclic, bicyclic, tricyclic, or tetracyclic ring systems, which can include fused or bridged ring systems. Aryl includes, but is not limited to, aryl radicals derived from acanthrylene, acenaphthylene, ac phenanthrylene, anthracene, azulene, benzene, biphenyl, chrysene, fluorene, indacene, indathrene, indane, indene, naphthalene, phenalene, phenanthracene, pyrene, and triphenylene.

[0066] “Aralkyl” refers to a radical of the formula -R b -R c where R b is alkylene or alkenylene as defined above and R cFor one or more aryl groups as defined above, for example, benzyl, diphenylmethyl, and the like. Unless specifically indicated otherwise in the specification, arylalkyl groups are optionally substituted.

[0067] "Heterocyclyl" or "heterocycle" refers to a stable 3- to 18-membered non-aromatic ring radical which has from one to twelve ring carbon atoms (e.g., two to twelve) and from one to six ring heteroatoms which are selected from the group consisting of nitrogen, oxygen, and sulfur. Unless specifically indicated otherwise in the specification, a heterocyclyl group is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can include fused, spiro (a "spiro-heterocyclyl") and / or bridged ring systems; and the nitrogen, carbon, or sulfur atoms in the heterocyclyl radical are optionally oxidized; the nitrogen atom is optionally quaternized; and the heterocyclyl radical is partially or fully saturated. Examples of such heterocyclyl groups include, but are not limited to, dioxolanyl, thienyl[l,3]dithianyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolizinyl, octahydroisoindolizinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless specifically indicated otherwise in the specification, a heterocyclyl group is optionally substituted.

[0068] The term "substituted" as used herein means any of the above groups (e.g., alkyl, alkylhydroxy, alkenyl, alkynyl, alkylidene, cycloalkyl, aryl, arylalkyl, or heterocyclyl) in which at least one hydrogen atom is replaced by a non-hydrogen atom such as, but not limited to: a halogen atom, e.g., F, Cl, Br, and I; an oxo group (=0); a hydroxyl group (-OH); an alkoxy group (-OR a wherein R a is C1-C 12 alkyl or cycloalkyl); a carboxyl group (-OC(=0)R a or -C(=0)OR a wherein R a is H, C1-C 12 alkyl or cycloalkyl); an amine group (-NR a R b wherein R a and R b are each independently H, C1-C 12 alkyl or cycloalkyl); a C1-C 12 alkyl group; and a cycloalkyl group. In some embodiments, the substituent is a C1-C 12Alkyl group. In other embodiments, the substituent is a cyclic alkyl group. In other embodiments, the substituent is a halogen group, such as fluorine. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkyloxy group. In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group.

[0069] The terms "optional" or "optionally" (e.g., optionally substituted) mean that the event described below may or may not occur, and the description includes both cases where the event or condition occurs and cases where the event or condition does not occur. For example, "optionally substituted alkyl" means that the alkyl group may or may not be substituted, and the description includes both substituted and unsubstituted alkyl groups. In some embodiments, "optionally substituted" means that a particular group is substituted by one or more substituents selected from: halogens (e.g., F, Cl, Br, and I), oxo (=O), hydroxyl (-OH), alkoxy (-OR). a , where R a It is C1-C 12 Alkyl), cyclic alkyl group (-OR) a , where R a It is a C3-C8 cyclic hydrocarbon group, a carboxyl group (-OC(=O)R) a or -C(=O)OR a , where R a It is H, C1-C 12 Alkyl or C3-C8 cyclic hydrocarbon groups), amines (-NR) a R b , where R a and R b Each independently constitutes H, C1-C 12 Alkyl or C3-C8 cyclic hydrocarbon groups), C1-C 12 Alkyl and C3-C8 cyclic hydrocarbon groups.

[0070] In some embodiments, "optionally substituted" means substituted with one or more halogen 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 some 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 some embodiments, "optionally substituted" means substituted with one or more carboxyl substituents. In some embodiments, "optionally substituted" means substituted with one or more amine substituents. In some embodiments, "optionally substituted" means substituted with one or more C1-C1 substituents. 12substituted with one or more C3-C8cycloalkyl substituents. In some embodiments, "optionally substituted" means substituted with one or more C3-C8heterocycloalkyl substituents.

[0071] When a functional group is described as being "optionally substituted" and, in turn, a substituent on that functional group is also "optionally substituted," then for the purpose of this disclosure, such iterative repetition is limited to five, preferably two. In some embodiments, such iterative repetition is limited to one. In some embodiments, such iterative repetition is limited to zero.

[0072] The present disclosure also contemplates all pharmaceutically acceptable compounds of the compounds of Structure (I) that are isotopically-labeled by replacement of one or more atoms with -atoms having different atomic masses or mass numbers. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, and iodine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 36 Cl, 123 I, and 125 I. These radiolabeled compounds can be used in 3 H, and carbon- 14 i.e. 14 C, incorporation can facilitate

[0073] Substitution with heavier isotopes such as deuterium, i.e. 2 H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence can be preferred in some circumstances.

[0074] Substitution with positron emitting isotopes such as 11 C, 18 F, 15 O, and 13N-substitution can be used in positron emission tomography (PET) studies to examine substrate receptor occupancy. Isotopically-labeled compounds of structure (I) typically can be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described below for the production of the non-labeled compounds using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.

[0075] The present disclosure also is intended to encompass in vivo metabolic products of the disclosed compounds. Such products can result for example from oxidative, reductive, hydrolytic, amidation, esterification, and like processes that primarily occur due to enzymatic processes. Accordingly, the present disclosure includes compounds produced by a process comprising administering a compound of the present disclosure to a mammal for a period of time sufficient to yield a metabolic product thereof. Such products are typically identified by administering a detectably labeled compound of the disclosure to an animal (e.g., rat, mouse, guinea pig, monkey, or human), allowing sufficient time for metabolism to occur, and isolating the products of metabolism from urine, blood, or other biological samples.

[0076] "Stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.

[0077] "Mammal" includes humans and both domestic animals such as laboratory test animals and household pets (e.g., cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and non-domestic animals (e.g., wildlife, etc.).

[0078] "Pharmaceutically acceptable carrier, diluent or excipient" includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or carrier materials, coatings, isotonic and absoibable salts, preservatives, buffers, antioxidants, or drugs, which are compatible with the disclosed compounds and are not deleterious to the recipients thereof.

[0079] "Pharmaceutically acceptable salt" includes both acid and base addition salts.

[0080] "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 not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1 -hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like.

[0081] "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 inorganic bases or from organic bases. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, 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, methylglucosamine, theobromine, triethanolamine, tromethamine, purines, piperizine, piperidine, N - ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0082] Crystallization generally produces solvates of the compounds 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 can be water, in which case the solvate can be a hydrate. Alternatively, the solvent can be an organic solvent. Thus, the compounds of the disclosure can exist in a hydrate form (including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, and the like) as well as corresponding solvated forms. The compounds of the disclosure can be true solvates, while in other instances, the compounds of the disclosure can only retain a mixture of the extraneous solvent or solvents with water.

[0083] A "pharmaceutical composition" refers to a formulation of a compound of the disclosure and a medium generally accepted for the delivery of biologically active compounds to mammals, e.g., humans. Such media include all pharmaceutically acceptable carriers, diluents or excipients.

[0084] An "effective amount" or "therapeutically effective amount" refers to the amount of a compound of the disclosure that, 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 that 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 in a routine manner by a person of ordinary skill in the art in view of his own knowledge and this disclosure.

[0085] As used herein, "treating" or "treatment" covers the treatment of a target disease or condition in a mammal, preferably a human, after the manifestation of the target disease or condition, and includes: (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 with the condition; (ii) inhibiting the disease or condition, i.e., arresting its development; (iii) relieving the disease or condition, i.e., causing regression of the disease or condition; or (iv) alleviating 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" can be used interchangeably, or can differ in that a particular disease or condition can not have a known causative agent (such that the etiology has not yet been worked out), and thus it has not yet been identified as a disease but only as an undesirable state or syndrome, in which a clinician has identified a more or less specific set of symptoms.

[0086] The compounds of the disclosure, or pharmaceutically acceptable salts thereof, can contain one or more asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms, which can be defined, in terms of absolute stereochemistry, as (R )- or ( S )-, or for amino acids is defined as (D)- or (L)-. The present disclosure is intended to include all such possible isomers, as well as, their racemic mixtures and optically pure forms. Optically active (+) and (-), ( R )- and ( S )-, or (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques such as chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of single enantiomers include chiral synthesis from a suitable optically pure precursor (e.g., an alcohol) or reso lution of the racemic form (or salt or derivative thereof) using, for example, chiral high pressure liquid chromatography. When the compounds described herein contain olefinic double bonds, other geometric isomers are intended unless otherwise stated. Likewise, when the compounds described herein contain other geometrically isomeric centers, for example, at aryl-alkenyl, or cistrans double bonds, both isomers, are intended to be included.

[0087] " Stereoisomers " refer to compounds composed of the same atoms in the same sequence, but having a different three-dimensional configuration, and which are not interchangeable. The present disclosure contemplates various stereo isomers and mixtures thereof, and includes " enantiomers ", which refers to two stereoisomers of a compound that are mirror images of one another and are not superimposable.

[0088] " Tautomers " refer to the transfer of a proton from one atom to another atom within the same molecule. The present disclosure includes any tautomers of the compounds described.

[0089] compound

[0090] In one aspect, the present disclosure provides novel lipid compounds that are capable of being combined with other lipid components such as neutral lipids, charged lipids, steroids, and / or polymer conjugated lipids to form lipid nanoparticles with oligonucleotides. Without wishing to be bound by theory, it is believed that these lipid nanoparticles shield the oligonucleotides from degradation in serum and provide for efficient delivery of the oligonucleotides to cells in vitro and in vivo.

[0091] One embodiment provides a compound having the following structure (I):

[0092] (I)

[0093] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: A is a 3- to 10-membered carbocyclic ring or a 3- to 10-membered oxygen-containing heterocyclic ring, the carbocyclic ring or oxygen-containing heterocyclic ring optionally substituted with one or more fluoro, hydroxyl, C1-C6 alkyl, and / or C1-C6 alkylhydroxyl substituents; R 1 is -NRa C(=O)R 3 or -C(=O)NR b R c ; R 2 is -NR d C(=O)R 4 or -C(=O)NR e R f ; R 3 and R 4 each independently is C6-C 24 alkyl or C6-C 24 alkenyl; R a , R b , R d and R e each independently is H, C1-C 16 alkyl or C2-C 16 alkenyl; R c and R f each independently is C1-C 16 alkyl or C2-C 16 alkenyl; L 1 and L 2 each independently is a direct bond or C1-C6alkylene; and L 2a and L 2b each independently is C4-C 12 alkylene; wherein each alkyl, alkylene and alkenyl is optionally substituted with fluorine.

[0094] One embodiment provides a compound having the following structure (I):

[0095] (I)

[0096] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: A is a 3- to 10-membered carbocyclic ring or a 3- to 10-membered oxygen-containing heterocyclic ring, which carbocyclic ring or oxygen-containing heterocyclic ring is optionally substituted with one or more fluorine, hydroxyl, C1-C6alkyl or C1-C6alkylhydroxyl substituents (including combinations thereof, i.e., one or more fluorine substituents and one or more hydroxyl substituents, etc.); R 1 is -NR a C(=O)R 3 or -C(=O)NR b R c ; R 2 is -NR d C(=O)R 4 or -C(=O)NR e R f ; R 3 and R 4 are each independently C6-C 24 alkyl or C6-C 24 alkenyl; R a , R b , R d , and R e are each independently H, C1-C 20 alkyl or C2-C 20 alkenyl; R c and R f are each independently C1-C 20 alkyl or C2-C 20 alkenyl; L 1 and L 2 are each independently a direct bond or C1-C6alkylene; and L 2a and L 2b are each independently C4-C 12 alkylene; wherein each alkyl, alkylene, and alkenyl is optionally substituted with one or more fluorines.

[0097] In some embodiments, R 1 is -C(=O)NR b R c In some embodiments, R 1 is -NR a C(=O)R 3 In some embodiments, R 2 is -C(=O)NR e R f In certain embodiments, -NR d C(=O)R 4 In certain embodiments, -C(=O)NR b R c and R 2 is -C(=O)NR e R f In certain embodiments, R 1 is -NR a C(=O)R 3 and R 2 is -NR d C(=O)R4 In certain embodiments, R 1 is -NR a C(=O)R 3 and R 2 is -C(=O)NR e R f .

[0098] In some embodiments, R a is C8-C 12 alkyl. In certain embodiments, R a is C8, C9, or C 10 alkyl. In some embodiments, R b is C8-C 12 alkyl. In certain embodiments, R b is C8, C9, or C 10 alkyl. In some embodiments, R c is C8-C 12 alkyl. In certain embodiments, R c is C8, C9, or C 10 alkyl. In some embodiments, R d is C8-C 12 alkyl. In certain embodiments, R d is C8, C9, or C 10 alkyl. In some embodiments, R e is C8-C 12 alkyl. In certain embodiments, R e is C8, C9, or C 10 alkyl. In some embodiments, R f is C8-C 12 alkyl. In certain embodiments, R f is C8, C9, or C 10 alkyl.

[0099] In some embodiments, R a , R b , R c , R d , R e , and R f are each independently C1-C 20 alkyl. In certain embodiments, R a , R b , R c , R d , R e , and R f are each independently C8-C 19 alkyl. In some embodiments, R a , Rb , R c , R d , R e , and R f are each independently C8, C9, C 10 , or C 19 alkyl. In certain embodiments, R 3 and R 4 are each independently C6-C 19 alkyl.

[0100] In some embodiments, R 3 is C6-C 12 alkyl. In certain embodiments, R 3 is C8-C 10 alkyl. In certain embodiments, R 3 is C8, C9, or C 10 alkyl. In some embodiments, R 4 is C6-C 12 alkyl. In certain embodiments, R 4 is C8-C 10 alkyl. In certain embodiments, R 4 is C8, C9, or C 10 alkyl.

[0101] In some embodiments, L 1 is a direct bond. In certain embodiments, L 1 is C1-C6 alkylene. In certain embodiments, L 1 is C1 or C2 alkylene. In some embodiments, L 2 is a direct bond. In certain embodiments, L 2 is C1-C6 alkylene. In certain embodiments, L 2 is C1 or C2 alkylene.

[0102] In some embodiments, L 2a is C6-C 10 alkylene. In certain embodiments, L 2a is C6, C7, C8, C9, or C 10 alkylene. In some embodiments, L 2b is C6-C 10 alkylene. In certain embodiments, L 2b is C6, C7, C8, C9, or C 10 alkylene.

[0103] In some embodiments, A is C3-C 10Cycloalkyl. In certain embodiments, A is monocyclic. In certain embodiments, A is bicyclic. In certain embodiments, A is spirocyclic.

[0104] In some embodiments, A is C3-C8 cycloalkyl optionally substituted with one or more hydroxyl and / or C1-C6 alkylhydroxyl substituents. In certain embodiments, A is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl.

[0105] In some embodiments, A is a 3- to 10-membered oxygen-containing heterocycle optionally substituted with one or more hydroxyl and / or C1-C6 alkylhydroxyl substituents. A is a 3- to 6-membered oxygen-containing heterocycle optionally substituted with one or more hydroxyl and / or C1-C6 alkylhydroxyl substituents. In certain embodiments, A is oxiranyl, oxetanyl, tetrahydrofuranyl, or tetrahydropyranyl.

[0106] In some embodiments, A is unsubstituted. In certain embodiments, A is substituted with hydroxyl.

[0107] In some embodiments, having one of the following structures: ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; , or a stereoisomer thereof.

[0108] In certain embodiments, having one of the following structures: ; ; ; ; ; ; ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ; ; ; ; ; ; ; or .

[0109] In some embodiments, R a substituted with one or more fluorine atoms. In some embodiments, R b substituted with one or more fluorine atoms. In some embodiments, R c substituted with one or more fluorine atoms. In some embodiments, R d substituted with one or more fluorine atoms. In some embodiments, R e substituted with one or more fluorine atoms. In some embodiments, R f substituted with one or more fluorine atoms. In some embodiments, L 1 substituted with one or more fluorine atoms. In some embodiments, L 2 substituted with one or more fluorine atoms. In some embodiments, L 2a substituted with one or more fluorine atoms. In some embodiments, L 2b substituted with one or more fluorine atoms.

[0110] In various embodiments, the compounds have one of the structures listed in Table 1 below.

[0111] Table 1

[0112] One embodiment provides a racemic mixture of one of the following pairs of enantiomers of structure (I): and ; and ; and ; and ; or and .

[0113] One embodiment provides a racemic mixture or combination of one of the following pairs of enantiomers of structure (I): and ; and ; and ; and ; and ; and ; and ; , , and ; and .

[0114] It should be understood that any embodiment of a compound of Structure (I) as described above, as well as any particular substituents and / or variables of a compound of Structure (I) as described above, can be independently combined with other embodiments and / or substituents and / or variables of a compound of Structure (I) to form embodiments of the disclosure not specifically set forth above. Additionally, where a list of substituents and / or variables for any particular A group, R group, or L group is set forth in a particular embodiment and / or claim, it should be understood that each individual substituent and / or variable can be deleted from the particular embodiment and / or claim, and the remaining list of substituents and / or variables will be considered to be within the scope of embodiments of the disclosure.

[0115] It should be understood that, in the present specification, combinations of substituents and / or variables depicted by a depicted general formula are permissible only if such contributions result in stable compounds.

[0116] For purposes of administration, the compounds of the disclosure, typically in the form of a lipid nanoparticle in combination with a therapeutic agent, can be administered as a chemical raw material or can be formulated into a pharmaceutical composition. The pharmaceutical compositions of the disclosure comprise a compound of Structure (I) and one or more pharmaceutically acceptable carriers, diluents, or excipients. The compound of Structure (I) is present in the composition in an amount effective to form a lipid nanoparticle and deliver a therapeutic agent, e.g., a therapeutic agent for treating a particular target disease or condition. Appropriate concentrations and dosages can be readily determined by one of skill in the art.

[0117] Embodiments provide compositions comprising a compound of Structure (I) and a therapeutic agent. In some embodiments, the composition further comprises one or more excipients selected from neutral lipids, steroids, and polymer-conjugated lipids.

[0118] In some embodiments, the therapeutic agent comprises a nucleic acid. In certain embodiments, the nucleic acid is selected from the group consisting of antisense RNA and messenger RNA.

[0119] In certain embodiments, the composition comprises one or more neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the neutral lipid is DSPC. In some embodiments, the molar ratio of the compound to the neutral lipid is 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 is about 2: 1 to 1: 1. In certain embodiments, the polymer-conjugated lipid is a pegylated lipid. In various embodiments, the polymer-conjugated lipid is a pegylated lipid. For example, some embodiments include a PEGylated diacylglycerol (PEG-DAG), such as l-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG); a PEGylated phosphatidylethanolamine (PEG-PE); a PEG succinic acid diacylglycerol (PEG-S-DAG), such as 4-0-(2',3'-ditetradecanoyloxy)propyl-l-0-(w-methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG); a PEGylated ceramide (PEG-cer); or a PEG dialkoxylpropyl carbamate, such as w-methoxy(polyethoxy)ethyl-N-(2,3-ditetradecyloxypropyl) carbamate or 2,3-ditetradecyloxypropyl-N-(w-methoxy(polyethoxy)ethyl) carbamate. In some embodiments, the molar ratio of the compound to the pegylated lipid is about 100: 1 to about 10: 1 or about 100: 1 to about 25: 1. In some embodiments, the PEGylated lipid is PEG-DMG. In certain embodiments, the pegylated lipid has the following structure (II):

[0120] (II)

[0121] or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein: R 10 and R 11 each independently is a straight chain or branched alkyl, alkenyl, or alkynyl, each containing 10 to 30 carbon atoms, wherein the alkyl, alkenyl, or alkynyl is optionally interrupted by one or more ester linkages; and w has a value of 30 to 60.

[0122] In some embodiments, R 10 and R 11 each independently is a straight chain alkyl chain containing 12 to 16 carbon atoms.

[0123] In some embodiments, the lipid nanoparticles or composition comprise a plurality of compounds of structure (II), and for the plurality of compounds, the average value of w is about 49. In some embodiments, the plurality of compounds of structure (II) have an average w of 40 to 55. In some embodiments, the plurality of compounds of structure (II) have an average w of 40 to 50, or 42 to 48. In some embodiments, the plurality of compounds of structure (II) have an average w of 30 to 55, 30 to 50, 30 to 45, 30 to 40, or 30 to 35. In some embodiments, the plurality of compounds of structure (II) have an average w of 35 to 55, 40 to 55, 42 to 55, 45 to 55, or 48 to 55. In some embodiments, the average w is about 45 (e.g., 43, 44, 45, 46, or 47). In some embodiments, the average w is about 43 to 47. In some embodiments, the average w is about 40-50. In some implementations, the average w is approximately 48 to 55.

[0124] The synthesis of PEGylated lipids can be found in U.S. Patent No. 9,738,593, the disclosure of which is incorporated herein by reference.

[0125] method of administration

[0126] The compositions of this disclosure can be administered via any acceptable method of administration of the pharmaceutical agent to achieve similar efficacy. The pharmaceutical compositions of this disclosure can be formulated into formulations in solid, semi-solid, liquid, or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalers, gels, microspheres, and aerosols. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal administration. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intradermal, intrasternal injection, or infusion techniques. The pharmaceutical compositions of this disclosure are formulated such that the active ingredient contained therein is bioavailable after administration of the composition to a patient. The composition to be administered to an individual or patient is in the form of one or more dose units, wherein, for example, a tablet may be a single dose unit, and a container of the compounds of this disclosure in aerosol form may contain multiple dose units. Practical methods for preparing such dosage forms are known to those skilled in the art or will be apparent; see, for example, [link to relevant documentation]. Remington: The Science and Practice of PharmacyRemington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). In accordance with the teachings of the present disclosure, in any case, the composition to be administered will contain a therapeutically effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof for the treatment of the disease or condition of interest.

[0127] One embodiment provides a method for administering a therapeutic agent to a patient in need thereof, the method comprising preparing or providing a lipid nanoparticle or composition of the present disclosure, and administering the composition to the patient.

[0128] One embodiment provides a pharmaceutical composition comprising a lipid nanoparticle of the present disclosure and a pharmaceutically acceptable diluent or excipient.

[0129] Another embodiment provides a method for inducing protein expression in a patient in need thereof, the method comprising administering to the patient a pharmaceutical composition of the present disclosure, wherein the lipid nanoparticle comprises mRNA encoding the protein. In some embodiments, the protein is an antigen, and the method is for inducing an immune response in a patient. In certain embodiments, the protein is an antigen, and the method is for vaccinating a patient against a pathogen. In some embodiments, the protein is for gene editing.

[0130] The pharmaceutical compositions of the present disclosure can be in the form of a solid or a liquid. In one aspect, the carrier is particulate, such that the composition is in the form of, for example, a tablet or a powder. The carrier can be a liquid, wherein the composition is, for example, an oral syrup, an injectable liquid, or an aerosol, which can be useful, for example, in inhalable administration.

[0131] When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, with semi-solid, semi-liquid, suspension, and gel forms being included within the forms considered herein as either solid or liquid.

[0132] As a solid composition for oral administration, the pharmaceutical composition can be formulated into the form of powders, granules, compressed tablets, pills, capsules, chewing gum, wafers, and the like. Such solid compositions generally include one or more inert diluents or edible carriers. Additionally, one or more of the following can be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starches, lactose, or dextrins; disintegrating agents such as alginic acid, sodium alginate, Primogel, cornstarch, and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; flavoring agents such as peppermint oil, methyl salicylate, or orange flavoring; and coloring agents.

[0133] When the pharmaceutical composition is in the form of a capsule, such as a gelatin capsule, it can contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.

[0134] The pharmaceutical composition can be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension. The liquid can be for oral administration or for delivery by injection as two examples. When intended for oral administration, preferred compositions comprise one or more of a sweetening agent, preservatives, dye / colorant and flavor enhancer, in addition to the compound of the present application. In compositions intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can be included.

[0135] The liquid pharmaceutical compositions of the present disclosure, whether they are solutions, suspensions or other like form, can include one or more of the following adjuvants: sterile diluent such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or digylcerides, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl parabens; 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 for the adjustment of pH such as hydrochloric acid or sodium hydroxide; and agents for the adjustment of isotonicity such as sodium chloride or dextrose. 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. The injectable pharmaceutical composition is preferably sterile.

[0136] The liquid pharmaceutical compositions of the present disclosure intended for either parenteral or oral administration should contain an amount of the compound of the present disclosure sufficient to produce the desired effects in combination with an appropriate diluent solution.

[0137] The pharmaceutical compositions of the present disclosure can be intended for topical administration, in which case the carrier will conveniently comprise a solution, emulsion, ointment or gel base. The base, for example, can comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents can be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition can include a transdermal patch or iontophoresis device.

[0138] The pharmaceutical compositions of the present disclosure can be intended for rectal administration, for example, in the form of suppositories, which melt in the rectum and release the drug. The compositions for rectal administration can contain an oleaginous base as a suitable nonirritating excipient. Such bases include, but are not limited to, lanolin, cocoa butter and polyethylene glycol.

[0139] The pharmaceutical compositions of the present disclosure can include various materials which alter the physical form of a solid or liquid dosage unit. For example, the composition can include materials which form a coating shell around the active ingredients. The materials which form the coating shell are typically inert and can be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients can be encased in a gelatin capsule.

[0140] The pharmaceutical compositions of the present disclosure in solid or liquid form can include an agent that binds the compound of the present disclosure to facilitate delivery of the compound. Suitable agents that can function in this capacity include monoclonal or polyclonal antibodies or proteins.

[0141] The pharmaceutical compositions of the present disclosure can consist of dosage units that can be administered as an aerosol. The term "aerosol" is used to mean a variety of systems ranging from those of a colloidal nature to systems consisting of pressurized packages. Delivery can be by a liquefied gas or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols of the compounds of the present disclosure can be delivered in a single phase, two phase, or three phase system to deliver the active ingredients. Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together can form a kit. One skilled in the art can determine preferred aerosols without undue experimentation.

[0142] The pharmaceutical compositions of the present disclosure can be prepared by methods well known in the pharmaceutical art. For example, a pharmaceutical composition intended to be administered by injection can be prepared by combining a lipid nanoparticle of the present disclosure with a sterile, distilled water or other carrier to form a solution. A surfactant can be added to facilitate the formation of a uniform solution or suspension. A surfactant is a compound that non-covalently interacts with the compound of the present disclosure to facilitate the dissolution or uniform suspension of the compound in an aqueous delivery system.

[0143] The compositions of the present disclosure, or pharmaceutically acceptable salts thereof, are administered in therapeutically effective amounts. The amount that will constitute a therapeutically effective dose will vary depending on a variety of factors including the activity of the specific therapeutic agent employed; the metabolic stability and length of action of the therapeutic agent; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the excitation rate; the drug combination; the severity of the particular disorder or condition; and the individual undergoing therapy.

[0144] The compositions of this disclosure may also be administered concurrently with, before, or after the administration of one or more other therapeutic agents. Such combination therapies include single-dose formulations of the compositions of this disclosure and one or more additional active agents, as well as separate drug-dose formulations of the compositions of this disclosure and each active agent. For example, the compositions of this disclosure and other active agents may be administered to a patient together as a single oral dose composition (such as tablets or capsules), or as each agent in separate oral dose formulations. When using separate dose formulations, the compounds of this disclosure and one or more additional active agents may be administered at substantially the same time, i.e., simultaneously, or at separately staggered times, i.e., sequentially; combination therapy is understood to include all of these regimens.

[0145] The methods for preparing the above compounds and compositions are described below and / or are known in the art.

[0146] Those skilled in the art will understand that, in the methods described herein, the functional groups of the intermediate compounds may require protection by suitable protecting groups. Such functional groups include hydroxyl, amino, mercapto, and carboxylic acids. Suitable protecting groups for hydroxyl groups include trialkylsilyl or diarylalkylsilyl (e.g., tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or trimethylsilyl), tetrahydropyranyl, benzyl, etc. Suitable protecting groups for amino, amidine, and guanidine groups include tert-butyloxycarbonyl, benzyloxycarbonyl, etc. Suitable protecting groups for mercapto groups include -C(O)-R″ (where R″ is alkyl, aryl, or arylalkyl), p-methoxybenzyl, triphenylmethyl, etc. Suitable protecting groups for carboxylic acids include alkyl esters, aryl esters, or arylalkyl esters. Protecting groups may be added or removed according to techniques known to those skilled in the art and as described herein, as well as standard techniques. The use of protecting groups is described in detail in Green, TW, and PGM Wutz. Protective Groups in Organic Synthesis (1999), 3rd edition, Wiley. As those skilled in the art will understand, the protecting group may also be a polymeric resin, such as Wang resin, Rink resin, or 2-chlorotriphenylmethyl-chloride resin.

[0147] Those skilled in the art will also understand that although such protected derivatives of the compounds of this disclosure may not be pharmacologically active on their own, they can be administered to mammals and subsequently metabolized in vivo to form pharmacologically active compounds of this disclosure. Therefore, such derivatives can be described as “prodrugs.” All prodrugs of the compounds of this disclosure are included within the scope of this disclosure.

[0148] Furthermore, all compounds of this disclosure, existing in free base or acid form, can be converted into their pharmaceutically acceptable salts by methods known to those skilled in the art, through treatment with suitable inorganic or organic bases or acids. Salts of the compounds of this disclosure can be converted into their free base or acid forms using standard techniques.

[0149] The following reaction scheme illustrates a method for preparing the compound disclosed herein, namely the compound of structure (I):

[0150] (I)

[0151] Where R 1 R 2 L 1 L 2 L 2a L 2b And A as defined herein. It should be understood that those skilled in the art can prepare these compounds by similar methods or by combining other methods known to them. It should also be understood that those skilled in the art will be able to prepare other compounds of structures (I) not specifically exemplified below in a similar manner by using appropriate starting components and changing the synthetic parameters as needed. Typically, starting components can be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, or synthesized from sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Edition (Wiley, December 2000)) or prepared as described in this disclosure.

[0152] General Reaction Scheme 1

[0153] The compound of structure (I) can be prepared according to general reaction scheme 1 (“Program A”).

[0154] Program A

[0155] Referring to General Reaction Scheme 1, compound starting material A1 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. To a mixture of starting material A1 is added A2 and the reaction mixture is stirred at room temperature for 1 h. The reaction mixture is concentrated and used without further purification for the next step. To the mixture is added A3 and the reaction mixture is stirred at room temperature for 17 h. The reaction mixture is concentrated and the crude material is re-suspended in hexanes. The resulting solid is filtered and the filtrate is purified via automated flash chromatography to yield A4.

[0156] A mixture of A4 and the appropriate amine is heated at 75 °C for 24 h to 72 h. The reaction mixture is concentrated and the resulting solid is filtered and the filtrate is purified by automated flash chromatography to yield the desired compound of structure (I).

[0157] It should be noted that various alternative strategies for preparing compounds of structure (I) are available to one of ordinary skill in the art. For example, other compounds of structure (I) can be prepared according to analogous methods using appropriate starting materials, and as shown in the following examples. The use of protecting groups and other modifications to the above general reaction schemes will be apparent to one of ordinary skill in the art as needed. The following examples are provided for purposes of illustration and not limitation.

[0158] Example 1

[0159] In vivo evaluation of luciferase mRNA using lipid nanoparticle compositions

[0160] Lipids of structure (I), DSPC, cholesterol, and PEG-lipid were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 or 47.5:10:40.7:1.8. Lipid nanoparticles (LNPs) were prepared at a total lipid to mRNA weight ratio of about 10:1 to 40:1. Briefly, mRNA was diluted to 0.2 mg / mL in 10 to 50 mM citrate buffer, pH 4-6, or 10 to 25 mM acetate buffer, pH 4. The ethanol lipid solution was mixed with the aqueous mRNA solution at a ratio of about 1:5 to 1:3 (vol / vol) using a syringe pump at a total flow rate higher than 15 mL / min. The ethanol was then removed and the external buffer was replaced with PBS by dialysis. Finally, the lipid nanoparticles were filtered through a 0.2 pm pore sterile filter.

[0161] Studies were performed in 6-8 week old female C57BL / 6 mice (Charles River) or 8-10 week old CD-1 mice (Charles River or Inotiv) in accordance with guidelines established by the Institutional Animal Care Committee (ACC) and the Canadian Council on Animal Care (CCAC). Various doses of mRNA-lipid nanoparticles were administered systemically via tail vein injection and animals were euthanized at specific time points post administration (e.g., 4 hours). Livers and spleens were collected in pre-weighed tubes, weighed, immediately snap-frozen in liquid nitrogen and stored at -80°C until processed for analysis.

[0162] For livers, approximately 50 mg were cut into 2 mL FastPrep tubes (MP Biomedicals, Solon OH) for analysis. A ¼” ceramic bead (MP Biomedicals) was added to each tube and 500 to 750 µL of Glo Lysis Buffer - GLB (Promega, Madison WI) equilibrated to room temperature was added to the liver tissue. The liver tissue was homogenized with a FastPrep 24 instrument (MP Biomedicals) at 2 x 6.0 m / s for 15 seconds. The homogenate was incubated at room temperature for 5 minutes, then diluted 1:4 to 1:6 in GLB and evaluated using the SteadyGlo Luciferase Assay System (Promega). Specifically, 50 µL of diluted tissue homogenate was reacted with 50 µL of SteadyGlo substrate, shaken for 10 seconds, followed by a 5 minute incubation, then luminescence was quantified using a FilterMax F5 Microplate Reader (Molecular Devices, USA). The amount of protein assayed was determined by using a BCA Protein Assay Kit (Pierce, Rockford, IL). Relative luminescence units (RLU) were then normalized to the total µg of protein assayed or the weight (g) of the tissue. To convert RLU to ng of luciferase, a standard curve was generated with QuantiLum Recombinant Luciferase (Promega).

[0163] FLuc mRNA (L-7202) from Trilink Biotechnologies will express luciferase originally from the firefly Photinus pyralis (P. pyralis) (GenBank Accession No. AY823993.1). The mRNA sequence is shown below: photinus pyralisLuciferase protein isolated in the Examples of the present disclosure. Fluc is commonly used in mammalian cell culture to measure gene expression and cell viability. In the presence of the substrate luciferin, it emits bioluminescence. This capped and polyadenylated mRNA is modified with 5-methoxyuridine and optimized for mammalian systems.

[0164] Example 2

[0165] In vivo evaluation of immunoglobulin G (IgG) mRNA using lipid nanoparticle compositions

[0166] Lipids of structure (I), DSPC, cholesterol, and PEG-lipid were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 or 47.5:10:40.7:1.8. Lipid nanoparticles (LNPs) were prepared at a total lipid to mRNA weight ratio of about 10:1 to 40:1. Briefly, mRNA was diluted to 0.2 mg / mL in 10 to 50 mM citrate buffer (pH 4-6) or 10 to 25 mM acetate buffer (pH 4-6). The ethanol lipid solution was mixed with the aqueous mRNA solution at a ratio of about 1:5 to 1:3 (vol / vol) using a syringe pump at a total flow rate higher than 15 mL / min. The ethanol was then removed and the external buffer was replaced with PBS by dialysis. Finally, the lipid nanoparticles were filtered through a 0.2 pm pore sterile filter.

[0167] Studies were performed in 6 to 8 week old CD-1 / ICR mice (Envigo, Charles River, or Inotiv) in accordance with the guidelines established by the Animal Care Committee (ACC) and the Canadian Council on Animal Care (CCAC). Different doses of mRNA-lipid nanoparticles were administered systemically by tail vein injection and animals were euthanized at specific time points post administration (e.g., 24 hours). Whole blood was collected and serum was subsequently isolated by centrifugation of whole blood tubes at 2000 x g for 10 minutes at 4°C and stored at -80°C until used for analysis.

[0168] For the immunoglobulin G (IgG) ELISA (Life Diagnostics Human IgG ELISA kit), serum samples were diluted 100 to 20,000 times with 1× diluent solution. 100 μL of diluted serum was dispensed into duplicate into anti-human IgG-coated 96-well plates, along with human IgG standards, and incubated at 25°C for 45 minutes at 150 rpm in a plate shaker. The wells were washed 5 times with 1× washing solution using a plate washer (400 μL / well). 100 μL of HRP conjugate was added to each well, and the plates were incubated under the same conditions as above in a plate shaker. The wells were washed again 5 times with 1× washing solution using a plate washer (400 μL / well). 100 μL of TMB reagent was added to each well, and the plates were incubated under the same conditions as above in a plate shaker. The reaction was terminated by adding 100 μL of stop solution to each well. The absorbance was read at 450 nm (A450) using a microplate reader. The amount of human IgG in mouse serum was determined by plotting the A450 values ​​of the assay standards against the concentration of human IgG.

[0169] Example 3

[0170] pK of the formulated lipids a Measurement

[0171] As described elsewhere, the pK of the formulated lipids a This is related to the efficacy of LNP in delivering nucleic acids (see Jayaraman et al., Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semil et al., Nature Biotechnology 28, 172–176 (2010)). In some implementations, pK a The preferred range is ~5 to ~7. The pK of each lipid in the lipid nanoparticles can be determined using a fluorescence assay based on 2-(p-toluidine)-6-naphthalenesulfonic acid (TNS). aLipid nanoparticles comprising the compound of structure (I) / DSPC / cholesterol / PEG-lipid (50 / 10 / 38.5 / 1.5 or 47.5:10:40.7:1.8 mol%) in PBS at a total lipid concentration of 0.4 mM were prepared using the in-line process as described in Example 1. TNS was prepared as a 100 μΜ stock solution in distilled water. The vesicles were diluted to 24 μΜ lipid in 2 mL buffer solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate and 130 mM NaCl, with a pH range of 2.5 to 11. An aliquot of the TNS solution was added to give a final concentration of 1 μΜ, and after vortex mixing, the fluorescence intensity was measured in a SLM Aminco Series 2 Luminescence spectrophotometer using excitation and emission wavelengths of 321 nm and 445 nm at room temperature. The sigmoidal best fit analysis was applied to the fluorescence data and the pK a was measured as the pH that produced half the maximum fluorescence intensity.

[0172] Example 4

[0173] Determination of efficacy of lipid nanoparticle formulations containing various cationic lipids using an in vivo luciferase / IgG mRNA expression rodent model

[0174] Representative compounds of the disclosure shown in Table 2 were formulated using the following molar ratios: 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG-lipid 2-[2-(ω-methoxy(polyethylene glycol 2000 )ethoxy]-N,N-ditetradecylacetamide) or 47.5% cationic lipid / 10% DSPC / 40.7% cholesterol / 1.8% PEG-lipid. Relative activity was determined by measuring luciferase expression in the liver 4 hours after administration via tail vein injection as described in Example 1, or by determining the amount of human IgG in mouse serum as described in Example 2. Activity was compared at a dose of 1.0 or 0.5 or 0.3 mg mRNA / kg, and expressed as ng luciferase / g liver measured 4 hours after administration as described in Example 1, or as μg IgG / mL serum measured 24 hours after administration as described in Example 2. Compound numbers in Table 2 refer to compound numbers in Table 1.

[0175] Table 2. novel cationic lipids and related activities thereof

[0176] Example 5

[0177] Synthetic route for compounds 1-1 to 1-18

[0178] Synthesis of 8-bromo-N,N-didecanyloctanamide (Intermediate 1)

[0179] To a mixture of 8-bromooctanoic acid (47.3 mmol, 10.5 g) and DMF (cat.) in DCM (100 mL) was added oxalyl chloride (142 mmol, 18.0 g) and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated to give 8-bromooctanoyl chloride which was used in the next step without further purification.

[0180] To a mixture of didecylamine (69.1 mmol, 20.6 g), triethylamine (377 mmol, 52.5 mL) and N,N-dimethylpyridin-4-amine (cat., 8.0 mg) in DCM (100 mL) was added 8-bromooctanoyl chloride (62.8 mmol, 15.2 g) in DCM (60 mL) and the reaction mixture was stirred at room temperature for 17 h. The reaction mixture was concentrated and the crude material was re-suspended in hexanes. The resulting solid was filtered and the filtrate was purified via automated flash chromatography (5% to 25% EtOAc in hexanes) to give 8-bromo-N,N-didecanyloctanamide (26 g, 82%).

[0181] General Procedure A

[0182] A mixture of 8-bromo-N,N-didecanyloctanamide (1.0 to 2.0 eq), the appropriate amine (1.0 eq), diisopropylethylamine (“DIEA” 3.0 to 5.0 eq) and potassium iodide (0.0 to 3.0 eq) in ACN (0.1 M) was heated at 75 °C for 24 h to 72 h. The reaction mixture was concentrated and the crude material was suspended in a mixture of hexanes:EtOAc:Et3N (95:5:1). The resulting solid was filtered and the filtrate was purified by automated flash chromatography to give the desired compound.

[0183] General Procedure B

[0184] A mixture of 8-bromo-N,N-didecyloctanamide (1.0 to 2.0 equivalents), the appropriate amine (1.0 equivalent), DIEA (3.0 to 5.0 equivalents), and potassium iodide (3.0 to 4.0 equivalents) in ACN (0.5 M) was heated via microwave irradiation at 140 °C to 160 °C for 30 minutes to 3 hours. The reaction mixture was concentrated and the crude material was suspended in a mixture of hexanes:EtOAc:Et3N (95:5:1). The resulting solid was filtered, and the filtrate was purified by automated flash chromatography to afford the desired compound.

[0185] Example 6

[0186] 8,8’-((2-((1s,3r)-3-hydroxycyclobutyl)ethyl)azanediyl)bis(N,N-didecyloctanamide) (Compound (1 s ,3 r )-i-1)

[0187] (1 S ,3 R )-I-1

[0188] Compound (1S,3R)-I-1 was synthesized according to general procedure A using (1r,3s)-3-(2- aminoethyl)cyclobutane-1-ol. Purification via automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N) afforded the title compound (yield 12%). 1 H NMR (400 MHz, CDCl3) δ 4.03 - 3.23 (m, 1H), 3.32 – 3.15 (m, 4H), 3.23 – 3.15 (m, 4H), 2.52 – 2.41 (m, 2H), 2.39 – 2.22 (m, 10H), 1.72 – 1.45 (m, 22H), 1.44 – 1.16 (m, 72H), 0.95 - 0.83 (m, 12H). Chemical Formula C 62 H 123 of N3O3 m / z Calculated = 958.0. Found [M+H] + = 959.1.

[0189] Example 7

[0190] 8,8’-((((1s,3s)-3-hydroxycyclobutyl)methyl)azanediyl)bis(N,N-didecyloctanamide) (Compound (1 s ,3 s )-I-2)

[0191] Synthesis of (1S,3S)-I-2

[0192] The compound (1S,3S)-I-2 was synthesized from (1S,3S)-3-(aminomethyl)cyclobutane-1-ol according to general procedure A. The title compound was purified by automated rapid chromatography (5% to 65% EtOAc in hexane solution and 1% Et3N in yield 44%). 1 H NMR (600 MHz, CDCl3) δ 4.18 – 4.10 (m, 1H), 3.33 – 3.27 (m, 4H), 3.24 – 3.19 (m, 4H), 2.52 – 2.44 (m, 4H), 2.40 – 2.34 (m, 4H), 2.32 – 2.26(m, 4H), 1.96 - 1.88 (m, 1H), 1.69 - 1.60 (m ,5H), 1.60 - 1.48 (m, 11H), 1.46– 1.38 (m, 4H), 1.38 - 1.19 (m, 69H), 0.93 - 0.87 (m, 12H). Chemical formula C 61 H 121 N3O3 m / z Calculated value = 943.9. Measured value [M+H] + = 945.0.

[0193] Example 8

[0194] 8,8'-((((1r,3r)-3-hydroxycyclobutyl)methyl)azadiyl)bis(N,N-didecyloctamide) (compound(1r,3r)-I-2)

[0195] (1 R ,3 R Synthesis of )-I-2

[0196] According to general procedure A, compound (1) is synthesized using (1s,3s)-3-(aminomethyl)cyclobutane-1-ol. R ,3 R )-I-2. The title compound was purified by automated rapid chromatography (5% to 65% EtOAc and 1% Et3N in hexane solution) to obtain the title compound (yield 40%). 1H NMR (600 MHz, CDCl3) δ 4.44 - 4.36 (m, 1H), 3.33 – 3.27 (m, 4H),3.24 - 3.19 (m, 4H), 2.41 (s, 3H), 2.39 – 2.33 (m, 4H), 2.32 – 2.26 (m, 4H),2.13 – 1.99 (m, 4H), 1.69 – 1.61 (m, 7H), 1.60 – 1.48 (m, 8H), 1.45 – 1.20(m, 74H), 0.94 - 0.87 (m, 12H). Chemical Formula C 61 H 121 N3O3 of m / z Calculated = 943.9. Found [M+H] + = 944.9.

[0197] Example 9

[0198] 8,8’-((((1r,3s)-3-hydroxycyclohexyl)methyl)azanediyl)bis(N,N-didecyloctanamide)

[0199] 8,8’-((((1s,3r)-3-hydroxycyclohexyl)methyl)azanediyl)bis(N,N-didecyloctanamide)

[0200] (rac)-(1 rs ,2 sr )-I-3

[0201] (rac)-(1 RS ,2 SR )-I-3

[0202] The mixture of enantiomers of compound I-3 was synthesized according to the general procedure A using a mixture of (1R,3S)-3-(aminomethyl)cyclohexan-1-ol and (1S,3R)-3-(aminomethyl)cyclohexan-1-ol. Purification via automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N), followed by a second purification via automated flash chromatography (10% to 100% EtOAc in hexanes) afforded the title compound (yield 27%). 1H NMR (400 MHz, CDC13) δ 3.63 - 3.51 (m, 1H), 3.33 - 3.24 (m, 4H), 3.25 - 3.14 (m, 4H), 2.40 - 2.21 (m, 8H), 2.20 - 2.08 (m, 3H), 2.01 - 1.93 (m, 1H), 1.77 (dt, J = 13.0, 3.4 Hz, 1H),1.71 - 1.59 (m, 10H), 1.57 – 1.44 (m, 10H), 1.42 - 1.06 (m, 80H), 0.97 – 0.64(m, 15H). Chemical Formula C 63 H 125 N3O3 of m / z Calculated = 972.0. Found [M+H] + = 973.1.

[0203] Example 10

[0204] 8,8’-(((3-hydroxycyclopentyl)methyl)azanediyl)bis(N,N-didecyloctanamide)

[0205] (Compound I-4)

[0206] Synthesis of I-4

[0207] Compound I-4 was synthesized according to general procedure A using 3- (aminomethyl)cyclopentan-1-ol. Purification via automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N), followed by a second purification via automated flash chromatography (5% to 100% EtOAc in hexanes) afforded the title compound (yield 39%). 1 H NMR (400 MHz, CDC13) δ 4.39 - 4.31 (m, 1H), 4.29 - 4.20 (m, 1H), 3.31 – 3.24 (m, 4H), 3.23 – 3.14 (m, 4H), 2.45 – 2.30 (m, 6H), 2.29 – 2.21 (m, 5H), 2.18 – 1.97 (m, 1H), 1.97 – 1.84 (m, 1H), 1.86 – 1.08 (m, 96H), 0.94 - 0.83 (m, 13H). Chemical Formula C 62 H 123 N3O3 of m / zCalcd = 958.0. Found [M+H] + = 959.1.

[0208] Example 11

[0209] 8,8’-(((1 r ,3 r )-3- hydroxycyclobutyl)azanediyl)bis(N,N-didecyloctanamide)

[0210] Synthesis of compound (1 r ,3 r )-I-5

[0211] (1 R ,3 R )-I-5

[0212] Compound (1 R ,3 R )-I-5 was synthesized according to general procedure A using (1r,3r)-3- aminocyclobutane-1-ol hydrochloride. Purification via automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N), followed by a second purification via automated flash chromatography (5% to 100% EtOAc in hexanes), afforded the title compound (yield 10%). 1 H NMR (400 MHz, CDC13) δ 4.43 - 4.34 (m, 1H), 3.49 - 3.37 (m, 1H), 3.32 - 3.23 (m, 4H), 3.23 - 3.15 (m, 4H), 2.30 - 2.22 (m, 4H), 2.22 - 2.12 (m, 2H), 2.08 - 1.97 (m, 2H), 1.79 - 1.69 (m, 1H), 1.68 - 1.56 (m, 7H), 1.57 - 1.43 (m, 8H), 1.44 - 1.14 (m, 75H), 0.92 - 0.83 (m, 12H). Chemical Formula C 60 H 119 N3O3 of m / z Calcd = 929.9. Found [M+H] + = 931.1.

[0213] Example 12

[0214] 8,8’-((((1S,2S)-2-hydroxycyclohexyl)methyl)azanediyl)bis(N,N-didecyloctanamide)

[0215] 8,8'-((((lR,2R)-2-hydroxycyclohexyl)methyl)azanediyl)bis(N,N-didecyloctanamide)

[0216] (Compound (rac)-(lRS,2RS)-l-6)

[0217] Synthesis of (rac)-(lRS,2RS)-l-6

[0218] According to general procedure A using (l R ,2 R )-2-(aminomethyl)cyclohexan-l-ol hydrochloride and a mixture of (l S ,2 S )-2-(aminomethyl)cyclohexan-l-ol hydrochloride. Purification was performed via automated flash chromatography (10% to 100% EtOAc in hexanes). A second purification was performed via automated flash chromatography (5% to 100% EtOAc in hexanes) to give the title compound (yield 11%). 1 H NMR (400 MHz, CDCl3) δ 3.84 - 3.75 (m, 1H), 3.32 – 3.23 (m, 4H), 3.23 – 3.15 (m, 4H), 2.96 (dd, J = 13.1,10.5 Hz, 1H), 2.60 - 2.48 (m, 2H), 2.31 – 2.20 (m, 7H), 2.17 - 2.07 (m, 1H),1.78 – 1.39 (m, 24H), 1.36 - 1.18 (m, 74H), 0.95 - 0.83 (m, 12H). Chemical Formula C 63 H 125 N3O3 of m / z Calculated = 972.0. Found [M+H] + = 973.1.

[0219] Example 13

[0220] 8,8'-((((l r ,4 r )-4-hydroxycyclohexyl)methyl)azanediyl)bis(N,N-didecyloctanamide)

[0221] (Compound (l r ,4 r )-l-7)

[0222] (1 R 4 R Synthesis of )-I-7

[0223] According to general procedure A, use (1) r 4 r Synthetic compound (1)-4-(aminomethyl)cyclohexyl-1-ol hydrochloride R 4 R )-I-7. Purified by automated rapid chromatography (5% to 100% EtOAc in hexane solution). A second purification was performed by automated rapid chromatography (2% to 10% MeOH in DCM solution) to give the title compound (yield 28%). 1 H NMR (400MHz, CDCl3) δ 3.59 – 3.49 (m, 1H), 3.32 – 3.23 (m, 4H), 3.23 – 3.15 (m, 4H), 2.35 – 2.23 (m, 8H), 2.11 (d, J = 6.9 Hz, 2H), 2.03 – 1.92 (m, 2H), 1.89 – 1.79 (m, 2H), 1.69 - 1.59 (m, 7H), 1.57 - 1.46 (m, 8H), 1.45 – 1.14 (m, 79H), 0.94 - 0.81 (m, 15H). Chemical formula C 63 H 125 N3O3 m / z Calculated value = 972.0. Measured value [M+H] + = 973.0.

[0224] Example 14

[0225] 8,8'-((((1s,4s)-4-hydroxycyclohexyl)methyl)azadiyl)bis(N,N-didecyloctamide)

[0226] (compound(1) s 4 s )-I-7)

[0227] (1 S 4 S Synthesis of )-I-7

[0228] According to general procedure A, use (1) s 4 s Synthetic compound (1)-4-(aminomethyl)cyclohexyl-1-ol hydrochlorideS ,4 S )-I-7. Purification via automated flash chromatography (5% to 100% EtOAc in hexanes) afforded the title compound (yield 26%). 1 H NMR (400 MHz, CDCl3) δ 3.95 (s, 1H), 3.32 – 3.23 (m, 4H), 3.23 – 3.15(m, 4H), 2.36 - 2.22 (m, 8H), 2.17 (d, J = 6.8 Hz, 2H), 1.73 – 1.43 (m, 24H),1.43 – 1.15 (m, 81H), 0.94 - 0.82 (m, 14H). Chemical Formula C 63 H 125 N3O3 of m / z Calculated = 972.0. Found [M+H] + = 973.1.

[0229] Example 15

[0230] 8,8’-((((1 R ,2 S )-2-hydroxycyclohexyl)methyl)azanediyl)bis(N,N-didecyloctanamide)

[0231] (Compound (1 R ,2 S )-I-8)

[0232] (1 R ,2 S )-I-8

[0233] Compound (1 R ,2 S )-I-8 was synthesized according to general procedure A using (1S,2R)-2-(aminomethyl)cyclohexan-1-ol hydrochloride. Purification via automated flash chromatography (5% to 65% EtOAc with 1% Et3N in hexanes). A second purification via automated flash chromatography (5% to 100% EtOAc in hexanes) afforded the title compound (yield 22%). 1H NMR (400 MHz, CDC13) δ 3.41 - 3.32 (m, 1H), 3.32 - 3.24 (m, 4H), 3.23 - 3.15 (m, 4H), 2.64 - 2.54 (m, 2H), 2.49 - 2.40 (m, 1H), 2.33 - 2.23 (m, 5H), 2.22 - 2.11 (m, 2H), 1.96 - 1.87 (m, 1H), 1.74 - 1.57 (m, 8H), 1.57 - 1.38 (m, 15H), 1.37 - 1.16 (m, 75H), 0.90 - 0.9 (m, 15H). Chemical Formula C 63 H 125 N3O3 of m / z Calculated = 972.0. Found [M+H] + = 973.1.

[0234] Example 16

[0235] 8,8’-(((1 r ,4 r )-4-hydroxycyclohexyl)azanediyl)bis(N,N-didecyloctanamide)

[0236] Synthesis of compound (1 r ,4 r )-I-9

[0237] Synthesis of compound (1 R ,4 R )-I-9

[0238] Compound (1 R ,4 R )-I-9 was synthesized according to general procedure A using (1r,4r)-4-aminocyclohexan-1-ol. Purification via automated flash chromatography (5% to 100% EtOAc in hexanes) afforded the title compound (yield 42%). 1H NMR (400 MHz, CDC13) δ 3.60 - 3.49 (m, 1H), 3.32 - 3.24 (m, 4H), 3.22 - 3.14 (m, 4H), 2.49 - 2.40 (m, 1H), 2.40 - 2.33 (m, 4H), 2.31 - 2.22 (m, 4H), 2.05 - 1.94 (m, 2H), 1.81 - 1.71 (m, 2H), 1.68 - 1.58 (m, 6H), 1.57 - 1.45 (m, 9H), 1.44 - 1.19 (m, 82H), 0.94 - 0.81 (m, 13H). Chemical Formula C 62 H 123 N3O3 of m / z Calculated = 958.0. Found [M+H] + = 959.0.

[0239] Example 17

[0240] 8,8’-(((1 s ,4 s )-4-hydroxycyclohexyl)azanediyl)bis(N,N-didecyloctanamide)

[0241] Synthesis of compound (1 s ,4 s )-I-9

[0242] Synthesis of (1 S ,4 S )-I-9

[0243] Compound (1 s ,4 s )-I-9 was synthesized according to general procedure A using (1 S ,4 S )-4-aminocyclohexan-1-ol. Purification via automated flash chromatography (5% to 100% EtOAc in hexanes) afforded the title compound (yield 42%). 1 H NMR (400 MHz, CDC13) δ 3.60 - 3.49 (m, 1H), 3.32 - 3.24 (m, 4H), 3.22 - 3.14 (m, 4H), 2.49 - 2.40 (m, 1H), 2.40 - 2.33 (m, 4H), 2.31 - 2.22 (m, 4H), 2.05 - 1.94 (m, 2H), 1.81 - 1.71 (m, 2H), 1.68 - 1.58 (m, 6H), 1.57 - 1.45 (m, 9H), 1.44 - 1.19 (m, 82H), 0.94 - 0.81 (m, 13H). Chemical Formula C J= 7.6 Hz, 4H), 1.89 – 1.77 (m, 2H), 1.71- 1.58 (m, 8H), 1.57 – 1.44 (m, 13H), 1.44 – 1.17 (m, 77H), 0.92 - 0.82 (m,13H). Chemical Formula C 62 H 123 N3O3 of m / z Calcd = 958.0. Found [M+H] + = 959.0.

[0244] Example 18

[0245] 8,8’-(((1 s ,3 s )-3-hydroxycyclobutyl)azanediyl)bis(N,N-didecyloctanamide)

[0246] Synthesis of compound (1 s ,3 s )-I-10

[0247] Synthesis of compound (1 S ,3 S )-I-10

[0248] Compound (1 s ,3 s )-I-10 was synthesized according to general procedure A using (1 S ,3 S )-3-aminocyclobutane-1-ol hydrochloride. Purification via automated flash chromatography (5% to 100% EtOAc in hexanes) afforded the title compound (yield 47%). 1 H NMR (400 MHz, CDCl3) δ 4.00 - 3.88 (m, 1H), 3.32 – 3.24 (m, 4H), 3.22 –3.15 (m, 4H), 2.64 - 2.45 (m, 3H), 2.40 – 2.32 (m, 4H), 2.31 - 2.22 (s, 4H),1.95 - 1.84 (m, 1H), 1.82 – 1.58 (m, 8H), 1.58 – 1.43 (m, 8H), 1.43 – 1.18(m, 74H), 0.94 - 0.81 (m, 12H). Chemical Formula C 60 H 119 N3O3 of m / z Calcd = 929.9. Found [M+H]+ = 931.0.

[0249] Example 19

[0250] 8,8’-(((1-hydroxycyclobutyl)methyl)azanediyl)bis(N,N-didecyloctanamide)

[0251] (Synthesis of Compound I-11)

[0252] Synthesis of I-12

[0253] Compound I-11 was synthesized according to general procedure A using 1- (aminomethyl)cyclobutane-1-ol. The crude material was purified via automated reverse phase flash chromatography (10% to 100% MeOH in 0.1% TFA in water). The collected fractions were concentrated and partitioned between EtOAc and saturated NaHCO3. Purification via automated flash chromatography (0% to 10% MeOH in DCM) afforded the title compound (yield 28%). 1 H NMR (400 MHz, CDCl3) δ 3.32 – 3.24 (m, 4H), 3.23 –3.15 (m, 4H), 2.57 (s, 2H), 2.52 – 2.42 (m, 4H), 2.31 – 2.21 (m, 4H), 2.20 -2.09 (m, 2H), 2.04 – 1.92 (m, 2H), 1.86 – 1.72 (m, 1H), 1.69 – 1.36 (m, 19H),1.36 – 1.17 (m, 67H), 0.92 - 0.84 (m, 12H). Chemical Formula C 61 H 121 m / z calculated for N3O3 = 943.9. Found [M+H] + = 945.0.

[0254] Example 20

[0255] 8,8’-(((3-hydroxyoxetan-3-yl)methyl)azanediyl)bis(N,N-didecyloctanamide)

[0256] (Synthesis of Compound I-12)

[0257] Synthesis of I-12

[0258] Compound 1-12 was synthesized according to general procedure A using 1-(aminomethyl)cyclobutan-1-ol. Purification via automated flash chromatography (0% to 10% MeOH in DCM) afforded the title compound (yield 54%). 1 H NMR (400 MHz, CDC13) δ 4.72 (d, J = 6.6 Hz, 2H), 4.47 (d, J = 6.7 Hz, 2H), 3.32 - 3.25 (m, 4H), 3.22 - 3.15 (m, 4H), 2.82 (s, 2H), 2.39 (t, J = 7.5 Hz, 4H), 2.26 (t, J = 7.6 Hz, 4H), 1.68 - 1.59 (m, 4H), 1.57 - 1.46 (m, 8H), 1.44 - 1.36 (m, 4H),1.36 - 1.18 (m, 67H), 0.92 - 0.83 (m, 12H). Formula C 60 H 119 N3O4 of m / z Calculated = 945.9. Found [M+H] + = 946.9.

[0259] Example 21

[0260] 8,8’-((2-(1-hydroxycyclobutyl)ethyl)azanediyl)bis(N,N-didecyloctanamide)

[0261] (Compound 1-13)

[0262] Synthesis of 1-13

[0263] Compound 1-13 was synthesized according to general procedure A using 1-(2- aminoethyl)cyclobutan-1-ol. Purification via automated flash chromatography (0% to 10% MeOH in DCM) afforded the title compound (yield 98%). 1 H NMR (400 MHz, CDC13) δ 3.31 - 3.23 (m, 4H), 3.22 - 3.14 (m, 4H), 2.27 (t, J= 7.4 Hz, 4H), 2.15 – 2.01 (m, 6H), 1.81 – 1.44 (m, 20H), 1.40 – 1.17 (m, 71H), 0.94 - 0.81 (m, 12H). Chemical formula C 62 H 123 N3O3 m / z Calculated value = 958.0. Measured value [M+H] + = 958.9.

[0264] Example 22

[0265] 8,8'-(((1R,3S)-3-hydroxycyclopentyl)azadiyl)bis(N,N-didecyloctamide)

[0266] 8,8'-(((1S,3R)-3-hydroxycyclopentyl)azadiyl)bis(N,N-didecyloctamide)

[0267] (compound (racemic)-(1) RS ,3 SR )-I-14)

[0268] (racemic)-(1) RS ,3 SR Synthesis of )-I-14

[0269] A mixture of enantiomers of compound I-14 was synthesized using a mixture of (1S,3R)-3-aminocyclopentane-1-ol hydrochloride and (1R,3S)-3-aminocyclopentane-1-ol hydrochloride according to general procedure A. The title compound was purified by automated rapid chromatography (5% to 65% EtOAc in hexane solution with 1% Et3N) to give the title compound (28% yield). 1 H NMR (400 MHz, CDCl3) δ 4.24 - 4.17 (m, 1H), 3.32 - 3.23 (m, 4H), 3.23 - 3.15 (m, 4H), 3.07- 3.00 (m, 1H), 2.55 - 2.41 (m, 4H), 2.26 (t, J = 7.6 Hz, 4H), 1.92 – 1.05 (m, 92H), 0.93 - 0.80 (m, 12H). Chemical formula C 61 H 121 N3O3 m / z Calculated value = 943.9. Measured value [M+H] + = 945.2.

[0270] Example 23

[0271] 8,8'-(((1R,3R)-3-hydroxycyclopentyl)azanediyl)bis(N,N-didecyloctanamide)

[0272] 8,8'-(((1S,3S)-3-hydroxycyclopentyl)azanediyl)bis(N,N-didecyloctanamide)

[0273] (S)-I-14 RS ,3 RS )-I-14

[0274] (S)-I-14 RS ,3 RS )-I-14

[0275] A mixture of (1R,3R)-3-aminocyclopentan-1-ol hydrochloride and (1S,3S)-3-aminocyclopentan-1-ol hydrochloride was used according to the general procedure A to synthesize a mixture of enantiomers of compound I-14. Purification via automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N) afforded the title compound (yield 33%). 1 H NMR (400 MHz, CDC13) δ 4.39 - 4.32 (m, 1H), 3.43 - 3.24 (m, 5H), 3.23 - 3.15 (m, 4H), 2.48- 2.40 (m, 4H), 2.31 - 2.22 (m, 4H), 2.07 - 1.89 (m, 2H), 1.88 - 1.77 (m, 1H), 1.70 - 1.37 (m, 23H), 1.37 - 1.16 (m, 68H), 0.93 - 0.83 (m, 12H). Chemical Formula C 61 H 121 of N3O3 m / z Calculated = 943.9. Found [M+H] + = 945.3.

[0276] Example 24

[0277] 8,8'-(((1 S ,2 S )-2-hydroxycyclopentyl)azanediyl)bis(N,N-didecyloctanamide)

[0278] (S)-I-14 S ,2S )-I-15

[0279] (1 S ,2 S Synthesis of (1

[0280] According to general procedure A, compound (1 S ,2 S )-2-aminocyclopentan-1-ol hydrochloride was used to synthesize compound (1 S ,2 S )-I-15. Purification via automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N), followed by a second automated flash chromatography (10% to 100% EtOAc in hexanes) afforded the title compound (yield 12%). 1 H NMR (400 MHz, CDC13) δ 3.94 - 3.83 (m, 1H), 3.32 - 3.23 (m, 4H), 3.23 - 3.15 (m, 4H), 2.87 - 2.76 (m, 1H), 2.52 - 2.33 (m, 4H), 2.27 (t, J = 7.6 Hz, 4H), 1.95 - 1.03 (m, 94H), 0.96 - 0.81 (m, 12H). Chemical Formula C 61 H 121 of N3O3 m / z Calculated = 943.9. Found [M+H] + = 945.3.

[0281] Example 25

[0282] 8,8'-(((1 S ,2 R )-2-hydroxycyclopentyl)azanediyl)bis(N,N-didecyloctanamide)

[0283] (compound (1 S ,2 R )-I-15)

[0284] (1 S ,2 R )-I-15

[0285] According to general procedure A, compound (1 S ,2R )-I-15. Purification via automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N), followed by a second automated flash chromatography (100% EtOAc) to afford the title compound (yield 16%). 1 H NMR (400 MHz, CDCl3) δ 4.01 - 3.97 (m, 1H), 3.32 – 3.24 (m, 4H), 3.23 – 3.15 (m, 4H), 2.77 - 2.63(m, 1H), 2.62 – 2.49 (m, 4H), 2.31 – 2.23 (m, 4H), 1.92 – 1.05 (m, 94H), 0.92- 0.81 (m, 12H). Chemical Formula C 61 H 121 N3O3 of m / z Calculated = 943.9. Found [M+H] + = 945.3.

[0286] Example 26

[0287] 8,8’-(((1 S ,2 S )-2-hydroxycyclohexyl)azanediyl)bis(N,N-didecyloctanamide)

[0288] (Compound (1 S ,2 S )-I-16)

[0289] (1 S ,2 S )-I-16

[0290] Compound (1 S ,2 S )-I-16 was synthesized according to general procedure A using (1 S ,2 S )-2-aminocyclohexan-1-ol. Purification via automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N), followed by a second automated flash chromatography (0% to 100% EtOAc), followed by a third automated flash chromatography (0% to 10% MeOH in DCM) to afford the title compound (yield 13%). 1H NMR (400 MHz, CDC13) δ 3.80 - 3.02 (m, 10H), 2.61 - 2.18 (m, 8H), 2.17 - 2.05 (m, 2H), 2.02 - 1.00 (m, 96H), 0.92 - 0.83 (m, 12H). Chemical Formula C 62 H 123 N3O3 of m / z Calculated = 958.0. Found [M+H] + = 959.3.

[0291] Example 27

[0292] 8,8’-(((1 S ,2 R )-2-hydroxycyclohexyl)azanediyl)bis(N,N-didecyloctanamide)

[0293] Synthesis of compound (1 S ,2 R )-I-16

[0294] Synthesis of compound (1 S ,2 R )-I-16

[0295] Compound (1 S ,2 R )-I-16 was synthesized according to general procedure A using (1R,2S)-2-aminocyclohexan-1-ol hydrochloride. Purification via automated flash chromatography (10% to 100% EtOAc in hexanes) followed by a second automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N) afforded the title compound (yield 17%). 1 H NMR (400 MHz, CDC13) δ 3.94 - 3.86 (m, 1H), 3.32 - 3.24 (m, 4H), 3.23 - 3.15 (m, 4H), 2.61 - 2.47 (m, 4H), 2.37 - 2.31 (m, 1H), 2.30 - 2.23 (m, 4H), 2.05 - 1.97 (m, 1H), 1.80 - 1.02 (m, 97H), 0.94 - 0.84 (m, 12H). Chemical Formula C 62 H 123 N3O3 of m / z Calculated = 958.0. Found [M+H] += 959.3.

[0296] Example 28

[0297] 8,8'-(((1 R ,3 R )-3-hydroxycyclohexyl)azadiyl)bis(N,N-didecyloctamide)

[0298] (compound(1) R ,3 R )-I-17)

[0299] (1 R ,3 R Synthesis of )-I-17

[0300] According to general procedure A, use (1) R ,3 R Synthetic compound (1)-3-aminocyclohexyl-1-ol hydrochloride R ,3 R )-I-17. The title compound was purified by automated rapid chromatography (5% to 65% EtOAc and 1% Et3N in hexane solution) to obtain the title compound (yield 29%). 1 H NMR (400 MHz, CDCl3) δ 4.27 - 4.19 (m, 1H), 3.32 - 3.23 (m, 4H), 3.23- 3.15 (m, 4H), 3.01 - 2.86 (m, 1H), 2.38 (t, J = 7.4 Hz, 4H), 2.31 - 2.22 (m, 4H), 1.88 – 1.05 (m, 97H), 0.92 - 0.83 (m, 12H). Chemical formula C 62 H 123 N3O3 m / z Calculated value = 958.0. Measured value [M+H] + = 959.4.

[0301] Example 29

[0302] 8,8'-(((1 S ,3 R )-3-hydroxycyclohexyl)azadiyl)bis(N,N-didecyloctamide)

[0303] (compound(1) S ,3 R )-I-17)

[0304] (1 S ,3 R Synthesis of )-I-17

[0305] According to general procedure A, compound (1R,3S)-3-aminocyclohexyl-1-ol hydrochloride was synthesized using (1R,3S)-3-aminocyclohexyl-1-ol hydrochloride. S ,3 R )-I-17. The title compound was purified by automated rapid chromatography (5% to 65% EtOAc and 1% Et3N in hexane solution) to obtain the title compound (yield 36%). 1 H NMR (400 MHz, CDCl3) δ 3.70 - 3.60 (m, 1H), 3.32 - 3.24 (m, 4H), 3.22- 3.14 (m, 4H), 2.61 - 2.51 (m, 1H), 2.48 - 2.38 (m, 4H), 2.31 - 2.22 (m,4H), 2.00 – 1.73 (m, 3H), 1.70 – 1.01 (m, 89H), 0.93 – 0.80 (m, 12H). Chemical formula C 62 H 123 N3O3 m / z Calculated value = 958.0. Measured value [M+H] + = 959.2.

[0306] Example 30

[0307] 8,8'-(((1R,2R)-2-hydroxycyclobutyl)azadiyl)bis(N,N-didecyloctamide)

[0308] 8,8'-(((1S,2S)-2-hydroxycyclobutyl)azadiyl)bis(N,N-didecyloctamide)

[0309] (compound (racemic)-(1) RS ,2 RS )-I-18)

[0310] (racemic)-(1) RS ,2 RS Synthesis of )-I-18

[0311] A mixture of (1R,2R)-2-aminocyclobutan-1-ol hydrochloride and (1S,2S)-2- aminocyclobutan-1-ol hydrochloride was synthesized according to the general procedure A. Purification via automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N) afforded the title compound (yield 30%). 1 H NMR (400 MHz, CDC13) δ 4.02 - 3.93 (m, 1H), 3.32 - 3.22 (m, 4H), 3.23 - 3.13 (m, 4H), 2.90 (q, J = 8.0 Hz, 1H), 2.60 - 2.35 (m, 4H), 2.26 (t, J = 7.5 Hz, 4H), 2.16 - 2.06 (m, 1H), 1.80 (q, J = 9.2 Hz, 1H), 1.74 - 1.05 (m, 90H), 0.94 - 0.80 (m, 12H). Formula C 60 H 119 N3O3 of m / z Calculated = 929.9. Found [M+H] + = 931.2.

[0312] Example 31

[0313] 8,8’-(((1S,2R)-2-hydroxycyclobutyl)azanediyl)bis(N,N-didecyloctanamide)

[0314] 8,8’-(((1R,2S)-2-hydroxycyclobutyl)azanediyl)bis(N,N-didecyloctanamide)

[0315] Synthesis of (rac)-(1 RS ,2 SR )-I-18

[0316] (rac)-(1 RS ,2 SR )-I-18

[0317] A mixture of enantiomers of compound I-18 was synthesized using a mixture of (1R,2S)-2-aminocyclobutane-1-ol hydrochloride and (1S,2R)-2-aminocyclobutane-1-ol hydrochloride according to general procedure A. Purification was performed by automated rapid chromatography (5% to 65% EtOAc in hexane solution with 1% Et3N), followed by a second purification by reversed-phase rapid chromatography (50% to 100% MeOH in aqueous solution with 0.1% TFA), and a third purification by automated rapid chromatography (5% to 65% EtOAc in hexane solution with 1% Et3N) to give the title compound (15% yield). 1 H NMR (400 MHz, CDCl3) δ4.08 - 4.00 (m, 1H), 3.32 - 3.23 (m, 4H), 3.23 - 3.15 (m, 4H), 3.12 - 3.02(m, 1H), 2.51 - 2.30 (m, 4H), 2.30 - 2.22 (m, 4H), 2.12 (p, J = 10.0 Hz, 1H), 2.01 – 1.89 (m, 1H), 1.87 – 1.76 (m, 1H), 1.72 – 1.05 (m, 90H), 0.92 - 0.83 (m, 12H). Chemical formula C 60 H 119 N3O3 m / z Calculated value = 929.9. Measured value [M+H] + = 931.2.

[0318] Example 32

[0319] 8,8'-((2-((1s,3r)-3-hydroxycyclobutyl)ethyl)azadiyl)bis(N,N-decyl-2-fluorooctamide) (compound(1 s ,3 r )-I-19)

[0320] Synthesis of intermediate 2

[0321] A mixture of 2-fluoromalonate diethyl ester (56.1 mmol, 10.0 g), 1,6- dibromohexane (168 mmol, 26 mL), and sodium methoxide (61.7 mmol, 3.3 g) in EtOH (110 mL) was stirred at room temperature for 24 h. The reaction mixture was concentrated and the crude material was partitioned between DCM and water. The organic layer was separated, dried over Na2SO4, filtered, and concentrated. Purification via automated flash chromatography (0% to 25% EtOAc in hexanes) afforded intermediate 2 (11.7 g, 61%).

[0322] Synthesis of Intermediate 3

[0323] A mixture of intermediate 2 (5.9 mmol, 2.0 g) and KOH (11.8 mmol, 660 mg) in MeOH (20 mL), THF (2 mL), and water (4 mL) was stirred at room temperature for 3 h. The reaction mixture was concentrated and the crude material was diluted with 0.1 M NaOH (20 mL). The aqueous layer was washed with DCM (3 x 10 mL), acidified with 1 M HC1, and then extracted with EtOAc (2 x 20 mL). The combined EtOAc layers were dried over Na2SO4, filtered, and concentrated to afford intermediate 3 (1.57 g, 94%) which was used in the next step without further purification.

[0324] Synthesis of Intermediate 4

[0325] A mixture of intermediate 3 (4.1 mmol, 1.2 g) and DMAP (cat.) in DMF (3 mL) was heated at 180 °C for 12 min. The reaction mixture was partitioned between EtOAc and 1 M HC1. The organic layer was separated, dried over Na2SO4, filtered, and concentrated to afford intermediate 4 (960 mg, 98%) which was used in the next step without further purification.

[0326] Synthesis of Intermediate 5

[0327] A mixture of intermediate 4 (4.0 mmol, 960 mg), oxalyl chloride (12 mmol, 1.0 mL), and DMF (cat.) in DCM (10 mL) was stirred at room temperature for 20 min. The reaction mixture was concentrated to afford intermediate 5 which was used in the next step without further purification.

[0328] Synthesis of Intermediate 6

[0329] To a mixture of didecylamine (4.0 mmol, 1.2 g), triethylamine (24 mmol, 3.4 mL) and DMAP (catalyst) in DCM (10 mL) was added a solution of crude intermediate 5 (4.0 mmol) in DCM (5 mL). The reaction mixture was stirred at room temperature for 1 h. The reaction was concentrated and purified via automated flash chromatography (5% to 25% EtOAc in hexanes) to give intermediate 6 (1.2 g, 58% over 2 steps).

[0330] (1 S ,3 R )-I-19. Purification via automated flash chromatography (5% to 65% EtOAc with 1% Et3N in hexanes) gave the title compound (yield 63%).

[0331] Compound (1 S ,3 R )-I-19 was synthesized according to general procedure A using intermediate 6 and (1R,3S)-3-(2-aminoethyl)cyclobutan-1-ol hydrochloride. Purification via automated flash chromatography (5% to 65% EtOAc with 1% Et3N in hexanes) gave the title compound (yield 63%). 1 H NMR (400 MHz, CDCl3) δ 5.03 (ddd, J = 49.4, 8.5,4.3 Hz, 2H), 4.14 – 4.04 (m, 1H), 3.38 - 3.12 (m, 8H), 2.51 - 2.42 (m, 2H),2.39 - 2.25 (m, 5H), 2.01 – 1.15 (m, 87H), 0.92 - 0.83 (m, 12H). Chemical Formula C 62 H 121 F2N3O3 of m / z Calculated = 993.9. Found [M+H] + = 995.0.

[0332] Example 33

[0333] Compound (1 r ,3 r )-I-22 and (1 s ,3 s )-I-22 was synthesized according to general procedure A using intermediate 6 and (1R,3S)-3-(2-aminoethyl)cyclobutan-1-ol hydrochloride. Purification via automated flash chromatography (5% to 65% EtOAc with 1% Et3N in hexanes) gave the title compound (yield 63%).

[0334]

[0335] General procedure for the preparation of Int 1

[0336] To a solution of 8-aminooctanol (1.0 eq) in ACN (7 mL / mmol) was added the appropriate bromide (1.0 eq) and the reaction mixture was refluxed under N2overnight. Then, the reaction mixture was concentrated to give the crude product. The solid crude product was purified by column chromatography (DCM / 3% NH3 in MeOH, 100:0 to 80:20).

[0337] General procedure for the preparation of Int 2

[0338] To a solution of the acid (0.9 eq) in DCM (2.5 mL / mmol) was added N-hydroxysuccinimide (0.9 eq), 4-dimethylaminopyridine (0.9 eq) and dicyclohexylcarbodiimide (0.9 eq) at room temperature under N2. The resulting mixture was stirred at room temperature until complete conversion. The precipitate was filtered off and the filtrate was added dropwise to a solution of Int 1 (1.0 eq) in DCM (2.3 mL mmol) at room temperature. The reaction mixture was stirred at room temperature under N2overnight. Then, the organic phase was washed with HC1 (aq. sol., 1 mol / L), Na2CO3 (aq. sol.) and dried over Na2SO4. The organic phase was concentrated. The light yellow solid was purified by column chromatography (Hex / EtOAc, 100:0 to 0:100).

[0339] General procedure for the preparation of Int 4

[0340] To a solution of Int 2 (1.0 eq) in diethyl ether (8.0 mL / mmol) was added dropwise PBr3 (2-3 eq) at 0 °C under N2. The reaction mixture was slowly warmed to room temperature and stirred at room temperature under N2until the reaction was complete. Then, ice-cold water was gradually added until a clear solution was obtained. The aqueous phase was extracted three times with ethyl acetate, dried over Na2SO4and concentrated under reduced pressure. The crude product was purified by column chromatography (Hex / EtOAc, 100:0 to 90:10).

[0341]

[0342] Synthesis of N-(8-bromooctyl)-N-decyldecanamide (Int 4-1)

[0343] Int 4-1 was prepared following the general procedure for the preparation of Int 4.

[0344] General procedure for the preparation of compounds (1 R ,3 R )-I-22 and (1 S ,3 S )-I-22

[0345] Add DIPEA (3.8 equivalents) and the desired alkylating agent (1.0 equivalent) to a solution of Int 4-1 (1.5 equivalents) in ACN (5.5 mL / mmol). Insulate the reaction mixture in a sealed tube at 80 °C for 24 h. Then, add more Int 4-1 (0.5 equivalents) in ACN (2.0 mL / mmol) to the reaction mixture and continue stirring at 80 °C for another 24 h. Cool the reaction mixture to room temperature and concentrate under reduced pressure. Purify the crude product by column chromatography (Hex / 1% NEt3 in EtOAc solution, 95:5 to 0:100).

[0346] N , N '-(((((1 S ,3 S )-3-hydroxycyclobutyl)methyl)azadiyl)bis(octane-8,1-diyl))bis(N-decyldecamide) ((1 S ,3 S Synthesis of )-I-22)

[0347] According to the general procedure, Int 4-1 (0.5 g, 0.994 mmol), (1 S , 3 S Compound (1) was prepared by reacting 3-(aminomethyl)cyclobutane-1-ol hydrochloride (0.047 g, 0.338 mmol) and DIPEA (0.83 mL, 5.97 mmol). S ,3 S )-I-22. A colorless oily product was obtained (0.2 g, 0.211 mmol, 21.3%). 1 H NMR (600 MHz, chloroform-) d ) δ4.12 (p, J = 7.4 Hz, 1H), 3.31 – 3.24 (t, J = 8.5 Hz, 4H), 3.19 (t, J = 6.3Hz, 4H), 2.45 (m, 4H), 2.35 (m, 4H), 2.29 – 2.23 (t, J = 7.1 Hz, 4H), 1.89(m, 1H), 1.63 (m, 4H), 1.51 (m, 10H), 1.39 (m, 4H), 1.35 – 1.19 (m, 70H), 0.87 (t, J = 6.0 Hz, 12H). ESI-MS: C 61H 121 [M+H] of N3O3 MW + Calculated value: 944.95; Measured value: 945.09.

[0348] N,N'-(((((1 R ,3 R )-3-hydroxycyclobutyl)methyl)azadiyl)bis(octane-8,1-diyl))bis(N-decyldecamide) ((1 R ,3 R Synthesis of )-I-22)

[0349] According to the general procedure, Int 4-1 (0.5 g, 0.994 mmol), (1 R , 3 R Compound (1) was prepared by reacting 3-(aminomethyl)cyclobutane-1-ol hydrochloride (0.047 g, 0.338 mmol) and DIPEA (0.83 mL, 5.97 mmol). R ,3 R )-I-22. A colorless oily product was obtained (0.22 g, 0.233 mmol, 23.4%). 1 H NMR (600 MHz, CDCl3) δ4.41 – 4.33 (p, J = 7.4 Hz, 1H), 3.28 (t, J = 8.5 Hz, 4H), 3.19 (t, J = 6.3Hz, 4H), 2.39 (m, 3H), 2.37 – 2.30 (m, 4H), 2.26 (t, J = 7.1 Hz, 4H), 2.07(m, 2H), 2.03 (m, 2H), 1.70 – 1.57 (m, 7H), 1.57 – 1.45 (m, 8H), 1.37 (m,4H), 1.34 – 1.21 (m, 70H), 0.90 – 0.85 (t, J = 6.0 Hz, 12H). ESI-MS: C 61 H 121 [M+H] of N3O3 MW + Calculated value: 944.95; Measured value: 945.02.

[0350] Example 34

[0351] N-decyl-N-(8-((8-(didecylamino)-8-oxooctyl)(((1S,3S)-3- hydroxycyclobutyl)methyl)amino)octyl)decanamide

[0352] (Compound (1 s ,3 s )-I-21)

[0353] Synthesis of N,N-didecyl-8-((((1S,3S)-3-hydroxycyclobutyl)methyl)amino)octanamide (Int-16)

[0354] N,N-didecyl-8-((((1S,3S)-3-hydroxycyclobutyl)methyl)amino)octanamide was prepared from 8-bromo-N,N-didecyloctanamide (Example 5, 0.4 g, 0.796 mmol), (1 S , 3 S )-3-(aminomethyl)cyclobutane-1-ol hydrochloride (0.384 g, 2.785 mmol) and DIPEA (0.44 mL, 3.183 mmol). The product was obtained as colorless oil (0.3 g, 0.574 mmol, 72%). ESI-MS: C 33 H 66 [M+H] of MW of N2O2 + Calcd 523.52, Found 523.61.

[0355]

[0356] Synthesis of N-decyl-N-(8-((8-(didecylamino)-8-oxooctyl)(((1S,3S)-3- hydroxycyclobutyl)methyl)amino)octyl)decanamide ((1 S ,3 S )-I-21)

[0357] Compound (1 S ,3 S )-I-21 was prepared from Int 4-1 (0.32 g, 0.631 mmol), Int 16 (0.3 g, 0.574 mmol) and DIPEA (0.4 mL, 2.296 mmol). The product was obtained as colorless oil (0.19 g, 0.201 mmol, 35%). 1 H NMR (600 MHz, CDCl3) δ 4.25 – 4.15 (p, J = 7.4 Hz, 1H), 3.31 – 3.23(t,J = 8.5 Hz, 4H), 3.19 (t, J = 6.3 Hz, 4H), 3.05 (m, 1H), 2.88 (m, 3H), 2.60 (m, 2H), 2.30 – 2.23 (m, 4H), 1.75 (s, 5H), 1.68 – 1.56 (m, 8H), 1.50 (m, 9H), 1.40 – 1.18 (m, 70H), 0.88 (t, J = 6.0 Hz, 12H). ESI-MS: C 61 H 121 [M+H] of N3O3 MW + Calculated value: 944.95; Measured value: 945.09.

[0358] Example 35

[0359] Compound (1) r ,3 r )-I-20 and (1 s ,3 s Synthetic pathway of )-I-20

[0360] Synthesis of 8-bromo-N,N-dioctyloctamide

[0361] 8-Bromo-N,N-Dioctyloctanoamide was prepared from 8-bromooctanoic acid (2.00 g, 9.0 mmol), DMF (1 drop), oxalyl chloride (2.30 mL, 27 mmol), dioctylamine (2.39 g, 9.9 mmol), triethylamine (7.50 mL, 54 mmol), DMAP (0.02 g, 0.16 mmol), and DCM (50 mL) according to the procedure described in Example 5. A colorless oily product, 8-bromo- N,N - Dioctyloctamide (3.12 g, 7.0 mmol, 78%). ESI-MS: MW for C 24 H 49 BrNO's MW [M+H] + Calculated values: 446.30 and 448.29; measured values: 446.36 and 448.38 (bromine mode).

[0362] 8,8'-((((1S,3S)-3-hydroxycyclobutyl)methyl)azadiyl)bis(N,N-dioctyloctamide) ((1 S ,3 S Synthesis of )-I-20)

[0363] Compound (1 S , 3 S )-3-(aminomethyl)cyclobutane-1 -ol hydrochloride (0.050 g, 0.36 mmol) and DIPEA (1 mL, 6 mmol) to give compound (1 S ,3 S )-I-20. The product was obtained as a light yellow oil (117 mg, 0.14 mmol, 39%). 1 H NMR (600 MHz, CDCl3) δ 4.11(p, J = 7.4 Hz, 1H), 3.31 – 3.25 (m, 4H), 3.22 – 3.16 (m, 4H), 2.50 – 2.42(m, 4H), 2.39 – 2.31 (m, 4H), 2.29 – 2.24 (m, 4H), 1.95 – 1.85 (m, 1H), 1.73– 1.58 (m, 7H), 1.57 – 1.45 (m, 11H), 1.43 – 1.36 (m, 4H), 1.35 – 1.20 (m,55H), 0.88 (dt, J = 9.4, 7.0 Hz, 12H)。ESI-MS:C 53 H 105 N3O3 of [M+H] + Calcd 832.83, Found 832.85.

[0364] 8,8’-((((1R,3R)-3-hydroxycyclobutyl)methyl)azanediyl)bis(N,N- dioctyldecanamide) ((1 R ,3 R )-I-20) was synthesized

[0365] Compound (1 R , 3 R )-3-(aminomethyl)cyclobutane-1 -ol hydrochloride (0.050 g, 0.36 mmol) and DIPEA (1 mL, 6 mmol) to give compound (1 R ,3 R )-I-20. The product was obtained as a light yellow oil (182 mg, 0.22 mmol, 60 %). 1H NMR (600 MHz, CDCl3) δ 4.37(p, J = 7.4 Hz, 1H), 3.32 – 3.24 (m, 4H), 3.21 – 3.15 (m, 4H), 2.38 (s, 3H), 2.36 – 2.31 (m, 4H), 2.30 – 2.23 (m, 4H), 2.10 – 2.04 (m, 2H), 2.04 – 1.96 (m, 3H), 1.69 – 1.57 (m, 8H), 1.56 – 1.46 (m, 8H), 1.38 (dt, J = 14.7, 7.5Hz, 4H), 1.35 – 1.19 (m, 54H), 0.88 (dt, J = 9.2, 7.0 Hz, 12H). ESI-MS:MWforC 53 H 105 [M+H] of N3O3 MW + Calculated value: 832.83; Measured value: 832.85.

[0366] Example 36

[0367] 8,8'-(((1 S 4 S )-4-(hydroxymethyl)cyclohexyl)azadiyl)bis(N,N-didecyloctamide)

[0368] (compound(1) s ,4s)-I-23)

[0369] (1 S Synthesis of ,4S)-I-23

[0370] According to general procedure A, compound (1) is synthesized using ((1s,4s)-4-aminocyclohexyl)methanol. S (4S)-I-23. The title compound (55 mg, 21%) was purified by automated rapid chromatography (5% to 65% EtOAc and 1% Et3N in hexane). 1 H NMR (400 MHz, CDCl3) δ 3.60 (d, J= 7.0 Hz, 2H), 3.32 – 3.23 (m, 4H),3.23 – 3.15 (m, 4H), 2.50 – 2.37 (m, 5H), 2.27 - 2.26 (m, 4H), 1.83 – 1.05(m, 99H), 0.92 - 0.83 (m, 12H). ESI-MS: C 63 H 125 N3O3 of m / z Calculated = 972.0, Found [M+H] + = 973.2.

[0371] Example 37

[0372] 8,8'-(((1r,4r)-4-(hydroxymethyl)cyclohexyl)azanediyl)bis(N,N-didecyloctanamide)

[0373] Synthesis of (1r,4r)-I-23

[0374] Synthesis of (1R,4R)-I-23

[0375] According to general procedure A using ((1r,4r)-4-aminocyclohexyl)methanol to synthesize compound (1 R ,4 R )-I-23. Purification via automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N) afforded the title compound (165 mg, 63%). 1 H NMR (400 MHz, CDCl3) δ 3.48 - 3.41 (m, 2H), 3.33 – 3.23 (m, 4H), 3.23– 3.15 (m, 4H), 2.44 - 2.36 (m, 5H), 2.27 - 2.26 (m, 4H) 1.89 – 1.78 (m, 4H),1.70 – 1.13 (m, 92H), 1.03 - 0.77 (m, 14H). ESI-MS: C 63 H 125 N3O3 of m / z Calculated = 972.0, Found [M+H] + = 973.3.

[0376] Example 38

[0377] 8,8'-(((1r,4r)-4-(hydroxymethyl)cyclohexyl)azanediyl)bis(N,N-didecyloctanamide)

[0378] (Synthesis of compound (1r,3r)-I-24)

[0379] Synthesis of (1R,3R)-I-24

[0380] Compound (1 was synthesized according to general procedure A using ((1r,3r)-3- aminocyclobutyl)methanol R ,3 R -I-24. Purification via automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N) afforded the title compound (198 mg, 77%). 1 H NMR (400 MHz, CDCl3) δ 3.72 - 3.62 (m, 2H), 3.32 – 3.23 (m, 4H), 3.23– 3.13 (m, 4H), 2.40 - 2.32 (m, 4H), 2.26 (t, J = 7.6 Hz, 5H), 2.09 - 1.95(m, 2H), 1.93 - 1.84 (m, 2H), 1.69 – 1.04 (m, 88H), 0.92 - 0.81 (m, 12H). ESI-MS: C 61 H 121 of N3O3 m / z Calculated = 943.9, Found [M+H] + = 945.2.

[0381] Example 39

[0382] 8,8'-(((1s,3s)-3-(hydroxymethyl)cyclobutyl)azanediyl)bis(N,N-didecyloctanamide)

[0383] (Synthesis of compound (1s,3s)-I-24)

[0384] Synthesis of (1S,3S)-I-24

[0385] Compound (1 was synthesized according to general procedure A using ((1s,3s)-3- aminocyclobutyl)methanol S ,3 S)-I-24. Purification via automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N) afforded the title compound (195 mg, 76%). 1 H NMR (400 MHz, CDC13) δ 3.58 (d, J = 4.1 Hz, 2H), 3.34 - 3.23 (m, 4H), 3.23 - 3.15 (m, 4H), 2.99 - 2.89 (m, 1H), 2.42 - 2.34 (m, 4H), 2.31 - 2.16 (m, 7H), 1.75 - 1.03 (m, 88H), 0.92 - 0.82 (m, 12H). ESI-MS: C 61 H 121 Calcd for C49H88N3O3 = 943.9, Found [M+H] + = 945.2.

[0386] Example 40

[0387] 8,8'-(((1r,4r)-4-hydroxy-4-methylcyclohexyl)azanediyl)bis(N,N-didecyloctanamide)

[0388] (Compound (1r,4r)-I-25)

[0389] Synthesis of (1R,4R)-I-25

[0390] Compound (1r,4r)-I-25 was synthesized according to general procedure A using (1r,4r)-4-amino-1-methylcyclohexan-1-ol. R ,4R)-I-25. Purification via automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N) afforded the title compound (150 mg, 34%). 1 H NMR (400 MHz, CDC13) δ 3.32 - 3.23 (m, 4H), 3.23 - 3.15 (m, 4H), 2.56 - 2.35 (m, 5H), 2.31 - 2.22 (m, 4H), 1.80 - 1.01 (m, 89H), 0.93 - 0.79 (m, 12H). ESI-MS: C 63 H 125 Calcd for C49H88N3O3 = 943.9, Found [M+H] + = 945.2.

[0391] Example 41

[0392] 8,8'-((6-hydroxyspiro[3.3]heptan-2-yl)azanediyl)bis(N,N-didecyloctanamide)

[0393] (Compound I-36)

[0394] Synthesis of I-36

[0395] Compound I-36 was synthesized according to general procedure A using 6- aminospiro[3.3]heptan-2-ol. Purification via automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N) afforded the title compound (185 mg, 42%). 1 H NMR (400 MHz, CDC13) δ 4.22 - 4.12 (m, 1H), 3.32 - 3.23 (m, 4H), 3.23 - 3.14 (m, 4H), 2.98 - 2.87 (m, 1H), 2.48 - 237 (m, 1H), 2.35 - 2.20 (m, 9H) 2.13 - 1.97 (m, 2H), 1.94 - 1.79 (m, 4H), 1.69 - 1.02 (m, 87H), 0.92 - 0.84 (m, 12H). ESI-MS: C 63 H 123 [M+H]+ = 971.2. found.

[0396] Example 42

[0397] 8,8'-(((1s,4s)-4-hydroxy-4-methylcyclohexyl)azanediyl)bis(N,N-didecyloctanamide)

[0398] (Compound (1s,4s)-I-25)

[0399] Synthesis of (1S,4S)-I-25

[0400] Compound (1S,4S)-I-25 was synthesized according to general procedure A using (1s,4s)-4-amino-1-methylcyclohexan-1-ol. Purification via automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N) afforded the title compound (145 mg, 33%). 1H NMR (400 MHz, CDC13) δ 3.32 - 3.23 (m, 4H), 3.23 - 3.15 (m, 4H), 2.45 - 2.37 (m, 5H), 2.30 - 2.22 (m, 4H), 1.74 - 1.04 (m, 99H), 0.92 - 0.84 (m, 12H). ESI-MS: C 63 H 125 m / z calcd for N3O3 = 972.0, found [M+H]+ = 973.2. + = 973.2.

[0401] Example 43

[0402] 8,8'-(((1r,3r,5s)-3,5-dihydroxycyclohexyl)azanediyl)bis(N,N-didecyloctanamide)

[0403] (Compound (1r,3R,5S)-I-34)

[0404] Synthesis of (1R,3R,5S)-I-34

[0405] Compound (1R,3R,5S)-I-34 was synthesized according to general procedure A using (1R,3S,5r)-5-aminocyclohexane-1,3-diol hydrochloride. Purification via automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N), followed by a second purification via automated flash chromatography (0% to 10% MeOH in chloroform), afforded the title compound (46 mg, 12%). 1 H NMR (400 MHz, CDC13) δ 3.32 - 3.23 (m, 4H), 3.23 - 3.15 (m, 4H), 2.45 - 2.37 (m, 5H), 2.30 - 2.22 (m, 4H), 1.74 - 1.04 (m, 99H), 0.92 - 0.84 (m, 12H). ESI-MS: C 62 H 123 m / z calcd for N3O4 = 974.0, found [M+H]+ = 975.2.

[0406] Example 44

[0407] 8,8'-(((1s,3R,5S)-3,5-dihydroxycyclohexyl)azanediyl)bis(N,N-didecyloctanamide)

[0408] (Compound (1S, 3R, 5S)-I-34)

[0409] Synthesis of (1S, 3R, 5S)-I-34

[0410] Compound (1S, 3R, 5S)-I-34 was synthesized according to general procedure A using (1R, 3S, 5s)-5-aminocyclohexane-1, 3-diol hydrochloride. Purification via automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N) afforded the title compound (85 mg, 22%). 1 H NMR (400 MHz, CDCl3) δ 4.28 (s, 2H), 3.74 (s, 2H), 3.51- 3.34 (m, 1H), 3.31 - 3.15 (m, 8H), 2.46 - 2.38 (m, 4H), 2.27 (t, J = 7.4 Hz, 4H), 2.09 - 1.97 (m, 3H), 1.72 – 1.02 (m, 92H), 0.92 - 0.83 (m, J = 6.9,2.9 Hz, 12H). ESI-MS: C 62 H 123 [M+H]+ = 975.2. Example 45

[0411] Example 45

[0412] 8,8'-(((1S, 2R)-2-(hydroxymethyl)cyclopropyl)azanediyl)bis(N,N-didecyloctanamide)

[0413] (Compound (1S, 2R)-I-46)

[0414] Synthesis of (1S, 2R)-I-46

[0415] Compound (1S, 2R)-I-46 was synthesized according to general procedure A using ((1R, 2S)-2-aminocyclopropyl)methanol hydrochloride. Purification via automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N) followed by a second purification via automated flash chromatography (0% to 10% MeOH in chloroform) afforded the title compound (107 mg, 29%). 1H NMR (400 MHz, CDCl3) δ 4.14 – 4.07 (m, 1H), 3.98 – 3.88 (m, 1H), 3.32 –3.22 (m, 4H), 3.23 – 3.14 (m, 4H), 2.60 – 2.47 (m, 4H), 2.31 – 2.23 (m, 4H), 2.05 – 1.98 (m, 1H), 1.72 – 1.00 (m, 90H), 0.96 – 0.84 (m, 14H), 0.70 – 0.59 (m, 1H). ESI-MS:C 60 H 119 The calculated m / z value for N3O3 is 929.9, while the measured value is [M+H]. + = 931.1.

[0416] Example 46

[0417] 8,8'-(((1S,2S)-2-(hydroxymethyl)cyclopropyl)azadiyl)bis(N,N-didecyloctamide)

[0418] (Compound (1S,2S)-I-46)

[0419] Synthesis of (1s,2s)-I-46

[0420] The compound (1S,2S)-I-46 was synthesized from ((1S,2S)-2-aminocyclopropyl)methanol according to general procedure A. Purification was performed by automated rapid chromatography (5% to 65% EtOAc in hexane solution and 1% Et3N), followed by a second purification by automated rapid chromatography (0% to 10% MeOH in chloroform solution) to give the title compound (65 mg, 18%). 1 H NMR(400 MHz, CDCl3) δ 3.57 – 3.37 (m, 2H), 3.32 – 3.23 (m, 4H), 3.23 – 3.15 (m,4H), 2.56 – 2.51 (m, 4H), 2.27 (t, J = 7.5 Hz, 4H), 1.89 – 1.01 (m, 91H), 0.92 – 0.83 (m, 12H), 0.73 – 0.60 (m, 1H), 0.51 – 0.38 (m, 1H). ESI-MS:C 60 H 119ESI-MS for C3H3N3O3of m / z calcd = 929.9, found [M+H] + = 931.1.

[0421] Example 47

[0422] 8,8'-(((1-(hydroxymethyl)cyclobutyl)methyl)azanediyl)bis(N,N-didecyloctanamide)

[0423] (Compound I-29)

[0424] Synthesis of I-29

[0425] Compound I-29 was synthesized according to general procedure A using (1- (aminomethyl)cyclobutyl)methanol. Purification via automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N) afforded the title compound (168 mg, 44%). 1 HNMR (400 MHz, CDCl3) δ 6.17 (broad s, 1H), 3.75 (s, 2H), 3.32 – 3.23 (m, 4H), 3.23 – 3.14 (m, 4H), 2.55 (s, 2H), 2.41 – 2.31 (m, 4H), 2.31 – 2.20 (m, 4H), 2.03 – 1.89 (m, 1H), 1.87 – 1.74 (m, 5H), 1.69 – 1.04 (m, 90H), 0.92 - 0.83 (m, 12H). ESI-MS: C 62 H 123 ESI-MS for C3H3N3O3of m / z calcd = 958.0, found [M+H] + = 959.2.

[0426] Example 48

[0427] 8,8'-(((3-(hydroxymethyl)oxetan-3-yl)methyl)azanediyl)bis(N,N-didecyloctanamide)

[0428] (Compound I-41)

[0429] Synthesis of I-41

[0430] Compound I-41 was synthesized from (3-(aminomethyl)oxetane-3-yl)methanol according to general procedure A. The title compound (150 mg, 39%) was purified by automated rapid chromatography (hexane solution of 5% to 65% EtOAc and 1% Et3N). 1 ¹H NMR (400 MHz, CDCl₃) δ 5.69 (wide s, 1H), 4.47 (d, J = 6.0 Hz, 2H), 4.37(d, J = 6.0 Hz, 2H), 4.04 (s, 2H), 3.32 – 3.24 (m, 4H), 3.23 – 3.15 (m, 4H), 2.84 (s, 2H), 2.36 – 2.22 (m, 8H), 1.72 – 0.99 (m, 90H), 0.92 - 0.83 (m,12H). ESI-MS:C 61 H 121 The calculated m / z value for N3O4 is 959.9, while the measured value is [M+H]. + = 961.2.

[0431] Example 49

[0432] 8,8'-(((3R,4R)-4-hydroxytetrahydrofuran-3-yl)azadiyl)bis(N,N-didecyloctamide)

[0433] 8,8'-(((3S,4S)-4-hydroxytetrahydrofuran-3-yl)azadiyl)bis(N,N-didecyloctamide)

[0434] (compound (racemic)-(3) RS 4 RS )-I-40)

[0435] (racemic)-(3) RS 4 RS Synthesis of )-I-40

[0436] A mixture of enantiomers of compound I-40 was synthesized using a mixture of (3R,4R)-4-aminotetrahydrofuran-3-ol and (3S,4S)-4-aminotetrahydrofuran-3-ol according to general procedure A. The title compound (94 mg, 25%) was purified by automated rapid chromatography (5% to 65% EtOAc in hexane solution and 1% Et3N). 1H NMR (400 MHz, CDCl3) δ4.14 - 4.07 (m, 1H), 4.00 – 3.91 (m, 2H), 3.89 - 3.81 (m, 1H), 3.71 (dd, J =9.7, 7.9 Hz, 1H), 3.32 – 3.23 (m, 4H), 3.21 – 3.17 (m, 4H), 3.16 - 3.07 (m,1H), 2.58 – 2.42 (m, 4H), 2.31 – 2.23 (m, 4H), 1.73 – 1.05 (m, 86H), 0.92 -0.81 (m, 12H). ESI-MS: C 60 H 119 N3O4 of m / z calculated = 945.9, found [M+H] + = 947.2.

[0437] Example 50

[0438] 8,8'-(((3R,4S)-4-hydroxytetrahydrofuran-3-yl)azanediyl)bis(N,N-didecyloctanamide)

[0439] 8,8'-(((3S,4R)-4-hydroxytetrahydrofuran-3-yl)azanediyl)bis(N,N-didecyloctanamide)

[0440] Synthesis of (rac)-(3 RS ,4 SR )-I-40

[0441] Synthesis of (rac)-(3 RS ,4 SR )-I-40

[0442] The mixture of enantiomers of compound I-40 was synthesized according to general procedure A using a mixture of (3S,4R)-4-aminotetrahydrofuran-3-ol hydrochloride and (3R,4S)-4-aminotetrahydrofuran-3-ol hydrochloride. Purification was carried out via automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N) followed by a second purification via automated flash chromatography (50% to 100% EtOAc in hexanes). This resulted in the title compound (90 mg, 24%). 1H NMR (400 MHz, CDC13) δ 4.34 - 4.29 (m, 1H), 4.05 - 3.19 (m, 2H), 3.70 - 3.59 (m, 2H), 3.32– 3.24 (m, 5H), 3.23 – 3.15 (m, 4H), 2.55 – 2.42 (m, 4H), 2.27 (t, J = 7.4Hz, 4H), 1.97 – 1.00 (m, 87H), 0.92 - 0.81 (m, 12H). ESI-MS: C 60 H 119 N3O4 m / z calcd = 945.9, found [M+H] + = 947.2.

[0443] Example 51

[0444] 8,8'-((2-((1R,2R)-2-hydroxycyclohexyl)ethyl)azanediyl)bis(N,N-didecyloctanamide)

[0445] 8,8'-((2-((1S,2S)-2-hydroxycyclohexyl)ethyl)azanediyl)bis(N,N-didecyloctanamide)

[0446] Synthesis of (rac)-(1 RS ,2 RS )-I-31

[0447] Synthesis of (rac)-(1 RS ,2 RS )-I-31

[0448] The mixture of enantiomers of compound I-31 was synthesized according to general procedure B using rac-(1R,2S)-2-(2-aminoethyl)cyclohexan-1-ol. Purification via automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N) afforded the title compound (86 mg, 29%). 1H NMR (400 MHz, CDC13) δ 7.95 (broad s, 1H), 3.32 - 3.23 (m, 4H), 3.23 - 3.14 (m, 4H), 3.10 - 2.99 (m, 1H), 2.60 - 2.38 (m, 4H), 2.36 - 2.22 (m, 6H), 2.01 - 1.93 (m, 1H), 1.77 - 0.97 (m, 97H), 0.92 - 0.83 (m, 12H). ESI-MS: C 64 H 127 N3O3 m / z calcd = 986.0, found [M+H] + = 987.2.

[0449] Example 52

[0450] 8,8'-((2-((1R,2S)-2-hydroxycyclohexyl)ethyl)azanediyl)bis(N,N-didecyloctanamide)

[0451] 8,8'-((2-((1S,2R)-2-hydroxycyclohexyl)ethyl)azanediyl)bis(N,N-didecyloctanamide) (Compound (rac)-(1 RS ,2 SR )-I-31)

[0452] (Compound (rac)-(1 RS ,2 SR )-I-31

[0453] Compound I-31 mixture of enantiomers was synthesized according to general procedure B using rac-(1R,2R)-2-(2-aminoethyl)cyclohexan-1-ol. Purification via automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N) afforded the title compound (92 mg, 25%). 1 H NMR (400 MHz, CDC13) δ 7.95 (broad s, 1H), 3.32 - 3.23 (m, 4H), 3.23 - 3.14 (m, 4H), 3.10 - 2.99 (m, 1H), 2.60 - 2.38 (m, 4H), 2.36 - 2.22 (m, 6H), 2.01 - 1.93 (m, 1H), 1.77 - 0.97 (m, 97H), 0.92 - 0.83 (m, 12H). ESI-MS: C 64 H127 m / z calcd for N3O3 = 986.0, found [M+H] + 987.2.

[0454] Example 53

[0455] 8,8'-((((1R,2S)-2-(hydroxymethyl)cyclopropyl)methyl)azanediyl)bis(N,N- didecyloctanamide)

[0456] 8,8'-((((1S,2R)-2-(hydroxymethyl)cyclopropyl)methyl)azanediyl)bis(N,N- didecyloctanamide) (Compound (rac)-(1 RS ,2 SR )-I-45)

[0457] (rac)-(1 RS ,2 SR )-I-45

[0458] Compound I-45 enantiomeric mixture was synthesized according to general procedure B using rac-((1R,2S)-2-(aminomethyl)cyclopropyl)methanol. Purification via automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N) afforded the title compound (112 mg, 31%). 1 H NMR (400 MHz, CDCl3) δ 6.19 (broad s, 1H), 4.03- 3.91 (m, 1H), 3.32 – 3.23 (m, 4H), 3.23 – 3.12 (m, 4H), 3.12 - 3.00 (m,1H), 2.75 – 2.58 (m, 3H), 2.34 – 2.13 (m, 7H), 1.73 – 0.96 (m, 88H), 0.92 –0.75 (m, 13H), 0.26 - 0.13 (m, 1H)。ESI-MS: C 61 H 121 m / z calcd for N3O3 = 943.9, found [M+H] + 945.2.

[0459] Example 54

[0460] 8,8'-((((1R,2R)-2-(hydroxymethyl)cyclopropyl)methyl)azanediyl)bis(N,N- didecyloctanamide)

[0461] 8,8'-((((1S,2S)-2-(hydroxymethyl)cyclopropyl)methyl)azanediyl)bis(N,N- didodecyloctanamide) (Compound (rac)-(1 RS ,2 RS )-I-45)

[0462] (Compound (rac)-(1 RS ,2 RS )-I-45

[0463] Compound I-45 enantiomeric mixture was synthesized according to general procedure B using rac-((1R,2R)-2-(aminomethyl)cyclopropyl)methanol. Purification via automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N) afforded the title compound (110 mg, 31%). 1 H NMR (400 MHz, CDCl3) δ 3.54 – 3.42 (m, 2H), 3.32 – 3.23 (m, 4H), 3.23 – 3.15 (m, 4H), 2.51 - 2.39 (m, 5H), 2.34 – 2.22 (m, 5H), 2.00 – 1.02 (m, 86H), 0.98 - 0.71 (m, 14H), 0.50 - 0.41 (m, 1H), 0.0.40 - 0.31 (m, 1H). ESI-MS: C 61 H 121 N3O3 m / z calc = 943.9, found [M+H] + = 945.2.

[0464] Example 55

[0465] 8,8'-(((1R,2S)-2-hydroxycyclopentyl)azanediyl)bis(N,N-didodecyloctanamide)

[0466] (Compound (1R,2S)-I-48)

[0467] Synthesis of (1R,2S)-I-48

[0468] Compound (1R,2S)-I-48 was synthesized according to general procedure B using (1S,2R)-2-aminocyclopentan-1-ol hydrochloride. Purification via automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N) followed by a second flash chromatography (100% EtOAc) afforded the title compound (61 mg, 18%). 1 H NMR (400 MHz, CDCl3) δ 3.95 - 3.83 (m, 1H), 3.32 – 3.23 (m,4H), 3.23 – 3.15 (m, 4H), 2.87 - 2.74 (m,1H), 2.55 – 2.20 (m, 9H), 1.95 -1.85 (m, 1H), 1.81 – 1.02 (m, 97H), 0.92 - 0.83 (m, 12H). ESI-MS: C 61 H 121 N3O3 of m / z calculated = 943.9, found [M+H] + = 945.3.

[0469] Example 56

[0470] 8,8'-(((1R,2R)-2-hydroxycyclopentyl)azanediyl)bis(N,N-didecyloctanamide)

[0471] (Compound (1R,2R)-I-48)

[0472] Synthesis of (1R,2R)-I-48

[0473] Compound (1R,2R)-I-48 was synthesized according to general procedure B using (1R,2R)-2-aminocyclopentan-1-ol hydrochloride. Purification via automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N) afforded the title compound (111 mg, 33%). 1 H NMR (400 MHz, CDCl3) δ 3.95 - 3.83 (m, 1H), 3.32 – 3.23 (m,4H), 3.23 – 3.15 (m, 4H), 2.87 - 2.74 (m,1H), 2.55 – 2.20 (m, 9H), 1.95 -1.85 (m, 1H), 1.81 – 1.02 (m, 97H), 0.92 - 0.83 (m, 12H). ESI-MS: C 61 H121 The calculated m / z value for N3O3 is 943.9, while the measured value is [M+H]. + = 945.3.

[0474] Example 57

[0475] 8,8'-(((1R,2S)-2-hydroxycyclopentyl)azadiyl)bis(N,N-didecyloctamide)

[0476] 8,8'-(((1S,2R)-2-hydroxycyclopentyl)azadiyl)bis(N,N-didecyloctamide)

[0477] (compound (racemic)-(1) RS ,2 SR )-I-48)

[0478] (racemic)-(1) RS ,2 SR Synthesis of )-I-48

[0479] A mixture of enantiomers of compound I-48 was synthesized using racemic-(1R,2S)-2-aminocyclopentane-1-ol hydrochloride according to general procedure A. The mixture was purified by automated rapid chromatography (5% to 65% EtOAc in hexane solution with 1% Et3N), followed by a second rapid chromatography (50% to 100% EtOAc in hexane solution) to give the title compound (68 mg, 20%). 1 H NMR (400 MHz, CDCl3) δ 4.04 - 3.95 (m, 1H), 3.31 - 3.24 (m, 4H), 3.23 - 3.15 (m, 4H), 2.73 - 2.67 (m, 1H), 2.62 - 2.49 (m, 4H), 2.31 - 2.22 (m, 4H), 1.91 – 1.00 (m, 96H), 0.92 – 0.83 (m, 12H). ESI-MS:C 61 H 121 N3O3 m / z Calculated value = 943.9, measured value [M+H] + = 945.3.

[0480] Example 58

[0481] 8,8'-(((1R,2R)-2-hydroxycyclopentyl)azadiyl)bis(N,N-didecyloctamide)

[0482] 8,8'-(((1S,2S)-2-hydroxycyclopentyl)azanediyl)bis(N,N-didecyloctanamide)

[0483] (S)-I-48 RS ,2 RS )-I-48

[0484] (S)-I-48 RS ,2 RS )-I-48

[0485] A mixture of the enantiomers of compound I-48 was synthesized according to the general procedure A using rac-(1R,2R)-2-aminocyclopentan-1-ol. Purification via automated flash chromatography (5% to 65% EtOAc in hexanes with 1% Et3N) afforded the title compound (130 mg, 39%). 1 H NMR (400 MHz, CDCl3) δ 3.94 - 3.81 (m, 1H), 3.32 –3.23 (m, 4H), 3.23 – 3.15 (m, 4H), 2.88 - 2.75 (m, 1H), 2.55 – 2.34 (m, 4H),2.31 - 2.26 (t, J = 7.6 Hz, 5H), 1.96 - 1.85 (m, 1H), 1.79 – 1.02 (m, 96H),0.92 - 0.83 (m, 12H). ESI-MS: C 61 H 121 [M+H] Calcd for C + = 945.2.

[0486] Example 59

[0487] 8,8'-((4-hydroxycyclohexyl)azanediyl)bis(N,N-didecyloctanamide)

[0488] Compound I-9

[0489] Synthesis of I-9

[0490] Compound I-9 was prepared by mixing compound (1S,4S)-I-9 (25 mg) and compound (1R,4R)-I-9 (25 mg) together to afford the title compound (47 mg, 94%). 1H NMR (400 MHz, CDC13) δ 3.98 (broad s, 0.5H), 3.54 (broad s, 0.5H), 3.32 - 3.23 (m, 4H), 3.23 - 3.15 (m, 4H), 2.46 -2.32 (m, 5H), 2.30 - 2.22 (m, 4H), 2.06 - 1.92 (m, 1H), 1.89 - 1.73 (m, 2H), 1.71 - 1.07 (m, 105H), 0.94 - 0.83 (m, 12H). ESI-MS: C 62 H 123 Calcd for m / z = 958.0, Found [M+H] + = 959.2.

[0491] Example 60

[0492] 8,8'-(((1s,4s)-4-hydroxycyclohexyl)azanediyl) bis(N,N-didecyl-2-fluorooctanamide)

[0493] Synthesis of (1s,4s)-l-49

[0494] Synthesis of (1s,4s)-l-49

[0495] Compound (1s,4s)-l-49 was synthesized according to general procedure A using intermediate 6 (Example 32) and (1s,4s)-4-aminocyclohexan-1-ol. Purification via automated flash chromatography (5% to 100% EtOAc in hexanes with 1% Et3N) afforded the title compound (155 mg, 54%). 1 H NMR (400 MHz, CDC13) δ 5.12 (dd, J = 8.4, 4.4 Hz,1H), 4.99 (dd, J = 8.5, 4.3 Hz, 1H), 4.01 (s, 1H), 3.41 - 3.14 (m, 8H), 2.52– 2.40 (m, 5H), 2.02 - 1.73 (m, 6H), 1.72 – 1.09 (m, 92H), 0.95 - 0.86 (m,12H). ESI-MS: C 62 H 121 Calcd for m / z = 993.9, Found [M+H] + = 995.3.

[0496] Example 61

[0497] 8,8'-(((1R,4R)-4-hydroxycyclohexyl)azanediyl) bis(N,N-didecyl-2-fluorooctanamide)

[0498] (Synthesis of compound (1R,4R)-I-49)

[0499] Synthesis of (1R,4R)-I-49

[0500] Compound (1R,4R)-I-49 was synthesized according to general procedure A using intermediate 6 (Example 32) and (1R,4R)-4-aminocyclohexan-1-ol. Purification via automated flash chromatography (5% to 100% EtOAc in hexanes with 1% Et3N) afforded the title compound (170 mg, 60%). 1 H NMR (400 MHz, CDCl3) δ 5.12 (dd, J = 8.4, 4.4 Hz,1H), 4.99 (dd, J = 8.5, 4.3 Hz, 1H), 3.57 (broad s, 1H), 3.41 - 3.14 (m, 8H), 2.54 – 2.31 (m, 5H), 2.09 – 1.72 (m, 8H), 1.69 – 1.08 (m, 90H), 0.95 - 0.86 (m, 12H). ESI-MS: C 62 H 121 F2N3O3 m / z calcd = 993.9, found [M+H] + = 995.2.

[0501] Example 62

[0502] 8,8'-(((1S,4S)-4-hydroxycyclohexyl)azanediyl) bis(N-(nonadecan-10-yl)octanamide)

[0503] (Synthesis of compound (1S,4S)-I-50)

[0504] Synthesis of intermediate 7

[0505] A mixture of (ls,4s)-4-aminocyclohexan-1-ol (4.34 mmol, 500 mg), ethyl 8- bromooctanoate (8.68 mmol, 2.18 g), DIEA (13.5 mmol, 2.34 mL) and potassium iodide (13.0 mmol, 2.16 g) in acetonitrile (8.7 mL) was stirred at 75 °C for 19 h. The reaction mixture was concentrated and the crude material was suspended in dichloromethane and filtered. The filtrate was purified via automated flash chromatography (10% to 100% EtOAc in hexanes with 1% Et3N) to give Intermediate 7 (1.28 g, 65%).

[0506]

[0507] Synthesis of Intermediate 8

[0508] A mixture of Intermediate 7 (0.44 mmol, 200 mg) and potassium hydroxide (1.34 mmol, 75 mg) in methanol (0.88 mL) and water (0.88 mL) was stirred at room temperature for 20 h. The reaction mixture was concentrated to give Intermediate 8 which was used in the next step without further purification.

[0509]

[0510] Synthesis of (ls,4s)-l-50

[0511] A mixture of Intermediate 8 (0.25 mmol, 100 mg), nonadecan-10-amine (0.5 mmol, 142 mg), DIEA (0.75 mmol, 0.13 mL) and HATU (0.5 mmol, 190 mg) in dichloromethane (1.25 mL) was stirred at room temperature for 20 h. The reaction mixture was concentrated and the crude material was partitioned between EtOAc and saturated NaHC03. The organic layer was separated, dried over Na2S04and concentrated. Purification via flash chromatography (5% to 100% EtOAc in hexanes with 1% Et3N) gave compound (ls,4s)-l-50 (75 mg, 32%). 1 H NMR (400 MHz, CDCl3) δ 5.25 (d, J = 9.2Hz, 2H), 4.05 – 3.88 (m, 3H), 2.58 - 2.39 (m, 5H), 2.18 (t, J= 7.6 Hz, 4H),1.92 - 1.83 (m, 2H), 1.77 – 1.05 (m, 105H), 0.95 - 0.84 (m, 15H). ESI-MS: C 60 H 119 N3O3 m / z calcd = 929.9, found [M+H] + = 931.2.

[0512] Example 63

[0513] 8,8'-(((1s,4s)-4-hydroxycyclohexyl)azanediyl)bis(N-(dec-9-en-1-yl)-N-decanyl octanamide)

[0514] Synthesis of (1s,4s)-I-51

[0515] Synthesis of Intermediate 9

[0516] A mixture of decanamine (13.7 mmol, 2.15 g), 10-bromo-1-decene (4.6 mmol, 1.0 g), and DIEA (13.7 mmol, 2.38 mL) in acetonitrile (9.1 mL) was stirred at room temperature for 72 h. The reaction mixture was concentrated and the crude material was purified via flash chromatography (0% to 20% EtOAc in hexanes with 1% Et3N). A second purification via flash chromatography (1% to 15% MeOH in DCM) afforded Intermediate 9 (538 mg, 40%).

[0517]

[0518] Synthesis of (1s,4s)-I-51

[0519] A mixture of Intermediate 8 (0.25 mmol, 100 mg), Intermediate 9 (0.5 mmol, 148 mg), DIEA (1.50 mmol, 0.26 mL), and HATU (0.65 mmol, 247 mg) in dichloromethane (2.5 mL) was stirred at room temperature for 20 h. The reaction mixture was concentrated and the crude material was partitioned between EtOAc and saturated NaHCO3. The organic layer was separated, dried over Na2SO4, and concentrated. Purification via flash chromatography (5% to 100% EtOAc in hexanes with 1% Et3N) afforded compound (1s,4s)-I-51 (95 mg, 40%). 1H NMR (400 MHz, CDC13) δ 5.88 - 5.73 (m, 2H), 5.04 - 4.88 (m, 4H), 4.01 - 3.92 (m, 1H), 3.32 - 3.23 (m, 4H), 3.23 - 3.15 (m, 4H), 2.49 - 2.38 (m, 5H), 2.30 - 2.22 (m, 4H), 2.09 - 1.97 (m, 4H), 1.92 - 1.75 (m, 2H), 1.76 - 1.07 (m, 89H), 0.93 - 0.81 (m, 6H). ESI-MS: C 62 H 119 N3O3 m / z calcd = 953.9, found [M+H] + = 955.2.

[0520] Example 64

[0521] 8,8'-(((1s,4s)-4-hydroxycyclohexyl)azanediyl)bis(N-dodecyl-N-octyloctanamide)

[0522] (Compound (1s,4s)-I-62)

[0523] Synthesis of Intermediate 10

[0524] A mixture of octylamine (24.1 mmol, 3.11 g), 1-bromododecane (8.0 mmol, 2.0 g), and DIEA (24.1 mmol, 4.2 mL) in acetonitrile (16 mL) was stirred at 50 °C for 20 h. The reaction mixture was concentrated and the crude material was purified via flash chromatography (1% to 15% MeOH in DCM) to give Intermediate 10 (1.75 g, 73%).

[0525]

[0526] Synthesis of (1s,4s)-I-62

[0527] Following the procedure outlined for compound (1s,4s)-I-51 (Example 63), compound (1S,4S)-I-62 was prepared from Intermediate 8 and Intermediate 10. Yield (67 mg, 28%). 1H NMR (400 MHz, CDC13) δ 4.02 - 3.92 (m, 1H), 3.32 - 3.23 (m, 4H), 3.23 - 3.15 (m, 4H), 2.50 - 2.38 (m, 5H), 2.31 - 2.22 (m, 4H), 1.86 - 1.79 (m, 2H), 1.72 - 1.10 (m, 96H), 0.92 - 0.83 (m, 12H). ESI-MS: C 62 H 123 N3O3 m / z calcd = 958.0, found [M+H] + = 959.3.

[0528] Example 65

[0529] 8,8'-(((1s,4s)-4-hydroxycyclohexyl)azanediyl)bis(N-decyl-N-hexyloctanamide)

[0530] (Compound (1s,4s)-I-52)

[0531] Synthesis of Intermediate 11

[0532] A mixture of hexylamine (24.1 mmol, 2.44 g), 1-bromododecane (8.0 mmol, 2.0 g), and DIEA (24.1 mmol, 4.2 mL) in acetonitrile (16 mL) was stirred at 50 °C for 20 h. The reaction mixture was concentrated and the crude material was purified via flash chromatography (1% to 15% MeOH in DCM) to give Intermediate 11 (1.7 g, 73%).

[0533]

[0534] Synthesis of (1S,4S)-I-52

[0535] Following the procedure outlined for compound (1s,4s)-I-51 (Example 63), compound (1S,4S)-I-52 was prepared from Intermediate 8 and Intermediate 11. Yield (33 mg, 15%). 1H NMR (400 MHz, CDC13) δ 4.02 - 3.91 (m, 1H), 3.32 - 3.24 (m, 4H), 3.23 - 3.15 (m, 4H), 2.51 - 2.38 (m, 5H), 2.31 - 2.22 (m, 4H), 1.89 - 1.79 (m, 2H), 1.76 - 1.05 (m, 88H), 0.94 - 0.83 (m, 12H). ESI-MS: C 58 H 115 N3O3 m / z calcd = 901.9, found [M+H] + = 903.2.

[0536] Example 66

[0537] 8,8'-(((1s,4s)-4-hydroxycyclohexyl)azanediyl)bis(N,N-di(dec-9-en-1-yl)octanamide)

[0538] (Synthesis of compound (1s,4s)-I-53)

[0539] Synthesis of intermediate 12

[0540] A mixture of dec-9-en-1-amine (6.84 mmol, 1.06 g), 10-bromodec-1-ene (2.28 mmol, 500 mg), and DIEA (6.84 mmol, 1.19 mL) in acetonitrile (4.6 mL) was stirred at room temperature for 20 h. The reaction mixture was concentrated and the crude material was purified via flash chromatography (1% to 15% MeOH in DCM). A second purification via flash chromatography (1% to 10% MeOH in CHCl3) afforded intermediate 12 (0.36 g, 54%).

[0541]

[0542] Synthesis of compound (1S,4S)-I-53

[0543] Compound (1S,4S)-I-53 was prepared from intermediate 8 and intermediate 12 following the procedure outlined for compound (1S,4S)-I-51 (Example 63). Yield (150 mg, 48%). 1H NMR (400 MHz, CDCl3) δ 5.83 (m,4H), 5.07 – 4.90 (m, 8H), 4.05 – 3.96 (m, 1H), 3.34 – 3.26 (m, 4H), 3.25 –3.17 (m, 4H), 2.51 – 2.40 (m, 5H), 2.33 – 2.25 (m, 4H), 2.12 – 2.00 (m, 8H), 1.90 – 1.81 (m, 2H), 1.74 – 1.06 (m, 84H). ESI-MS:C 62 H 115 The calculated m / z value for N3O3 is 949.9, while the measured value is [M+H]. + = 951.2.

[0544] Example 67

[0545] 8,8'-(((1s,4s)-4-hydroxycyclohexyl)azadiyl)bis(N-butyl-N-(heptadecane-9-yl)octamide)(compound(1s,4s)-I-54)

[0546] Synthesis of intermediate 13

[0547] A mixture of heptadecano-9-one (5.89 mmol, 1.5 g), butylamine (8.84 mmol, 0.87 mL), acetic acid (8.84 mmol, 0.51 mL), and sodium triacetoxyborohydride (23.6 mmol, 5.0 g) in dichloroethane (29.5 mL) was stirred at room temperature for 20 hours. An additional portion of sodium triacetoxyborohydride (9.4 mmol, 2.0 g) was added, and the reaction mixture was stirred at room temperature for 20 hours. The reaction mixture was concentrated, and the crude material was partitioned between EtOAc and saturated NaHCO3. The organic layer was separated, dried over Na2SO4, and concentrated. Purification by rapid chromatography (1% to 10% MeOH in CHCl3 solution) gave intermediate 13 (1.29 g, 70%).

[0548]

[0549] Synthesis of (1S,4S)-I-54

[0550] Compound (1S,4S)-I-54 was prepared from intermediates 8 and 13 according to the procedure outlined for compound (1S,4S)-I-51 (Example 63). Yield (130 mg, 35%). 1H NMR (400 MHz, CDCl3) δ 4.45 (width, 1H), 4.05 - 3.96 (m, 1H), 3.70 - 3.58 (m, 1H), 3.12 - 3.00 (m, 4H), 2.58 -2.38 (m, 5H), 2.35 - 2.26 (m, 4H), 1.89 - 1.81 (m, 2H), 1.77 - 1.06 (m, 97H), 1.02 - 0.85 (m, 18H). ESI-MS:C 64 H 127 The calculated m / z value for N3O3 is 986.0, while the measured value is [M+H]. + = 987.2.

[0551] Example 68

[0552] N,N-Dicoyl-8-((8-(dodecyl(octyl)amino)-8-oxooctyl)((1s,4s)-4-hydroxycyclohexyl)amino)octylamide

[0553] (Compound (1s,4s)-I-55)

[0554] Synthesis of intermediate 14

[0555] A mixture of (1s,4s)-4-aminocyclohexane-1-ol (18.3 mmol, 2.11 g), intermediate 1 (4.58 mmol, 2.3 g), DIEA (14.2 mmol, 2.47 mL), and potassium iodide (13.7 mmol, 2.28 g) in acetonitrile (9.2 mL) was heated at 75 °C for 20 h. The reaction mixture was concentrated and the resulting crude product was suspended in 95:5:1 (hexane:EtOAc:Et3N) and filtered. The filtrate was purified by rapid chromatography (100% EtOAc) followed by a second rapid chromatography (1% to 10% MeOH in CHCl3 solution) to give intermediate 14 (1.0 g, 41%).

[0556]

[0557] Synthesis of Intermediate 15

[0558] A mixture of intermediate 14 (1.6 mmol, 850 mg), ethyl 8-bromooctanoate (1.6 mmol, 400 mg), DIEA (4.9 mmol, 0.86 mL), and potassium iodide (4.8 mmol, 790 mg) was heated at 75 °C for 20 hours. The reaction mixture was concentrated, and the resulting crude product was suspended in 95:5:1 (hexane:EtOAc:Et3N) and filtered. The filtrate was purified by rapid chromatography (5% to 100% hexane solution of EtOAc and 1% Et3N) to give intermediate 15 (700 mg, 63%).

[0559]

[0560] Synthesis of intermediate 16

[0561] A mixture of intermediate 15 (0.99 mmol, 0.70 g) and sodium hydroxide (25 mmol, 1 g) in methanol:water (4 mL:0.4 mL) was heated at 60 °C for 2 hours. The reaction mixture was diluted with water and extracted with EtOAc. The organic layer was separated, dried over Na2SO4, and concentrated to give intermediate 16 (600 mg, 89%).

[0562]

[0563] Synthesis of (1S,4S)-I-55

[0564] A mixture of intermediate 16 (0.12 mmol, 80 mg), intermediate 10 (0.12 mmol, 35 mg), DIEA (0.47 mmol, 0.082 mL), and HATU (0.15 mmol, 58 mg) in dichloromethane (1.2 mL) was stirred at room temperature for 20 hours. The reaction mixture was concentrated, and the crude material was partitioned between EtOAc and saturated NaHCO3. The organic layer was separated, dried over Na2SO4, and concentrated. Purification by rapid chromatography (5% to 100% EtOAc in hexane solution of 1% Et3N) gave the title compound (33 mg, 29%). 1H NMR (400 MHz, CDCl3) δ 4.05 - 3.98 (m, 1H), 3.35 - 3.27 (m, 4H), 3.26 - 3.18 (m, 4H), 2.57 - 2.40 (m, 5H), 2.34 - 2.26(m, 4H), 1.92 - 1.82 (m, 2H), 1.78 – 1.09 (m, 95H), 0.96 – 0.87 (m, 12H). ESI-MS:C 62 H 123 The calculated m / z value for N3O3 is 958.0, while the measured value is [M+H]. + = 959.2.

[0565] Example 69

[0566] N,N-Dicoyl-8-((8-(dodecyl(hexyl)amino)-8-oxooctyl)((1s,4s)-4-hydroxycyclohexyl)amino)octylamide

[0567] (Compound (1s,4s)-I-56)

[0568] Synthesis of (1s,4s)-I-56

[0569] Compound (1S,4S)-I-56 was prepared from intermediates 16 and 11 according to the procedure outlined for compound (1S,4S)-I-55 (Example 68). Yield (26 mg, 24%). 1 H NMR (400 MHz, CDCl3) δ 4.05 -3.98 (m, 1H), 3.35 - 3.27 (m, 4H), 3.27 - 3.18 (m, 4H), 2.57 - 2.40 (m, 5H), 2.34 - 2.26 (m, 4H), 1.92 - 1.82 (m, 2H), 1.77 – 1.09 (m, 91H), 0.91 (td, J =6.9, 2.8 Hz, 12H). ESI-MS:C 60 H 119 The calculated m / z value for N3O3 is 929.9, while the measured value is [M+H]. + = 931.2.

[0570] Example 70

[0571] N,N-Di(dec-9-en-1-yl)-8-((8-(didecylamino)-8-oxooctyl)((1s,4s)-4-hydroxycyclohexyl)amino)octylamide

[0572] (Compound (1s,4s)-I-57)

[0573] Synthesis of (1S,4S)-I-57

[0574] Compound (1S,4S)-I-57 was prepared from intermediates 16 and 12 according to the procedure outlined for compound (1S,4S)-I-55 (Example 68). Yield (30 mg, 21%). 1 H NMR (400 MHz, CDCl3) δ 5.92 -5.77 (m, 2H), 5.08 – 4.92 (m, 4H), 4.01 (s, 1H), 3.35 – 3.27 (m, 4H), 3.27 –3.18 (m, 4H), 2.59 – 2.39 (m, 4H), 2.34 – 2.26 (m, 4H), 2.13 – 2.01 (m, 4H), 1.93 – 1.80 (m, 2H), 1.79 – 1.07 (m, 85H), 0.91 (td, J = 6.8, 3.1 Hz, 6H). ESI-MS:C 62 H 119 The calculated m / z value for N3O3 is 953.9, while the measured value is [M+H]. + = 955.2.

[0575] Example 71

[0576] N,N-Didecyl-8-(((1s,4s)-4-hydroxycyclohexyl)(8-(nonadecan-10-ylamino)-8-oxooctyl)amino)octylamide

[0577] (Compound (1s,4s)-I-58)

[0578] Synthesis of (1S,4S)-I-58

[0579] Compound (1S,4S)-I-58 was prepared from intermediate 16 and nonadecanoamine according to the procedure outlined for compound (1S,4S)-I-55 (Example 68). Yield (36 mg, 26%). 1H NMR (400 MHz, CDCl3) δ 5.22(d, J = 9.1 Hz, 1H), 4.03 - 3.84 (m, 2H), 3.28 (t, J = 7.7 Hz, 2H), 3.20 (t, J = 7.8 Hz, 2H), 2.55 - 2.37 (m, 5H), 2.27 (t, J = 7.6 Hz, 2H), 2.16 (t, J =7.6 Hz, 2H), 1.88 - 1.80 (m,2H), 1.75 - 1.03 (m, 93H), 0.93 - 0.84 (m, 12H). ESI-MS:C 61 H 121 The calculated m / z value for N3O3 is 943.9, while the measured value is [M+H]. + = 945.3.

[0580] Example 72

[0581] N-Butyl-8-((8-(decylamino)-8-oxooctyl)((1s,4s)-4-hydroxycyclohexyl)amino)-N-(heptadec-9-yl)octamide

[0582] (Compound (1s,4s)-I-59)

[0583] Synthesis of (1S,4S)-I-59

[0584] Compound (1S,4S)-I-59 was prepared from intermediates 16 and 13 according to the procedure outlined for compound (1S,4S)-I-55 (Example 68). Yield (39 mg, 27%). 1 H NMR (400 MHz, CDCl3) δ 4.02 -3.96 (m, 1H), 3.67 - 3.57 (m, 1H), 3.28 (t, J = 7.7 Hz, 2H), 3.20 (t, J= 7.8Hz, 2H), 3.12 – 2.97 (m, 2H), 2.55 – 2.36 (m, 5H), 2.33 – 2.23 (m, 4H), 1.90- 1.78 (m, 2H), 1.76 – 1.05 (m, 97H), 1.00 – 0.84 (m, 15H). ESI-MS:C 63 H 125 The calculated m / z value for N3O3 is 972.0, while the measured value is [M+H]. + = 973.3.

[0585] Example 73

[0586] 10,10'-(((1s,4s)-4-hydroxycyclohexyl)azadiyl)bis(N,N-dicecyldecanoamide)

[0587] (Compound (1s,4s)-I-60)

[0588] Synthesis of intermediate 17

[0589] Intermediate 17 was prepared from 10-bromodecanoic acid according to the procedure outlined for intermediate 1 (Example 5). Yield (5.07 g, 40%).

[0590]

[0591] Synthesis of (1s,4s)-I-60

[0592] Compound (1S,4S)-I-60 was synthesized using a mixture of intermediate 17 and (1S,4S)-4-aminocyclohexane-1-ol according to general procedure A. The title compound (yield 97 mg, 33%) was obtained by automated rapid chromatography (5% to 100% EtOAc in hexane solution with 1% Et3N). 1 H NMR (400 MHz, CDCl3) δ 4.04 - 3.98 (m, 1H), 3.34 –3.26 (m, 4H), 3.25 – 3.17 (m, 4H), 2.55 – 2.38 (m, 5H), 2.33 – 2.25 (m, 4H),1.92 – 1.80 (m, 2H), 1.77 – 1.05 (m, 106H), 0.95 – 0.86 (m, 12H). ESI-MS:C 66 H 131 The calculated m / z value for N3O3 is 1014.0, while the measured value is [M+H].+ = 1015.3.

[0593] Example 74

[0594] N,N-Didecyl-10-((6-(didecylamino)-6-oxohexyl)((1s,4s)-4-hydroxycyclohexyl)amino)decanoamide(compound(1s,4s)-I-61)

[0595] Synthesis of intermediate 18

[0596] A mixture of (1s,4s)-4-aminocyclohexane-1-ol (3.77 mmol, 434 mg), intermediate 17 (0.94 mmol, 0.50 g), DIEA (2.92 mmol, 0.510 mL), and potassium iodide (2.83 mmol, 469 mg) in acetonitrile (1.9 mL) was heated at 140 °C for 1 hour by microwave irradiation. The reaction mixture was concentrated and the resulting crude product was suspended in 95:5:1 (hexane:EtOAc:Et3N) and filtered. The filtrate was purified by rapid chromatography (25% to 100% EtOAc in hexane solution) followed by a second rapid chromatography (1% to 10% MeOH in CHCl3 solution) to give intermediate 18 (440 mg, 83%).

[0597] Synthesis of Intermediate 19

[0598] Intermediate 19 was prepared from 6-bromohexanoic acid according to the procedure outlined for intermediate 1 (Example 5). Yield (4.6 g, 76%).

[0599]

[0600] Synthesis of (1s,4s)-I-61

[0601] The compound (1S,4S)-I-61 was prepared from intermediates 18 and 19 according to the procedure outlined for intermediate 15 (Example 68). Yield (90 mg, 53%). 1H NMR (400 MHz, CDCl3) δ 4.02 - 3.94 (m, 1H), 3.32 - 3.23 (m, 4H), 3.23 - 3.15 (m, 4H), 2.52 - 2.37 (m, 5H), 2.31 - 2.22(m, 4H), 1.90 - 1.77 (m, 2H), 1.76 – 1.04 (m, 97H), 0.92 – 0.83 (m, 12H). ESI-MS:C 62 H 123 The calculated m / z value for N3O3 is 958.0, while the measured value is [M+H]. + = 959.3.

[0602] Example 75

[0603] N,N'-((((1s,4s)-4-hydroxycyclohexyl)azadiyl)bis(octane-8,1-diyl))bis(N-decyldecanoamide) (compound(1s,4s)-I-63)

[0604] Synthesis of (1s,4s)-I-63

[0605] Compound (1S,4S)-I-63 was prepared from Int 4-1 (Example 33) and (1S,4S)-4-aminocyclohexane-1-ol according to general procedure B. The title compound (122 mg, 59%) was purified by automated rapid chromatography (5% to 100% EtOAc in hexane solution with 1% Et3N). 1 H NMR (400 MHz, CDCl3) δ 4.02 - 3.95 (m, 1H), 3.32 -3.23 (m, 4H), 3.23 - 3.15 (m, 4H), 2.54 - 2.34 (m, 5H), 2.31 - 2.22 (m, 4H),1.89 - 1.77 (m, 2H), 1.74 – 1.04 (m, 94H), 0.92 – 0.84 (m, 12H). ESI-MS:C 62 H 123 The calculated m / z value for N3O3 is 958.0, while the measured value is [M+H]. + = 959.3.

[0606] Example 76

[0607] N-decyl-N-(8-((8-(didecylamino)-8-oxooctyl)((1s,4s)-4-hydroxycyclohexyl)amino)octyl)decamide(compound(1s,4s)-I-47)

[0608] Synthesis of (1s,4s)-I-47

[0609] A mixture of intermediate 14 (0.21 mmol, 112 mg), Int 4-1 (0.14 mmol, 70 mg), DIEA (0.43 mmol, 0.075 mL), and potassium iodide (0.42 mmol, 69 mg) was heated at 160 °C for 3 hours by microwave irradiation. The reaction mixture was concentrated, and the resulting crude product was suspended in 95:5:1 (hexane:EtOAc:Et3N) and filtered. The filtrate was purified by rapid chromatography (5% to 100% hexane solution of EtOAc and 1% Et3N) to give the title compound (45 mg, 34%). 1 H NMR (400 MHz, CDCl3) δ 4.03 - 3.94 (m, 1H), 3.32 - 3.23 (m, 4H), 3.23 - 3.15 (m, 4H), 2.52 - 2.35 (m, 5H), 2.31 - 2.22 (m, 4H), 1.89 - 1.78 (m, 2H), 1.73 – 1.02 (m, 97H), 0.92 – 0.84 (m, 12H). ESI-MS:C 62 H 123 The calculated m / z value for N3O3 is 958.0, while the measured value is [M+H]. + = 959.2.

[0610] Example 77

[0611] N-decyl-N-(8-((8-(didecylamino)-8-oxooctyl)((1R,4R)-4-hydroxycyclohexyl)amino)octyl)decamide(compound(1r,4r)-I-47)

[0612] Synthesis of intermediate 20

[0613] A mixture of (1r,4r)-4-aminocyclohexane-1-ol (3.99 mmol, 458 mg), intermediate 1 (1.0 mmol, 500 mg), DIEA (3.1 mmol, 0.54 mL), and potassium iodide (3.0 mmol, 495 mg) in acetonitrile (2.0 mL) was heated at 140 °C for 40 min by microwave irradiation. The reaction mixture was concentrated and the resulting crude product was suspended in 95:5:1 (hexane:EtOAc:Et3N) and filtered. The filtrate was purified by rapid chromatography (10% to 100% EtOAc in hexane solution) followed by a second rapid chromatography (1% to 12% MeOH in dichloromethane solution) to give intermediate 20 (363 mg, 68%).

[0614] Synthesis of (1r,4r)-I-47

[0615] A mixture of intermediate 20 (0.24 mmol, 130 mg), Int 4-1 (0.29 mmol, 146 mg), DIEA (0.75 mmol, 0.13 mL), and potassium iodide (0.73 mmol, 121 mg) was heated at 140 °C for 40 min by microwave irradiation. The reaction mixture was concentrated, and the resulting crude product was suspended in 95:5:1 (hexane:EtOAc:Et3N) and filtered. The filtrate was purified by rapid chromatography (5% to 100% hexane solution of EtOAc and 1% Et3N) to give the title compound (160 mg, 69%). 1 H NMR (400 MHz, CDCl3) δ 3.60 - 3.49 (m, 1H), 3.32 - 3.23 (m,4H), 3.23 - 3.15 (m, 4H), 2.51 - 2.31 (m, 5H), 2.31 - 2.21 (m, 4H), 2.06 –1.93 (m, 2H), 1.84 – 1.02 (m, 97H), 0.92 – 0.81 (m, 12H). ESI-MS:C 62 H 123 The calculated m / z value for N3O3 is 958.0, while the measured value is [M+H]. + = 959.2.

[0616] The various embodiments described above can be combined to provide other embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referenced in this specification, including U.S. Provisional Patent Application No. 63 / 500,511, filed May 5, 2023, are incorporated herein by reference in their entirety. If necessary, aspects of the embodiments can be modified to provide other embodiments using the concepts of various patents, applications and publications.

[0617] These and other changes can be made to the embodiments based on the detailed description above. Generally, the terminology used in the appended claims should not be construed as limiting the claims to the specific embodiments disclosed in this specification and claims, but rather as encompassing all possible embodiments and the full scope of equivalents conferred by such claims. Therefore, the claims are not limited to this disclosure.

Claims

1. A compound having the following structure (I): (I) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: A is a 3- to 10-membered carbocyclic ring or a 3- to 10-membered oxygen-containing heterocyclic ring, the carbocyclic ring or oxygen-containing heterocyclic ring optionally substituted with one or more fluoro, hydroxyl, C1-C6 alkyl, or C1-C6 alkylhydroxyl substituents; B is a 3- to 10-membered carbocyclic ring or a 3- to 10-membered oxygen-containing heterocyclic ring, the carbocyclic ring or oxygen-containing heterocyclic ring optionally substituted with one or more fluoro, hydroxyl, C1-C6 alkyl, or C1-C6 alkylhydroxyl substituents; each R1is independently selected from the group consisting of hydrogen, C1-C6 alkyl, C1-C6 alkylhydroxyl, C1-C6 alkylhalo, C1-C6 alkylhydroxyhalo, C1-C6 alkylhydroxyalkyl, C1-C6 alkylhydroxyalkylhalo, C1-C6 alkylhydroxyalkylhydroxyl, C1-C6 alkylhydroxyalkylhydroxylhalo, C1-C6 alkylhydroxyalkylhydroxylalkyl, C1-C6 alkylhydroxyalkylhydroxylalkylhalo, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxyl, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylhalo, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkyl, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhalo, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkyl, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhalo, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkyl, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhalo, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkyl, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhalo, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkyl, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhalo, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkyl, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhalo, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkyl, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhalo, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkyl, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhalo, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkyl, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhalo, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkyl, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhalo, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkyl, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhalo, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkyl, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhalo, C1-C6 alkylhydroxyalkylhydroxylalkyl, C1-C6 alkylhydroxyalkyl, C1-C6 alkylhydroxyl, C1-C6 alkylhalo, C1-C6 alkylhydroxyhalo, C1-C6 alkylhydroxyalkyl, C1-C6 alkylhydroxyalkylhalo, C1-C6 alkylhydroxyalkylhydroxyl, C1-C6 alkylhydroxyalkylhydroxylhalo, C1-C6 alkylhydroxyalkylhydroxylalkyl, C1-C6 alkylhydroxyalkylhydroxylalkylhalo, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxyl, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylhalo, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkyl, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhalo, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkyl, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhalo, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkyl, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhalo, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkyl, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhalo, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkyl, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkyl, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhalo, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkyl, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkyl, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhalo, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkyl, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkylhydroxylalkyl, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkylhydroxylalkylhalo, C1-C6 alkylhydroxyalkylhydroxylalkylhydroxylalkyl, C1-C6 alkylhydroxyalkyl, C1-C6 alkylhydrox (I) ​ ​ R 1 is -NR a C(=O)R 3 or -C(=O)NR b R c ; R 2 is -NR d C(=O)R 4 or -C(=O)NR e R f ; R 3 and R 4 each independently is C6-C 24 alkyl or C6-C 24 alkenyl; R a , R b , R d , and R e are each independently H, C1-C 20 alkyl or C2-C 20 alkenyl; R c and R f each independently is C1-C 20 alkyl or C2-C 20 alkenyl; L 1 and L 2 each independently is a direct link or C1-C6alkylene; and L 2a and L 2b each independently is C4-C 12 alkylene; ​ 2. The compound of claim 1, wherein R 1 is -C(=0)NR b R c and R 2 is -C(=0)NR e R f .

3. The compound of claim 1, wherein R 1 is -NR a C(=O)R 3 and R 2 is -NR d C(=O)R 4 .

4. The compound of claim 1, wherein R 1 is -NR a C(=O)R 3 and R 2 is -C(=O)NR e R f .

5. The compound of any one of claims 1 to 4, wherein R a , R b , R c , R d , R e , and R f are each independently C1-C 20 alkyl.

6. The compound of claim 5, wherein R a , R b , R c , R d , R e , and R f are each independently C8-C 19 alkyl.

7. The compound of claim 6, wherein R a , R b , R c , R d , R e , and R f are each independently C8, C9, C 10 , or C 19 alkyl.

8. The compound of any one of claims 1 or 3 to 7, wherein R 3 and R 4 are each independently C6-C 19 alkyl.

9. The compound of claim 8, wherein R 3 and R 4 are each independently C8-C 10 alkyl.

10. The compound of claim 9, wherein R 3 and R 4 are each independently C8, C9, or C 10 alkyl.

11. The compound of any one of claims 1 to 10, wherein L 1 is a direct bond.

12. The compound of any one of claims 1 to 10, wherein L 1 is Ci-C6alkylene.

13. The compound of any one of claims 1 to 10, wherein L 1 is Ci or C2 alkylene.

14. The compound of any one of claims 1 to 13, wherein L 2 is a direct bond.

15. The compound of any one of claims 1 to 13, wherein L 2 is Ci-C6alkylene.

16. The compound of any one of claims 1 to 13, wherein L 2 is Ci or C2 alkylene.

17. The compound of any one of claims 1 to 16, wherein L 2a and L 2b each independently is C6-C 10 alkylene.

18. The compound of any one of claims 1 to 16, wherein L 2a and L 2b each independently is C6, C7, C8, C9, or C 10 alkylene.

19. The compound of any one of claims 1 to 18, wherein A is C3-C6carbocycle optionally substituted with one or more hydroxyl or C1-C6alkylhydroxyl substituents. 10 carbocycle. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 30. The compound of any one of claims 1 to 29, wherein one of the following structures: ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ， ​ 31. The compound of any one of claims 1 to 30, wherein one of the following structures: ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ; ; ; ; ; ; ; ; ; ; ; ; ; or .

32. The compound of any one of claims 1 to 31, wherein one or more of R a , R b , R c , R d , R e , R f , L 1 , L 2 , L 2a , and L 2b are substituted with one or more fluorine atoms. ​ and ; and ; and ; and ; and ; and ; and ; ， , and ； and . ​ ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； , ， , or . ​ ​ ​ ​ ​ ​ ​ 42. The lipid nanoparticle or composition of claim 41, wherein the molar ratio of the compound to cholesterol is 5: 1 to 1: 1 or 2: 1 to 1:

1.

43. The lipid nanoparticle or composition of any one of claims 37-42, wherein the polymer-conjugated lipid is a pegylated lipid.

44. The lipid nanoparticle or composition of claim 43, wherein the molar ratio of the compound to pegylated lipid is about 100: 1 to about 20: 1 or about 100: 1 to about 10:

1.

45. The lipid nanoparticle or composition of claim 43 or 44, wherein the pegylated lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, or PEG dialkyloxypropyl carbamate.

46. The lipid nanoparticle or composition of claim 43 or 44, wherein the pegylated lipid has the following structure (II): (II) or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein: w has a value of 30 to 60. R 10 and R 11 each independently is a linear or branched alkyl, alkenyl, or alkinyl of 10 to 30 carbon atoms, wherein the alkyl, alkenyl, or alkinyl is optionally interrupted by one or more ester bonds; and 48. The lipid nanoparticle or composition of claim 46 or 47, wherein the lipid nanoparticle or composition comprises a plurality of compounds of structure (II), and the average value of w for the plurality of compounds is about 49.

47. The lipid nanoparticle or composition of claim 46, wherein R 10 and R 11 each independently is a straight alkyl chain containing 12 to 16 carbon atoms.

49. The lipid nanoparticle or composition of any one of claims 35-48, wherein the therapeutic agent comprises a nucleic acid.

50. The lipid nanoparticle or composition of claim 49, wherein the nucleic acid is selected from the group consisting of an antisense RNA and a messenger RNA.

51. A method of administering a therapeutic agent to a patient in need thereof, the method comprising preparing or providing a lipid nanoparticle or composition of any one of claims 35-50, and administering the composition to the patient.

52. A pharmaceutical composition comprising a lipid nanoparticle of claim 35 and a pharmaceutically acceptable diluent or excipient.

53. A method for inducing protein expression in a patient in need thereof, comprising administering a pharmaceutical composition of claim 52 to the patient, wherein the lipid nanoparticle comprises an mRNA encoding the protein.

54. The method of claim 53, wherein the protein is an antigen, and the method is for inducing an immune response in the patient.

55. The method of claim 53, wherein the protein is an antigen, and the method is for vaccinating the patient against a pathogen.

56. The method of claim 53, wherein the protein is for gene editing. ​

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