Lipids for Delivering Active Agents by Lipid Nanoparticles

Abstract

Compounds having the following structure are provided: or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein R 1 , R 2 , R 3 , R 4 , R 5 , L 1 , L 2 , L 3 , G 1 , G 2 and G 3 are as defined herein. Also provided are uses of the compounds as components of lipid nanoparticle formulations for delivering therapeutic agents, compositions comprising the compounds, and methods of using and preparing them.

Description

Background Art Technical Field

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

[0003] Description of related technologies

[0004] Many challenges associated with nucleic acid delivery affect the desired response in biological systems. Nucleic acid-based therapies have great potential, but there is still a need to more effectively deliver nucleic acids to appropriate sites within cells or organisms to realize this potential. Therapeutic nucleic acids include, for example, messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, immunostimulatory nucleic acids, antagomir, antimir, mimics, supermir, and aptamers. Some nucleic acids, such as mRNA or plasmids, can be used to achieve the expression of specific cellular products, which would be useful in treating diseases, for example, related to protein or enzyme deficiencies. The therapeutic applications of translatable nucleotide delivery are extremely broad because constructs can be synthesized to produce any selected protein sequence, whether or not the sequence is native to the system. The expression products of nucleic acids can increase the existing levels of proteins, replace missing or non-functional forms of proteins, or introduce new proteins and related functions in cells or organisms.

[0005] Some nucleic acids, such as miRNA inhibitors, can be used to achieve the expression of specific cellular products regulated by miRNA, which would be useful in treating diseases, for example, related to protein or enzyme deficiencies. The therapeutic applications of miRNA inhibition are extremely broad because constructs can be synthesized to inhibit one or more miRNAs, and the one or more miRNAs in turn regulate the expression of mRNA products. Inhibiting endogenous miRNAs can increase the expression of their downstream target endogenous proteins and restore appropriate functions in cells or organisms as a means of treating diseases related to specific miRNAs or a group of miRNAs.

[0006] Other nucleic acids can downregulate the intracellular levels of specific mRNAs and thus can downregulate the synthesis of the corresponding proteins by processes such as RNA interference (RNAi) or complementary binding of antisense RNA. The therapeutic applications of antisense oligonucleotides and RNAi are also very extensive because oligonucleotide constructs with any nucleotide sequence targeting the target mRNA can be synthesized. The targets can include mRNAs from normal cells, mRNAs associated with disease states such as cancer, and mRNAs of infectious agents such as viruses. To date, in vitro and in vivo models have shown the ability of antisense oligonucleotide constructs to specifically downregulate target proteins by degrading homologous mRNAs. Additionally, antisense oligonucleotide constructs are currently being evaluated in clinical studies.

[0007] However, there are two problems currently associated 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 translation machinery is located. Lipid nanoparticles formed from cationic lipids and oligonucleotides together with other lipid components such as neutral lipids, cholesterol, PEG, and pegylated lipids 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 delivering oligonucleotides. Preferably, these lipid nanoparticles will provide an optimal drug:lipid ratio, protect the nucleic acid from degradation and clearance in the serum, be suitable for systemic or local delivery, and provide intracellular delivery of the nucleic acid. Additionally, these lipid-nucleic acid particles should be well tolerated and provide a sufficient therapeutic index such that treatment of patients with an effective dose of the nucleic acid is not associated with unacceptable toxicity and / or risk to the patient. The present disclosure provides these and related advantages.

[0009] Summary

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

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

[0012]

[0013] or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein R 1 , R 2 , R 3 , R 4 , R 5 , L 1 , L 2 , L 3 , G 1 , G 2 and G 3 are as defined herein.

[0014] Also provided are pharmaceutical compositions comprising a compound of structure (I) as described above and a therapeutic agent. In some embodiments, the pharmaceutical composition further comprises 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 delivery of therapeutic agents.

[0015] In other embodiments, the present disclosure provides methods for administering a therapeutic agent to a patient in need thereof, the method comprising preparing or providing a composition of lipid nanoparticles comprising a compound of structure (I) and a therapeutic agent, and delivering the composition to the patient.

[0016] These and other aspects of the present disclosure will become apparent after 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 the various embodiments of the present disclosure. However, one skilled in the art will understand that the present disclosure may be practiced without these details.

[0019] The present disclosure is in part based on the discovery of novel cationic (amino) lipids that provide advantages when used in lipid nanoparticles for in vivo delivery of active agents or therapeutic agents such as nucleic acids to mammalian cells. In particular, embodiments of the present disclosure provide nucleic acid-lipid nanoparticle compositions comprising one or more of the novel cationic lipids described herein, which 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 the therapeutic index. In other embodiments, the disclosed lipids and lipid nanoparticles comprising them have increased safety and / or tolerability when used to deliver active agents such as nucleic acids.

[0020] In certain embodiments, the present disclosure provides novel cationic lipids that enable the formulation of 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 miRNA inhibitors that target a specific miRNA or a set of miRNAs that regulate a target mRNA or multiple mRNAs. In other embodiments, these improved lipid nanoparticle compositions can be used to downregulate (e.g., silence) the protein level and / or mRNA level 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 the production of red blood cells by delivering a suitable erythropoietin mRNA, or protecting against infection by delivering mRNA encoding a suitable antigen or antibody.

[0021] The lipid nanoparticles and compositions of the embodiments of the present disclosure can be used for a variety of purposes, including delivering 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 a subject in need thereof by contacting the subject with a lipid nanoparticle encapsulating a suitable therapeutic agent or associated with a suitable therapeutic agent, wherein the lipid nanoparticle comprises one or more of the 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, for example, mRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomir / antimir), messenger RNA-interfering complementary RNA (micRNA), DNA, multivalent RNA, Dicer substrate RNA, complementary DNA (cDNA), and the like. Thus, the lipid nanoparticles and compositions of certain embodiments of the present disclosure can be used to induce the expression of a desired protein in vivo and in vitro by contacting cells with lipid nanoparticles comprising one or more of the novel cationic lipids described herein, wherein the lipid nanoparticles encapsulate or associate with a nucleic acid that is expressed to produce the desired protein (e.g., messenger RNA or plasmid encoding the desired protein) or inhibits the process of terminating mRNA (e.g., miRNA inhibitor) expression. Optionally, the lipid nanoparticles and compositions of embodiments of the present disclosure can be used to reduce the expression of target genes and proteins in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more of the novel cationic lipids described herein, wherein the lipid nanoparticles encapsulate or associate with a nucleic acid that reduces target gene expression (e.g., antisense oligonucleotide or small interfering RNA (siRNA)). The lipid nanoparticles and compositions of embodiments of the present disclosure can also be used to co-deliver different nucleic acids (e.g., mRNA and plasmid DNA) separately or in combination, such as can be used to provide the effect of co-localization of the need for different nucleic acids (e.g., mRNA encoding a suitable gene-modifying enzyme and a DNA segment for incorporation into the host genome).

[0023] Nucleic acids for use with embodiments of the present disclosure can be prepared according to any available technology. For mRNA, the main preparation methods are, but not limited to, enzymatic synthesis (also known as in vitro transcription), which represents the currently most efficient method for generating long sequence-specific mRNA. In vitro transcription describes the process of template-directed synthesis of RNA molecules from engineered DNA templates that contain upstream phage promoter sequences (such as, including but not limited to promoter sequences from T7, T3, and SP6 Escherichia coli phages) linked to downstream sequences encoding the target gene. 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. (ed.), New York, N.Y. Humana Press, 2012).

[0024] RNA transcription is carried out in vitro using a linearized DNA template in the presence of the corresponding RNA polymerase and adenosine, guanosine, uridine, and cytidine ribonucleoside triphosphates (rNTPs) under conditions that support polymerase activity while minimizing potential degradation of the resulting mRNA transcript. In vitro transcription can be carried out using a variety of commercially available kits as well as commercially available reagents (including RNA polymerase and rNTPs), including but not limited to the RiboMax Large Scale RNA Production System (Promega), the 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 of which are incorporated herein by reference).

[0025] Then, the desired in vitro transcribed mRNA is purified from the undesired components of the transcription or related 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 an alcohol (ethanol, isopropanol) (in the presence of a monovalent cation 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 carried out 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] In addition, although reverse transcription can generate large amounts of mRNA, the product can contain many abnormal RNA impurities associated with undesired polymerase activities, and these impurities may need to be removed from the full-length mRNA preparation. These include short RNAs generated by abortive transcription initiation, as well as double-stranded RNA (dsRNA) generated by RNA-dependent RNA polymerase activity, RNA-primed transcription from an RNA template, and self-complementary 3′ extensions. It has been demonstrated that these contaminants with dsRNA structures can lead to undesired immune-stimulatory activities by interacting with various innate immune sensors in eukaryotic cells that function to recognize specific nucleic acid structures and induce an effective immune response. This in turn can significantly reduce mRNA translation because protein synthesis is decreased during the innate cellular immune response. Thus, additional techniques for removing these dsRNA contaminants have been developed and are known in the art, including but not limited to scalable HPLC purification (see, e.g., Kariko, K., Muramatsu, H., Ludwig, J. and Weissman, D., 2011, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA, Nucl Acid Res, v. 39 e142; Weissman, D., Pardi, N., Muramatsu, H. and Kariko, K., HPLC Purification of in vitro transcribed long RNA in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v. 969 (Rabinovich, P.H. ed.), 2013). It has been reported that HPLC-purified mRNA translates at much higher levels, particularly in primary cells and in vivo.

[0027] A variety of modifications have been described in the art for altering the specific properties of in vitro transcribed mRNA and enhancing its utility. These include but are not limited to modifications to the 5' and 3' termini of the mRNA. Endogenous eukaryotic mRNAs typically contain a cap structure at the 5'-terminus 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 the 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 5'-most nucleotides at the 2'-hydroxyl group.

[0028] A variety of different cap structures can be used to generate the 5'-cap of in vitro transcribed synthetic mRNA. 5'-capping of synthetic mRNA can be carried out cotranscriptionally with a chemical cap analogue (i.e., capping during in vitro transcription). For example, the anti-reverse cap analogue (ARCA) cap contains a 5'-5'-triphosphate guanine-guanine linkage, where one guanine contains an N7 methyl group as well as a 3'-O-methyl group. However, during this cotranscriptional process, up to 20% of the transcripts remain uncapped, and the synthetic cap analogue is different from the 5'-cap structure of authentic cellular mRNA, which may reduce translatability and cellular stability. Optionally, synthetic mRNA molecules can also be enzymatically capped post-transcriptionally. These can generate more authentic 5'-cap structures that structurally or functionally more closely mimic the endogenous 5'-cap, which have enhanced binding of cap-binding proteins, an extended half-life, and reduced sensitivity to 5′-endonucleases and / or reduced 5'-decapping. Many synthetic 5'-cap analogues have been developed, and it is known in the art that the synthetic 5'-cap analogues 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 stretches of adenine nucleotides (poly-A tail) are typically added to mRNA molecules during RNA processing. Immediately after transcription, the 3′-end of the transcript is cleaved to release a 3′ hydroxyl group, and in a process called polyadenylation, poly-A polymerase adds a chain of adenine nucleotides to the RNA at this 3′ hydroxyl group. The poly-A tail has been widely shown to enhance the translational efficiency and stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly(A), poly(A) binding protein and the regulation of mRNA stability, Trends Bio Sci v.14 373-377; Guhaniyogi, J. and Brewer, G., 2001, Regulation of mRNA stability in mammalian cells, Gene, v.265, 11-23; Dreyfus, M. and Regnier, P., 2002, The poly(A) tail of mRNAs: Bodyguard in eukaryotes, scavenger in bacteria, Cell, v.111, 611-613).

[0030] Poly(A) tailing of in vitro transcribed mRNA can be achieved using a variety of methods, including but not limited to cloning a poly(T) tract into the DNA template, or by post-transcriptional addition using poly-A polymerase. The first case allows for the in vitro transcription of mRNA with a defined length of poly(A) tail depending on the size of the poly(T) tract, but requires additional manipulation of the template. The latter case involves the enzymatic addition of a poly(A) tail to in vitro transcribed mRNA using a poly-A polymerase that catalyzes the incorporation of adenine residues at the 3’-end of the RNA, which does not require additional manipulation of the DNA template, but results in mRNA with a poly(A) tail of heterogeneous length. 5’-capping and 3’-poly(A) tailing can be performed using a variety of commercially available kits as well as commercially available reagents, various ARCA caps, poly-A polymerases, etc., including but not limited to the Poly(A) Polymerase Tailing kit (EpiCenter), mMESSAGE mMACHINE T7 Ultra kit, and Poly(A) Tailing kit (Life Technologies).

[0031] It has been reported that, in addition to the 5’ cap and 3’ polyadenylation, other modifications of in vitro transcripts provide benefits related to translation efficiency and stability. It is well known in the art that pathogenic DNA and RNA can be recognized by multiple sensors within eukaryotes and trigger an effective innate immune response. 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, since most nucleic acids from natural sources contain modified nucleosides. In contrast, in vitro synthesized RNA lacks these modifications, making it immunostimulatory and thus capable of inhibiting the efficient mRNA translation outlined above. The introduction of modified nucleosides into in vitro transcribed mRNA can be used to prevent the recognition and activation of RNA sensors, thereby alleviating this undesired 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 variety of nucleoside modifications can be obtained, which can be incorporated into in vitro transcribed mRNA to some extent either alone or in combination with other modified nucleosides (see, e.g., US 2012 / 0251618). It has been reported that the in vitro synthesis of nucleoside-modified mRNA reduces the ability to activate immune sensors, while accompanied by enhanced translational ability.

[0032] Other components of mRNA that can be modified to provide benefits in terms of translatability and stability include the 5' and 3' untranslated regions (UTRs). Optimization of the UTRs (favorable 5' and 3' UTRs can be obtained from cellular or viral RNA), either simultaneously or individually, has been shown to improve the 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 also be used in the present disclosure. For oligonucleotides, the preparation methods include but are not limited to chemical synthesis and enzymatic, chemical cleavage of longer precursors, in vitro transcription as described above, etc. Methods for 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 used in conjunction with the embodiments of the present disclosure typically utilize, but are not limited to, in vitro amplification and isolation of plasmid DNA in a liquid culture of bacteria containing the target plasmid. The presence of genes encoding resistance to specific antibiotics (penicillin, kanamycin, etc.) in the target plasmid allows those bacteria containing the target plasmid to grow selectively 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., S., 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 US6,197,553B1). Plasmid isolation can be performed using a variety of commercially available kits as well as commercially available reagents, including but not limited to the Plasmid Plus (Qiagen), GenJET Plasmid MaxiPrep (Thermo) and PureYield MaxiPrep (Promega) kits.

[0035] Various exemplary embodiments of the cationic lipids, lipid nanoparticles and compositions containing them of the present disclosure, and their use in delivering active agents (such as therapeutic agents) such as nucleic acids to modulate gene and protein expression are described in further detail below.

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

[0037] Unless the context otherwise requires, throughout the specification and claims, the word “comprise” and its variations (such as “comprises” and “comprising”) shall be interpreted in an open and inclusive sense, i.e., “including but not limited to”.

[0038] Throughout this specification, references to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the phrases "in one embodiment" or "in an embodiment" appearing throughout the specification in various places are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[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. As used in the specification and claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0040] The phrase "inducing the expression of a desired protein" means that a nucleic acid is capable of increasing the expression of a desired protein. To examine the degree 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 a nucleic acid (e.g., a nucleic acid combined with a lipid of the present disclosure). The expression of the desired protein in the test sample or test animal is compared with the expression of the desired protein in a control sample (e.g., a sample of 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 or not administered the nucleic acid. When the desired protein is present in the control sample or control mammal, the expression of the desired protein in the control sample or control mammal can be assigned a value of 1.0. In a particular embodiment, induction of the expression of the desired protein is achieved when the ratio of the expression level of the desired protein in the test sample or test mammal to the expression level of the desired protein in the control sample or control mammal is greater than 1, such as 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, induction of the expression of the desired protein is achieved when any measurable level of the desired protein is detected in the test sample or test mammal. Those of ordinary skill in the art will understand the appropriate assays for determining the level of protein expression in a sample, such as dot blot, Northern blot, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays, or assays based on a reporter protein that can produce fluorescence or luminescence under appropriate conditions.

[0041] The phrase "inhibiting the expression of a target gene" means that a nucleic acid can silence, reduce or inhibit the expression of a target gene. To examine 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 the expression of the target gene. The expression of the target gene in the test sample or test animal is compared with the expression of the target gene in a control sample (e.g., a sample of 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 certain embodiments, relative to the level of target gene expression in the control sample or control mammal, 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%, silencing, inhibition or reduction of the expression of the target gene is achieved. In other words, relative to the level of target gene expression in a control sample or control mammal that has not been contacted or administered the nucleic acid, the nucleic acid can silence, reduce or inhibit the expression of the target gene 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%. Suitable assays for determining the level of target gene expression include, but are not limited to, examining protein or mRNA levels using techniques known to those of skill in the art, such as dot blot, Northern blot, in situ hybridization, ELISA, immunoprecipitation, 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 sufficient to produce a desired effect, such as an increase or inhibition of target sequence expression as compared to the normal expression level detected in the absence of the nucleic acid. An increase in target sequence expression is achieved when any measurable level of an expression product that is not present in the absence of the nucleic acid is detected. When the expression product is present at some level prior to contact with the nucleic acid, an increase in expression is achieved when the fold increase in the value obtained with the nucleic acid, such as mRNA, relative to the control is about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000 or greater. Inhibition of target gene or target sequence expression is achieved when the value obtained with the nucleic acid, such as an antisense oligonucleotide, relative to the control is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 0%. Suitable assays for measuring target gene or target sequence expression include examining protein or RNA levels using techniques known to those of skill in the art, such as dot blot, Northern blot, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of a suitable reporter protein, and phenotypic assays known to those of skill in the art.

[0043] As used herein, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form, and includes DNA, RNA, and their hybrids. DNA can be in the form of antisense molecules, plasmid DNA, cDNA, PCR products, or vectors. RNA can be in the form of small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA, Dicer substrate RNA, or viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring and have binding properties similar to those of a reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNA). Unless specifically defined otherwise, the term encompasses nucleic acids containing known analogs of natural nucleotides having binding properties similar to those of the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences, as well as the explicitly recited sequences. 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 bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). A "nucleotide" contains the following sugars: deoxyribose (for DNA) or ribose (for RNA); a base; and a phosphate group. Nucleotides are linked together by phosphate groups. A "base" includes 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 alkyl halides).

[0044] The term "gene" refers to a nucleic acid (such as DNA or RNA) sequence that contains a coding sequence of a partial length or the entire length necessary to produce a polypeptide or a precursor polypeptide.

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

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

[0047] "Steroid" is a compound that contains the following carbon skeleton:

[0048]

[0049] Non-limiting examples of steroids include cholesterol and the like.

[0050] "Cationic lipid" refers to a lipid that can carry a positive charge. Exemplary cationic lipids include one or more amine groups with a positive charge. Preferred cationic lipids are ionizable such that they can exist in a positively charged or neutral form depending on the pH. Under different pH conditions, the ionization of cationic lipids affects the surface charge of lipid nanoparticles. This charge state can affect plasma protein uptake, blood clearance, and tissue distribution (Semple, S.C., et al., Adv. Drug Deliv Rev 32:3-17 (1998)), which are crucial for intracellular delivery of nucleic acids, as well as the ability to form endosomolytic non-bilayer structures (Hafez, I.M., et al., Gene Ther 8:1188-1196 (2001)).

[0051] The term "polymer-conjugated lipid" refers to a molecule that contains a lipid moiety and a polymer moiety. An example of a polymer-conjugated lipid is a polyethylene glycolylated lipid. The term "polyethylene glycolylated lipid" refers to a molecule that contains a lipid moiety and a polyethylene glycol moiety. Polyethylene glycolylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-DMG) and the like.

[0052] The term "neutral lipid" refers to any one of a variety of lipid substances that exist in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, but are not limited to: phosphatidylcholines such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM), ceramides, steroids such as sterols and their derivatives. Neutral lipids can be synthetic or of natural origin.

[0053] The term "charged lipid" refers to any one of a variety of lipid substances that exist in a positively or negatively charged form independent of pH within a useful physiological range (such as pH ~3 to pH ~9). Charged lipids can be synthetic or of natural origin. Examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, diacyltrimethylammonium propane (such as DOTAP, DOTMA), diacyl dimethylaminopropane, ethylphosphocholine, dimethylaminoethane carbamoyl sterol (such as DC-Chol).

[0054] The term "lipid nanoparticle" refers to a particle having at least one dimension in the nanometer range (such as 1 - 1,000 nm) that contains one or more of the compounds of structure (I) or other specified cationic lipids. In some embodiments, the lipid nanoparticles are included in a formulation that can be used to deliver an active agent or therapeutic agent (such as a nucleic acid (e.g., mRNA)) to a target site (such as a cell, tissue, organ, tumor, etc.). In some embodiments, the lipid nanoparticles contain a compound of structure (I) and a nucleic acid. Such lipid nanoparticles typically contain a compound of structure (I) and one or more excipients selected from: neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. In some embodiments, the active agent or therapeutic agent, such as a nucleic acid, can be encapsulated within the lipid portion of the lipid nanoparticle or in an aqueous space enclosed by some or all of the lipid portions of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cell such as an adverse immune response.

[0055] In various embodiments, the average diameter of the lipid nanoparticles is from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm or 150 nm. In some embodiments, the lipid nanoparticles are substantially non-toxic. In certain embodiments, when present in the lipid nanoparticles, the nucleic acid is resistant to degradation by nucleases in an aqueous solution. Lipid nanoparticles comprising a nucleic acid and methods for their preparation are disclosed in, for example, 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 herein by reference in their entirety for all purposes.

[0056] As used herein, "encapsulating lipid" refers to a lipid nanoparticle that provides complete encapsulation, partial encapsulation, or both for an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA). In an embodiment, a nucleic acid (e.g., mRNA) is completely encapsulated within the lipid nanoparticle.

[0057] As used herein, the term "aqueous solution" refers to a composition that contains water.

[0058] With respect to nucleic acid-lipid nanoparticles, "serum-stable" means that the nucleotide does not significantly degrade after exposure to serum or a nuclease assay that would significantly degrade free DNA or RNA. Suitable assays include, for example, a standard serum assay, a DNase assay, or an RNase assay.

[0059] As used herein, "systemic delivery" refers to the delivery of a therapeutic product that can result in widespread exposure of the active agent within an organism. Some administration techniques can result in systemic delivery of certain agents, while others cannot. Systemic delivery means that a useful, preferably therapeutic amount of the agent is exposed to a majority of the body. Systemic delivery of lipid nanoparticles can be effected by any means known in the art, including, for example, intravenous, intra-arterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of the lipid nanoparticles is effected by intravenous delivery.

[0060] "Local delivery" as used herein 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 diseased site such as a tumor, other target sites such as an inflamed site, or a target organ (such as the 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 exclude systemic pharmacological effects.

[0061] "Hydrocarbyl" refers to a straight or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, which is saturated or unsaturated (i.e., contains one or more double bonds (alkenyl) and / or triple bonds (alkyne)), having, for example, from one to twenty-four carbon atoms (C1-C 24 hydrocarbyl), from four to twenty carbon atoms (C4-C 20 hydrocarbyl), from six to sixteen carbon atoms (C6-C 16 hydrocarbyl), from six to nine carbon atoms (C6-C9 hydrocarbyl), from one to fifteen carbon atoms (C1-C 15 hydrocarbyl), from one to twelve carbon atoms (C1-C 12 hydrocarbyl), from one to eight carbon atoms (C1-C8 hydrocarbyl) or from one to six carbon atoms (C1-C6 hydrocarbyl), and is linked to the remainder of the molecule by a single bond, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, vinyl, prop-1-enyl, but-1-enyl, pent-1-enyl, pent-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc. Unless otherwise specifically stated in the specification, the hydrocarbyl group is optionally substituted.

[0062] "Hydrocarbylene" or "hydrocarbylene chain" refers to a straight or branched divalent hydrocarbon chain consisting only of carbon and hydrogen that links the remainder of the molecule to a group, which is saturated or unsaturated (i.e., contains one or more double bonds (alkenylene) and / or triple bonds (alkynylene)) and has, for example, from one to twenty-four carbon atoms (C1-C 24 hydrocarbylene), from one to fifteen carbon atoms (C1-C 15 hydrocarbylene), from one to twelve carbon atoms (C1-C 12(alkylene), a C1-C8 alkylene, a C1-C6 alkylene, a C2-C4 alkylene, a C1-C2 alkylene, such as methylene, ethylene, propylene, n-butylene, vinylidene, propenylene, n-butenylene, propynylene, n-butynylene, etc. The alkylene chain is connected to the rest of the molecule through a single bond or a double bond and is connected to the group through a single bond or a double bond. The connection points of the alkylene chain to the rest of the molecule and to the group can be through one carbon or any two carbons within the chain. Unless otherwise specifically stated in the specification, the alkylene chain can be optionally substituted.

[0063] "Cycloalkyl" or "carbocyclic ring" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon group consisting only of carbon and hydrogen atoms, which may include a fused or bridged ring system having 3 to 15 carbon atoms, preferably 3 to 10 carbon atoms, is saturated or unsaturated and is connected to the rest of the molecule through a single bond. Monocyclic groups include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. Polycyclic groups include, for example, adamantyl, norbornyl, decahydronaphthyl, 7,7-dimethyl-bicyclo[2.2.1]heptyl, etc. Unless otherwise specifically stated in the specification, the cycloalkyl group can be optionally substituted.

[0064] "Sub-cycloalkyl" is a divalent cycloalkyl group. Unless otherwise specifically stated in the specification, the sub-cycloalkyl group can be optionally substituted.

[0065] "Heterocyclic group" or "heterocycle" refers to a stable 3- to 18-membered (such as 5-membered, 6-membered or 7-membered) non-aromatic ring group having 1 to 12 ring carbon atoms (for example, 2 to 12) and 1 to 6 ring heteroatoms selected from nitrogen, oxygen and sulfur. Examples of such heterocyclic groups include, but are not limited to, azetidinyl, pyrrolidinyl, piperidinyl, imidazolidinyl, tetrahydropyrimidinyl, etc. Unless otherwise specifically stated in the specification, the heterocyclic group can be optionally substituted.

[0066] "Heteroaryl" refers to a 5- to 14-membered ring system group containing a hydrogen atom, 1 to 13 ring carbon atoms, 1 to 6 ring heteroatoms selected from nitrogen, oxygen and sulfur and at least one aromatic ring. Examples include, but are not limited to, pyrrolyl, imidazolyl, triazolyl, thiazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, etc. Unless otherwise specifically stated in the specification, the heteroaryl can be optionally substituted.

[0067] As used herein, the term "substituted" means any of the above groups (such as hydrocarbon group, alkylene, cycloalkyl, sub-cycloalkyl), wherein at least one hydrogen atom is replaced by a bond to a non-hydrogen atom such as, but not limited to, the following: halogen atoms such as F, Cl, Br or I; oxo group (=O); hydroxy group (-OH); C1-C 12Alkyl group; cycloalkyl group; -(C=O)OR'; –O(C=O)R'; -C(=O)R'; -OR'; -S(O) x R'; -S-SR'; -C(=O)SR'; -SC(=O)R'; -NR'R'; -NR'C(=O)R'; -C(=O)NR'R'; -NR'C(=O)NR'R'; -OC(=O)NR'R'; -NR'C(=O)OR'; -NR'S(O) x NR'R'; -NR'S(O) x R'; and -S(O) x NR'R', where R' is independently H, C1-C 15 alkyl or cycloalkyl each time it appears, and x is 0, 1, or 2. In some embodiments, the substituent is a C1-C 12 alkyl group. In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halogenated group, such as fluoro. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxy group. In other embodiments, the substituent is an alkoxy group (-OR'). In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group (-NR'R').

[0068] "Optional" or "optionally" (e.g., optionally substituted) means that the subsequently described event or circumstance may or may not occur, and the description includes both the case where the event or circumstance occurs and the case where the event or circumstance does not occur. For example, "optionally substituted alkyl" means that the alkyl group may or may not be substituted, and the description includes both the substituted alkyl group and the unsubstituted alkyl group.

[0069] The disclosure herein also intends to cover all pharmaceutically acceptable compounds of the compounds of structure (I) that are isotopically labeled by replacing one or more atoms with atoms having different atomic weights or mass numbers. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, 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 to help determine or measure the effectiveness of a compound by characterizing, for example, the site or mode of action or the binding affinity to a pharmacologically important site of action. Certain isotopically labeled compounds of structure (I), (IA), or (IB), such as those incorporating a radioactive isotope, can be used in drug and / or substrate tissue distribution studies. Given that the radioactive isotopes tritium, i.e., 3 H, and carbon-14, i.e., 14 C, are readily incorporated and detection means are readily available, they can be particularly useful for this purpose.

[0070] Replacement with a heavier isotope such as deuterium (i.e., 2 H) may provide certain therapeutic advantages due to higher metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and may thus be preferred in certain cases.

[0071] Replacement with a positron-emitting isotope such as 11 C, 18 F, 15 O, and 13 N can be used in positron emission tomography (PET) studies to examine substrate receptor occupancy. Isotopically labeled compounds of structure (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes similar to those described in the preparations and examples set forth below using appropriate isotopically labeled reagents in place of the previously used unlabeled reagents.

[0072] The embodiments disclosed herein are also intended to cover the in vivo metabolites of the disclosed compounds. Such products may be produced, for example, by oxidation, reduction, hydrolysis, amidation, esterification, etc. of the administered compound mainly due to enzymatic processes. Accordingly, embodiments of the disclosure include compounds produced by a method comprising administering a compound of the present disclosure to a mammal for a period of time sufficient to produce its metabolites. Such products are generally identified by administering a detectable dose of a radiolabeled compound of the present disclosure to an animal such as a rat, mouse, guinea pig, monkey, or human, allowing sufficient time for metabolism, and isolating the conversion products from urine, blood, or other biological samples.

[0073] "Stable compound" and "stable structure" are intended to mean a compound that is sufficiently robust to withstand isolation to a useful degree of purity from a reaction mixture and formulated into an effective therapeutic agent.

[0074] "Mammal" includes humans as well as domestic animals such as laboratory animals and household pets (such as cats, dogs, pigs, cows, sheep, goats, horses, rabbits) and non-domestic animals (such as wild animals, etc.).

[0075] "Pharmaceutically acceptable carriers, diluents or excipients" include, but are not limited to, any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavorant, surfactant, wetting agent, dispersing agent, suspending agent, stabilizing agent, isotonic agent, solvent or emulsifying agent that has been approved by the US Food and Drug Administration and is acceptable for use in humans or domestic animals.

[0076] "Pharmaceutically acceptable salts" include acid addition salts and base addition salts.

[0077] "Pharmaceutically acceptable acid addition salts" refer to those salts that retain the biological potency and properties of the free base, are not biologically or otherwise undesirable, and are formed with inorganic acids (such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.) or with 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, etc.).

[0078] "Pharmaceutically acceptable base addition salts" refer to salts that retain the biological potency and properties of the free acid and are not biologically or otherwise undesirable. These salts are prepared by adding an inorganic base or an organic base to the free acid. 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, etc. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines, which include 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, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0079] Crystallization generally produces solvates of the compounds of the present disclosure (i.e., compounds of structure (I)). As used herein, the term "solvate" refers to an aggregate comprising one or more molecules of a compound of the present disclosure and one or more solvent molecules. The solvent can be water, in which case the solvate can be a hydrate. Optionally, the solvent can be an organic solvent. Thus, the compounds of the present disclosure can exist in the form of hydrates (including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc.) and the corresponding solvated forms. The solvates of the compounds of the present disclosure can be true solvates, while in other cases, the compounds of the present disclosure may only retain adventitious water or be a mixture of water and some adventitious solvent.

[0080] "Pharmaceutical composition" refers to a preparation of a compound of the present disclosure and a medium generally accepted in the art for delivering a bioactive compound to a mammal such as a human. Such media include all pharmaceutically acceptable carriers, diluents, or excipients therefor.

[0081] "Effective amount" or "therapeutically effective amount" refers to the amount of a compound of the present disclosure sufficient to effect treatment in a mammal, preferably a human, when administered to the mammal, preferably a human. The amount of the lipid nanoparticles of the embodiments of the present disclosure that constitutes a "therapeutically effective amount" will vary depending on the compound, the condition and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one of ordinary skill in the art based on their knowledge and the present disclosure.

[0082] As used herein, "treating" or "treatment" encompasses treatment of a target disease or condition in a mammal, preferably a human, having the target disease or condition and includes:

[0083] (i) preventing the disease or condition from occurring in a mammal, particularly when such mammal is predisposed to the condition but has not been diagnosed as having the condition;

[0084] (ii) inhibiting the disease or condition, i.e., arresting its development;

[0085] (iii) alleviating the disease or condition, i.e., causing regression of the disease or condition; or

[0086] (iv) relieving the symptoms caused by the disease or condition, i.e., relieving pain without addressing the underlying disease or condition. As used herein, the terms "disease" and "condition" may be used interchangeably or may be different, as a particular disease or condition may not have a known pathogen (such that the cause has not been determined) and thus has not been identified as a disease but is only regarded as an undesirable condition or syndrome in which a clinician has identified a more or less specific group of symptoms.

[0087] The compounds of the present disclosure or pharmaceutically acceptable salts thereof may contain one or more stereocenters and thus may give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined in terms of absolute stereochemistry as (R)- or (S)- or, for amino acids, as (D)- or (L)-. The present disclosure is intended to include all such possible isomers, as well as their racemic and optically pure forms. The 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 / separation of individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of the racemate (or racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC). When the compounds described herein contain an olefinic double bond or other geometrically asymmetric center, and unless otherwise specified, the compounds are intended to include the E and Z geometric isomers. Similarly, all tautomeric forms are also intended to be included.

[0088] A "stereoisomer" refers to a compound composed of the same atoms bonded by the same bonds but having different three-dimensional structures that are non-interchangeable. The present disclosure contemplates various stereoisomers and mixtures thereof and includes "enantiomers", which refers to two stereoisomers that are non-overlapping mirror images of each other.

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

[0090] Compound

[0091] In one aspect, the present disclosure provides novel lipid compounds that are capable of conjugating with other lipid components such as neutral lipids, charged lipids, steroids, and / or lipid-conjugated polymers to form lipid nanoparticles having oligonucleotides. Without wishing to be bound by theory, it is believed that these lipid nanoparticles shelter the oligonucleotides from degradation in serum and provide for the effective delivery of oligonucleotides to cells in vitro and in vivo.

[0092] In one embodiment, the compound has the following structure (I):

[0093]

[0094] or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:

[0095] R 1 is optionally substituted C1-C 24 alkyl or optionally substituted C2-C 24 alkenyl;

[0096] R 2 and R 3 are each independently optionally substituted C1-C 36 hydrocarbyl;

[0097] R 4 and R 5 are each independently optionally substituted C1-C6 hydrocarbyl, or R 4 and R 5 together with the N to which they are attached form a heterocyclic group or heteroaryl;

[0098] L 1 、L 2 and L 3 are each independently optionally substituted C1-C 18 hydrocarbylene;

[0099] G 1 is a direct bond, -(CH2) n O(C=O)-, -(CH2) n (C=O)O-, or –(C=O)-;

[0100] G 2 and G 3 are each independently -(C=O)O- or -O(C=O)-; and

[0101] n is an integer greater than 0.

[0102] In some embodiments, the compound has the following structure (IA):

[0103]

[0104] In some embodiments, the compound has the following structure (IB):

[0105]

[0106] In some embodiments, R 1 is an optionally substituted C6-C 18 alkyl or C 14 -C 18 alkenyl. In certain embodiments, R 1 is C8 alkyl, C9 alkyl, C 10 alkyl, C 12 alkyl, C 14 alkyl or C 16 alkyl. In some more specific embodiments, R 1 is C 16 alkenyl. In certain more specific embodiments, R 1 is unbranched. In some embodiments, R 1 is branched. In certain embodiments, R 1 is unsubstituted.

[0107] In some embodiments, G 1 is a direct bond, -(CH2) n O(C=O)- or -(CH2) n (C=O)O-. In certain embodiments, G 1 is a direct bond. In some more specific embodiments, G 1 is -(CH2) n (C=O)O- and n is greater than 1. In some embodiments, n is 1-20. In some embodiments, n is 1-10. In some embodiments, n is 5-11. In some embodiments, n is 6-10. In certain more specific embodiments, n is 5, 6, 7, 8, 9, or 10. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In certain embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10.

[0108] In some embodiments, L 1 is a C1-C6 alkylene group. In certain embodiments, L 1is a C2 alkylene, C3 alkylene or C4 alkylene. In some more specific embodiments, L 1 is unbranched. In certain more specific embodiments, L 1 is unsubstituted.

[0109] In some embodiments, R 2 is a C8-C 24 hydrocarbyl. In some embodiments, R 3 is a C8-C 24 hydrocarbyl. In some more specific embodiments, R 2 and R 3 are both C8-C 24 hydrocarbyl. In some embodiments, R 2 and R 3 are each independently a C 11 hydrocarbyl, C 12 hydrocarbyl, C 13 hydrocarbyl, C 14 hydrocarbyl, C 15 hydrocarbyl, C 16 hydrocarbyl, C 18 hydrocarbyl or C 20 hydrocarbyl. In certain embodiments, R 2 is branched. In more specific embodiments, R 3 is branched. In some more specific embodiments, R 2 and R 3 each independently have one of the following structures:

[0110]

[0111] wherein:

[0112] R 6 and R 7 are each independently a C2-C 12 hydrocarbyl.

[0113] In some embodiments, R 2 and R 3 each independently have one of the following structures:

[0114]

[0115] In some embodiments, L 2 and L 3 are each independently a C4-C 10 alkylene. In certain embodiments, L 2 and L 3 are both C5 alkylene. In some more specific embodiments, L 2 and L 3are both C6 alkylene groups. In certain embodiments, L 2 and L 3 are both C8 alkylene groups. In some more specific embodiments, L 2 and L 3 are both C9 alkylene groups. In some embodiments, L 2 is unbranched. In some embodiments, L 3 is unbranched. In more specific embodiments, L 2 is unsubstituted. In some embodiments, L 2 is unsubstituted.

[0116] In some embodiments, R 4 and R 5 are each independently a C1-C6 hydrocarbyl group. In more specific embodiments, R 4 and R 5 are both methyl. In certain embodiments, R 4 and R 5 are both ethyl. In certain embodiments, R 4 is methyl and R 5 is n-butyl. In some embodiments, R 4 and R 5 are both n-butyl. In different embodiments, R 4 is methyl, and R 5 is n-hexyl.

[0117] In some embodiments, R 4 and R 5 together with the N to which they are attached are joined to form a heterocyclic group. In certain embodiments, the heterocyclic group is a 5-membered heterocyclic group. In some embodiments, the heterocyclic group has the following structure:

[0118]

[0119] In various different embodiments, the compound has one of the structures listed in Table 1 below.

[0120] Table 1: Compounds included in embodiments of compounds of structure (I)

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127] It should be understood that any embodiment of the compounds of structure (I) as shown above, and any particular substituents and / or variables of the compound structure (I) as shown above, can be independently combined with other embodiments and / or substituents and / or variables of the compounds of structure (I) to form embodiments of the present invention not specifically shown above. Additionally, if a list of substituents and / or variables for any particular R group, G group, L group, or variable n is listed 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 the present disclosure.

[0128] It should be understood that in this specification, combinations of substituents and / or variables of the depicted formula are permitted only if such a combination results in a stable compound.

[0129] In some embodiments, lipid nanoparticles comprising a compound of structure (I) are provided. The lipid nanoparticles optionally comprise excipients selected from neutral lipids, steroids, and polymer-conjugated lipids.

[0130] In some embodiments, a composition comprising any one or more of the compounds of structure (I) and a therapeutic agent is provided. For example, in some embodiments, the composition comprises any one of the compounds of structure (I) and a therapeutic agent and one or more excipients selected from neutral lipids, steroids, and polymer-conjugated lipids. Other pharmaceutically acceptable excipients and / or carriers are also included in various embodiments of the composition.

[0131] In some embodiments, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the neutral lipid is DSPC. In various embodiments, the molar ratio of the compound to the neutral lipid ranges from about 2:1 to about 8:1.

[0132] In various embodiments, the composition further comprises a steroid or steroid analogue. In certain embodiments, the steroid or steroid analogue is cholesterol. In some of these embodiments, the molar ratio of the compound to cholesterol ranges from about 5:1 to 1:1.

[0133] In various embodiments, the polymer-conjugated lipid is a polyethylene glycolylated lipid. For example, some embodiments include polyethylene glycolylated diacylglycerol (PEG-DAG) such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), polyethylene glycolylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2’,3’-bis(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG), polyethylene glycolylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamate such as ω-methoxy(polyethoxy)ethyl-N-(2,3-bis(tetradecyloxy)propyl)carbamate or 2,3-bis(tetradecyloxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of the compound to the polyethylene glycolylated lipid ranges from about 100:1 to about 20:1.

[0134] In some embodiments, the composition comprises a polyethylene glycolylated lipid having the following structure (II):

[0135]

[0136] or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:

[0137] R 8 and R 9 are each independently a straight-chain or branched, saturated or unsaturated hydrocarbon chain containing 10 to 30 carbon atoms, wherein the hydrocarbon chain is optionally interrupted by one or more ester bonds; and

[0138] The average value of w ranges from 30 to 60.

[0139] In some embodiments, R 8 and R 9 are each independently a straight-chain saturated hydrocarbon chain containing 12 to 16 carbon atoms. In other embodiments, the average value of w ranges from about 42 to 55, such as about 49.

[0140] In some embodiments of the above composition, the therapeutic agent comprises a nucleic acid. For example, in some embodiments, the nucleic acid is selected from antisense RNA and messenger RNA. In some of the foregoing embodiments, the composition comprises lipid nanoparticles.

[0141] Some related embodiments provide lipid nanoparticles comprising a compound (e.g., a compound of structure (I)) of any of the foregoing embodiments. In certain embodiments, the lipid nanoparticles further comprise a therapeutic agent (e.g., a nucleic acid, such as antisense RNA and messenger RNA).

[0142] In some embodiments, the lipid nanoparticles further comprise one or more excipients selected from: neutral lipids, steroids, and polymer-conjugated lipids. In some embodiments, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In more specific embodiments, the neutral lipid is DSPC.

[0143] In some more specific embodiments, the molar ratio of the compound to the neutral lipid ranges from about 2:1 to about 8:1. In some embodiments, the steroid is cholesterol. In some embodiments, the molar ratio of the compound to cholesterol ranges from 5:1 to 1:1.

[0144] In certain embodiments, the polymer-conjugated lipid is a polyethylene glycolated lipid. In certain more specific embodiments, the molar ratio of the compound to the polyethylene glycolated lipid ranges from about 100:1 to about 20:1.

[0145] In some embodiments, the polyethylene glycolated lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, or PEG dialkoxypropyl carbamate. In other embodiments, the polyethylene glycolated lipid has the following structure (II):

[0146]

[0147] or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:

[0148] R 8 and R 9 are each independently a straight-chain or branched, saturated or unsaturated hydrocarbon chain containing 10 to 30 carbon atoms, wherein the hydrocarbon chain is optionally interrupted by one or more ester bonds; and

[0149] the average value of w ranges from 30 to 60.

[0150] In some more specific embodiments of structure (II), R 8 and R 9 are each independently a straight-chain saturated hydrocarbon chain containing 12 to 16 carbon atoms. In more specific embodiments, the average value of w is about 49.

[0151] In other different embodiments, the present disclosure relates to a method for administering a therapeutic agent to a patient in need thereof, the method comprising preparing or providing any one of the foregoing compositions and administering the composition to the patient.

[0152] For administration purposes, embodiments of the compounds of the present disclosure (commonly in the form of lipid nanoparticles in combination with a therapeutic agent) can be administered as a chemical starting material or formulated into a pharmaceutical composition. The pharmaceutical compositions of embodiments of the present disclosure comprise a compound of structure (I) and one or more pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the compound of structure (I) is present in the composition in an amount effective to form lipid nanoparticles and deliver a therapeutic agent, such as for treating a particular target disease or condition. Appropriate concentrations and dosages can be readily determined by those skilled in the art.

[0153] The administration of the compositions of embodiments of the present disclosure can be effected by any acceptable mode of administration of agents for similar utilities. The pharmaceutical compositions of embodiments of the present disclosure can be formulated into preparations in solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes of administering such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalational, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. As used herein, the term "parenteral" includes subcutaneous injection, intravenous, intramuscular, intradermal, intra-sternal injection, or infusion techniques. The pharmaceutical compositions of embodiments of the present disclosure are formulated such that the active ingredient contained therein is bioavailable after the composition is administered to a patient. In some embodiments, the composition to be administered to a subject or patient takes the form of one or more dosage units, where, for example, a tablet can be a single dosage unit, and a container of a compound of an embodiment of the present disclosure in aerosol form can contain multiple dosage units. The actual methods of preparing such dosage forms are known to those skilled in the art or will be apparent; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). In some embodiments, 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 treating a target disease or condition.

[0154] The pharmaceutical compositions of embodiments of the present disclosure can be in solid or liquid form. In one aspect, the carrier is particulate, such that the composition is in the form of, for example, a tablet or powder. The carrier can be liquid, where the composition is, for example, an oral syrup, an injectable liquid, or an aerosol, which can be used, for example, for inhalational administration.

[0155] When intended for oral administration, the pharmaceutical compositions of certain embodiments are preferably in solid or liquid form, where semi-solid, semi-liquid, suspension, and gel forms are included within the forms considered solid or liquid herein.

[0156] As solid compositions for oral administration, the pharmaceutical compositions of some embodiments can be formulated in the form of powders, granules, compressed tablets, pills, capsules, chewing gums, wafers, etc. Such solid compositions generally contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, tragacanth, or gelatin; excipients such as starch, lactose, or dextrin, disintegrants such as alginic acid, sodium alginate, Primogel, corn starch, etc.; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavoring agents such as peppermint, methyl salicylate, or orange essence; and coloring agents.

[0157] When the pharmaceutical compositions of some embodiments are in the form of capsules, such as gelatin capsules, in addition to the materials of the above types, it can also contain liquid carriers such as polyethylene glycol or oil.

[0158] The pharmaceutical compositions of some embodiments can be in liquid form, such as elixirs, syrups, solutions, emulsions, or suspensions. As two examples, the liquid can be for oral administration or for delivery by injection. When intended for oral administration, in addition to the compound of structure (I), the preferred compositions also contain one or more of sweeteners, preservatives, dyes / coloring agents, and flavor enhancers. In compositions intended to be administered by injection, one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, and isotonic agents can be included.

[0159] The liquid pharmaceutical compositions of the embodiments of the present disclosure, whether they are solutions, suspensions, or other similar forms, can contain one or more of the following adjuvants: sterile diluents such as water for injection, saline solutions preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic monoglycerides or diglycerides of glycerol that can be used as solvents or suspension media, polyethylene glycol, glycerol, propylene glycol, or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate, or phosphate, and agents for adjusting tonicity such as sodium chloride or dextrose; agents used as cryoprotectants, such as sucrose or trehalose. Parenteral preparations can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. Physiological saline is the preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

[0160] Liquid pharmaceutical compositions of embodiments of the present disclosure intended for parenteral or oral administration should contain an amount of the compounds of the present disclosure to obtain a suitable dosage.

[0161] The pharmaceutical compositions of embodiments of the present disclosure can be intended for topical administration, in which case the carrier can suitably include solutions, emulsions, ointments or gel matrices. The matrix can for example include one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents (such as water and alcohol), and emulsifying and stabilizing agents. Thickeners can be present in the pharmaceutical composition for topical administration. If intended for transdermal administration, the composition can include a transdermal patch or an iontophoresis device.

[0162] The pharmaceutical compositions of embodiments of the present disclosure can be intended for rectal administration, for example in the form of suppositories, which will melt in the rectum and release the drug. Compositions for rectal administration can contain an oily matrix as a suitable non-irritating excipient. Such matrices include but are not limited to lanolin, cocoa butter and polyethylene glycol.

[0163] The pharmaceutical compositions of embodiments of the present disclosure can contain various materials that alter the physical form of solid or liquid dosage units. For example, the composition can contain materials that form a coating shell around the active ingredient. The materials that form the coating shell are generally inert and can be selected from, for example, sugars, shellac and other enteric coating agents. Optionally, the active ingredient can be enclosed in a gelatin capsule.

[0164] The pharmaceutical compositions of embodiments of the present disclosure in solid or liquid form can contain reagents that bind to the compounds of the present disclosure and thus assist in the delivery of the compounds. Suitable reagents that can function in this capacity include monoclonal antibodies or polyclonal antibodies or proteins.

[0165] The pharmaceutical compositions of embodiments of the present disclosure can consist of dosage units that can be administered as an aerosol. The term "aerosol" is used to denote a variety of systems ranging from those of a colloidal nature to those consisting of pressurized packages. Delivery can be effected by liquefied or compressed gases or by a suitable pump system for dispensing the active ingredient. The compounds of embodiments of the present disclosure can be delivered as an aerosol in a single-phase, two-phase or three-phase system in order to deliver one or more active ingredients. Delivery of the aerosol includes the necessary container, activator, valve, sub-container, etc., which together can form a kit. A person skilled in the art can determine the preferred aerosol without undue experimentation.

[0166] The pharmaceutical compositions of the embodiments of the present disclosure can be prepared by methods well known in the pharmaceutical art. For example, a pharmaceutical composition intended for administration by injection can be prepared by combining the lipid nanoparticles of the present disclosure with sterile distilled water or other carriers to form a solution. Surfactants can be added to facilitate the formation of a homogeneous solution or suspension. A surfactant is a compound that non-covalently interacts with the compounds of the present disclosure to facilitate the dissolution or homogeneous suspension of the compounds in an aqueous delivery system.

[0167] The compositions of the embodiments of the present disclosure or their pharmaceutically acceptable salts are administered in a therapeutically effective amount, which will vary depending on a variety of factors, including the activity of the specific therapeutic agent employed; the metabolic stability and duration 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 rate of excretion; drug combinations; the severity of the particular disorder or condition; and the subject being treated.

[0168] The compositions of the embodiments of the present disclosure can also be administered concurrently with, prior to, or following the administration of one or more other therapeutic agents. Such combination therapies include single pharmaceutical dosage formulations that administer a composition of the embodiments of the present disclosure and one or more additional active agents, as well as the administration of the composition of the present invention and each active agent in its own separate pharmaceutical dosage formulations. For example, a composition of the embodiments of the present disclosure and other active agents can be administered to a patient together in a single oral dosage composition (such as a tablet or capsule) or each agent administered in separate oral dosage formulations. When separate dosage formulations are used, the compounds of the embodiments of the present disclosure and one or more additional active agents can be administered at substantially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially; combination therapy is understood to include all such regimens.

[0169] Methods for preparing the above compounds and compositions are described below and / or are known in the art.

[0170] Those skilled in the art will understand that in the processes described herein, the functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxyl, amino, mercapto and carboxylic acid. Suitable hydroxyl protecting groups include trialkylsilyl or diarylalkylsilyl (e.g., tert-butyldimethylsilyl, tert-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, etc. Suitable protecting groups for amino, amidino and guanidino groups include tert-butoxycarbonyl, benzyloxycarbonyl, etc. Suitable mercapto protecting groups include -C(O)-R″ (wherein R″ is alkyl, aryl or arylalkyl), p-methoxybenzyl, triphenylmethyl, etc. Suitable carboxylic acid protecting groups include alkyl, aryl or arylalkyl esters. The protecting groups can be added or removed according to standard techniques known to those skilled in the art and as described herein. The use of protecting groups is described in detail in Green, T.W. and P.G.M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Edition, Wiley. As those skilled in the art will understand, the protecting group can also be a polymer resin, such as Wang resin, Rink resin or 2-chlorotrityl chloride resin.

[0171] Those skilled in the art will also understand that although such protected derivatives of the compounds of the present disclosure may not themselves have pharmacological activity, they can be administered to a mammal and then metabolized in vivo to form the compounds of the present disclosure having pharmacological activity. Therefore, such derivatives can be described as "prodrugs". All prodrugs of the compounds of the present disclosure are included within the scope of the present disclosure.

[0172] In addition, the compounds of the embodiments of the present disclosure in the form of free base or acid can be converted into their pharmaceutically acceptable salts by treatment with appropriate inorganic or organic bases or acids by methods known to those skilled in the art. The salts of the compounds of the embodiments of the present disclosure can be converted into their free base or acid form by standard techniques.

[0173] The following general reaction scheme 1 illustrates an exemplary method for preparing the compounds of the present disclosure, i.e., the compounds of structure (I) or their pharmaceutically acceptable salts, tautomers or stereoisomers:

[0174]

[0175]

[0176] wherein R 1 、R 2 、R 3 、R 4 、R 5 、L1 , L 2 , L 3 , G 1 , G 2 and G 3 as defined herein. It is understood that those skilled in the art will be able to prepare these compounds by similar methods or by combining other methods known to those skilled in the art. It should also be understood that those skilled in the art will be able to prepare other compounds of structure (I) not specifically described below in a similar manner by using appropriate starting components and varying the synthetic parameters as needed. Generally, the starting components can be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, or synthesized according to sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Edition (Wiley, December 2000)) or prepared as described in the present disclosure.

[0177] General reaction scheme 1

[0178]

[0179] General reaction scheme I provides an exemplary method for preparing compounds of structure (I). The R in general reaction scheme 1 1 , R 2 , R 3 , R 4 , R 5 , L 1 , L 2 , L 3 , G 1 , G 2 and G 3 as defined herein. X 1 and X 2 are reactive moieties (e.g., halogens) selected to facilitate the desired reaction. Compounds of structure A1 are purchased or prepared according to methods known in the art. A1 is reacted under appropriate reducing conditions (e.g., sodium triacetoxyborohydride) to give the reductive amination product A3 between A1 and A2. Then A3 is reacted with A4 under appropriate basic conditions (e.g., using triethylamine and DMAP) to give compound A5. Then A5 is reacted with amine A6 under appropriate conditions (e.g., heating) to produce the compound of structure (I) shown.

[0180] It should be noted that various alternative strategies for preparing the compounds of structure (I) are available to those of ordinary skill in the art. For example, other compounds of structure (I) can be prepared using appropriate starting materials according to similar methods. The use of protecting groups and other modifications to the general reaction scheme as needed will be apparent to those of ordinary skill in the art.

[0181] The following examples are provided for illustrative purposes only and not for purposes of limitation.

[0182] Example 1

[0183] In vivo evaluation of luciferase mRNA using lipid nanoparticle compositions

[0184] Lipid nanoparticles were prepared and tested according to the general procedures described in PCT Publication Nos. WO 2015 / 199952 and WO 2017 / 004143, the entire disclosures of which are incorporated herein by reference. Briefly, the cationic lipid, DSPC, cholesterol, and PEG-lipid were dissolved in ethanol at a molar ratio of about 50:10:38.5:1.5 or about 47.5:10:40.8:1.7. Lipid nanoparticles (LNPs) were prepared at a total lipid to mRNA weight ratio of about 10:1 to 30:1. The mRNA was diluted to 0.2 mg / mL in 10 to 50 mM citrate or acetate buffer (pH 4). The ethanol lipid solution was mixed with the mRNA aqueous solution at a ratio of about 1:5 to 1:3 (vol / vol) using a syringe pump, where the total flow rate was greater 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 μm pore sterile filter. The diameter of the lipid nanoparticle size was about 55 - 95 nm, and in some cases about 70 - 90 nm, as determined by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK).

[0185] Studies were conducted in female C57BL / 6 mice (Charles River) at 6 - 8 weeks of age or CD-1 (Harlan) mice (Charles River) at 8 - 10 weeks of age according to the 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 the animals were euthanized at specific time points after administration, such as 4 hours. The liver and spleen were collected in pre-weighed tubes, the weights were determined, and they were immediately snap-frozen in liquid nitrogen and stored at -80 °C until processed for analysis.

[0186] For the liver, approximately 50 mg was excised for analysis in 2 mL FastPrep tubes (MP Biomedicals, Solon OH). To each tube was added 1 1 / 4” ceramic beads (MP Biomedicals), and 500 μL of Glo Lysis Buffer – GLB (Promega, Madison WI) equilibrated to room temperature was added to the liver tissue. The liver tissue was homogenized for 15 seconds at 2 × 6.0 m / s using a FastPrep24 instrument (MP Biomedicals). The homogenate was incubated for 5 minutes at room temperature and then diluted 1:4 in GLB and evaluated using the SteadyGlo luciferase assay system (Promega). Specifically, 50 μL of the diluted tissue homogenate was reacted with 50 μL of SteadyGlo substrate, shaken for 10 seconds, followed by a 5-minute incubation, and then quantified using a CentroXS 3 LB 960 luminometer (Berthold Technologies, Germany). The amount of protein assayed was determined using a BCA protein assay kit (Pierce, Rockford, IL). The relative light units (RLU) were then normalized to the total μg protein assayed. To convert RLU to ng luciferase, a standard curve was generated using QuantiLum recombinant luciferase (Promega).

[0187] FLuc mRNA (L-6107 or L-7202) from Trilink Biotechnologies will express the luciferase protein originally isolated from the firefly Photinus pyralis. 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. Completely replace the uridine and / or cytidine nucleosides in this capped and polyadenylated mRNA.

[0188] Example 2

[0189] Determine the pK of the formulated lipid a

[0190] As described elsewhere, the pKa of formulated cationic lipids correlates with the potency of LNP-mediated nucleic acid delivery (see Jayaraman et al., Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al., Nature Biotechnology 28, 172–176 (2010)). The preferred range of pKa is ~5 to ~7. The pKa of each cationic lipid was determined in lipid nanoparticles using a fluorescence-based assay with 2-(p-toluidino)-6-naphthalene sulfonic acid (TNS). a . Lipid nanoparticles containing cationic lipid / DSPC / cholesterol / PEG-lipid (50 / 10 / 38.5 / 1.5 mol%) in PBS at a total lipid concentration of 0.4 mM were prepared using the in-line process described in Example 1. A stock solution of TNS was prepared at 100 μM in distilled water. Vesicles were diluted to 24 μM lipid in 2 mL of buffer containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl, where the pH ranged from 2.5 to 11. Aliquots of the TNS solution were added to give a final concentration of 1 μM, and after vortex mixing, the fluorescence intensity was measured in an SLM Aminco Series 2 Luminescence spectrophotometer at excitation and emission wavelengths of 321 nm and 445 nm at room temperature. A sigmoidal best-fit analysis was applied to the fluorescence data, and the pK a was measured as the pH that produced half of the maximum fluorescence intensity.

[0191] Example 3

[0192] The efficacy of lipid nanoparticle formulations containing various cationic lipids was determined using an in vivo luciferase mRNA expression rodent model.

[0193] The cationic lipids shown in Table 2 have previously been tested with nucleic acids. For purposes of comparison, using an in-line mixing method, these lipids were also used to formulate lipid nanoparticles containing FLuc mRNA (L-6107), as described in Example 1 and PCT / US10 / 22614, which is hereby incorporated by reference in its entirety. Lipid nanoparticles were formulated using the following molar ratios: 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid (“PEG-DMG”, i.e., 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol, with an average PEG molecular weight of 2000). In an alternative embodiment, the cationic lipid, DSPC, cholesterol, and PEG-lipid were formulated in a molar ratio of approximately 47.5:10:40.8:1.7. As described in Example 1, relative activity was determined by measuring luciferase expression in the liver 4 hours after administration via tail vein injection. Activity was compared at doses of 0.3 and 1.0 mg mRNA / kg and expressed as ng luciferase / g liver measured 4 hours after administration, as described in Example 1.

[0194] Table 2: Comparative lipids showing activity with mRNA

[0195]

[0196]

[0197] Representative compounds of the present disclosure shown in Table 3 were formulated using the following molar ratios: 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid (“PEG-DMA” 2-[2-(ω-methoxy(polyethylene glycol 2000 )ethoxy]-N,N-ditetradecylethanamide) or 47.5% cationic lipid / 10% DSPC / 40.7% cholesterol / 1.8% PEG lipid. As described in Example 1, relative activity was determined by measuring luciferase expression in the liver 4 hours after administration via tail vein injection. Activity was compared at a dose of 0.5 mg mRNA / kg and expressed as ng luciferase / g liver measured 4 hours after administration, as described in Example 1. The compound numbers in Table 3 refer to the compound numbers in Table 1.

[0198] Table 3: Novel cationic lipids and related activities

[0199]

[0200]

[0201]

[0202]

[0203] Example 4

[0204] Synthesis of bis(2-butyl octyl) 10-(N-decyl-5-(dimethylamino)pentanamido)nonanedioate (Compound I-7)

[0205]

[0206] Synthesis of Compound 4-2

[0207] A solution of ketone 4-1 (1.10 g, 1.62 mmol) and 1-decylamine (2.43 mmol, 382 mg, 0.486 mL) in DCE (10 mL) was stirred at room temperature for 15 minutes, then sodium triacetoxyborohydride (2.43 mmol, 515 mg) and acetic acid (2.43 mmol, 146 mg; 0.138 mL) were added. After stirring the mixture at room temperature for 2 days, the reaction mixture was concentrated. The residue was diluted with a mixture of hexanes and washed with dilute NaOH, saturated NaHCO3, and brine. The organic phase was separated, dried over sodium sulfate, and concentrated (colorless oil, 1.41 g). The crude product was purified by silica gel column chromatography (hexanes / EtOAC / Et3N, 95:5:0 to 80:20:1). The desired product was obtained as a colorless oil (863 mg colorless oil, 1.05 mmol, 65% yield). 1 1H NMR (400 MHz, CDCl3) δ: 3.98 (d, 5.8 Hz, 4H), 2.54 (t, 7.1 Hz, 2H), 2.43 (quintet, 5.5 Hz, 1H), 2.30 (t, 7.5 Hz, 4H), 1.68 - 1.57 (m, 6H), 1.50 - 1.41 (m, 2H), 1.41 - 1.08 (70H), 0.92 - 0.86 (m, 15H), 0.86 - 0.77 (br.1H).

[0208] Synthesis of Compound 4-3

[0209] At room temperature, within 1 minute, a solution of thionyl chloride (3.36 mmol, 400 mg, 0.25 mL) in CH2Cl2 (5 mL) was added to a stirred solution of 5-bromopentanoic acid (1.12 mmol, 204 mg) in CH2Cl2 (1 mL), and then DMF (about 16 mg) was added. Then the mixture was heated to reflux for 2 hours. Then the reaction mixture was concentrated under vacuum. The acyl chloride was used directly in the next step.

[0210] At room temperature, a solution of the above-mentioned 5-bromovaleryl chloride in benzene (5 mL) was added dropwise within 2 minutes to a solution of 4-2 (230 mg, 0.28 mmol), triethylamine (5.6 mmol, 565 mg, 0.780 mL), and DMAP (5 mg) in benzene (5 mL). After addition, the reactants were stirred at room temperature for 1 hour. Methanol (1 mL) was added, and the mixture was stirred for 2 hours. The reaction mixture was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane / EtOAC, 98:2 to 85:15). The desired product was obtained as a colorless oil (250 mg, 0.25 mmol, 91%, colorless oil).

[0211] Synthesis of Compound I-7

[0212] Dimethylamine (2 M in THF, 10 mL) was added to 4-2 (250 mg, 0.25 mmol). The solution was stirred at 64 °C overnight. The reaction mixture was concentrated. The residue was dissolved in a mixture of hexane, ethyl acetate, and Et3N (80:20:1), filtered through a silica gel pad, and washed with the same solvent mixture. The filtrate was concentrated to give a brown oil (ca. 233 mg). The crude product (233 mg) was purified by flash silica gel dry column chromatography (chloroform containing 0 to 6% methanol). The desired product was obtained as a colorless oil (194 mg,, 0.20 mmol, 82%). 1 H NMR (400 MHz, CDCl3) δ: 4.60 - 4.20 (br, estimated 0.3H, due to slow isomerization about the amide bond), 3.97, 3.96 (2 sets of doublets, 5.8 Hz, 4H), 3.60 (quintet-like, 7.0 Hz, 0.7H), 3.06 - 2.99 (m, 2H), 2.34 - 2.24 (m, 8H), 2.21 (singlet, 6H), 1.72 - 1.56 (m, 8H), 1.56 - 1.37 (m, 8H), 1.37 - 1.10 (66H), 0.91 - 0.85 (m, 15H).

[0213] Example 5

[0214] Synthesis of bis(2-butyl octyl) 10-(N-decyl-4-(dimethylamino) butanamido) nonanedioate (Compound I-18)

[0215]

[0216] Synthesis of Compound I-18 (Method A)

[0217] At room temperature, oxalyl chloride (5.6 mmol, 722 mg, 0.496 mL) was added to a stirred solution of 4-(dimethylamino)butyric acid hydrochloride (1.12 mmol, 188 mg) and DMF (10 - 20 μL) in CH2Cl2 (10 mL). The resulting mixture was stirred overnight at room temperature. The reaction mixture (dark red) was concentrated under reduced pressure. The resulting acyl chloride (brick red solid) was used directly in the next step.

[0218] At room temperature, a solution of the above acyl chloride in CH2Cl2 (10 mL) was added dropwise to a solution of 4-2 (230 mg, 0.28 mmol), triethylamine (5.6 mmol, 565 mg, 0.780 μL) and DMAP (5 mg) in CH2Cl2 (5 mL). The resulting mixture was stirred overnight at room temperature and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane / EtOAC / Et3N, from 80:20:0.1 to 75:25:1) and further purified by silica gel flash dry column chromatography (chloroform containing 0 to 5% methanol). The desired product (92 mg, colorless oil, 0.10 mmol, 35%) was obtained. 1 1H NMR (400 MHz, CDCl3) δ: 4.57 - 4.29 (br. 0.4H), 3.97, 3.96 (two doublets, 5.8 Hz, 5.8 Hz, 4H), 3.63 (quintet-like, 6.8 Hz, 0.6H), 3.06 - 3.00 (m, 2H), 2.35 - 2.26 (m, 8H), 2.213, 2.211 (two singlets, 6H), 1.82 (sextet-like, 7.6 Hz, 2H), 1.65 - 1.56 (m, 6H), 1.54 - 1.48 (m, 2H), 1.47 - 1.37 (m, 4H), 1.36 - 1.06 (66H), 0.91 - 0.86 (m, 15H).

[0219] Synthesis of Compound I-18 (Method B)

[0220] To a solution of 4-dimethylaminobutyric acid hydrochloride (2 equiv, 3.04 mmol, 510 mg) and 4-dimethylaminopyridine (3 equiv, DMAP, 4.56 mmol, 557 mg) in acetonitrile (30 mL) was added DCC (2.2 equiv, 3.34 mmol, 690 mg), and the mixture was stirred at room temperature for 45 minutes. A solution of 4-2 (1.25 g, 1.52 mmol) in CH2Cl2 (6 mL) was added, and the resulting mixture was stirred overnight. The next day, more DCC (450 mg) was added and the mixture was stirred for another day. The mixture was then concentrated under reduced pressure. The residue was dissolved in a mixture of hexane, ethyl acetate, and Et3N (80:20:1), filtered through a short silica gel column, and washed with the same solvent mixture. The filtrate was concentrated to give a colorless oil. The product was further purified by flash dry column chromatography on silica gel (chloroform containing 0 to 5% methanol with a trace of Et3N). The desired product was obtained as a colorless oil (1.14 g, 81%).

[0221] Example 6

[0222] Synthesis of bis(2-butyl octyl) 10-(N-decyl-5-(diethylamino)pentanamido)nonanedioate (Compound I-8)

[0223]

[0224] Synthesis of Compound I-8

[0225] A mixture of 4-3 (179 mg, 0.18 mmol), diethylamine (0.90 mmol, 66 mg, 0.093 mL), and N,N-diisopropylethylamine (0.36 mmol, 46 mg, 0.063 mL) in acetonitrile (6 mL) was sealed and heated at 83 °C for 24 hours. The reaction mixture was concentrated. The residue was dissolved in a mixture of hexane, ethyl acetate, and Et3N (80:20:1), filtered through a silica gel pad, and washed with the same solvent mixture. The filtrate was concentrated to give a brown oil (153 mg). The crude product (233 mg) was purified by flash dry column chromatography on silica gel (chloroform containing 0 to 6% MeOH). The desired product was obtained (136 mg, colorless oil, 0.14 mmol, 77%).

[0226] Example 7

[0227] Synthesis of bis(2-butyl octyl) 10-(N-decyl-5-(pyrrolidin-1-yl)pentanamido)nonanedioate (Compound I-9)

[0228]

[0229] Synthesis of Compound I-9

[0230] A mixture of 4-3 (200 mg, 0.20 mmol), pyrrolidine (50 equivalents, 0.83 mL, 10 mmol) in THF (10 mL) was sealed and heated at 64 °C for 24 h. The reaction mixture was concentrated. The residue was dissolved in a mixture of hexane, ethyl acetate and Et3N (80:20:1), filtered through a silica gel pad and washed with the same solvent mixture. The filtrate was concentrated to give a brown oil. The crude product (233 mg) was purified by flash dry column chromatography on silica gel (chloroform containing 0 to 6% MeOH). The desired product (158 mg, colorless oil, 0.16 mmol, 80%) was obtained.

[0231] Example 8

[0232] Synthesis of bis(2-hexyldecyl) 7-(N-decyl-4-(dimethylamino)butanamido)tridecanedioate (Compound I-16)

[0233]

[0234] Synthesis of 8-2

[0235] A solution of 8-1 (1 equivalent, 1.15 g, 1.62 mmol) and 1-decylamine (1.5 equivalents, 2.43 mmol, 382 mg, 0.486 mL) in DCE (10 mL) was stirred at room temperature for about 15 min. Sodium triacetoxyborohydride (1.5 equivalents, 2.43 mmol, 515 mg) and ACOH (1.5 equivalents, 2.43 mmol, 146 mg, 0.14 mL) were added to the solution. The mixture was stirred at room temperature for 3 days. Then the reaction mixture was concentrated. The residue was diluted with hexane / EtOAC (99:1) and washed with dilute NaOH solution, saturated NaHCO3 and brine. The organic extract was dried over sodium sulfate and poured onto a short silica gel column. The column was eluted with a mixture of hexane, EtOAC and Et3N (95:5:0 to 80:20:1). The fractions containing the pure product were combined and concentrated. The desired product (1.28 g, 1.51 mmol, 93%) was obtained as a colorless oil. 1 1H NMR (400 MHz, CDCl3) δ: 3.97 (d, 5.8 Hz, 4H), 2.53 (t, 7.2 Hz, 2H), 2.43 (quintet-like, 5.5 Hz, 1H), 2.30 (t, 7.5 Hz, 4H), 1.68 - 1.57 (m, 6H), 1.49 - 1.40 (m, 2H), 1.40 - 1.08 (74H), 0.91 - 0.85 (m, 15H), 0.83 - 0.74 (br. 1H).

[0236] Synthesis of Compound I-16

[0237] Under Ar, at room temperature, oxalyl chloride (3 equiv, 6.3 mmol, 800 mg, 0.55 mL) was added to a stirred solution of 4-(dimethylamino)butyric acid hydrochloride (2.1 mmol, 352 mg) and DMF (about 13 mg) in CH2Cl2 (15 mL). The resulting mixture was stirred overnight at room temperature. The reaction mixture (light orange solution) was concentrated in vacuo. The resulting acyl chloride (8-3, light brown solid) was used directly in the following reaction.

[0238] At room temperature, a solution of the above acyl chloride in CH2Cl2 (10 mL) was added to a solution of 8-2 (300 mg, 0.35 mmol), triethylamine (10.5 mmol, 1.06 g, 1.5 mL) and DMAP (5 mg) in CH2Cl2 (5 mL). After addition, the reaction mixture was stirred overnight at room temperature. After concentrating the mixture, the product was separated by silica gel column chromatography (hexane, EtOAC and Et3N, from 80:20:0.1 to 70:30:1), and the product was further purified by silica gel flash dry column chromatography (chloroform containing 0-5% MeOH). The desired product as a pale yellow oil was obtained (110 mg, 0.11 mmol, 32%). 1 HNMR (400 MHz, CDCl3, at 7.26 ppm) δ: 4.57 - 4.34 (br. 0.4H), 3.98 - 3.94 (m, 4H), 3.64 (quintet-like, 6.8 Hz, 0.6H), 3.06 - 3.00 (m, 2H), 2.35 - 2.25 (m, 8H), 2.213, 2.210 (2 singlets, 6H), 1.82 (sextet-like, 7.4 Hz, 2H), 1.65 - 1.56 (m, 6H), 1.54 - 1.48 (m, 2H), 1.48 - 1.37 (m, 4H), 1.37 - 1.06 (70H), 0.91 - 0.86 (m, 15H).

[0239] Example 9

[0240] Synthesis of bis(2-butyl octyl) 10-(4-(dimethylamino)-N-(2-ethylhexyl)butanamido)nonanedioate (Compound I-20)

[0241]

[0242] Synthesis of 9-2

[0243] A solution of 9-1 (1 eq., 0.82 g, 1.21 mmol) and 2-ethyl-1-hexylamine (1.5 eq., 1.81 mmol, 234 mg) in DCE (8 mL) was stirred at room temperature for about 15 minutes. Sodium triacetoxyborohydride (1.5 eq., 1.81 mmol, 384 mg) and AcOH (1.5 eq., 1.81 mmol, 109 mg) were added to the solution. The mixture was stirred at room temperature for 2 days. Then the reaction mixture was concentrated. The residue was diluted with hexane and EtOAC (ca. 99:5) and washed with dilute NaOH, saturated NaHCO3 and brine. The extract was filtered through a short silica gel column. The mixture was washed with a mixture of hexane and EtOAC (95:5), then with a mixture of hexane, EtOAC and Et3N (80:20:0.5). The filtrate from the latter wash was concentrated to dryness. This gave the pure product as a colorless oil (888 mg, 1.12 mmol, 93%). 1 1H NMR (400 MHz, CDCl3) δ: 3.98 (d, 5.8 Hz, 4H), 2.45 (d, 5.1 Hz, 2H), 2.39 (quintet-like, 5.5 Hz, 1H), 2.30 (t, 7.5 Hz, 4H), 1.68 - 1.57 (m, 6H), 1.41 - 1.08 (65H), 0.92 - 0.85 (m, 18H), 0.84 - 0.78 (br. 1H).

[0244] Synthesis of 9-4

[0245] At room temperature, within 1 minute, a solution of thionyl chloride (3 eq., 6.72 mmol, 800 mg, 0.49 mL) in CH2Cl2 (5 mL) was slowly added to a stirred solution of 5-bromovaleric acid (2.24 mmol, 405 mg) in CH2Cl2 (2 mL). Two drops of DMF (ca. 16 mg) were added to the reaction mixture. Then the mixture was heated to reflux for 2 hours. The reaction mixture was concentrated in vacuo. The resulting acyl chloride 9-3 was used directly in the next step.

[0246] At room temperature, within 2 minutes, a solution of the above 5-bromovaleryl chloride in benzene (8 mL) was added to a solution of 9-2 (444 mg, 0.56 mmol), triethylamine (1.56 mL) and DMAP (5 mg) in benzene (5 mL). After addition, the mixture was stirred at room temperature overnight. After evaporation of the solvent in vacuo, the product was separated by silica gel column chromatography (hexane / EtOAC, 99:1 to 90:10). The desired product was pure enough for the next step (colorless oil, 527 mg, 0.55 mmol, 98%).

[0247] Synthesis of Compound I-20

[0248] To a pressure flask containing 9-4 (260 mg, 0.27 mmol) was added dimethylamine (2 M in THF, 10 mL). The solution was stirred overnight at 64 °C (oil bath temperature). The excess amine and solvent were evaporated. The residue was dissolved in a mixture of ethyl acetate and hexane (95:5) and filtered through a silica gel pad. The pad was washed with a mixture of hexane and EtOAC (95:5), and then with a mixture of hexane, EtOAC and Et3N (80:20:1). The filtrate from the latter wash was concentrated to dryness. This gave the crude product (233 mg) as a brown oil. The crude product was further purified by flash dry column chromatography on silica gel (chloroform containing 0 - 5% MeOH). The desired product was obtained as a colorless oil (204 mg, 0.22 mmol, 82%). 1 HNMR (400 MHz, CDCl3, at 7.27 ppm) δ: 3.97 (d, 5.8 Hz, 4H), 3.66 - 3.57 (m, ca 1H), 3.19 - 2.99 (two sets of peaks, 2H), 2.38 - 2.24 (m, 8H), 2.22 (singlet, 6H), 1.72 - 1.37 (m, 15H), 1.37 - 1.10 (60H), 0.91 - 0.85 (m, 18H).

[0249] Example 10

[0250] Synthesis of bis(2-butyl octyl) 10-(3-(dimethylamino)-N-nonyl propanamido) nonanedioate (Compound I-25)

[0251]

[0252] Synthesis of 10-1

[0253] At room temperature, oxalyl chloride (5.05 mmol, 641 mg, 0.44 mL) was added to a solution of 3-bromopropionic acid (2.02 mmol, 311 mg) in CH2Cl2 (5 mL) and DMF (0.01 mL). The resulting mixture was stirred overnight at room temperature. Then the mixture was concentrated under vacuum. The remaining liquid / solid (yellow) was dissolved in 10 mL of CH2Cl2 and added, within 4 minutes at room temperature, to a solution of 4-2 (833 mg, 1.02 mmol), triethylamine (5.05 mmol, 0.7 mL) and DMAP (5 mg) in CH2Cl2 (10 mL). After addition, the mixture was stirred at room temperature for 2 h. After evaporation of the solvent in vacuo, the product was separated by silica gel column chromatography (hexane / EtOAC, 99:1 to 90:10). The desired product was obtained as a colorless oil (794 mg, 0.83 mmol, 81%).

[0254] Synthesis of Compound I-25

[0255] A mixture of 10-1 (283 mg, 0.30 mmol) and dimethylamine (2 M in THF, 12 mL) in a pressure flask was stirred overnight at 68 °C (oil bath temperature). The reaction mixture was concentrated. The residue was dissolved in a mixture of hexane, ethyl acetate and Et3N (80:20:1), filtered through a silica gel pad and washed with the same solvent mixture. The filtrate was concentrated to give a brown oil (302 mg). The crude product (302 mg) was purified by flash dry column chromatography on silica gel (chloroform containing 0 - 5% MeOH with a trace of Et3N). The desired product was obtained as a colorless oil (169 mg, 0.18 mmol, 61%). 1 1H NMR (400 MHz, CDCl3, at 7.26) δ: 4.50 - 4.31 (br, estimated 0.3H, due to slow isomerization about the amide bond), 3.97 (shouldered doublet, 5.8 Hz, 4H), 3.60 (quintet-like, 7.0 Hz, 0.7H), 3.07 - 3.00 (m, 2H), 2.65 (q-like, 7.6 Hz, 2H), 2.48 (q-like, 7.6 Hz, 2H), 2.29 (shouldered triplet, 7.6 Hz, 4H), 2.26, 2.25 (2 singlets, 6H), 1.66 - 1.56 (m, 6H), 1.56 - 1.48 (m, 2H), 1.48 - 1.37 (m, 4H), 1.37 - 1.10 (66H), 0.91 - 0.85 (m, 15H).

[0256] Example 11

[0257] Synthesis of Bis(2-hexyldecyl) 7-(N-decyl-4-(pyrrolidin-1-yl)butanamido)tridecanedioate (Compound I-29)

[0258]

[0259] Synthesis of 11-1

[0260] At room temperature, oxalyl chloride (3 eq, 2.91 mmol, 370 mg, 0.3 mg) was added to a solution of 4-bromobutyric acid (0.97 mmol, 161 mg) in CH2Cl2 (3 mL) and DMF (0.01 mL). The mixture was stirred overnight at room temperature. The reaction mixture was then concentrated under vacuum. The residual liquid / solid (pale yellow) was dissolved in 5 mL of CH2Cl2 and added thereto, within 2 minutes at room temperature, to a solution of 8-2 (410 mg, 0.48 mmol), triethylamine (0.4 mL) and DMAP (2 mg) in CH2Cl 2 (20 mL). After addition, the resulting mixture was stirred at room temperature for 2.5 h. TLC (hexane / ethyl acetate = 9:1) showed two major spots. The reaction mixture was then concentrated at room temperature under reduced pressure. The residue was used in the next reaction without any purification.

[0261] Synthesis of Compound I-29

[0262] The residue containing 11-1 above was dissolved in a mixture of pyrrolidine (2.10 mL, 25 mmol) and THF (15 mL). The mixture was transferred to a pressure flask and heated overnight at 68 °C. The mixture was cooled and concentrated under reduced pressure. The residue was dissolved in a mixture of hexane, ethyl acetate and Et3N (80:20:1), filtered through a short silica column, and washed with the same solvent mixture. The filtrate was concentrated to give a yellow oil / solid. The crude product (300 mg) was purified by silica column chromatography (0% to 10% MeOH and 0% to 0.5% Et3N in CH2Cl2). The desired product as a yellow oil (215 mg) was obtained. The product (215 mg) was further purified by silica flash dry column chromatography (chloroform containing 0 - 5% MeOH). The desired product as a colorless oil (162 mg, 0.16 mmol, 34%) was obtained. 1 HNMR (400 MHz, CDCl3, at 7.26 ppm) δ: 4.57 - 4.34 (br. 0.4H), 3.98 - 3.94 (m, 4H), 3.64 (quintet-like, 6.8 Hz, 0.6H), 3.06 - 3.00 (m, 2H), 2.51 - 2.44 (m, 6H), 2.37 - 2.21 (m, 6H), 1.86 (sextet-like, 7.6 Hz, 2H), 1.80 - 1.71 (m, 4H), 1.65 - 1.48 (m, 8H), 1.48 - 1.37 (m, 4H), 1.37 - 1.06 (70H), 0.91 - 0.86 (m, 15H).

[0263] Example 12

[0264] Synthesis of bis(2-butyl octyl) 10-(4-(dimethylamino)-N-(2-ethylhexyl) butanamido) nonadecanedioate (Compound I-30)

[0265]

[0266] Synthesis of Compound I-30

[0267] To a solution of 4-dimethylaminobutyric acid hydrochloride (0.50 mmol, 85 mg) and 4-dimethylaminopyridine (3 eq., DMAP, 0.75 mmol, 92 mg) in acetonitrile (5 mL) was added DCC (1.1 mmol x 2, 226 mg), and the mixture was stirred at room temperature for 45 minutes. A solution of 9-2 (160 mg, 0.20 mmol) in CH2Cl2 (1 mL) was added to the reaction mixture, and the resulting mixture was stirred over the weekend. More DCC (135 mg) was added and stirred for another day. No progress was observed based on TLC analysis. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in a mixture of hexane and EtOAC (ca. 99:5) and filtered through a short silica gel column. The column was washed with a mixture of hexane and EtOAC (95:5), and then with a mixture of hexane, EtOAC and Et3N (80:20:1). The filtrate from the latter wash was concentrated to dryness (133 mg). The crude product (133 mg) was further purified by silica gel flash dry column chromatography (chloroform with 0 - 5% MeOH, with a trace of Et3N). The desired product (48 mg, colorless oil, 0.053 mmol, 27%) was obtained. 1 HNMR (400 MHz, CDCl3, at 7.27 ppm) δ: 3.97 (d, 5.8 Hz, 4H), 3.70 - 3.60 (m, ca 1H), 3.19 - 2.99 (two sets of peaks, 2H), 2.39 - 2.25 (m, 8H), 2.22 (singlet, 6H), 1.86 - 1.76 (m, 2H), 1.72 - 1.37 (m, ca 11H), 1.37 - 1.10 (60H), 0.91 - 0.85 (m, 18H).

[0268] Example 13

[0269] Synthesis of bis(2-butyl octyl) 10-(N-decyl-5-(dibutylamino) pentanamido) nonadecanedioate (Compound I-31)

[0270]

[0271] Synthesis of Compound I-31

[0272] A mixture of 4-3 (284 mg, 0.29 mmol), THF (10 mL), sodium iodide (5 mg), and dibutylamine (10 mmol, 1.29 g, 1.68 mL) in a pressure flask was stirred overnight at 78 °C. The reaction mixture was concentrated. The residue was dissolved in a mixture of hexane, ethyl acetate, and Et3N (80:20:1), filtered through a silica gel pad, and washed with the same solvent mixture. The filtrate was concentrated to give a brown oil (the product and dibutylamine). The oil was diluted with hexane, washed twice with dilute aqueous HCl (0.5 M), washed with saturated NaHCO3 and brine, and dried over sodium sulfate. The extract was concentrated under reduced pressure. The crude product (309 mg) was purified by flash dry column chromatography on silica gel (chloroform containing 0 - 5% MeOH with a trace of Et3N). The desired product was obtained as a colorless oil (216 mg, 0.21 mmol, 72%). 1 1H NMR (400 MHz, CDCl3) δ: 4.50 - 4.35 (br, estimated 0.3H, due to slow isomerization about the amide bond), 3.97, 3.96 (2 sets of doublets, 5.8 Hz, 4H), 3.61 (quintet-like, 7.0 Hz, 0.7H), 3.07 - 2.99 (m, 2H), 2.45 - 2.36 (m, 6H), 2.34 - 2.27 (m, 6H), 1.70 - 1.56 (m, 8H), 1.56 - 1.36 (m, 12H), 1.37 - 1.10 (70H), 0.97 - 0.85 (m, 21H).

[0273] Example 14

[0274] Synthesis of 10-(4-(dimethylamino)-N-nonylbutanamido)nonadecane-1,19-diyl bis(2-butyl octanoate) (Compound I-32)

[0275]

[0276] Synthesis of Compound I-32

[0277] To a solution of 4-dimethylaminobutyric acid hydrochloride (2 equiv, 1.08 mmol, 181 mg) and 4-dimethylaminopyridine (3 equiv, DMAP, 1.62 mmol, 198 mg) in acetonitrile (10 mL) was added DCC (2.2 equiv, 1.18 mmol, 245 mg), and the mixture was stirred at room temperature for 45 minutes. Then a solution of 14-1 (442 mg, 0.54 mmol) in CH2Cl2 (2 mL) was added. The resulting mixture was stirred overnight. More DCC (140 mg) was added and the mixture was stirred for another day. Then the mixture was concentrated under reduced pressure. The residue was dissolved in a mixture of hexane, ethyl acetate and Et3N (80:20:1), filtered through a short silica gel column, and washed with the same solvent mixture. The filtrate was concentrated to give a yellow oil (381 mg). The crude product (381 mg) was purified by flash dry column chromatography on silica gel (chloroform containing 0 - 5% MeOH with a trace of Et3N). The desired product was obtained as a colorless oil (345 mg, 0.38 mmol, 70%). 1 HNMR (400 MHz, CDCl3, at 7.26 ppm) δ: 5.30 - 4.34 (br., 0.3H), 4.061, 4.056 (two sets of triplets, 6.7 Hz, 4H), 3.64 (quintet-like, 6.8 Hz, 0.7H), 3.07 - 3.01 (m, 2H), 2.35 - 2.26 (m, 6H), 2.214, 2.211 (two sets of singlets, 6H), 1.82 (sextet-like, 7.6 Hz, 2H), 1.65 - 1.48 (m, 10H), 1.48 - 1.37 (m, 8H), 1.37 - 1.02 (60H), 0.90 - 0.85 (m, 15H).

[0278] Example 15

[0279] Synthesis of bis(2-butyl octyl) 10-(N-decyl-3-(pyrrolidin-1-yl)propanamido)nonanedioate (Compound I-33)

[0280]

[0281] Synthesis of Compound I-33

[0282] A mixture of 10-1 (283 mg, 0.30 mmol), pyrrolidine (1.25 mL, 15 mmol) and THF (10 mL) in a pressure tube was heated overnight at 64 °C. The mixture was cooled and concentrated under reduced pressure. The residue was dissolved in a mixture of hexane, ethyl acetate and Et3N (80:20:1), filtered through a short silica gel column, and washed with the same solvent mixture. The filtrate was concentrated to give a yellow oil. The crude product (314 mg) was further purified by flash dry column chromatography on silica gel (chloroform containing 0 - 5% MeOH with a trace of Et3N). The desired product (113 mg, 0.12 mmol, 40%) was obtained as a colorless oil. 1 1H NMR (400 MHz, CDCl3, at 7.26) δ: 4.55 - 4.30 (br, estimated 0.3H, due to slow isomerization about the amide bond), 3.97 (shoulder doublet, 5.8 Hz, 4H), 3.61 (quintet-like, 7.0 Hz, 0.7H), 3.06 - 3.00 (t-like, 2H), 2.81 (q-like, 7.6 Hz, 2H), 2.58 - 2.51 (m, 6H), 2.292, 2.285 (2 sets of triplets, 7.5 Hz, 4H), 1.83 - 1.73 (m, 4H), 1.65 - 1.56 (m, 6H), 1.56 - 1.48 (m, 2H), 1.48 - 1.37 (m, 4H), 1.37 - 1.10 (66H), 0.91 - 0.85 (m, 15H).

[0283] Example 16

[0284] Synthesis of bis(2-butyl octyl) 10-(N-decyl-5-(hexyl(methyl)amino)pentanamido)nonanedioate (Compound I-34)

[0285]

[0286] Synthesis of 16-1

[0287] A mixture of 4-3 (200 mg, 0.20 mmol), hexylamine (20 mmol, 2 g), N,N-diisopropylethylamine (5 equivalents, 1.0 mmol, 0.17 mL)) and sodium iodide (10 mg) in acetonitrile (6 mL) was sealed and heated at 70 °C for 24 h. The reaction mixture was concentrated at 75 - 85 °C under reduced pressure (about 30 mmHg). TLC showed that most of the excess hexylamine was removed. The residue was dissolved in a mixture of hexane, ethyl acetate and Et3N (80:20:1), filtered through a short silica gel column, and washed with the same solvent mixture. The filtrate was concentrated to give a brown oil (192 mg), which was used in the next step without further purification.

[0288] Synthesis of Compound I-34

[0289] At room temperature, a solution of 16-1 (192 mg, 0.19 mmol) in THF (5 mL) was added with a solution of formaldehyde HCHO (500 mg, 37 wt.% aqueous solution). The resulting mixture was stirred for 30 minutes and then sodium triacetoxyborohydride (1.2 mmol, 243 mg) was introduced. The resulting mixture was stirred overnight at room temperature. The reaction mixture was concentrated. The residue was dissolved in a mixture of hexane and washed with dilute NaOH solution, saturated sodium bicarbonate solution and brine. After drying over sodium sulfate, the solution was concentrated to dryness (yellow oil). The residue was dissolved in a mixture of hexane, ethyl acetate and Et3N (80:20:1), filtered through a short silica gel column and washed with the same solvent mixture. The filtrate was concentrated to give a yellow oil (220 mg). The crude product (220 mg) was further purified by silica gel flash dry column chromatography (chloroform containing 0-5% MeOH with a trace of Et3N). The desired product was obtained as a colorless oil (113 mg, 0.13 mmol, 69%). 1 HNMR (400 MHz, CDCl3, at 7.26 ppm) δ: 4.50 - 4.35 (br, estimated 0.3H, due to slow isomerization about the amide bond), 3.97, 3.96 (2 doublets, 5.8 Hz, 4H), 3.60 (quintet-like, 7.0 Hz, 0.7H), 3.06 - 2.98 (m, 2H), 2.36 - 2.26 (m, 10H), 2.191, 2.189 (2 singlets, 3H), 1.70 - 1.56 (m, 8H), 1.56 - 1.36 (m, 10H), 1.37 - 1.10 (72H), 0.91 - 0.85 (m, 18H).

[0290] Example 17

[0291] Synthesis of Bis(2-hexyldecyl) 7-(N-decyl-3-(dimethylamino)propanamido)tridecanedioate (Compound I-35)

[0292]

[0293] Synthesis of Compound I-35

[0294] To a solution of 3-dimethylaminopropionic acid hydrochloride (2 eq., 0.62 mmol, 95 mg) and 4-dimethylaminopyridine (3 eq., DMAP, 0.93 mmol, 114 mg) in acetonitrile (10 mL) was added DCC (2.2 eq., 0.68 mmol, 141 mg), and the mixture was stirred at room temperature for 45 min. A solution of 8-2 (262 mg, 0.31 mmol) in CH2Cl2 (2 mL) was added, and the resulting mixture was stirred over the weekend. TLC (chloroform / MeOH, 9:1) showed a major spot at the solvent front, which could be the elimination product, with no starting material and a small amount of the desired product. The reaction mixture was concentrated. The possible elimination product (107 mg, colorless oil) was separated by column chromatography (hexane-EtOAC, 95:5) and treated with a solution of dimethylamine in THF (2 M, 9 mL) at room temperature for 4 days. The mixture was concentrated. The residue was dissolved in a mixture of hexane, ethyl acetate, and Et3N (80:20:1), filtered through a short silica column, and washed with the same solvent mixture. The filtrate was concentrated to give a yellow oil. The crude product was further purified by flash dry column chromatography on silica gel (chloroform containing 0 - 5% MeOH with a trace of Et3N). The desired product (62 mg) was obtained as a colorless oil. 1 HNMR (400 MHz, CDCl3, at 7.26) δ: 4.50 - 4.36 (br, estimated 0.3H, due to slow isomerization about the amide bond), 3.97, 3.96 (two doublets, 5.8 Hz, 4H), 3.61 (quintet-like, 7.0 Hz, 0.7H), 3.07 - 3.00 (m, 2H), 2.65 (q-like, 7.2 Hz, 2H), 2.53 - 2.41 (m, 2H), 2.31 - 2.26 (m, 4H), 2.26, 2.25 (two singlets, 6H), 1.66 - 1.56 (m, 6H), 1.56 - 1.48 (m, 2H), 1.48 - 1.37 (m, 4H), 1.37 - 1.10 (70H), 0.91 - 0.85 (m, 15H).

[0295] Example 18

[0296] Synthesis of bis(2-hexyldecyl) 7-(3-(dimethylamino)-N-(6-((2-ethylhexyl)oxy)-6-oxohexyl)propanamido)tridecanedioate (Compound I-36)

[0297]

[0298] Synthesis of 18-2

[0299] At room temperature, oxalyl chloride (0.86 mmol, 109 mg, 74 μL) was added to a solution of 3-bromopropionylic acid (0.34 mmol, 52 mg) in CH2Cl2 (3 mL) and DMF (1 drop from a fine needle). The mixture was stirred overnight at room temperature. Then the mixture was concentrated under reduced pressure at room temperature for 60 minutes. The residual liquid / solid (yellow) was dissolved in 5 mL of CH2Cl2 and added thereto within 1 minute at room temperature to a solution of 18-1 (160 mg, 0.17 mmol), triethylamine (0.86 mmol, 0.12 mL) and DMAP (1 mg) in CH2Cl2 (5 mL). After the addition, the mixture was stirred at room temperature for 3 h and then concentrated. The product was separated by silica gel column chromatography (hexane, EtOAC and Et3N, 95:5 to 85:15). The desired product was obtained as a colorless oil (99 mg, 0.09 mmol, 53%).

[0300] Synthesis of Compound I-36

[0301] Dimethylamine (2 M in THF, 5 mL) was added to a pressure flask containing 18-2 (99 mg, 0.09 mmol). The solution was stirred at 68 °C (oil bath temperature) for 2 days. The mixture was cooled and concentrated under reduced pressure. The residue was dissolved in a mixture of hexane, ethyl acetate and Et3N (80:20:1), filtered through a short silica gel column and washed with the same solvent mixture. The filtrate was concentrated to give a brown oil (94 mg). The product (94 mg) was further purified by silica gel flash dry column chromatography (chloroform containing 0 - 5% MeOH with a trace of Et3N). The desired product was obtained as a colorless oil (71 mg, 0.069 mmol, 76%). 1 HNMR (400 MHz, CDCl3, at 7.26) δ: 4.53 - 4.35 (br, estimated 0.3H, due to slow isomerization about the amide bond), 4.02 - 3.93 (m, 6H), 3.62 (quintet-like, 7.0 Hz, 0.7H), 3.07 - 3.01 (m, 2H), 2.65 (q-like, 7.6 Hz, 2H), 2.50 - 2.44 (m, 2H), 2.34 - 2.26 (m, 6H), 2.26, 2.25 (2 singlets, 6H), 1.69 - 1.56 - 1.48 (m, estimated 11H, overlapping with water peak), 1.49 - 1.37 (m, 4H), 1.37 - 1.10 (66H), 0.92 - 0.86 (m, 18H).

[0302] Example 19

[0303] Synthesis of bis(tridecan-7-yl) 10-(N-decyl-4-(dimethylamino)butanamido)nonadecanedioate (Compound I-37)

[0304]

[0305] Synthesis of 19-2

[0306] A solution of 19-1 (1 equiv, 0.493 g, 0.70 mmol) and 1-decylamine (1.5 equiv, 1.05 mmol, 165 mg, 0.21 mL) in DCE (10 mL) was stirred at room temperature for about 15 minutes. Sodium triacetoxyborohydride (1.5 equiv, 1.05 mmol, 222 mg) and AcOH (1.5 equiv, 1.05 mmol, 63 mg, 0.059 mL) were added to the solution. The mixture was stirred at room temperature for 2 days. Then the reaction mixture was concentrated. The residue was diluted with hexane and washed with dilute NaOH, saturated NaHCO3, and brine. The organic extract was dried over sodium sulfate and the solvent was removed under reduced pressure. The residue was dissolved in a mixture of hexane, ethyl acetate, and Et3N (80:20:1), filtered through a short silica gel column, and washed with the same solvent mixture. Concentration of the filtrate gave the desired product as a colorless oil (582 mg, 0.69 mmol, 98%). The product was used in the next step without further purification.

[0307] Synthesis of Compound I-37

[0308] To a solution of 4-dimethylaminobutyric acid hydrochloride (1.71 mmol, 287 mg) and 4-dimethylaminopyridine (3 equiv, DMAP, 2.07 mmol, 253 mg) in acetonitrile (15 mL) was added DCC (2.2 equiv, 1.52 mmol, 313 mg), and the mixture was stirred at room temperature for 45 minutes. A solution of 19-2 (582 mg, 0.69 mmol) in CH2Cl2 (3 mL) was added, and the resulting mixture was stirred overnight. The next day, more DCC (200 mg) was added and stirring was continued for one week (4 days). Then the mixture was concentrated under reduced pressure. The residue was dissolved in a mixture of hexane, ethyl acetate, and Et3N (80:20:1), filtered through a short silica gel column, and washed with the same solvent mixture. Concentration of the filtrate gave a yellow oil. The product was further purified by flash dry column chromatography on silica gel (chloroform containing 0 - 5% MeOH with a trace of Et3N). The desired product was obtained as a colorless oil.

[0309] Example 20

[0310] Synthesis of Bis(2-hexyldecyl) 10-(N-decyl-4-(dimethylamino)butanamido)nonadecanedioate (Compound I-38)

[0311]

[0312] Synthesis of 20-2

[0313] A solution of ketone 20-1 (0.92 g, 1.16 mmol) and 1-decylamine (2.03 mmol, 319 mg, 0.40 mL) in DCE (6 mL) was stirred at room temperature for 15 minutes, then sodium triacetoxyborohydride (2.03 mmol, 429 mg) and AcOH (2.03 mmol, 121 mg, 0.115 mL) were added. The mixture was stirred at room temperature for 2 days and then the reaction mixture was concentrated. The residue was diluted with hexane and washed with dilute NaOH, saturated NaHCO3 and brine. The organic phase was separated and dried over sodium sulfate. The extract was filtered through a short silica gel column and the column was washed with a mixture of hexane / EtOAC / Et3N (95:5:0 to 80:20:1). The desired product was obtained as a colorless oil (814 mg colorless oil, 0.87 mmol, 75% yield).

[0314] Synthesis of I-38

[0315] To a solution of 4-dimethylaminobutyric acid hydrochloride (2 equiv, 0.76 mmol, 127 mg) and 4-dimethylaminopyridine (3 equiv, 1.14 mmol, 139 mg) in CH3CN (5 mL) was added DCC (2.2 equiv, 0.84 mmol, 172 mg), and the mixture was stirred at room temperature for 45 minutes. A solution of 20-2 (350 mg, 0.38 mmol) in DCM (1 mL) was added, and the resulting mixture was stirred overnight. On the next day, more DCC (50 mg) was added and stirred for another day. Then the mixture was concentrated under reduced pressure. The residue was dissolved in a mixture of hexane, ethyl acetate and Et3N (80:20:1), filtered through a short silica gel column and washed with the same solvent mixture. The filtrate was concentrated to give a colorless oil. The product was further purified by silica gel flash dry column chromatography (chloroform containing 0 - 5% MeOH with a trace of Et3N). The desired product was obtained as a colorless oil (260 mg).

[0316] Example 21

[0317] Synthesis of Bis(2-hexyldecyl) 10-(N-decyl-4-(pyrrolidin-1-yl)butanamido)nonadecanedioate (Compound I-39)

[0318]

[0319] Synthesis of 21-1

[0320] At room temperature, oxalyl chloride (3 equivalents, 3.00 mmol, 381 mg, 0.26 mL) was added to a solution of 4-bromobutyric acid (1.00 mmol, 167 mg) in DCM (3 mL) and a small drop of DMF. The mixture was stirred overnight at room temperature. Then the reaction mixture was concentrated under vacuum. The residual liquid / solid (pale yellow) was dissolved in 5 mL of DCM, and a solution of 20-2 (464 mg, 0.50 mmol), triethylamine (0.42 mL), and DMAP (2 mg) in DCM (5 mL) was added within 2 minutes at room temperature. After addition, the resulting mixture was stirred at room temperature for 2.5 h. TLC (hexane / ethyl acetate = 9:1) showed two main spots. Then the reaction mixture was concentrated under reduced pressure at room temperature. The residue was used in the next reaction without any purification.

[0321] Synthesis of I-39

[0322] The above residue was dissolved in a mixture of pyrrolidine (2.20 mL, 26 mmol) and THF (15 mL). The mixture was transferred to a pressure bottle and heated overnight at 68 °C. The mixture was cooled and concentrated under reduced pressure. The residue was dissolved in a mixture of hexane, ethyl acetate, and Et3N (80:20:1), filtered through a short silica gel column, and washed with the same solvent mixture. The filtrate was concentrated to give a yellow oil / solid (359 mg). The crude product (359 mg) was further purified by flash dry column chromatography on silica gel (chloroform containing 0 - 5% MeOH). The desired product was obtained as a colorless oil (165 mg).

[0323] The various embodiments described above can be combined to provide additional embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications (including U.S. 62 / 791,566 and 62 / 890,469) cited in this specification and / or listed in the application data sheet are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified to provide additional embodiments if the concepts of various patents, applications, and publications are required. These and other changes can be made to the embodiments in accordance with the above detailed description. Generally, in the appended claims, the terms used should not be construed as limiting the claims to the specific embodiments disclosed in this specification and the claims, but should be construed to include all possible embodiments and the full scope of equivalents given by such claims. Thus, the claims are not limited by the present disclosure.

Claims

1. A compound having the following structure (I): or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: R 1 is an optionally substituted C6-C 18 alkyl or an optionally substituted C 14 -C 18 alkenyl; R 2 and R 3 each independently is an optionally substituted C8-C 36 hydrocarbyl group; R 4 and R 5 each independently is an optionally substituted C1-C6 hydrocarbyl group, or R 4 and R 5 together with the N to which they are attached form an optionally substituted heterocyclic group; L 1 、L 2 and L 3 each independently is an optionally substituted C1-C 18 alkylene; G 1 is a direct connection key, -(CH2) n O(C=O)-, -(CH2) n (C=O)O- or –(C=O)-; G 2 and G 3 each independently is -(C=O)O- or -O(C=O)-; and n is an integer from 1 to 10, wherein the term "optionally substituted" means that the group is optionally substituted with one or more halogens, and the heterocyclic group is a 3- to 7-membered non-aromatic ring group.

2. The compound according to claim 1, which has the following structure (IA):

3. The compound according to claim 1, which has the following structure (IB):

4. The compound according to any one of claims 1-3, wherein R 1 is an optionally substituted C6-C 18 alkyl group.

5. The compound according to any one of claims 1 - 3, wherein R 1 is C8 alkyl, C9 alkyl, C 10 alkyl, C 12 alkyl, C 14 alkyl or C 16 alkyl.

6. The compound according to any one of claims 1-3, wherein R 1 is C 16 alkenyl.

7. The compound according to any one of claims 1-3, wherein R 1 is unbranched.

8. A compound according to any one of claims 1-3, wherein R 1 is branched.

9. The compound according to any one of claims 1 - 3, wherein R 1 is unsubstituted.

10. The compound according to any one of claims 1-3, wherein G 1 is a direct bond, -(CH2) n O(C=O)- or -(CH2) n (C=O)O-.

11. The compound according to claim 10, wherein G 1 is a direct bond.

12. The compound according to claim 10, wherein G 1 is -(CH2) n (C=O)O- and n is an integer from 1 to 10.

13. The compound according to claim 12, wherein n is 5, 6, 7, 8, 9 or 10.

14. The compound according to claim 13, wherein n is 7.

15. The compound according to claim 13, wherein n is 8.

16. The compound according to any one of claims 1-3, wherein L 1 is a C1-C6 alkylene group.

17. The compound according to claim 16, wherein L 1 is a C2 alkylene group, a C3 alkylene group or a C4 alkylene group.

18. The compound according to any one of claims 1 - 3, wherein L 1 is unbranched.

19. The compound according to any one of claims 1-3, wherein L 1 is unsubstituted.

20. The compound according to any one of claims 1-3, wherein R 2 is a C8-C 24 hydrocarbyl group.

21. The compound according to any one of claims 1-3, wherein R 3 is a C8-C 24 hydrocarbyl group.

22. The compound according to any one of claims 1-3, wherein R 2 and R 3 are both C8-C 24 hydrocarbyl groups.

23. The compound according to any one of claims 1 - 3, wherein R 2 and R 3 are each independently C 11 hydrocarbyl, C 12 hydrocarbyl, C 13 hydrocarbyl, C 14 hydrocarbyl, C 15 hydrocarbyl, C 16 hydrocarbyl, C 18 hydrocarbyl or C 20 hydrocarbyl.

24. The compound according to any one of claims 1-3, wherein R 2 is branched.

25. The compound according to any one of claims 1 - 3, wherein R 3 is branched.

26. The compound according to any one of claims 1 - 3, wherein R 2 and R 3 each independently has one of the following structures: wherein: R 6 and R 7 each independently is a C2-C 12 hydrocarbyl group.

27. The compound according to claim 26, wherein R 2 and R 3 each independently has one of the following structures:

28. The compound according to any one of claims 1-3, wherein L 2 and L 3 are each independently a C4-C 10 alkylene group.

29. The compound according to any one of claims 1-3, wherein L 2 and L 3 are both C5 lower alkylene groups.

30. A compound according to any one of claims 1 - 3, wherein L 2 and L 3 are each C6 lower alkylene groups.

31. The compound according to any one of claims 1-3, wherein L 2 and L 3 are each C8 alkylene.

32. The compound according to any one of claims 1-3, wherein L 2 and L 3 are each C9 alkylene groups.

33. The compound according to any one of claims 1-3, wherein L 2 is unbranched.

34. A compound according to any one of claims 1 - 3, wherein L 3 is unbranched.

35. The compound according to any one of claims 1 - 3, wherein L 2 is unsubstituted.

36. The compound according to any one of claims 1-3, wherein L 2 is unsubstituted.

37. The compound according to any one of claims 1-3, wherein R 4 and R 5 are each independently a C1-C6 hydrocarbyl group.

38. The compound according to claim 37, wherein R 4 and R 5 are both methyl groups.

39. The compound according to claim 37, wherein R 4 and R 5 are both ethyl.

40. The compound according to claim 37, wherein R 4 is methyl and R 5 is n-butyl.

41. The compound according to any one of claims 1-3, R 4 and R 5 together with the N to which they are attached form a heterocyclic group, wherein the heterocyclic group is a 3- to 7-membered non-aromatic ring group.

42. The compound according to claim 41, wherein the heterocyclic group is a 5-membered heterocyclic group.

43. The compound according to claim 42, wherein the heterocyclic group has the following structure:

44. The compound according to claim 1, wherein the compound has one of the following structures:

45. A composition comprising the compound according to any one of claims 1-44 and a therapeutic agent.

46. The composition according to claim 45, which further comprises one or more excipients selected from the following: neutral lipids, steroids and polymer-conjugated lipids.

47. The composition according to claim 46, wherein the composition comprises one or more neutral lipids selected from the following: DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.

48. The composition according to claim 47, wherein the neutral lipid is DSPC.

49. The composition according to any one of claims 46-48, wherein the molar ratio of the compound to the neutral lipid ranges from 2:1 to 8:

1.

50. The composition according to any one of claims 46-48, wherein the steroid is cholesterol.

51. The composition according to claim 50, wherein the molar ratio of the compound to the cholesterol ranges from 5:1 to 1:

1.

52. The composition according to any one of claims 46-48, wherein the polymer-conjugated lipid is a polyethylene glycolated lipid.

53. The composition according to claim 52, wherein the molar ratio of the compound to the polyethylene glycolated lipid ranges from 100:1 to 20:

1.

54. The composition according to claim 52, wherein the polyethylene glycolated lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer or PEG dialkoxypropyl carbamate.

55. The composition according to claim 52, wherein the polyethylene glycolated lipid has the following structure (II): or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: R 8 and R 9 each independently is a straight-chain or branched, saturated or unsaturated hydrocarbon group chain having 10 to 30 carbon atoms, wherein the hydrocarbon group chain is optionally interrupted by one or more ester bonds; and The average value of w ranges from 30 to 60.

56. The composition according to claim 55, wherein R 8 and R 9 are each independently a straight-chain saturated hydrocarbon group chain having 12 to 16 carbon atoms.

57. The composition according to any one of claims 55 or 56, wherein the average value of w is 49.

58. A composition according to any one of claims 45 - 48, wherein the therapeutic agent comprises a nucleic acid.

59. The composition according to claim 58, wherein the nucleic acid is selected from antisense RNA and messenger RNA.

60. A composition according to any one of claims 45 - 48, wherein the composition comprises lipid nanoparticles.

61. Use of a composition according to any one of claims 45 - 60 in the manufacture of a medicament for administering a therapeutic agent to a patient in need thereof, wherein the compound is for delivering the therapeutic agent.

62. A lipid nanoparticle comprising a compound according to any one of claims 1 - 44.

63. The lipid nanoparticle according to claim 62, which further comprises a therapeutic agent.

64. The lipid nanoparticle according to claim 63, wherein the therapeutic agent comprises a nucleic acid.

65. The lipid nanoparticle according to claim 64, wherein the nucleic acid is selected from antisense RNA and messenger RNA.

66. The lipid nanoparticle according to any one of claims 62 - 65, which further comprises one or more excipients selected from: neutral lipids, steroids, and polymer - conjugated lipids.

67. The lipid nanoparticle according to claim 66, wherein the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

68. The lipid nanoparticle according to claim 67, wherein the neutral lipid is DSPC.

69. The lipid nanoparticle according to claim 66, wherein the molar ratio of the compound to the neutral lipid ranges from 2:1 to 8:

1.

70. The lipid nanoparticle according to claim 66, wherein the steroid is cholesterol.

71. The lipid nanoparticle according to claim 70, wherein the molar ratio of the compound to cholesterol ranges from 5:1 to 1:

1.

72. The lipid nanoparticle according to claim 66, wherein the polymer - conjugated lipid is a polyethylene glycol - conjugated lipid.

73. The lipid nanoparticle according to claim 72, wherein the molar ratio of the compound to the polyethylene glycol - conjugated lipid ranges from 100:1 to 20:

1.

74. The lipid nanoparticle according to claim 72 or 73, wherein the polyethylene glycol - conjugated lipid is PEG - DAG, PEG - PE, PEG - S - DAG, PEG - cer, or PEG dialkoxypropyl carbamate.

75. The lipid nanoparticle according to claim 72 or 73, wherein the polyethylene glycol - conjugated lipid has the following structure (II): or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein: R 8 and R 9 each independently is a straight-chain or branched, saturated or unsaturated hydrocarbon group chain having 10 to 30 carbon atoms, wherein said hydrocarbon group chain is optionally interrupted by one or more ester bonds; and The average value of w ranges from 30 to 60.

76. The lipid nanoparticle according to claim 75, wherein R 8 and R 9 are each independently a straight-chain saturated hydrocarbon group chain containing 12 to 16 carbon atoms.

77. The lipid nanoparticle according to claim 75, wherein the average value of w is 49.

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