Lipids for lipid nanoparticle delivery of active agents

Abstract

To provide novel cationic lipids that can be used in combination with other lipid components, such as neutral lipids, cholesterol and polymer conjugated lipids, to form lipid nanoparticles with oligonucleotides, to facilitate the intracellular delivery of therapeutic nucleic acids both in vitro and in vivo.SOLUTION: The present invention provides a compound represented by the following structure. [R1 represents a substituted / unsubstituted C1-C24 alkyl or the like; R2 and R3 each represent a substituted / unsubstituted C1-C36 alkyl; R4 and R5 each represent a substituted / unsubstituted C1-C6 alkyl or the like; L1, L2 and L3 each represent a substituted / unsubstituted C1-C18 alkylene; G1 represents a direct bond, -(CH2)nO(C=O)- or the like; G2 and G3 each represent -(C=O)O- or -O(C=O)-.]SELECTED DRAWING: None

Description

[Technical Field]

[0001] 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 bearing oligonucleotides, which can facilitate intracellular delivery of therapeutic nucleic acids (e.g., oligonucleotides, messenger RNA) both in vivo and in vitro. [Background technology]

[0002] Numerous efforts have been made to deliver nucleic acids to affect desired responses in biological systems. Nucleic acid-based therapeutics hold great promise, but to realize this potential, more effective delivery of nucleic acids to appropriate sites within cells or organisms is needed. Therapeutic nucleic acids include, for example, messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, immunostimulatory nucleic acids, antagomirs, antimirs, mimics, supermirs, and aptamers. Some nucleic acids, such as mRNA or plasmids, can be used to induce the expression of specific cellular products, useful for treating diseases associated with protein or enzyme deficiencies, for example. The therapeutic applications of translatable nucleotide delivery are extremely broad, due to the ability to synthesize constructs and generate selective protein sequences, whether native to the system or not. The expression products of nucleic acids can enhance the levels of existing proteins, replace missing or non-functioning proteins, or introduce new proteins and associated functions into cells or organisms.

[0003] Some nucleic acids, such as miRNA inhibitors, can be used to induce the expression of specific cellular products regulated by miRNAs, for example, to treat diseases associated with protein or enzyme deficiencies. Because constructs can be synthesized to inhibit one or more miRNAs that regulate the expression of mRNA products, the therapeutic applications of miRNA inhibition are extremely broad. Inhibition of endogenous miRNAs can enhance the expression of their downstream target endogenous proteins, restoring the proper function of cells or organisms as a means of treating diseases associated with specific miRNAs or groups of miRNAs.

[0004] Other nucleic acids can downregulate the intracellular levels of specific mRNAs, thereby downregulating the synthesis of the corresponding proteins through processes such as RNA interference (RNAi) or complementary binding of antisense RNA. Because oligonucleotide constructs can be synthesized with nucleotide sequences directed against target mRNAs, the therapeutic applications of antisense oligonucleotides and RNAi are also very broad. Targets can include mRNAs in normal cells, mRNAs associated with disease states such as cancer, and mRNAs from infectious agents such as viruses. Antisense oligonucleotide constructs have previously demonstrated the ability to specifically downregulate target proteins through the degradation of similar mRNAs in both in vitro and in vivo models. Furthermore, antisense oligonucleotide constructs are currently being evaluated in clinical studies.

[0005] However, currently, the use of oligonucleotides in therapeutic applications faces two problems. First, free RNA is susceptible to nuclease digestion in plasma. Second, free RNA has limited ability to access the intracellular compartment where the relevant translation machinery is located. Lipid nanoparticles formed from cationic lipids and other lipid components, such as neutral lipids, cholesterol, PEG, PEGylated lipids, and oligonucleotides, have been used to inhibit the degradation of RNA in plasma and promote the cellular uptake of oligonucleotides.

[0006] There is a need for improved cationic lipids and lipid nanoparticles for the delivery of oligonucleotides. Preferably, these lipid nanoparticles provide an optimal drug-lipid ratio, protect the nucleic acid from degradation and clearance in serum, are suitable for systemic or local delivery, and provide intracellular delivery of nucleic acid. Furthermore, these lipid-nucleic acid particles are well tolerated and provide an appropriate therapeutic index, so that treatment of patients with effective doses of nucleic acid is not associated with unacceptable toxicity and / or risk to the patient. The present disclosure provides these and related advantages. Summary of the Invention

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

[0008] In one embodiment, the following structure (I): [ka] (I) [In the formula, 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.] or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

[0009] Also provided is a pharmaceutical composition comprising one or more compounds of structure (I) and a therapeutic agent. In some embodiments, the pharmaceutical composition further comprises one or more components selected from neutral lipids, charged lipids, steroids, and polymer-bound lipids. Such compositions are useful for forming lipid nanoparticles for delivery of therapeutic agents.

[0010] In other embodiments, the present disclosure provides a method of administering a therapeutic agent to a patient in need thereof, comprising preparing or providing a lipid nanoparticle composition comprising a compound of structure (I) and a therapeutic agent, and delivering or administering the composition to the patient.

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

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

[0013] The present disclosure is based, in part, on the discovery of novel cationic (amino) lipids that offer advantages when used in lipid nanoparticles for the in vivo delivery of active or therapeutic agents, such as nucleic acids, to mammalian cells. In particular, embodiments of the present disclosure provide nucleic acid-lipid nanoparticle compositions comprising one or more of the novel cationic lipids described herein, which provide enhanced nucleic acid activity in vivo and improved composition tolerance, resulting in a significant increase in therapeutic index compared to previous nucleic acid-lipid nanoparticle compositions. In other embodiments, the disclosed lipids and lipid nanoparticles comprising them have improved safety and / or tolerance when used to deliver active agents, such as nucleic acids.

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

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

[0016] As described herein, lipid nanoparticle embodiments of the present 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 interference complementary RNA (micRNA), DNA, multivalent RNA, Dicer substrate RNA, complementary DNA (cDNA), etc. Accordingly, certain lipid nanoparticle embodiments and compositions of the present disclosure can be used to induce expression of a desired protein both in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more of the novel cationic lipids described herein, where the lipid nanoparticles encapsulate or associate with a nucleic acid to be expressed to produce the desired protein (e.g., messenger RNA or a plasmid encoding the desired protein) or to inhibit a process that silences mRNA expression (e.g., an miRNA inhibitor). Alternatively, the lipid nanoparticles and compositions of the present disclosure can be used to reduce target gene and protein expression both in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more of the novel cationic lipids described herein, where the lipid nanoparticles encapsulate or are associated with a nucleic acid (e.g., an antisense oligonucleotide or small interfering RNA (siRNA)) that reduces target gene expression. The lipid nanoparticles and compositions of the present disclosure can also be used for the co-delivery of different nucleic acids (e.g., mRNA and plasmid DNA), either separately or in combination, and are useful, for example, for providing effects where co-localization of different nucleic acids (e.g., mRNA encoding an appropriate gene-modifying enzyme and a DNA segment for integration into the host genome) is desired.

[0017] Nucleic acids for use in the embodiments of the present disclosure can be prepared according to any available technique. In the case of mRNA, the primary method of preparation is, but is not limited to, enzymatic synthesis (also called in vitro transcription), which is currently the most efficient method for generating specific mRNAs of long sequences. In vitro transcription describes the process of template-directed synthesis of RNA molecules from an engineered DNA template composed of an upstream bacteriophage promoter sequence (e.g., including but not limited to those from T7, T3, and SP6 coliphages) linked to a downstream sequence encoding a gene of interest. Template DNA can be prepared for in vitro transcription from many sources using suitable techniques well known in the art, including, but not limited to, plasmid DNA and polymerase chain reaction amplification (see Linpinsel, JL and Conn, GL, General protocols for preparation of plasmid DNA template and Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LD in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941 Conn GL (ed), New York, NY Humana Press, 2012).

[0018] RNA transcription is carried out in vitro using a linearized DNA template in the presence of the corresponding RNA polymerase and ribonucleoside triphosphates (rNTPs) of adenosine, guanosine, uridine, and cytidine under conditions that support polymerase activity while minimizing potential degradation of the resulting mRNA transcript. In vitro transcription can be performed using a variety of commercially available kits and reagents, including, but not limited to, the RiboMax Large Scale RNA Production System (Promega) and MegaScript Transcription kits (Life Technologies), including commercially available RNA polymerases and rNTPs. Methods for in vitro transcription of mRNA are well known in the art (e.g., Losick, R., 1972, In vitro transcription, Ann Rev Biochem v.41 409-46; Kamakaka, RT and Kraus, WL 2001. In Vitro Transcription. Current Protocols in Cell Biology. 2:11.6:11.6.1-11.6.17; Beckert, B. And Masquida, B., (2010) Synthesis of RNA by In Vitro Transcription in RNA in Methods in Molecular Biology v. 703 (Neilson, H. Ed), New York, NY Humana Press, 2010; Brunelle, JL and Green, R., 2013, Chapter Five - In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology v. 530, 101-114, all of which are incorporated herein by reference).

[0019] The desired in vitro transcribed mRNA is then purified from undesired components of the transcription or related reaction, including unincorporated rNTPs, protein enzymes, salts, short RNA oligos, etc. Techniques for isolating mRNA transcripts are well known in the art. Well-known methods include phenol / chloroform extraction or precipitation with either alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride. Further examples of usable purification methods include, but are not limited to, size exclusion chromatography (Lukavsky, PJ and Puglisi, JD, 2004, Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides, RNA v.10, 889-893), silica-based affinity chromatography and polyacrylamide gel electrophoresis (Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LD in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA synthesis Methods v. 941 Conn GL (ed), New York, NY Humana Press, 2012). Purification can be performed using various commercially available kits, including, but not limited to, the SV Total Isolation System (Promega) and the In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).

[0020] Furthermore, while reverse transcription can generate large amounts of mRNA, the product may contain numerous aberrant RNA impurities associated with undesired polymerase activity that may need to be removed from full-length mRNA preparations. These include short RNAs resulting from aberrant transcription initiation, as well as double-stranded RNA (dsRNA) generated by RNA-dependent RNA polymerase activity, RNA-primed transcription from RNA templates, and self-complementary 3' extensions. It has been demonstrated that these contaminants, including dsRNA structures, can result in unwanted immunostimulatory activity by interacting with various innate immune sensors in eukaryotic cells that recognize specific nucleic acid structures and induce potent immune responses. Furthermore, mRNA translation can be dramatically reduced due to reduced protein synthesis during the innate cellular immune response. Accordingly, 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, PH Ed), 2013).HPLC-purified mRNA has been reported to be translated to very high levels, especially in primary cells and in vivo.

[0021] A wide variety of modifications have been described in the art to alter specific properties of in vitro transcribed mRNA and improve its utility. These include, but are not limited to, modifications to the 5' and 3' ends of mRNA. Endogenous eukaryotic mRNAs typically contain a cap structure at the 5' end of the mature molecule, which plays an important role in mediating the binding of mRNA cap-binding protein (CBP), which contributes to intracellular mRNA stability and improved efficiency of mRNA translation. Therefore, the highest levels of protein expression are achieved with capped mRNA transcripts. The 5' cap contains a 5'-5' triphosphate linkage between the 5'-most nucleotide and a guanine nucleotide. The conjugated guanine nucleotide is methylated at the N7 position. Additional modifications include methylation of the last and penultimate 5'-most nucleotides at the 2'-hydroxyl group.

[0022] Several different cap structures can be used to generate 5' caps for in vitro transcribed synthetic mRNAs. 5'-capping of synthetic mRNAs can be performed co-transcriptionally (i.e., capping during in vitro transcription) with chemical cap analogs. For example, the anti-reverse cap analog (ARCA) cap contains a 5'-5'-triphosphate guanine-guanine linkage, in which one guanine contains an N7 methyl group and a 3'-O-methyl group. However, during this co-transcription process, up to 20% of transcripts remain uncapped, and synthetic cap analogs are not identical to the 5' cap structure of actual cellular mRNAs, potentially resulting in reduced translatability and cellular stability. Alternatively, synthetic mRNA molecules can also be enzymatically capped post-transcriptionally. These can generate more realistic 5'-cap structures that structurally or functionally mimic endogenous 5' caps, enhancing cap-binding protein binding, extending half-life, reducing susceptibility to 5' endonucleases, and / or reducing 5' decapping. Numerous synthetic 5' cap analogs have been developed and are known in the art to enhance mRNA stability and translatability (see, e.g., Grudzien-Nogalska, E., Kowalska, J., Su, W., Kuhn, AN, Slepenkov, SV, Darynkiewicz, E., Sahin, U., Jemielity, J., and Rhoads, RE, Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH Ed), 2013).

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

[0024] Poly(A) tailing of in vitro transcribed mRNA can be achieved using various methods, including, but not limited to, cloning a poly(T) tract into a DNA template or post-transcriptional addition using poly(A) polymerase. In the first case, mRNAs with poly(A) tails of defined lengths can be in vitro transcribed, depending on the size of the poly(T) tract, but additional template manipulation is required. In the latter case, poly(A) tails are enzymatically added to in vitro transcribed mRNA using poly(A) polymerase, which catalyzes the incorporation of adenine residues into the 3' end of RNA. This does not require additional DNA template manipulation, but can result in mRNAs with poly(A) tails of heterogeneous lengths. 5'-capping and 3'-poly(A) tailing can be performed using various commercially available kits, including, but not limited to, the Poly(A) Polymerase Tailing kit (EpiCenter), the mMESSAGE mMACHINE T7 Ultra kit, and the Poly(A)Tailing kit (Life Technologies), as well as commercially available reagents, various ARCA caps, poly(A) polymerases, etc.

[0025] In addition to 5' caps and 3' polyadenylation, other modifications of in vitro transcription have been reported to provide advantages in terms of translation efficiency and stability. It is well known in the art that pathogenic DNA and RNA can be recognized by various sensors in eukaryotes and elicit strong innate immune responses. The ability to distinguish between pathogenic and self DNA and RNA has been shown to be based, at least in part, on structural and nucleoside modifications, as most nucleic acids from natural sources contain modified nucleosides. In contrast, in vitro-synthesized RNA lacks these modifications, which can be immunostimulatory and inhibit effective mRNA translation, as discussed above.Introduction of modified nucleosides into in vitro transcribed mRNA can be used to prevent recognition and activation of RNA sensors, thereby reducing this undesirable immunostimulatory activity and improving translational competence (e.g., Kariko, K. And Weissman, D. 2007, Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: Implication for therapeutic RNA development, Curr Opin Drug Discov Devel, v.10 523-532; Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH Ed), 2013; Kariko, K., Muramatsu, H., Welsh, F.A., Ludwig, J., Kato, H., Akira, S., Weissman, (See, e.g., D., 2008, Incorporation of Pseudouridine Into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability, Mol Ther v.16, 1833-1840). The modified nucleosides and nucleotides used in the synthesis of modified RNA can be prepared, monitored, and utilized using common methods and techniques known in the art. A wide variety of nucleoside modifications are available that can be incorporated into in vitro transcribed mRNA, either alone or in some combination with other modified nucleosides (see, e.g., US2012 / 0251618).In vitro synthesis of nucleoside-modified mRNA has been reported to have enhanced translational potential but reduced ability to activate immune sensors.

[0026] Other components of mRNA that can be modified to benefit translatability and stability include the 5' and 3' untranslated regions (UTRs). Optimization of UTRs (preferable 5' and 3' UTRs can be derived from cellular or viral RNA) has been shown to increase mRNA stability and translation efficiency of in vitro transcribed mRNAs, both independently and together (see, e.g., Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in synthetic messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH Ed), 2013).

[0027] In addition to mRNA, other nucleic acid payloads can be used for the present disclosure.For oligonucleotides, preparation methods include, but are not limited to, chemical synthesis, enzymatic and chemical cleavage of longer precursors, in vitro transcription as described above, etc.The synthesis method of DNA and RNA nucleotides is widely used and well known in the art (for example, Gait, MJ (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, DC: IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, NJ) Totowa, NJ: Humana Press, 2005; which are both incorporated herein by reference).

[0028] For plasmid DNA, formulations used in embodiments of the present disclosure generally, but are not limited to, utilize in vitro propagation and isolation of plasmid DNA in liquid cultures of bacteria containing the plasmid of interest. The presence of a gene in the plasmid of interest that encodes resistance to a particular antibiotic (penicillin, kanamycin, etc.) allows bacteria containing the plasmid of interest to selectively grow 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, KL and Brent, R., (2001), Large-Scale Preparation of Plasmid DNA, Current Protocols in Molecular Biology, 41:11:1.7:1.7.1-1.7.16; Rozkov, A., Larsson, B., Gillstroem, S., Bjoernestedt, R. and Schmidt, SR, (2008), Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture, Biotechnol. Bioeng., 99: 557-566; and U.S. Pat. No. 6,197,553 B1). Plasmid isolation can be performed using a variety of commercially available kits, including but not limited to Plasmid Plus (Qiagen), GenJET plasmid MaxiPrep (Thermo), and Pure Yield MaxiPrep (Promega) kits, as well as commercially available reagents.

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

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

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

[0032] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0034] The phrase "inducing the expression of a desired protein" refers to the ability of a nucleic acid to increase the expression of a desired protein.To determine the level of protein expression, a test sample (e.g., a sample of cells in culture that express the desired protein) or a test mammalian (e.g., a mammalian, such as a human or animal) model, such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model, is contacted with a nucleic acid (e.g., a nucleic acid combined with a lipid of the present 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 cells in culture that express the desired protein) or a control mammalian (e.g., a mammalian, such as a human or animal) model, such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model, which has not been contacted or administered with the nucleic acid.If the desired protein is present in the control sample or control mammal, the expression of the desired protein in the control sample or control mammal can be assigned a value of 1.0. In certain embodiments, inducing expression of a desired protein is achieved when the ratio of the expression of the desired protein in a test sample or test mammal to the level of expression of the desired protein in a control sample or control mammal is greater than 1, e.g., about 1.1, 1.5, 2.0, 5.0, or 10.0. If the desired protein is not present in a control sample or control mammal, inducing expression of a desired protein is achieved when measurable levels of the desired protein are detected in the test sample or test mammal. Those skilled in the art will recognize suitable assays for determining the expression level of a protein in a sample, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzymatic function, and phenotypic assays, or assays based on reporter proteins capable of generating fluorescence or luminescence under appropriate conditions.

[0035] The phrase "inhibit the expression of a target gene" refers to the ability of a nucleic acid to silence, reduce, or inhibit the expression of a target gene.To determine the degree of gene silencing, a test sample (e.g., a sample of cells in culture that express the target gene) or a test mammalian (e.g., a mammalian, such as a human or animal) model, for example, 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 cells in culture that express the target gene) or a control mammalian (e.g., a mammalian, such as a human or animal), for example, a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model that has not been contacted or administered with 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, silencing, inhibition, or reduction of expression of a target gene is achieved when the level of target gene expression in a test sample or test mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% of the level of target gene expression in a control sample or control mammal. In other words, the nucleic acid can silence, reduce, or inhibit expression of the target gene by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in a test sample or test mammal relative to the level of target gene expression in a control sample or control mammal not contacted or administered with the nucleic acid.Suitable assays for determining the level of target gene expression include, but are not limited to, testing protein or mRNA levels using techniques known to those of skill in the art, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those of skill in the art.

[0036] An "effective amount" or "therapeutically effective amount" of an active agent or therapeutic agent, such as a therapeutic nucleic acid, is an amount sufficient to produce a desired effect, such as an increase or inhibition of expression of a target sequence compared to the normal expression level detected in the absence of the nucleic acid. In the case of an expression product that is not present in the absence of the nucleic acid, an increase in expression of the target sequence is achieved when a measurable level is detected. In the case where the expression product is present at a certain level before 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, compared 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 more. Inhibition of expression of a target gene or target sequence is achieved when the nucleic acid, such as an antisense oligonucleotide, achieves about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% inhibition of expression of a target gene or target sequence compared to a control. Suitable assays for measuring expression of a target gene or target sequence include testing protein or RNA levels using techniques known to those of skill in the art, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of appropriate reporter proteins, and phenotypic assays known to those of skill in the art.

[0037] As used herein, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form, including DNA, RNA, and hybrids thereof. DNA can be in the form of an antisense molecule, plasmid DNA, cDNA, PCR product, or vector. RNA can be in the form of small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, polyvalent RNA, Dicer substrate RNA, or viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which occur synthetically, naturally, and non-naturally, and which have similar binding properties to the reference nucleic acid. Such analogs include, but are not limited to, phosphorothioates, phosphoramidites, methyl phosphonates, chiral methyl phosphonates, 2'-0-methyl ribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise specified, this term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid.Unless otherwise specified, a specific 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 stated sequence.Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate group."Base" includes purines and pyrimidines, and further includes the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and naturally occurring analogues, and synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications that place new reactive groups such as, for example, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

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

[0039] As used herein, "gene product" means a product of a gene, such as an RNA transcript or a polypeptide.

[0040] The term "lipid" refers to a group of organic compounds, including but not limited to esters of fatty acids, which are generally characterized by poor solubility in water but solubility in many organic solvents. They are usually divided into at least three classes: (1) "simple lipids" (which include fats, oils, and waxes); (2) "complex lipids" (which include phospholipids and glycolipids); and (3) "derived lipids" (e.g., steroids).

[0041] "Steroids" are compounds with the following carbon skeleton: [ka] Examples of steroids include, but are not limited to, cholesterol.

[0042] "Cationic lipid" refers to a lipid that can be positively charged. For example, cationic lipids contain one or more amine groups that are positively charged. Preferred cationic lipids are ionizable, so that they can exist in a positively charged or neutral form depending on pH. The ionization of cationic lipids affects the surface charge of lipid nanoparticles under various pH conditions. This charge state can affect the absorption of plasma proteins, blood clearance, and tissue distribution (Semple, SC, et al., Adv. Drug Deliv Rev 32:3-17 (1998)), and the ability to form endosomolytic non-bilayer structures that are important for intracellular delivery of nucleic acids (Hafez, IM, et al., Gene Ther 8:1188-1196 (2001)).

[0043] The term "polymer-conjugated lipid" refers to a molecule that contains both a lipid portion and a polymer portion. An example of a polymer-conjugated lipid is a PEGylated lipid. The term "PEGylated lipid" refers to a molecule that contains both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG).

[0044] The term "neutral lipid" refers to any of several lipid species that exist in either uncharged or neutral zwitterionic form at selected pH levels. At physiological pH, such lipids include, but are not limited to, phosphotidylcholines such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), steroids such as sphingomyelin (SM), ceramides, sterols, and derivatives thereof. Neutral lipids can be synthetic or naturally occurring.

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

[0046] The term "lipid nanoparticle" refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) that comprises one or more of the compounds of structure (I) or other specific cationic lipids. In some embodiments, lipid nanoparticles comprising the disclosed cationic lipids (e.g., compounds of structure (I)) are included in formulations that can be used to deliver active or therapeutic agents, such as nucleic acids (e.g., mRNA), to a desired target site (e.g., a cell, tissue, organ, tumor, etc.). In some embodiments, the lipid nanoparticle comprises a compound of structure (I) and a nucleic acid. Such lipid nanoparticles typically comprise a compound of structure (I) and one or more excipients selected from neutral lipids, charged lipids, steroids, and polymer-bound lipids. In some embodiments, the active or therapeutic agent, such as a nucleic acid, can be encapsulated in the lipid portion of the lipid nanoparticle or in the aqueous space enclosed by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects elicited by mechanisms of the host organism or cells, such as a harmful immune response.

[0047] In various embodiments, the lipid nanoparticles have an average particle size of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, lipid nanoparticles are substantially non-toxic.In certain embodiments, when nucleic acid is present in lipid nanoparticles, it is resistant to degradation by nuclease in aqueous solution.The lipid nanoparticles containing nucleic acid and the method for producing the same are disclosed in, for example, US Patent Publication No. 2004 / 0142025, 2007 / 0042031, and PCT Publication No. WO2013 / 016058 and WO2013 / 086373 (the disclosures of which are all incorporated herein by reference in their entirety for all purposes).

[0048] As used herein, "lipid encapsulation" refers to lipid nanoparticles that provide an active or therapeutic agent, such as a nucleic acid (e.g., mRNA), with complete encapsulation, partial encapsulation, or both. In one embodiment, the nucleic acid (e.g., mRNA) is completely encapsulated in the lipid nanoparticle.

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

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

[0051] As used herein, "systemic delivery" refers to the delivery of a therapeutic product that allows widespread exposure of an active agent within an organism. Some techniques of administration can allow certain agents to be delivered systemically while others cannot. Systemic delivery means that a useful, preferably therapeutic, amount of the agent is exposed to most parts of the body. Systemic delivery of lipid nanoparticles can be by any means known in the art, including, for example, intravenous, intraarterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery.

[0052] As used herein, "local delivery" refers to the direct delivery of an active agent to a target site within an organism. For example, a drug can be delivered locally by direct injection into a disease site such as a tumor, another target site such as an inflammatory site, or a target organ such as the liver, heart, pancreas, or kidney. 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.

[0053] "Alkyl" means a radical of a straight or branched hydrocarbon chain consisting solely of carbon and hydrogen atoms, which is saturated or unsaturated (i.e., contains one or more double bonds (alkenyl) and / or triple bonds (alkynyl)), e.g., 1 to 24 carbon atoms (C1-C 24 alkyl), 4 to 20 carbon atoms (C4-C 20 alkyl), 6 to 16 carbon atoms (C6-C 16 alkyl), 6 to 9 carbon atoms (C6-C9 alkyl), 1 to 15 carbon atoms (C1-C 15 alkyl), 1 to 12 carbon atoms (C1-C 12alkyl), 1 to 8 carbon atoms (C-C alkyl), or 1 to 6 carbon atoms (C-C alkyl), which is attached 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 (t-butyl), 3-methylhexyl, 2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Unless stated otherwise in the specification, alkyl groups may be optionally substituted.

[0054] "Alkylene" or "alkylene chain" means a straight or branched divalent hydrocarbon chain, consisting solely of carbon and hydrogen, that is saturated or unsaturated (i.e., one or more double bonds (alkenylene) and / or triple bonds (alkynylene)), that connects the rest of the molecule to another group, e.g., from 1 to 24 carbon atoms (C1-C 24 Alkylene, 1 to 15 carbon atoms (C1-C 15 Alkylene, 1 to 12 carbon atoms (C1-C 12 The alkylene chain has 1 to 8 carbon atoms (C1-C8 alkylene), 1 to 6 carbon atoms (C1-C6 alkylene), 2 to 4 carbon atoms (C2-C4 alkylene), and 1 to 2 carbon atoms (C1-C2 alkylene), for example, methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, and the like. The alkylene chain is attached to the rest of the molecule through a single or double bond and to other groups through a single or double bond. The points of attachment of the alkylene chain to the rest of the molecule and to other groups can be through one carbon or any two carbons within the chain. Unless stated otherwise in the specification, the alkylene chain may be optionally substituted.

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

[0056] "Cycloalkylene" means a divalent cycloalkyl group. Unless stated otherwise in the specification, a cycloalkylene group may be optionally substituted.

[0057] "Heterocyclyl" or "heterocycle" refers to a stable 3- to 18-membered (e.g., 5-, 6-, or 7-membered) non-aromatic ring group having 1 to 12 ring carbon atoms (e.g., 2 to 12) and 1 to 6 ring heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Examples of such heterocyclyl groups include, but are not limited to, azetidinyl, pyrrolidinyl, piperidinyl, imidazolidinyl, tetrahydropyrimidinyl, and the like. Unless otherwise specified in this specification, heterocyclyl groups may be optionally substituted.

[0058] "Heteroaryl" refers to a 5- to 14-membered ring system containing a hydrogen atom, 1 to 13 ring carbon atoms, 1 to 6 ring heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, and at least one aromatic ring. Examples include, but are not limited to, pyrrolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, and the like. Unless otherwise specified herein, heteroaryl groups may be optionally substituted.

[0059] As used herein, the term "substituted" refers to any of the above groups (e.g., alkyl, alkylene, cycloalkyl, or cycloalkylene) in which at least one hydrogen atom has been replaced by a bond to a non-hydrogen atom, including, but not limited to, a halogen atom such as F, Cl, Br, or I; an oxo group (=O); a hydroxyl group (-OH); a C1-C2 12 Alkyl group; Cycloalkyl group; -(C=O)OR'; -O(C=O)R'; -C(=O)R'; -OR'; -S(O) x R';-S-SR';-C(=O)SR';-SC(=O)R';-NR'R';-NR'C(=O)R';-C(=O)NR'R';-NR'C(=O)NR'R';-OC(=O)NR'R';-NR'C(=O)OR';-NR'S(O) x NR'R';-NR'S(O) x R'; and -S(O) x NR'R', where R' at each occurrence is independently H, C-C 15 alkyl or cycloalkyl, and x is 0, 1, or 2. In some embodiments, the substituent is C-C 12 In another embodiment, the substituent is an alkyl group. In another embodiment, the substituent is a cycloalkyl group. In another embodiment, the substituent is a halo group, such as fluoro. In another embodiment, the substituent is an oxo group. In another embodiment, the substituent is a hydroxyl group. In another embodiment, the substituent is an alkoxy group (-OR). In another embodiment, the substituent is a carboxyl group. In another embodiment, the substituent is an amine group (-NR'R').

[0060] "Optional" or "optionally" (e.g., optionally substituted) means that the subsequently described circumstance event may or may not occur, and the description includes cases where said event or circumstance occurs and cases where it does not occur. For example, "optionally substituted alkyl" means that the alkyl group may be substituted or unsubstituted, and the description includes both substituted alkyl groups and alkyl groups that have no substituents.

[0061] The disclosure disclosed herein is also intended to encompass all pharmaceutically acceptable compounds of the compound of structure (I) that are isotopically labeled by replacing one or more atoms with atoms of different atomic mass or mass number.Isotopes that can be incorporated into the disclosed compounds include, for example, isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, for example, 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 may be useful to help determine or measure the efficacy of compounds, for example, by characterizing the site or mechanism of action, or binding affinity to a pharmacologically important site of action. Certain isotopically labeled compounds of structure (I), (IA), or (IB), e.g., compounds incorporating a radioisotope, are useful in drug and / or substrate tissue distribution studies. The radioisotope tritium, i.e., 3 H, and carbon-l4, i.e. 14 C is particularly useful for this purpose in view of its ease of incorporation and ready means for detection.

[0062] Deuterium i.e. 2 Substitution with heavier isotopes, such as H, may confer certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced required dosage, and therefore may be preferable in some circumstances.

[0063] 11 C. 18 F,15 O. 13 Substitution with positron emitting isotopes, such as N, can be useful in positron emission tomography (PET) studies to examine substrate receptor occupancy. Isotopically labeled compounds of structure (I) can generally be prepared by conventional techniques known to those skilled in the art, or by processes analogous to those described in the preparations and examples below, by substituting a suitable isotopically labeled reagent for a previously employed unlabeled reagent.

[0064] The embodiments disclosed herein are also meant to encompass in vivo metabolic products of the disclosed compounds. Such products may result, for example, from oxidation, reduction, hydrolysis, amidation, esterification, etc., of the administered compound, primarily due to enzymatic processes. Accordingly, embodiments of the present disclosure include compounds produced by a process comprising administering a compound of the present disclosure to a mammal for a period of time sufficient to produce its metabolic products. Such products are typically 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 to occur, and isolating the conversion products from urine, blood, or other biological sample.

[0065] "Stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.

[0066] "Mammal" includes both humans and domestic animals, such as laboratory animals and household pets (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits), and non-domestic animals, such as wildlife.

[0067] A "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, an adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier, which is approved by the U.S. Food and Drug Administration as acceptable for use in humans or domestic animals.

[0068] "Pharmaceutically acceptable salt" includes both acid and base addition salts.

[0069] "Pharmaceutically acceptable acid addition salts" means salts formed with inorganic and organic acids, including, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, which retain the biological effectiveness and properties of the free bases and are not biologically or otherwise undesirable, and organic acids, including, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, and the like. Acids that can be used include carboxylic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid.

[0070] "Pharmaceutically acceptable base addition salt" means a salt that retains the biological effectiveness and properties of the free acid and is not biologically or otherwise undesirable. These salts are prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0071] Crystallization often 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 may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of the present disclosure may exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc., and their corresponding solvate forms. The solvates of the compounds of the present disclosure may be true solvates, while in other cases, the compounds of the present disclosure may simply retain incidental water or may be a mixture of water and some incidental solvents.

[0072] "Pharmaceutical composition" means a formulation of a compound of the present disclosure and a vehicle generally accepted in the art for delivering the biologically active compound to a mammal (e.g., a human). Such a vehicle includes any pharmaceutically acceptable carrier, diluent, or excipient therefor.

[0073] "Effective amount" or "therapeutically effective amount" refers to the amount of a compound of the present disclosure that, when administered to a mammal, preferably a human, is sufficient to effect treatment in the mammal, preferably a human. The amount of lipid nanoparticles of the present disclosure that constitutes a "therapeutically effective amount" varies depending on the compound, the disease and its severity, the method of administration, and the age of the mammal being treated, but can be determined by one skilled in the art in the course of routine, given their knowledge and this disclosure.

[0074] As used herein, "treating" or "treatment" encompasses the treatment of a disease or condition of interest in a mammal (preferably a human) having the disease or condition of interest, and includes: (i) To prevent a disease or illness from occurring in a mammal (especially if the mammal is susceptible to the disease but has not yet been diagnosed as having it); (ii) inhibiting a disease or illness (i.e., arresting its development); (iii) alleviating the disease or illness (i.e., causing regression of the disease or illness); or (iv) Relieving symptoms caused by a disease or illness (i.e., relieving pain without addressing the underlying disease or illness). As used herein, the terms "disease" and "illness" may be used interchangeably or may differ in that a particular illness or condition may not have a known causative agent (and thus the etiology has not yet been elucidated), and therefore is not yet recognized as a disease, but only as an undesirable illness or syndrome in which more or less specific symptoms have been identified by a clinician.

[0075] The compounds of the present disclosure, or pharmaceutically acceptable salts thereof, may contain one or more stereocenters and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms, which may be defined with respect to absolute stereochemistry as (R)- or (S)-, or, in the case of 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. Optically active (+)- and (-), (R)- and (S)-, or (D)- and (L)-isomers can be prepared using chiral synthons or chiral reagents or separated using conventional techniques, such as chromatography and fractional crystallization. Conventional techniques for preparing / isolating individual enantiomers include chiral synthesis from appropriate optically pure precursors or separation of the racemate (or racemate of a salt or derivative) using, for example, chiral high-performance liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, unless specified otherwise, the compounds are intended to include both E and Z geometric isomers, as well as all tautomeric forms.

[0076] "Stereoisomers" refer to compounds consisting of the same atoms joined by the same bonds, but having different, incompatible three-dimensional structures. The present disclosure contemplates various stereoisomers and mixtures thereof, and includes "enantiomers," which refer to two stereoisomers whose molecules are non-superimposable mirror images of one another.

[0077] "Tautomer" means a shift 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 aforementioned compounds.

[0078] compound In one aspect, the present disclosure provides novel lipid compounds that can be combined with other lipid components, such as neutral lipids, charged lipids, steroids, and / or polymer-bound lipids, to form lipid nanoparticles with oligonucleotides.Without being bound by theory, it is believed that these lipid nanoparticles protect oligonucleotides from degradation in serum and provide effective cellular delivery of oligonucleotides in vitro and in vivo.

[0079] In one embodiment, the compound has the following structure (I): [ka] (I) [In the formula, R 1 is an optionally substituted C1-C 24 alkyl, or optionally substituted C-C 24 is alkenyl; R 2 and R 3 are each independently an optionally substituted C-C 36 is alkyl; R 4 and R 5 are each independently an optionally substituted C1-C6 alkyl, or R 4 and R 5together with the N to which they are attached form a heterocyclyl or heteroaryl; L 1 , L 2 , and L 3 are each independently an optionally substituted C-C 18 is alkylene; G 1 is a direct bond, -(CH2) n O(C=O)-, -(CH2) n (C=O)O- or -(C=O)-; G 2 and G 3 are each independently —(C═O)O—, or —O(C═O)—; and n is an integer greater than 0.] or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

[0080] In some embodiments, the compound has the following structure (IA): [ka] (IA) It is a compound represented by the formula:

[0081] In some embodiments, the compound has the following structure (IB): [ka] (IB) It is a compound represented by the formula:

[0082] In some embodiments, R 1 is an optionally substituted C-C 18 Alkyl, or C 14 -C 18 In one embodiment, R is alkenyl. 1 is C8 alkyl, C9 alkyl, C 10 Alkyl, C 12 Alkyl, C 14 Alkyl, or C16 In some embodiments, R 1 is C 16 In one embodiment, R is alkenyl. 1 is unbranched. In some embodiments, R 1 is branched. In one embodiment, R 1 is non-substituted.

[0083] In some embodiments, G 1 is a direct bond, -(CH2) n O(C=O)- or -(CH2) n (C═O)O—. In one embodiment, G 1 is a direct bond. In some embodiments, G 1 Ha-(CH2) n (C=O)O-, and n is greater than 1. In some embodiments, n is 1 to 20. In some embodiments, n is 1 to 10. In some embodiments, n is 5 to 11. In some embodiments, n is 6 to 10. In 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 some embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10.

[0084] In some embodiments, L 1 is C1-C6 alkylene. 1 is a C2 alkylene, a C3 alkylene, or a C4 alkylene. 1 is unbranched. In a more specific embodiment, L 1 is non-substituted.

[0085] In some embodiments, R 2 is C8-C 24In some embodiments, R 3 is C8-C 24 In some more specific embodiments, R 2 and R 3 are both C8-C 24 In some embodiments, R 2 and R 3 are each independently 11 Alkyl, C 12 Alkyl, C 13 Alkyl, C 14 Alkyl, C 15 Alkyl, C 16 Alkyl, C 18 Alkyl, or C 20 In one embodiment, R 2 is branched. In more specific embodiments, R 3 In some embodiments, R 2 and R 3 are each independently of the following structure: [ka] [In the formula, R 6 and R 7 are independently C2-C 12 It is alkyl. It is indicated by one of the following.

[0086] In some embodiments, R 2 and R 3 are each independently of the following structure: [ka] It is indicated by one of the following.

[0087] In some embodiments, L 2 and L 3 are independently C4-C 10 In one embodiment, L is alkylene. 2 and L 3and are both C5 alkylene. In some embodiments, L 2 and L 3 and are both C alkylene. In some embodiments, L 2 and L 3 and are both C alkylene. In some embodiments, L 2 and L 3 and are both C alkylene. In some embodiments, L 2 In some embodiments, L 3 is unbranched. In a more specific embodiment, L 2 is unsubstituted. In some embodiments, L 2 is non-substituted.

[0088] In some embodiments, R 4 and R 5 are each independently C1-C6 alkyl. In a more specific embodiment, R 4 and R 5 and R are both methyl. 4 and R 5 and R are both ethyl. 4 is methyl and R 5 is n-butyl. In some embodiments, R 4 and R 5 are both n-butyl. In a different embodiment, R 4 is methyl and R 5 is n-hexyl.

[0089] In some embodiments, R 4 and R 5 taken together with the N to which they are attached form a heterocyclyl. In certain embodiments, the heterocyclyl is a 5-membered heterocyclyl. In some embodiments, the heterocyclyl has the following structure: [ka] It is shown as follows.

[0090] In various different embodiments, the compound is a compound having one of the structures set forth in Table 1 below.

[0091] Table 1. Compounds included in embodiments of the compound of structure (I) [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7]

[0092] It is understood that any embodiment of the compounds of structure (I) above, and any particular substituents and / or variables in the compounds of structure (I) above, can be independently combined with other embodiments of the compounds of structure (I) and / or substituents and / or variables to form embodiments of the present disclosure not specifically set forth above. Furthermore, when a list of substituents and / or variables is recited in a particular embodiment and / or claim for a particular R group, G group, L group, or variable n, it is understood that the individual substituents and / or variables can be deleted from the particular embodiment and / or claim, respectively, and the remaining list of substituents and / or variables is considered to be within the scope of the present disclosure.

[0093] It is understood that combinations of substituents and / or variables within the depicted formulae herein are permissible only if such combinations result in stable compounds.

[0094] In some embodiments, lipid nanoparticles are provided comprising a compound of structure (I). The lipid nanoparticles may optionally include an excipient selected from a neutral lipid, a steroid, and a polymer-bound lipid.

[0095] In some embodiments, compositions are provided that include any one or more of the compounds of structure (I) and a therapeutic agent. For example, in some embodiments, the compositions include any one of the compounds of structure (I), a therapeutic agent, and one or more excipients selected from neutral lipids, steroids, and polymer-bound lipids. Other pharmaceutically acceptable excipients and / or carriers are also included in various embodiments of the compositions.

[0096] In some embodiments, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the neutral lipid is DSPC. In various embodiments, the molar ratio of the compound to the neutral lipid is in the range of about 2:1 to about 8:1.

[0097] In various embodiments, the composition further comprises a steroid or steroid analog. In certain embodiments, the steroid or steroid analog is cholesterol. In some of these embodiments, the molar ratio of the compound to cholesterol ranges from about 5:1 to 1:1.

[0098] In various embodiments, the polymer-bound lipid is a PEGylated lipid. For example, some embodiments include PEGylated diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG diacylglycerol succinate (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate. PEG-S-DMG), PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamate, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of the compound to the PEGylated lipid ranges from about 100:1 to about 20:1.

[0099] In some embodiments, the composition has the following structure (II): [ka] (II) [In the formula, R 8 and R 9 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain may optionally be interrupted by one or more ester linkages; and The average value of w is in the range of 30 to 60. or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

[0100] In some embodiments, R 8 and R 9are each independently a linear saturated alkyl chain containing 12 to 16 carbon atoms. In other embodiments, the average value of w is in the range of about 42 to 55, e.g., about 49.

[0101] In some embodiments of the above compositions, the therapeutic agent comprises a nucleic acid. For example, in some embodiments, the nucleic acid is selected from antisense RNA and messenger RNA. In some of the above embodiments, the composition comprises lipid nanoparticles.

[0102] In some related embodiments, a lipid nanoparticle is provided that includes a compound of any one of the above embodiments (e.g., a compound of structure (I)). In some embodiments, the lipid nanoparticle further includes a therapeutic agent (e.g., a nucleic acid such as an antisense RNA or a messenger RNA).

[0103] In some embodiments, the lipid nanoparticle further comprises one or more excipients selected from neutral lipids, steroids, and polymer-bound 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.

[0104] 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 the cholesterol ranges from 5:1 to 1:1.

[0105] In certain embodiments, the polymer-bound lipid is a pegylated lipid. In more specific embodiments, the molar ratio of the compound to the pegylated lipid ranges from about 100:1 to about 20:1.

[0106] In some embodiments, the pegylated lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, or PEG dialkyloxypropylcarbamate. In other embodiments, the pegylated lipid has the following structure (II): [ka] (II) [In the formula, R 8 and R 9 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain may optionally be interrupted by one or more ester linkages; and The average value of w is in the range of 30 to 60. or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

[0107] In some more specific embodiments of structure (II), R 8 and R 9 are each independently a linear saturated alkyl chain containing 12 to 16 carbon atoms. In a more specific embodiment, the average value of w is about 49.

[0108] In other different embodiments, the present disclosure relates to a method of administering a therapeutic agent to a patient in need thereof, comprising preparing or providing any of the compositions described above and administering the composition to the patient.

[0109] For purposes of administration, embodiments of the compounds of the present disclosure (typically in the form of lipid nanoparticles combined with a therapeutic agent) can be administered as raw chemicals or formulated as pharmaceutical compositions. Pharmaceutical compositions of embodiments of the present disclosure include 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, for example, a therapeutic agent for treating a particular disease or condition of interest. Appropriate concentrations and dosages can be readily determined by those skilled in the art.

[0110] Administration of the compositions of the presently disclosed embodiments can be carried out via any of the accepted methods of administering pharmaceuticals to provide similar benefits. Pharmaceutical compositions of the presently disclosed embodiments can be formulated into solid, semisolid, liquid, or gaseous formulations, including, for example, tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and nasal. As used herein, the term parenteral includes subcutaneous, intravenous, intramuscular, intradermal, and intrasternal injection or infusion techniques. Pharmaceutical compositions of the presently disclosed embodiments are formulated so that the active ingredient contained therein is bioavailable upon administration of the composition to a patient. In some embodiments, the composition administered to a subject or patient takes the form of one or more unit dosage forms; for example, a tablet may be one unit dosage form, and a container of the compound of the presently disclosed embodiments in aerosol form may hold multiple unit dosage forms. Actual methods for preparing such dosage forms are known or apparent to those skilled in the art. See, e.g., Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). In some embodiments, the composition administered comprises a therapeutically effective amount of a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, in each case for treating the disease or condition of interest according to the teachings of the present disclosure.

[0111] The pharmaceutical compositions of the present disclosure may be in solid or liquid form. In one aspect, the carrier is particulate, with the composition being, for example, in tablet or powder form. Alternatively, the carrier may be liquid, with the composition being, for example, an oral syrup, an injectable liquid, or an aerosol useful, for example, in inhalation administration.

[0112] When intended for oral administration, pharmaceutical compositions of certain embodiments are preferably in either solid or liquid form, with semi-solid, semi-liquid, suspension, and gel forms being included within the scope of forms considered herein as either solid or liquid.

[0113] As a solid composition for oral administration, some embodiments of the pharmaceutical composition can be formulated into the form of powder, granules, compressed tablets, pills, capsules, chewing gum, wafers, etc. Such solid compositions typically contain one or more inert diluents or edible carriers. Additionally, one or more of the following may be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch, lactose, or dextrin; disintegrants such as alginic acid, sodium alginate, Primogel, corn starch, etc.; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavorings such as peppermint, methyl salicylate, or orange flavor; and coloring agents.

[0114] When the pharmaceutical composition of some embodiments is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.

[0115] The pharmaceutical composition of some embodiments can be in the form of a liquid, such as an elixir, syrup, solution, emulsion, or suspension. The liquid can be for oral administration or for delivery by injection, as two examples. For oral administration, preferred compositions contain, in addition to the compound of structure (I), one or more of a sweetener, a preservative, a dye / colorant, and a flavoring agent. For compositions intended for injection administration, one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, and an isotonic agent can be included.

[0116] Liquid pharmaceutical compositions of the present disclosure, whether in solution, suspension, or other form, may contain one or more of the following adjuvants: sterile diluents, such as water for injection, saline, preferably saline, Ringer's solution, or isotonic sodium chloride; fixed oils, such as synthetic monoglycerides or diglycerides, polyethylene glycol, glycerin, propylene glycol, or other solvents that can serve as solvents or suspending media; antibacterial agents, such as benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates, and tonicity adjusters, such as sodium chloride or dextrose; and agents that act as cryoprotectants, such as sucrose or trehalose. Parenteral formulations can be placed in glass or plastic ampoules, disposable syringes, or multiple-dose vials. Saline is a preferred adjuvant. Pharmaceutical compositions for injection are preferably sterile.

[0117] Liquid pharmaceutical compositions of embodiments of the present disclosure intended for either parenteral or oral administration will contain an amount of a compound of the present disclosure such that a suitable dosage will be obtained.

[0118] The pharmaceutical composition of the present disclosure can be intended for topical administration, and in this case, the carrier can comprise a solution, emulsion, ointment, or gel base as appropriate.The base can comprise, for example, one or more of petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers.A thickener can also be present in the pharmaceutical composition for topical administration.When intended for transdermal administration, the composition can comprise a transdermal patch or iontophoresis device.

[0119] The pharmaceutical composition of the present disclosure can be intended for rectal administration, for example, in the form of a suppository that melts in the rectum and releases the drug.The composition for rectal administration can contain an oily base as a suitable non-irritating excipient.Such bases include, but are not limited to, lanolin, cocoa butter, and polyethylene glycol.

[0120] The pharmaceutical compositions of the present disclosure may contain various materials that modify the physical form of the solid or liquid dosage unit. For example, the compositions may contain materials that form a coating shell around the active ingredient. The coating shell material is usually inert and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredient can be encapsulated in a gelatin capsule.

[0121] Pharmaceutical compositions of embodiments of the present disclosure, in solid or liquid form, may include an agent that binds to the compounds of the present disclosure, thereby assisting in the delivery of the compounds. Suitable agents that may act in this capacity include monoclonal or polyclonal antibodies, or proteins.

[0122] The pharmaceutical compositions of the present disclosure may be comprised of dosage units that can be administered as an aerosol. The term aerosol is used to describe a variety of systems, ranging from colloidal systems to systems consisting of pressurized packaging. Delivery can be achieved by a liquefied or compressed gas that disperses the active ingredient, or by a suitable pump system. The aerosols of the compounds of the present disclosure may be delivered in single-phase, two-phase, or three-phase systems to deliver the active ingredient. Aerosol delivery includes the necessary containers, activators, valves, subcontainers, etc., which may be combined to form a kit. One skilled in the art can obtain a suitable aerosol without undue experimentation.

[0123] The pharmaceutical composition of the present disclosure can be prepared by methods well known in the pharmaceutical field.For example, pharmaceutical compositions intended for administration by injection can be prepared by combining the liquid lipid nanoparticles of the present disclosure with sterile distilled water or other carriers to form a solution.Surfactants may be added to promote the formation of a uniform solution or suspension.Surfactants are compounds that interact non-covalently with the compounds of the present disclosure to promote the dissolution or uniform suspension of the compound in aqueous delivery systems.

[0124] The compositions of the presently disclosed embodiments, or pharmaceutically acceptable salts thereof, are administered in a therapeutically effective amount, which will vary depending on a variety of factors, including the activity of the particular therapeutic agent used: the metabolic stability and duration of action of the therapeutic agent; the patient's age, weight, general health, sex, and diet; the mode and time of administration; excretion rate; drug combinations; the severity of the particular disorder or disease; and the subject being treated.

[0125] The composition of the present disclosure's embodiments can also be administered simultaneously with, before, or after the administration of one or more other therapeutic agents.Such combination therapy includes the administration of a single pharmaceutical dosage formulation of the composition of the present disclosure's embodiments and one or more additional active agents, as well as the administration of a composition of the present disclosure's embodiments and each active agent in a separate pharmaceutical dosage formulation.For example, the composition of the present disclosure's embodiments 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 can be administered in a separate oral dosage formulation.When separate dosage formulations are used, the compound of the present disclosure's embodiments and one or more additional active agents can be administered essentially simultaneously, i.e., together, or at different times, i.e., consecutively, and combination therapy is understood to include all of these regimens.

[0126] Methods for preparing the above compounds and compositions are described herein below and / or known in the art.

[0127] It will be understood by those skilled in the art that in the processes described herein, functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxy, amino, mercapto, and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl, or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino, and guanidino include t-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include -C(0)-R" (where R" is alkyl, aryl, or arylalkyl), p-methoxybenzyl, trityl, and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl, or arylalkyl esters. Protecting groups can be added or removed according to standard techniques known to those skilled in the art and described herein. The use of protecting groups is described in Green, TW and PGM Wutz, Protective Groups in Organic Synthesis (1999), 3 rd Ed., Wiley, As one skilled in the art will appreciate, the protecting group may also be a polymer resin such as a Wang resin, a Rink resin, or a 2-chlorotrityl chloride resin.

[0128] Such protected derivatives of the compounds of the present disclosure may not themselves have pharmacological activity, but they may be administered to a mammal and then metabolized in the body to form a pharmacologically active compound of the present disclosure. Such derivatives may therefore be described as "prodrugs." All prodrugs of the compounds of the present disclosure are included within the scope of the present disclosure.

[0129] Additionally, compounds of the disclosed embodiments that exist in a free base or acid form can be converted to their pharmaceutically acceptable salts by treatment with an appropriate inorganic or organic base or acid by methods known in the art. Salts of compounds of the disclosed embodiments can be converted to their free base or acid forms by standard techniques.

[0130] The following General Reaction Scheme 1 illustrates the synthesis of compounds of the present disclosure, i.e., structure (I): [ka] (I) [In the formula, 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.] The following provides exemplary methods for preparing a compound of the formula (I), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. It will be understood that one skilled in the art can prepare these compounds by similar methods or by combining other methods known to those skilled in the art. It will also be understood that one skilled in the art can prepare other compounds of structure (I) not specifically shown below by using the appropriate starting components and modifying the synthetic parameters as needed in a manner similar to that described below. In general, the starting components can be obtained from commercial sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, or can be synthesized according to sources known to those skilled in the art (see, e.g., Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)), or can be prepared as described in this disclosure.

[0131] General Reaction Scheme 1 [ka]

[0132] General Reaction Scheme 1 provides an exemplary method for preparing compounds of structure (I). 1 , R 2 , R 3 , R 4 , R 5 , L 1 , L 2 , L 3 , G 1 , G 2 , and G 3 is as defined herein. X 1 and X 2is a reactive moiety (e.g., halo) selected to facilitate the desired reaction. Compounds of structure A1 are purchased or prepared according to methods known in the art. Reaction of A1 under appropriate reducing conditions (e.g., sodium triacetoxyborohydride) produces the product A3 of the reductive amination of A1 and A2. A3 then reacts with A4 under appropriate basic conditions (e.g., using triethylamine and DMAP) to give compound A5. A5 then reacts with amine A6 using appropriate conditions (e.g., heat) to produce the depicted compound of structure (I).

[0133] It should be noted that various alternative strategies for preparing compounds of structure (I) are available to those skilled in the art. For example, other compounds of structure (I) can be prepared according to similar methods using appropriate starting materials. The use of protecting groups as necessary and other modifications to the above general reaction scheme will be readily apparent to those skilled in the art. [Example]

[0134] The following examples are offered by way of illustration and not by way of limitation.

[0135] Example 1 In vivo assessment of luciferase mRNA using lipid nanoparticle compositions Lipid nanoparticles were prepared and tested according to the general methods described in PCT Publication Nos. WO2015 / 199952 and WO2017 / 004143, the disclosures of which are incorporated herein by reference in their entirety. Briefly, cationic lipid, DSPC, cholesterol, and PEG lipids were solubilized in ethanol at a molar ratio of approximately 50:10:38.5:1.5 or approximately 47.5:10:40.8:1.7. Lipid nanoparticles (LNPs) were prepared at a total lipid to mRNA weight ratio of approximately 10:1 to 30:1. The mRNA was diluted to 0.2 mg / mL in 10-50 mM citrate or acetate buffer (pH 4). Using a syringe pump, the ethanolic lipid solution was mixed with the aqueous mRNA solution at a ratio of approximately 1:5 to 1:3 (vol / vol) at a total flow rate of 15 mL / min or greater. 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 particle size of the lipid nanoparticles was approximately 55–95 nm, and in some cases, approximately 70–90 nm, as determined by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK).

[0136] Studies were conducted in 6-8 week-old female C57BL / 6 mice (Charles River) or 8-10 week-old CD-1 (Harlan) mice (Charles River) in accordance with guidelines established by the Institutional Animal Care Committee (ACC) and the Canadian Council on Animal Care (CCAC). Various doses of mRNA-lipid nanoparticles were administered systemically via tail vein injection, and animals were euthanized at specific time points (e.g., 4 hours) post-administration. Livers and spleens were collected in pre-weighed tubes, weighed, immediately flash-frozen in liquid nitrogen, and stored at -80°C until analytical processing.

[0137] Approximately 50 mg of liver was processed for analysis in a 2 mL FastPrep tube (MP Biomedicals, Solon, OH). A 1 / 4-inch ceramic ball (MP Biomedicals) was added to each tube, 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 twice in a FastPrep24 instrument (MP Biomedicals) at 6.0 m / s for 15 seconds. The homogenate was incubated for 5 minutes at room temperature, then diluted 1:4 with GLB and measured using the SteadyGlo Luciferase Assay System (Promega). Specifically, 50 μL of the homogenate-diluted tissue was reacted with 50 μL of SteadyGlo substrate, shaken for 10 seconds, incubated for 5 minutes, and then assayed using a CentroXS. 3 Quantification was performed using an LB960 luminometer (Berthold Technologies, Germany). The amount of assayed protein was measured using a BCA protein assay kit (Pierce, Rockford, IL). Relative luminescence units (RLU) were then normalized to μg of total protein assayed. To convert RLU to ng of luciferase, a standard curve was generated with QuantiLum Recombinant Luciferase (Promega).

[0138] Trilink Biotechnologies' FLuc mRNA (L-6107 or L-7202) expresses the luciferase protein originally isolated from the firefly (Photinus pyralis). FLuc is commonly used in mammalian cell culture to measure both gene expression and cell viability. It emits bioluminescence in the presence of the substrate luciferin. This capped, polyadenylated mRNA is fully substituted for uridine and / or cytidine nucleosides.

[0139] Example 2 PK of formulated lipids A Measurement of As described elsewhere, the pK of the formulated cationic lipids a pK correlates with the efficacy of LNPs for 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)). a The preferred range of pK of each cationic lipid is from about 5 to about 7. a was measured in lipid nanoparticles using a fluorescence-based assay of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). Lipid nanoparticles containing cationic lipid / DSPC / cholesterol / PEG lipid (50 / 10 / 38.5 / 1.5 mol%) at a concentration of 0.4 mM total lipid in PBS were prepared using the in-line process described in Example 1. TNS was prepared as a 100 mM stock solution in distilled water. The vehicle was diluted to 24 mM lipid with 2 mL of buffer containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl, with a pH ranging from 2.5 to 11. An aliquot of TNS solution was added to a final concentration of 1 μM, followed by vortex mixing, and fluorescence intensity was measured at room temperature using an SLM Aminco Series 2 Luminescence Spectrophotometer at excitation and emission wavelengths of 321 nm and 445 nm. A sigmoidal best-fit analysis was applied to the fluorescence data and pKa was determined as the pH that gave rise to half the maximum fluorescence intensity.

[0140] Example 3 Determining the efficacy of lipid nanoparticle formulations containing various cationic lipids using an in vivo luciferase mRNA-expressing rodent model The cationic lipids shown in Table 2 have been previously tested with nucleic acids. For comparison purposes, these lipids were used to formulate lipid nanoparticles containing FLuc mRNA (L-6107) using the in-line mixing method described in Example 1 and PCT / US10 / 22614 (which is incorporated herein by reference in its entirety). The lipid nanoparticles were formulated with the following molar ratio: 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid ("PEG-DMG", i.e., 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol, average PEG molecular weight 2000). In an alternative embodiment, the cationic lipid, DSPC, cholesterol, and PEG lipid were formulated at a molar ratio of approximately 47.5:10:40.8:1.7. Relative activity was determined by measuring luciferase expression in the liver 4 hours after administration via tail vein injection, as described in Example 1. Activity was compared at doses of 0.3 mg and 1.0 mg mRNA / kg and expressed as ng luciferase / g liver measured 4 hours after administration as described in Example 1.

[0141] Table 2. Comparative lipids showing activity on mRNA [Table 2]

[0142] Representative compounds of the disclosure shown in Table 3 were formulated with the following molar ratio: 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-ditetradecylacetamide), or 47.5% cationic lipid / 10% DSPC / 40.7% cholesterol / 1.8% PEG lipid. Relative activity was determined by measuring luciferase expression in the liver 4 hours after administration via tail vein injection, as described in Example 1. 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. Compound numbers in Table 3 refer to compound numbers in Table 1.

[0143] Table 3. Novel cationic lipids and their activities [Table 3-1] [Table 3-2] [Table 3-3]

[0144] Example 4 Synthesis of bis(2-butyloctyl) 10-(N-decyl-5-(dimethylamino)pentanamido)nonadecanedioate (Compound I-7) [ka]

[0145] Synthesis of compound 4-2 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 min, followed by the addition of sodium triacetoxyborohydride (2.43 mmol, 515 mg) and acetic acid (2.43 mmol, 146 mg; 0.138 mL). 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 column chromatography on silica gel (hexane / EtOAc / Et3N, 95:5:0 to 80:20:1). The desired product was obtained as a colorless oil (863 mg of colorless oil, 1.05 mmol, 65% yield). 1 H 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).

[0146] Synthesis of compound 4-3 To a stirred solution of 5-bromovaleric acid (1.12 mmol, 204 mg) in CHCl (1 mL) at room temperature, a solution of thionyl chloride (3.36 mmol, 400 mg, 0.25 mL) in CHCl (5 mL) was added over 1 min, followed by the addition of DMF (approximately 16 mg). The mixture was then heated to reflux for 2 h. The reaction mixture was then concentrated in vacuo. The acid chloride was used directly in the next step. The benzene solution (5 mL) of 5-bromopentanoyl chloride from above was added dropwise over 2 min 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) at room temperature. After the addition, the reaction was stirred at room temperature for 1 h. Methanol (1 mL) was added, and the mixture was stirred for 2 h. The reaction mixture was concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (hexane / EtOAc, 98:2 to 85:15). The desired product was obtained as a colorless oil (250 mg, 0.25 mmol, 91%, colorless oil).

[0147] Synthesis of compound I-7 To 4-2 (250 mg, 0.25 mmol) was added dimethylamine (2 M in THF, 10 mL). The solution was stirred at 64 °C overnight. The reaction mixture was concentrated. The residue was taken up in a mixture of hexane, ethyl acetate, and EtN (80:20:1) and filtered through a pad of silica gel, washing with the same solvent mixture. The filtrate was concentrated to give a brownish oil (approximately 233 mg). The crude product (233 mg) was purified by flash column chromatography on silica gel (0–6% methanol in chloroform). The desired product was obtained as (194 mg, colorless oil, 0.20 mmol, 82%). 1 H NMR (400 MHz, CDCl) δ: 4.60-4.20 (br, estimated 0.3H, due to slow isomerization around the amide bond), 3.97, 3.96 (two sets of doublets, 5.8 Hz, 4H), 3.60 (quintet, 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).

[0148] Example 5 Synthesis of bis(2-butyloctyl) 10-(N-decyl-4-(dimethylamino)butanamido)nonadecanedioate (Compound I-18) [ka]

[0149] Synthesis of Compound I-18 (Method A) To a stirred solution of 4-(dimethylamino)butyric acid hydrochloride (1.12 mmol, 188 mg) and DMF (10-20 μL) in CHCl (10 mL) at room temperature, oxalyl chloride (5.6 mmol, 722 mg, 0.496 mL) was added. The resulting mixture was stirred at room temperature overnight. The reaction mixture (dark red) was concentrated under reduced pressure. The resulting acid chloride (blocky red solid) was used directly in the next step. A solution of the above acyl chloride in CHCl (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 CHCl (5 mL) at room temperature. 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 / EtN, 80:20:0.1 to 75:25:1) and further purified by silica gel flash column chromatography (0 to 5% methanol in chloroform). The desired product was obtained as a colorless oil (92 mg, 0.10 mmol, 35%). 1H NMR (400 MHz, CDCl3) δ: 4.57-4.29 (br. 0.4H), 3.97, 3.96 (two sets of doublets, 5.8 Hz, 5.8 Hz, 4H), 3.63 (quintet, 6.8 Hz, 0.6H), 3.06-3.00 (m, 2H), 2.35-2.26 (m, 8H), 2.213, 2.211 (two sets of singlets, 6H), 1.82 (sextet, 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).

[0150] Synthesis of Compound I-18 (Method B) 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 min. A solution of 4-2 (1.25 g, 1.52 mmol) in CHCl (6 mL) was added, and the resulting mixture was stirred overnight. The next day, additional DCC (450 mg) was added, and the mixture was stirred for another day. The mixture was then concentrated under reduced pressure. The residue was taken up in a mixture of hexane, ethyl acetate, and EtN (80:20:1) and filtered through a short column of silica gel, washing with the same solvent mixture. The filtrate was concentrated to give a colorless oil. The product was further purified by flash column chromatography on silica gel (0–5% methanol in chloroform containing a trace of EtN). The desired product was obtained as a colorless oil (1.14 g, 81%).

[0151] Example 6 Synthesis of bis(2-butyloctyl) 10-(N-decyl-5-(diethylamino)pentanamido)nonadecanedioate (Compound I-8) [ka]

[0152] Synthesis of compound I-8 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 h. The reaction mixture was concentrated. The residue was taken up in a mixture of hexane, ethyl acetate, and EtN (80:20:1) and filtered through a pad of silica gel, washing with the same solvent mixture. The filtrate was concentrated to give a brownish oil (153 mg). The crude product (233 mg) was purified by flash column chromatography on silica gel (0–6% MeOH in chloroform). The desired product was obtained as a colorless oil (136 mg, 0.14 mmol, 77%).

[0153] Example 7 Synthesis of bis(2-butyloctyl) 10-(N-decyl-5-(pyrrolidin-1-yl)pentanamido)nonadecanedioate (Compound I-9) [ka]

[0154] Synthesis of compound I-9 A mixture of 4-3 (200 mg, 0.20 mmol), pyrrolidine (50 equiv, 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 taken up in a mixture of hexane, ethyl acetate, and EtN (80:20:1) and filtered through a pad of silica gel, washing with the same solvent mixture. The filtrate was concentrated to give a brownish oil. The crude product (233 mg) was purified by flash column chromatography on silica gel (0–6% MeOH in chloroform). The desired product was obtained as a colorless oil (158 mg, 0.16 mmol, 80%).

[0155] Example 8 Synthesis of bis(2-hexyldecyl) 7-(N-decyl-4-(dimethylamino)butanamido)tridecanedioate (Compound I-16) [ka]

[0156] Synthesis of 8-2 A solution of 8-1 (1 equiv., 1.15 g, 1.62 mmol) and 1-decylamine (1.5 equiv., 2.43 mmol, 382 mg, 0.486 mL) in DCE (10 mL) was stirred at room temperature for approximately 15 min. To the solution, sodium triacetoxyborohydride (1.5 equiv., 2.43 mmol, 515 mg) and AcOH (1.5 equiv., 2.43 mmol, 146 mg, 0.14 mL) were added. The mixture was stirred at room temperature for 3 days. The reaction mixture was then 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 column of silica gel. 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 was obtained as a colorless oil (1.28 g, 1.51 mmol, 93%). 1 HNMR (400 MHz, CDCl3) δ: 3.97 (d, 5.8 Hz, 4H), 2.53 (t, 7.2 Hz, 2H), 2.43 (quintet, 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).

[0157] Synthesis of compound I-16 To a solution of 4-(dimethylamino)butyric acid hydrochloride (2.1 mmol, 352 mg) and DMF (approximately 13 mg) in CHCl (15 mL) stirred at room temperature under Ar, oxalyl chloride (3 equiv., 6.3 mmol, 800 mg, 0.55 mL) was added. The resulting mixture was stirred at room temperature overnight. The reaction mixture (a pale orange solution) was concentrated in vacuo. The resulting acid chloride (8-3, a pale brown solid) was used directly in the next reaction. A solution of the above acyl chloride in CHCl (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 CHCl (5 mL) at room temperature. After the addition, the reaction mixture was stirred overnight at room temperature. After concentration, the product was isolated by silica gel column chromatography (hexane, EtOAc, and EtN, 80:20:0.1 to 70:30:1). The product was further purified by flash dry column chromatography on silica gel (0 to 5% MeOH in chloroform). The desired product was obtained as a pale yellow oil (110 mg, 0.11 mmol, 32%). 1 HNMR (400 MHz, CDCl3, 7.26 ppm) δ: 4.57-4.34 (br. 0.4H), 3.98-3.94 (m, 4H), 3.64 (quintet, 6.8 Hz, 0.6H), 3.06-3.00 (m, 2H), 2.35-2.25 (m, 8H), 2.213, 2.210 (two sets of singlets, 6H), 1.82 (sextet, 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).

[0158] Example 9 Synthesis of bis(2-butyloctyl) 10-(4-(dimethylamino)-N-(2-ethylhexyl)butanamido)nonadecanedioate (Compound I-20) [ka]

[0159] Synthesis of 9-2 A solution of 9-1 (1 equiv., 0.82 g, 1.21 mmol) and 2-ethyl-1-hexylamine (1.5 equiv., 1.81 mmol, 234 mg) in DCE (8 mL) was stirred at room temperature for approximately 15 min. To the solution were added sodium triacetoxyborohydride (1.5 equiv., 1.81 mmol, 384 mg) and AcOH (1.5 equiv., 1.81 mmol, 109 mg). The mixture was stirred at room temperature for 2 days. The reaction mixture was then concentrated. The residue was diluted with hexane and EtOAc (approximately 99:5) and washed with dilute NaOH, saturated NaHCO3, and brine. The extract was filtered through a short column of silica gel. The column was washed with a mixture of hexane and EtOAc (95:5) and then with a mixture of hexane, EtOAc, and Et3N (80:20:0.5). 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 HNMR (400 MHz, CDCl3) δ: 3.98 (d, 5.8 Hz, 4H), 2.45 (d, 5.1 Hz, 2H), 2.39 (quintet, 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).

[0160] Synthesis of 9-4 To a stirred solution of 5-bromovaleric acid (2.24 mmol, 405 mg) in CHCl (2 mL) at room temperature, a solution of thionyl chloride (3 equiv., 6.72 mmol, 800 mg, 0.49 mL) in CHCl (5 mL) was added slowly over 1 min. DMF (2 small drops, ca. 16 mg) was added to the reaction mixture. The mixture was then heated to reflux for 2 h. The reaction mixture was concentrated in vacuo. The resulting acid chloride 9-3 was used directly in the next step. The above 5-bromopentanoyl chloride solution in 8 mL of benzene was added to a solution of 9-2 (444 mg, 0.56 mmol), triethylamine (1.56 mL), and DMAP (5 mg) in 5 mL of benzene over 2 min at room temperature. After the addition, the mixture was stirred overnight at room temperature. After evaporation of the solvent in vacuo, the product was isolated by column chromatography on silica gel (hexane / EtOAc, 99:1 to 90:10). The desired product was sufficiently pure for the next step (colorless oil, 527 mg, 0.55 mmol, 98%).

[0161] Synthesis of compound I-20 To a pressure flask containing 9-4 (260 mg, 0.27 mmol), dimethylamine (2 M in THF, 10 mL) was added. The solution was stirred overnight at 64 °C (oil bath temperature). Excess amine and solvent were evaporated. The residue was taken up in a mixture of ethyl acetate and hexane (95:5) and filtered through a pad of silica gel. 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 as a brown oil (233 mg). The crude product was further purified by flash-dry column chromatography on silica gel (0–5% MeOH in chloroform). The desired product was obtained as a colorless oil (204 mg, 0.22 mmol, 82%). 1 HNMR (400 MHz, CDCl, 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).

[0162] Example 10 Synthesis of bis(2-butyloctyl) 10-(3-(dimethylamino)-N-nonylpropanamido)nonadecanedioate (Compound I-25) [ka]

[0163] Synthesis of 10-1 To a solution of 3-bromopropionic acid (2.02 mmol, 311 mg) in CHCl (5 mL) and DMF (0.01 mL) was added oxalyl chloride (5.05 mmol, 641 mg, 0.44 mL) at room temperature. The resulting mixture was stirred at room temperature overnight. The mixture was then concentrated in vacuo. The remaining liquid / solid (yellow) was dissolved in 10 mL of CHCl and added to a solution of 4-2 (833 mg, 1.02 mmol), triethylamine (5.05 mmol, 0.7 mL), and DMAP (5 mg) in CHCl (10 mL) at room temperature over 4 min. After the addition, the mixture was stirred at room temperature for 2 h. After evaporation of the solvent in vacuo, the product was isolated 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%).

[0164] Synthesis of compound I-25 A mixture of 10-1 (283 mg, 0.30 mmol) and dimethylamine (2 M in THF, 12 mL) was stirred overnight at 68 °C (oil bath temperature) in a pressure flask. The reaction mixture was concentrated. The residue was taken up in a mixture of hexane, ethyl acetate, and EtN (80:20:1) and filtered through a pad of silica gel, washing with the same solvent mixture. The filtrate was concentrated to give a brownish oil (302 mg). The crude product (302 mg) was purified by flash dry column chromatography on silica gel (0–5% MeOH in chloroform containing a trace of EtN). The desired product was obtained as a colorless oil (169 mg, 0.18 mmol, 61%). 1HNMR (400 MHz, CDCl3, 7.26) δ: 4.50-4.31 (br, estimated 0.3H, due to slow isomerization around the amide bond), 3.97 (shoulder doublet, 5.8 Hz, 4H), 3.60 (quintet, 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 (shoulder triplet, 7.6 Hz, 4H), 2.26, 2.25 (two sets of 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).

[0165] Example 11 Synthesis of bis(2-hexyldecyl) 7-(N-decyl-4-(pyrrolidin-1-yl)butanamido)tridecanedioate (Compound I-29) [ka]

[0166] Synthesis of 11-1 To a solution of 4-bromobutyric acid (0.97 mmol, 161 mg) in CHCl (3 mL) and DMF (0.01 mL) was added oxalyl chloride (3 equiv., 2.91 mmol, 370 mg, 0.25 mL) at room temperature. The mixture was stirred at room temperature overnight. The reaction mixture was then concentrated in vacuo. The remaining liquid / solid (light yellow) was dissolved in 5 mL of CHCl, and a solution of 8-2 (410 mg, 0.48 mmol), triethylamine (0.4 mL), and DMAP (2 mg) in CHCl (20 mL) was added over 2 min at room temperature. After the 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 under reduced pressure at room temperature. The residue was used in the next reaction without purification.

[0167] Synthesis of compound I-29 The above residue containing 11-1 was taken up in a mixture of pyrrolidine (2.10 mL, 25 mmol) and THF (15 mL). The mixture was transferred to a pressure flask and heated at 68 °C overnight. The mixture was cooled and concentrated under reduced pressure. The residue was taken up in a mixture of hexane, ethyl acetate, and EtN (80:20:1) and filtered through a short column of silica gel, washing with the same solvent mixture. The filtrate was concentrated to give a yellow oil / solid. The crude product (300 mg) was purified by column chromatography on silica gel (0% to 10% MeOH and 0% to 0.5% EtN in CHCl). The desired product was obtained as a yellow oil (215 mg). The product (215 mg) was further purified by flash-dry column chromatography on silica gel (0% to 5% MeOH in chloroform). The desired product was obtained as a colorless oil (162 mg, 0.16 mmol, 34%). 1 HNMR (400 MHz, CDCl3, 7.26 ppm) δ: 4.57-4.34 (br. 0.4H), 3.98-3.94 (m, 4H), 3.64 (quintet, 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, 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).

[0168] Example 12 Synthesis of bis(2-butyloctyl) 10-(4-(dimethylamino)-N-(2-ethylhexyl)butanamido)nonadecanedioate (Compound I-30) [ka]

[0169] Synthesis of compound I-30 To a solution of 4-dimethylaminobutyric acid hydrochloride (0.50 mmol, 85 mg) and 4-dimethylaminopyridine (3 equiv., DMAP, 0.75 mmol, 92 mg) in acetonitrile (5 mL) was added DCC (1.1 mmol × 2, 226 mg), and the mixture was stirred at room temperature for 45 min. A solution of 9-2 (160 mg, 0.20 mmol) in CHCl (1 mL) was added to the reaction mixture, and the resulting mixture was stirred over the weekend. Additional 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 taken up in a mixture of hexane and EtOAc (approximately 99:5) and filtered through a short column of silica gel. The column was washed with a mixture of hexane and EtOAc (95:5), followed by a mixture of hexane, EtOAc, and EtN (80:20:1). The filtrate from the latter wash was concentrated to dryness (133 mg). The crude product (133 mg) was further purified by flash dry column chromatography on silica gel (0-5% MeOH in chloroform containing a trace of EtN) to give the desired product (48 mg, colorless oil, 0.053 mmol, 27%). 1 HNMR (400 MHz, CDCl, 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).

[0170] Example 13 Synthesis of bis(2-butyloctyl) 10-(N-decyl-5-(dibutylamino)pentanamido)nonadecanedioate (Compound I-31) [ka]

[0171] Synthesis of compound I-31 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) was stirred overnight at 78 °C in a pressure flask. The reaction mixture was concentrated. The residue was taken up in a mixture of hexane, ethyl acetate, and EtN (80:20:1) and filtered through a pad of silica gel, washing with the same solvent mixture. The filtrate was concentrated to give a brownish oil (product and dibutylamine). The oil was diluted with hexane, washed twice with dilute aqueous HCl (0.5 M), washed with saturated NaHCO 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 (0–5% MeOH in chloroform containing a trace of EtN). The desired product was obtained as a colorless oil (216 mg, 0.21 mmol, 72%). 1 HNMR (400 MHz, CDCl) δ: 4.50-4.35 (br, estimated 0.3H, due to slow isomerization around the amide bond), 3.97, 3.96 (two sets of doublets, 5.8 Hz, 4H), 3.61 (quintet, 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).

[0172] Example 14 Synthesis of 10-(4-(dimethylamino)-N-nonylbutanamido)nonadecane-1,19-diyl bis(2-butyloctanoate) (Compound I-32) [ka]

[0173] Synthesis of compound I-32 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 min. Next, a solution of 14-1 (442 mg, 0.54 mmol) in CHCl (2 mL) was added. The resulting mixture was stirred overnight. Additional DCC (140 mg) was added, and the mixture was stirred for another day. The mixture was then concentrated under reduced pressure. The residue was taken up in a mixture of hexane, ethyl acetate, and EtN (80:20:1) and filtered through a short column of silica gel, washing 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 (0-5% MeOH in chloroform containing a trace of EtN) to give the desired product as a colorless oil (345 mg, 0.38 mmol, 70%). 1 HNMR (400 MHz, CDCl3, 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, 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, 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).

[0174] Example 15 Synthesis of bis(2-butyloctyl) 10-(N-decyl-3-(pyrrolidin-1-yl)propanamido)nonadecanedioate (Compound I-33) [ka]

[0175] Synthesis of compound I-33 A mixture of 10-1 (283 mg, 0.30 mmol), pyrrolidine (1.25 mL, 15 mmol), and THF (10 mL) was heated in a pressure tube at 64 °C overnight. The mixture was cooled and concentrated under reduced pressure. The residue was taken up in a mixture of hexane, ethyl acetate, and EtN (80:20:1) and filtered through a short column of silica gel, washing 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 (0–5% MeOH in chloroform containing a trace of EtN). The desired product was obtained as a colorless oil (113 mg, 0.12 mmol, 40%). 1 HNMR (400 MHz, CDCl3, 7.26) δ: 4.55-4.30 (br, estimated 0.3H, due to slow isomerization around the amide bond), 3.97 (shoulder doublet, 5.8 Hz, 4H), 3.61 (quintet, 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 (two 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).

[0176] Example 16 Synthesis of bis(2-butyloctyl) 10-(N-decyl-5-(hexyl(methyl)amino)pentanamido)nonadecanedioate (Compound I-34) [ka]

[0177] Synthesis of 16-1 A mixture of 4-3 (200 mg, 0.20 mmol), hexylamine (20 mmol, 2 g), N,N-diisopropylethylamine (5 equiv., 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 under reduced pressure (approximately 30 mmHg) at 75–85 °C. TLC showed that most of the excess hexylamine had been removed. The residue was taken up in a mixture of hexane, ethyl acetate, and EtN (80:20:1) and filtered through a short column of silica gel, washing 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.

[0178] Synthesis of compound I-34 To a solution of 16-1 (192 mg, 0.19 mmol) in THF (5 mL) was added formaldehyde HCl solution (500 mg, 37 wt% aqueous solution) at room temperature. The resulting mixture was stirred for 30 min, followed by the addition of sodium triacetoxyborohydride (1.2 mmol, 243 mg). The resulting mixture was stirred at room temperature overnight. The reaction mixture was concentrated. The residue was taken up in a mixture of hexanes 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 taken up in a mixture of hexanes, ethyl acetate, and Et3N (80:20:1) and filtered through a short column of silica gel, washing 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 flash column chromatography on silica gel (0–5% MeOH in chloroform containing a trace of Et3N). The desired product was obtained as a colorless oil (113 mg, 0.13 mmol, 69%). 1HNMR (400 MHz, CDCl, 7.26 ppm) δ: 4.50-4.35 (br, estimated 0.3H, due to slow isomerization around the amide bond), 3.97, 3.96 (two sets of doublets, 5.8 Hz, 4H), 3.60 (quintet, 7.0 Hz, 0.7H), 3.06-2.98 (m, 2H), 2.36-2.26 (m, 10H), 2.191, 2.189 (two sets of 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).

[0179] Example 17 Synthesis of bis(2-hexyldecyl) 7-(N-decyl-3-(dimethylamino)propanamido)tridecanedioate (Compound I-35) [ka]

[0180] Synthesis of compound I-35 To a solution of 3-dimethylaminopropionic acid hydrochloride (2 equiv., 0.62 mmol, 95 mg) and 4-dimethylaminopyridine (3 equiv., DMAP, 0.93 mmol, 114 mg) in acetonitrile (10 mL) was added DCC (2.2 equiv., 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 CHCl (2 mL) was added, and the resulting mixture was stirred over the weekend. TLC (chloroform / MeOH, 9:1) showed a major spot before the solvent, which may be the elimination product, not the starting material, but a small amount of the desired product. The reaction mixture was concentrated. The possible elimination product (107 mg of a colorless oil) was isolated by column chromatography (hexane-EtOAc, 95:5) and treated with a 2 M solution of dimethylamine in THF (9 mL) at room temperature for 4 days. The mixture was concentrated. The residue was taken up in a mixture of hexane, ethyl acetate, and Et3N (80:20:1) and filtered through a short column of silica gel, washing 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 (0-5% MeOH in chloroform containing a trace of Et3N). The desired product was obtained as a colorless oil (62 mg). 1 HNMR (400 MHz, CDCl3, 7.26) δ: 4.50-4.36 (br, estimated 0.3H, due to slow isomerization around the amide bond), 3.97, 3.96 (two sets of 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 sets of 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)

[0181] Example 18 Synthesis of bis(2-hexyldecyl) 7-(3-(dimethylamino)-N-(6-((2-ethylhexyl)oxy)-6-oxohexyl)propanamido) tridecanedioate (Compound I-36) [ka]

[0182] Synthesis of 18-2 To a solution of 3-bromopropionic acid (0.34 mmol, 52 mg) in CHCl (3 mL) and DMF (1 drop from a fine needle), oxalyl chloride (0.86 mmol, 109 mg, 74 μL) was added at room temperature. The mixture was stirred overnight at room temperature. The mixture was then concentrated under reduced pressure at room temperature for 60 min. The remaining liquid / solid (yellow) was dissolved in 5 mL of CHCl and added to a solution of 18-1 (160 mg, 0.17 mmol), triethylamine (0.86 mmol, 0.12 mL), and DMAP (1 mg) in CHCl (5 mL) at room temperature over 1 min. After the addition, the mixture was stirred at room temperature for 3 h and then concentrated. The product was isolated by silica gel column chromatography (hexane, EtOAc, and EtN, 95:5 to 85:15). The desired product was obtained as a colorless oil (99 mg, 0.09 mmol, 53%).

[0183] Synthesis of compound I-36 To a pressure flask containing 18-2 (99 mg, 0.09 mmol) was added dimethylamine (2 M in THF, 5 mL). 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 taken up in a mixture of hexane, ethyl acetate, and EtN (80:20:1) and filtered through a short column of silica gel, washing with the same solvent mixture. The filtrate was concentrated to give a brown oil (94 mg). The product (94 mg) was further purified by flash dry column chromatography on silica gel (0–5% MeOH in chloroform containing a trace of EtN). The desired product was obtained as a colorless oil (71 mg, 0.069 mmol, 76%). 1HNMR (400 MHz, CDCl3, 7.26) δ: 4.53-4.35 (br, estimated 0.3H, due to slow isomerization around 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 (two sets of 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).

[0184] Example 19 Synthesis of di(tridecan-7-yl) 10-(N-decyl-4-(dimethylamino)butanamido)nonadecanedioate (Compound I-37) [ka]

[0185] Synthesis of 19-2 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 approximately 15 min. To this solution, 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. The mixture was stirred at room temperature for 2 days. The reaction mixture was then 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 taken up in a mixture of hexane, ethyl acetate, and Et3N (80:20:1) and filtered through a short column of silica gel, washing with the same solvent mixture. The filtrate was concentrated to give the desired product as a colorless oil (582 mg, 0.69 mmol, 98%), which was used in the next step without further purification.

[0186] Synthesis of compound I-37 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 min. A solution of 19-2 (582 mg, 0.69 mmol) in CHCl (3 mL) was added, and the resulting mixture was stirred overnight. The next day, additional DCC (200 mg) was added, and stirring continued over the weekend (4 days). The mixture was then concentrated under reduced pressure. The residue was taken up in a mixture of hexane, ethyl acetate, and EtN (80:20:1) and filtered through a short column of silica gel, washing with the same solvent mixture. The filtrate was concentrated to give a yellow oil. The product was further purified by flash column chromatography on silica gel (0–5% MeOH in chloroform containing a trace of EtN). The desired product was obtained as a colorless oil.

[0187] Example 20 Synthesis of bis(2-hexyldecyl) 10-(N-decyl-4-(dimethylamino)butanamido)nonadecanedioate (Compound I-38) [ka]

[0188] Synthesis of 20-2 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 min, followed by the addition of sodium triacetoxyborohydride (2.03 mmol, 429 mg) and AcOH (2.03 mmol, 121 mg, 0.115 mL). After stirring at room temperature for 2 days, 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 column of silica gel, 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).

[0189] Synthesis of I-38 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 min. A solution of 20-2 (350 mg, 0.38 mmol) in DCM (1 mL) was added, and the resulting mixture was stirred overnight. The next day, additional DCC (50 mg) was added and stirred for another day. The mixture was then concentrated under reduced pressure. The residue was taken up in a mixture of hexane, ethyl acetate, and Et3N (80:20:1) and filtered through a short column of silica gel, washing with the same solvent mixture. The filtrate was concentrated to give a colorless oil. The product was further purified by flash column chromatography on silica gel (0–5% MeOH in chloroform containing a trace of Et3N). The desired product was obtained as a colorless oil (260 mg).

[0190] Example 21 Synthesis of bis(2-hexyldecyl) 10-(N-decyl-4-(pyrrolidin-1-yl)butanamido)nonadecanedioate (Compound I-39) [ka]

[0191] Synthesis of 21-1 To a solution of 4-bromobutyric acid (1.00 mmol, 167 mg) in DCM (3 mL) and one small drop of DMF was added oxalyl chloride (3 equiv., 3.00 mmol, 381 mg, 0.26 mL) at room temperature. The mixture was stirred at room temperature overnight. The reaction mixture was then concentrated in vacuo. The remaining liquid / solid (light 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 over 2 min at room temperature. After the 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 under reduced pressure at room temperature. The residue was used in the next reaction without purification.

[0192] Synthesis of I-39 The above residue was taken up in a mixture of pyrrolidine (2.20 mL, 26 mmol) and THF (15 mL). The mixture was transferred to a pressure flask and heated at 68 °C overnight. The mixture was cooled and concentrated under reduced pressure. The residue was taken up in a mixture of hexane, ethyl acetate, and EtN (80:20:1) and filtered through a short column of silica gel, washing 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 (0-5% MeOH in chloroform). The desired product was obtained as a colorless oil (165 mg).

[0193] The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referenced herein and / or listed in the Application Data Sheet (including U.S. Patent Nos. 62 / 791,566 and 62 / 890,469) are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified, if necessary, to employ concepts from the various patents, applications, and publications to provide further embodiments. These and other changes can be made to the embodiments in light of the above detailed description. Generally, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments, along with the full range of equivalents to which such claims are entitled. Accordingly, the scope of the claims is not limited by the present disclosure.

Claims

1. i) A compound having the following structure (I): 【Chemistry 1】 (I) [In the formula, R 1 is optionally substituted C 1 -C 24 alkyl or optionally substituted C 2 -C 24 alkenyl; R 2 and R 3 are each independently optionally substituted C 1 -C 36 alkyl; R 4 and R 5 are each independently optionally substituted C 1 -C 6 alkyl, or R 4 and R 5 together with the N to which they are attached form a heterocyclyl or heteroaryl; L 1 , L 2 , and L 3 are each independently an optionally substituted C 1 -C 18 alkylene; G 1 is a direct bond, —(CH 2 ) n O(C═O)—, —(CH 2 ) n (C═O)O—, or —(C═O)—; G 2 and G 3 are each independently —(C═O)O— or —O(C═O)—; and n is an integer greater than 0. or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof; and ii) nucleic acid; 10. A lipid nanoparticle for use in a method for inducing protein expression in a subject, comprising:

2. A compound having the following structure (IA): 【Chemistry 2】 (IA) The lipid nanoparticle for use according to claim 1, which is a compound represented by the formula:

3. The compound having the following structure (IB): 【Transformation 3】 (IB) The lipid nanoparticle for use according to claim 1, which is a compound represented by the formula:

4. R 1 is i) optionally substituted C 6 -C 18 alkyl or C 14 -C 18 alkenyl; ii) is a C8 alkyl, C9 alkyl, C10 alkyl, C12 alkyl, C14 alkyl, or C16 alkyl; iii) is C 16 alkenyl; iv) whether it is branched; v) is branched; or vi) is unsubstituted; Lipid nanoparticles for use according to any one of claims 1 to 3.

5. G 1 is i) is a direct bond, -(CH 2 ) n O(C═O)-, or -(CH 2 ) n (C═O)O-; ii) is a direct bond; or iii) —(CH 2 ) n (C═O)O—, where n is greater than 1, preferably n is 5, 6, 7, 8, 9, or 10, or n is 7 or 8; Lipid nanoparticles for use according to any one of claims 1 to 4.

6. L 1 is i) C 1 -C 6 alkylene; ii) is a C2 alkylene, a C3 alkylene, or a C4 alkylene; iii) is unbranched; or iv) is unsubstituted; Lipid nanoparticles for use according to any one of claims 1 to 5.

7. i) R 2 is C 8 -C 24 alkyl; ii) R 3 is C 8 -C 24 alkyl; iii) R 2 and R 3 are both C 8 -C 24 alkyl; iv) R 2 and R 3 are each independently C 11 alkyl, C 12 alkyl, C 13 alkyl, C 14 alkyl, C 15 alkyl, C 16 alkyl, C 18 alkyl, or C 20 alkyl; v) R 2 is branched; vi) R 3 is branched; vii) R 2 and R 3 are each independently selected from the following structures: 【Chemistry 4】 wherein R 6 and R 7 are each independently C 2 -C 12 alkyl. or viii) R 2 and R 3 are each independently selected from the following structures: 【Transformation 5】 indicated by one of Lipid nanoparticles for use according to any one of claims 1 to 6.

8. i) L 2 and L 3 are each independently a C 4 -C 10 alkylene; ii) L 2 and L 3 are both C 5 alkylene; iii) L 2 and L 3 are both C 6 alkylene; iv) L 2 and L 3 are both C 8 alkylene; v) L 2 and L 3 are both C 9 alkylene; vi) L 2 is unbranched; vii) L 3 is unbranched; viii) L 2 is unsubstituted; or ix) L2 is unsubstituted; Lipid nanoparticles for use according to any one of claims 1 to 7.

9. i) R 4 and R 5 are each independently C 1 -C 6 alkyl; ii) R 4 and R 5 are both methyl; iii) R 4 and R 5 are both ethyl; iv) R 4 is methyl and R 5 is n-butyl; or v) R 4 and R 5 together with the N to which they are attached form a heterocyclyl, preferably said heterocyclyl is a 5-membered heterocyclyl or said heterocyclyl has the following structure: 【Transformation 6】 Denoted by Lipid nanoparticles for use according to any one of claims 1 to 8.

10. The compound of claim 1, wherein the compound has the following structure: 【Chemistry 7-1】 【Chemistry 7-2】 【Transformation 7-3】 【Chemistry 7-4】 【Transformation 7-5】 【Transformation 7-6】 2. The lipid nanoparticle for use according to claim 1, wherein the lipid nanoparticle is one of:

11. A lipid nanoparticle for use according to any one of claims 1 to 10, wherein the nucleic acid is messenger RNA.

12. A lipid nanoparticle for use as described in claim 11, wherein the mRNA encodes an antigen or antibody.

13. Lipid nanoparticles for use as described in claim 12, wherein the antigen or antibody provides protection against infection.

14. A lipid nanoparticle for use as described in claim 11, wherein the messenger RNA encodes a gene-modifying enzyme.

15. A lipid nanoparticle for use as described in claim 14, wherein the lipid nanoparticle further comprises a DNA segment for integration into the host genome, or the lipid nanoparticle is used in combination with a DNA segment for integration into the host genome.

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