lipids for the delivery of lipid nanoparticles of active agents

Abstract

A compound having the following chemical formula I or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof is provided: Chemical Formula I In the above chemical formula I, R1, R2, R3, R4, R5, L1, L2, L3, G1, G2, and G3 are as defined herein. The use of a compound as a component of a lipid nanoparticle formulation for the delivery of a therapeutic agent, a composition comprising said compound, and a method for the use and preparation thereof are also provided.

Description

Technology Field

[0001] The present disclosure 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 oligonucleotides and lipid nanoparticles, thereby facilitating the intracellular delivery of therapeutic nucleic acids (e.g., oligonucleotides, messenger RNA) in both in vitro and in vivo. Background Technology

[0002] There are many challenges associated with the delivery of nucleic acids that influence desired responses in biological systems. Nucleic acid-based therapeutics hold immense potential, but realizing this potential requires more effective delivery of nucleic acids to appropriate sites within cells or organisms. Therapeutic nucleic acids include, for example, messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, immunostimulatory nucleic acids, antagomir, antimir, mimics, supermir, and aptamers. Some nucleic acids, such as mRNA or plasmids, can be used to perform the expression of specific cellular products useful for treating diseases associated with, for example, protein or enzyme deficiencies. The therapeutic applications of translatable nucleotide delivery are very broad because constructs can be synthesized to produce any selected protein sequence, regardless of whether they are system-specific. The expression products of nucleic acids can increase existing levels of proteins, replace missing or non-functional versions of proteins, or introduce new proteins and related functions into cells or organisms.

[0003] Some nucleic acids, such as miRNA inhibitors, can be used to inhibit the expression of specific cellular products regulated by miRNAs, which are useful for treating diseases associated with deficiencies in proteins or enzymes, for example. The therapeutic applications of miRNA inhibition are very broad because constructs can be synthesized to inhibit one or more miRNAs that, in turn, regulate the expression of mRNA products. Inhibition of endogenous miRNAs can increase the expression of downstream target endogenous proteins and restore proper function in cells or organisms as a means to treat diseases associated with specific miRNAs or groups of miRNAs.

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

[0005] However, there are currently two problems facing the use of oligonucleotides in a therapeutic context. First, free RNA is vulnerable to nuclease digestion in plasma. Second, free RNA has limited access to intracellular compartments containing the relevant translation machinery. Lipid nanoparticles formed from cationic lipids and other lipid components such as neutral lipids, cholesterol, PEG, PEGylated lipids, and oligonucleotides have been used to block RNA degradation in plasma and promote the cellular uptake of oligonucleotides. The problem to be solved

[0006] There is a need for improved cationic lipids and lipid nanoparticles for the delivery of oligonucleotides. Preferably, these lipid nanoparticles will provide an optimal drug-to-lipid ratio, protect nucleic acids from degradation and clearance in serum, be suitable for systemic or local delivery, and provide intracellular delivery of nucleic acids. Furthermore, these lipid-nucleic acid particles must provide excellent tolerability and an appropriate therapeutic index so that patient treatment with an effective dose of nucleic acid is not associated with unacceptable toxicity and / or risk to the patient. The present disclosure provides these and related advantages. means of solving the problem

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

[0008] In one embodiment, a compound having the following formula I or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof is provided:

[0009] [Chemical Formula I]

[0010]

[0011] In the above chemical formula I,

[0012] R 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.

[0013] A pharmaceutical composition comprising one or more compounds of Formula I and a therapeutic agent is also provided. In some embodiments, the pharmaceutical composition further comprises one or more components selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. Such compositions are useful for forming lipid nanoparticles for the delivery of therapeutic agents.

[0014] In another embodiment, the present disclosure provides a method for administering a therapeutic agent to a patient in need, comprising the steps of preparing or providing a composition of lipid nanoparticles comprising a compound of Formula I and a therapeutic agent, and delivering or administering the composition to a patient.

[0015] These and other aspects of the present disclosure will become apparent by referring to the following detailed description. Specific details for implementing the invention

[0016] In the following description, specific details are provided to provide a complete understanding of various embodiments of the present disclosure. However, those skilled in the art will understand that the present disclosure may be practiced without such details.

[0017] The present disclosure is partially about activators or therapeutic agents, such as nucleic acids, into mammalian cells. In vivo It is based on the discovery of novel cationic (amino) lipids that provide advantages when used in lipid nanoparticles for delivery. In particular, embodiments of the present disclosure provide increased activity of nucleic acids and In vivo The present invention provides a nucleic acid-lipid nanoparticle composition comprising one or more of the novel cationic lipids described herein, which provides improved tolerability of the composition and results in a significant increase in therapeutic index compared to previously described nucleic acid-lipid nanoparticle compositions. In another embodiment, the disclosed lipids and lipid nanoparticles comprising them have increased safety and / or tolerability when used for the delivery of active agents such as nucleic acids.

[0018] In certain embodiments, the present disclosure relates to mRNA and / or other oligonucleotides. In vitro and In vivoA novel cationic lipid is provided that enables the formulation of an improved composition for delivery. In some embodiments, this improved lipid nanoparticle composition is useful for the expression of a protein encoded by mRNA. In another embodiment, this improved lipid nanoparticle composition is useful for the upregulation of endogenous protein expression by delivering a miRNA inhibitor that targets a single target mRNA or a specific miRNA or group of miRNAs that regulate multiple mRNAs. In another embodiment, this improved lipid nanoparticle composition is useful for downregulating (e.g., silencing) the protein and / or mRNA levels of a target gene. In some other embodiments, the lipid nanoparticles are also useful for the delivery of mRNA and plasmids for the expression of transplanted genes. In another embodiment, the lipid nanoparticle composition is useful for inducing pharmacological effects resulting from protein expression, for example, increased red blood cell production through the delivery of an appropriate erythropoietin mRNA, or protection against infection through the delivery of mRNA encoding an appropriate antigen or antibody.

[0019] The lipid nanoparticles and compositions of the embodiments of the present disclosure In vitro and In vivo In both cases, they may be used for various purposes, including the delivery of encapsulated or conjugated (e.g., complexed) therapeutic agents, such as nucleic acids, into cells. Accordingly, embodiments of the present disclosure provide a method for treating or preventing a disease or disorder in a subject in need by bringing lipid nanoparticles encapsulated or conjugated with a suitable therapeutic agent into contact with the subject, wherein the lipid nanoparticles comprise one or more of the novel cationic lipids described herein.

[0020] As described herein, embodiments of the lipid nanoparticles of the present disclosure are particularly useful for the delivery of nucleic acids, including, for example, mRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomir / antimir), messenger RNA-interference complementary RNA (micRNA), DNA, polyvalent RNA, Dicer substrate RNA, complementary DNA (cDNA), etc. Accordingly, lipid nanoparticles and compositions of specific embodiments of the present disclosure are used by contacting cells with lipid nanoparticles comprising one or more novel cationic lipids described herein. In vitro and In vivo In both cases, they can be used to induce the expression of a desired protein, wherein the lipid nanoparticles encapsulate or bind to a nucleic acid (e.g., messenger RNA or plasmid encoding the desired protein) expressed to produce the desired protein, or inhibit the process of terminating mRNA expression (e.g., miRNA inhibitor). Alternatively, the lipid nanoparticles and compositions of embodiments of the present disclosure are used by contacting a cell with lipid nanoparticles comprising one or more novel cationic lipids described herein. In vitro and In vivo Both can be used to reduce the expression of target genes and proteins, wherein the lipid nanoparticles encapsulate or bind to a nucleic acid (e.g., antisense oligonucleotide or small interfering RNA (siRNA)) that reduces target gene expression. The lipid nanoparticles and compositions of embodiments of the present disclosure may also be used to co-transport other nucleic acids (e.g., mRNA and plasmid DNA) individually or in combination, such as to be useful for providing effects requiring the co-localization of other nucleic acids (e.g., mRNA encoding a suitable genetic modification enzyme and DNA segment(s) for integration into the host genome).

[0021] Nucleic acids for use in embodiments of the present disclosure may be prepared according to any available technology. In the case of mRNA, the primary methodology for preparation is enzymatic synthesis, which represents the most efficient method currently for generating long sequence-specific mRNA ( In vitro (also called a warrior) but is not limited to this. In vitro Transcription describes the template-oriented synthesis process of an RNA molecule from an engineered DNA template consisting 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 the gene of interest. The template DNA is obtained from multiple sources using appropriate techniques well known in the art, including, but not limited to, plasmid DNA and polymerase chain reaction amplification. In vitro It can be prepared for transcription (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).

[0022] RNA transcription is performed using a linearized DNA template in the presence of the corresponding RNA polymerase and adenosine, guanosine, uridine, and cytidine ribonucleoside triphosphates (rNTPs) under conditions that support polymerase activity while minimizing potential degradation of the generated mRNA transcript. In a test tube It occurs. In vitroTranscription can be performed using various commercially available kits, including but not limited to the RiboMax large-scale RNA production system (Promega) and the MegaScript transcription kit (Life Technologies), as well as commercially available reagents including RNA polymerase and rNTPs of mRNA. In vitro Methodologies for transcription are well known in the art. (See, for example; 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 these are incorporated herein by reference.)

[0023] After that, what you want In vitroTranscribed mRNA is purified from unwanted components of transcription or related reactions (including unintegrated rNTPs, proteolytic enzymes, salts, short RNA oligos, etc.). Techniques for isolating mRNA transcripts are well known in the art. Well-known processes include phenol / chloroform extraction or precipitation using alcohols (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride. Additional non-limiting examples of purification processes that may be used include 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 syntheses 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 purification and concentration kit (Norgen Biotek).

[0024] Furthermore, while reverse transcription can generate large amounts of mRNA, the product may contain many abnormal RNA impurities associated with unwanted polymerase activity that must be removed from full-length mRNA preparations. These include short RNA resulting from lactic transcription initiation, as well as double-stranded RNA (dsRNA) generated by RNA-dependent RNA polymerase activity, RNA-primed transcription from RNA templates, and self-complementary 3' extension. It has been demonstrated that these contaminants with dsRNA structures can induce unwanted immunostimulatory activity through interactions with various innate immune sensors in eukaryotic cells that function to recognize specific nucleic acid structures and induce potent immune responses. This can ultimately lead to a dramatic reduction in mRNA translation because protein synthesis is decreased during innate cellular immune responses. Accordingly, additional techniques for removing these dsRNA contaminants, including but not limited to scalable HPLC purification, have been developed and are known in the art (e.g., see 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, Nucleoside 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 is particularly primary cells and. In vivo It was reported to be translated at a much higher level.

[0025] In vitro A wide variety of modifications used to alter specific characteristics of transcribed mRNA and improve its utility have been described in the art. These include, but are not limited to, modifications to the 5' and 3' ends of the mRNA. Endogenous eukaryotic mRNA typically contains a cap structure at the 5'-terminus of the mature molecule; this plays a crucial role in mediating the binding of mRNA cap-binding proteins (CBPs), which are ultimately responsible for improving mRNA stability and the efficiency of mRNA translation in the cell. High levels of protein expression are achieved with capped mRNA transcripts. The 5'-cap contains a 5'-5'-triphosphate linkage between a 5'-most nucleotide and a guanine nucleotide. The conjugated guanine nucleotide is methylated at the N7 position. Additional modifications include the methylation of the 2'-hydroxyl group at the end and the second-to-end most 5'-nucleotide.

[0026] Several separate cap structures In vitro It can be used to generate the 5'-cap of transcribed synthetic mRNA. 5'-capping of synthetic mRNA can be performed co-transcribed with chemical cap analogs (i.e., In vitro(Capping during transcription). For example, the ARCA (Anti-Reverse Cap Analog) cap contains a 5'-5'-guanine-triphosphate bond, where one guanine contains an N7 methyl group and a 3'-O methyl group. However, up to 20% of transcripts remain uncapped during this co-transcription process, and synthetic cap analogs are not identical to the 5'-cap structure of true cellular mRNA, potentially reducing translational potential and cellular stability. Alternatively, synthetic mRNA molecules can also be enzymatically capped after transcription. These can generate more distinct 5'-cap structures that structurally or functionally mimic the endogenous 5'-cap more closely, enhancing the binding of cap-binding proteins, increasing half-life, decreasing sensitivity to 5' endonucleases, and / or reducing 5' decapping. Numerous synthetic 5'-cap analogs have been developed and are known in the art to improve mRNA stability and translation potential (e.g., see 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).

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

[0028] In vitro Poly(A) tailing of transcribed mRNA can be achieved using various approaches, including but not limited to cloning poly(T) tubules into a DNA template or post-transcriptional addition using poly(A) polymerase. The first case involves mRNA having a poly(A) tail of a length defined by the size of the poly(T) tubule. In vitro Transcription is allowed, but additional manipulation of the template is required. In the latter case, poly(A) polymerase is used. In vitroIt involves enzymatically adding a poly(A) tail to transcribed mRNA, which catalyzes the incorporation of adenine residues at the 3' end of RNA without the need for further manipulation of the DNA template, but results in mRNA with poly(A) tails of non-uniform length. 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), mMESSAGE mMACHINE T7 Ultra kit, and poly(A) tailing kit (Life Technologies), as well as commercially available reagents, various ARCA caps, poly(A) polymerases, etc.

[0029] In addition to the 5' cap and 3' polyadenylation, In vitro Other modifications of transcripts have been reported to provide benefits related to translation efficiency and stability. It is well known in the art that pathogenic DNA and RNA can be recognized by various sensors within eukaryotes and trigger a potent innate immune response. The ability to distinguish between pathogenic and autogenic 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, thereby conferring immunostimulatory properties that can inhibit effective mRNA translation as summarized above. In vitroBy introducing modified nucleosides into transcribed mRNA, it is possible to mitigate these unwanted immunostimulatory activities and enhance translational capacity by preventing the recognition and activation of RNA sensors (e.g., see; Kariko, K. And Weissman, D. 2007, Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: implication for therapeutic RNA development, Curr Opin Drug Discovery, 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, FA, Ludwig, J., Kato, H., Akira, S., Weissman, D., 2008, Incorporation of Pseudouridine Into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability, Mol Ther v.16, 1833-1840). Modified nucleosides and nucleotides used in the synthesis of modified RNA may be prepared, monitored, and used using general methods and procedures known in the art. Alone or in combination with other modified nucleosides In vitroA wide variety of nucleoside modifications are available that can be incorporated to some extent into transcribed mRNA (see, for example, US No. 2012 / 0251618). of nucleoside-modified mRNA In vitro Synthesis was reported to have reduced ability to activate immune sensors, along with accompanying enhanced translation ability.

[0030] Other components of mRNA that can be modified to provide advantages in terms of translatability and stability include the 5' and 3' untranslated regions (UTRs). Optimization of UTRs (advantageous 5' and 3' UTRs can be obtained from cellular or viral RNA) involves, either together or independently, mRNA stability and In vitro It has been shown to increase the translation efficiency of transcribed mRNA (e.g., see 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).

[0031] In addition to mRNA, other nucleic acid payloads may be used for the present disclosure. In the case of oligonucleotides, the manufacturing method involves chemical synthesis and enzymatic or chemical cleavage of longer precursors, as described above. In vitroMethods for synthesizing DNA and RNA nucleotides include, but are not limited to, transcription, etc. Methods for synthesizing DNA and RNA nucleotides are widely used and well known in the art (e.g., see 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; both are incorporated herein by reference).

[0032] In the case of plasmid DNA, preparation for use in embodiments of the present disclosure is generally performed in a liquid culture of bacteria containing the plasmid of interest. In vitroThe expansion and isolation of plasmid DNA are utilized, but are not limited thereto. If a gene encoding resistance to a specific antibiotic (penicillin, kanamycin, etc.) is present in the plasmid of interest, these bacteria containing the plasmid of interest can be selectively grown in antibiotic-containing cultures. Methods for isolating plasmid DNA are widely used and well known in the art (e.g., see Heilig, J., Elbing, KL and Brent, R., (2001), Large-Scale Preparation of Plasmid DNA, Current Protocols in Molecular Biology, 41:II:1.7:1.7.1-1.7.16; Rozkov, A., Larsson, B., Gillstrφm, S., Bjφrnestedt, 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. No. 6,197,553 B1). Plasmid isolation can be performed using various commercially available kits and commercially available reagents, including but not limited to Plasmid Plus (Qiagen), GenJET plasmid MaxiPrep (Thermo), and PureYield MaxiPrep (Promega) kits.

[0033] Various exemplary embodiments of the cationic lipids of the present disclosure, lipid nanoparticles and compositions containing the same, and their use for delivering active agents (e.g., therapeutic agents) such as nucleic acids to regulate gene and protein expression are described in more detail below.

[0034] As used herein, the following terms have the meanings assigned to them unless otherwise specified.

[0035] Unless the context otherwise requires, throughout this specification and claims, the word “comprise” and variations thereof, e.g., “comprises” and “comprising”, shall be interpreted in an open and inclusive sense, namely, “including, but not limited to.”

[0036] Throughout this specification, references to "one embodiment" or "an embodiment" mean that specific features, structures, or characteristics described in relation to an embodiment are included in at least one embodiment of this disclosure. Accordingly, the appearance of the phrases "in one embodiment" or "in an embodiment" at various locations throughout this specification does not necessarily refer to the same embodiment. Furthermore, specific features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which this disclosure pertains. As used in the specification and claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise.

[0038] The phrase “induces expression of a desired protein” refers to the ability of a nucleic acid to increase the expression of a desired protein. To investigate the degree of protein expression, a test sample (e.g., a sample of cells in a culture expressing the desired protein) or a test mammalian model (e.g., a human or animal mammalian model), such as a rodent (e.g., mouse) or a non-human primate (e.g., monkey) model, is brought into contact with a nucleic acid (e.g., a nucleic acid combined with the lipids of the present disclosure). The expression of the desired protein in the test sample or test animal is compared to the expression of the desired protein in a control sample (e.g., a sample of cells in a culture expressing the desired protein) or a control mammalian model (e.g., a human or animal mammalian model), such as a rodent (e.g., mouse) or a non-human primate (e.g., monkey) model, which is not brought into contact with or administered with the nucleic acid. When the desired protein is present in the control sample or control mammalian, a value of 1.0 may be assigned to the expression of the desired protein in the control sample or control mammalian. In certain embodiments, induction of expression of the desired protein is achieved when the ratio of the expression of the desired protein in the test sample or test mammal to the level of expression of the desired protein in the control sample or control mammal is 1 or greater, e.g., 1.1, 1.5, 2.0, 5.0, or 10.0. If the desired protein is not present in the control sample or control mammal, induction of expression of the desired protein is achieved when any measurable level of the desired protein is detected in the test sample or test mammal. A person skilled in the art will understand appropriate assays for determining the level of protein expression in a sample, e.g., dot blot, northern blot, in-situ hybridization, ELISA, immunoprecipitation, enzymatic function, and phenotypic analysis, or assays based on reporter proteins capable of producing fluorescence or luminescence under appropriate conditions.

[0039] The phrase “repressing the expression of a target gene” refers to the ability of a nucleic acid to silence, reduce, or suppress the expression of a target gene. To investigate the degree of gene silencing, a test sample (e.g., a sample of cells in a culture expressing the target gene) or a test mammalian model (e.g., a human or animal-like mammal) such as a rodent (e.g., mouse) or non-human primate (e.g., monkey) model is brought into contact with a nucleic acid that silences, reduces, or suppresses the expression of the target gene. The expression of the target gene in the test sample or test animal is compared to the expression of the target gene in a control sample (e.g., a sample of cells in a culture expressing the target gene) or a control mammalian model (e.g., a human or animal-like mammal) such as a rodent (e.g., mouse) or non-human primate (e.g., monkey) model that is not brought into contact with or administered the nucleic acid. A value of 100% may be assigned to the expression of the target gene in the control sample or control mammalian. In certain embodiments, silencing, suppressing, or reducing the expression of a target gene is achieved when the level of the target gene expression in the test sample or test mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% compared to the level of the target gene expression in the control sample or control mammal. That is, nucleic acid can silence, reduce, or inhibit the expression of the target gene in the test sample or test mammal by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the level of target gene expression in the control sample or control mammal that was not in contact with or administered nucleic acid.Suitable assays for determining the level of target gene expression include, without limitation, tests for protein or mRNA levels using techniques known to those skilled in the art, such as dot blot, northern blot, in situ hybridization, ELISA, immunoprecipitation, and enzymatic function, as well as phenotypic analyses known to those skilled in the art.

[0040] The "effective dose" or "therapeutic effective dose" of an activator or therapeutic agent, such as a therapeutic nucleic acid, is an amount sufficient to produce a desired effect, for example, an increase or inhibition of the expression of a target sequence compared to the normal expression level detected in the absence of the nucleic acid. An increase in the expression of the target sequence is achieved when any measurable level is detected in the absence of the nucleic acid for the expression product that is not present. When the expression product is present at a certain level before contact with the nucleic acid, an increase in expression is achieved when the multiple 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. Suppression of the expression of a target gene or target sequence is achieved when the value obtained with a nucleic acid, such as an antisense oligonucleotide, relative to the control is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring the expression of a target gene or target sequence include protein or RNA level testing using techniques known to those skilled in the art, such as dot blot, Northern blot, in situ hybridization, ELISA, immunoprecipitation, enzymatic function, and fluorescence or luminescence of a suitable reporter protein, as well as phenotypic analyses known to those skilled in the art.

[0041] As used herein, the term “nucleic acid” refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in a single- or double-stranded form, and includes DNA, RNA, and hybrids thereof. DNA may be in the form of an antisense molecule, plasmid DNA, cDNA, PCR product, or vector. RNA may 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 known nucleotide analogs or nucleic acids containing modified backbone residues or bindings that are synthetic, naturally occurring, and non-naturally occurring and have binding properties similar to those of a reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2'-O-ribonucleotides, and peptide-nucleic acids (PNA). Unless specifically limited, the above terms include nucleic acids containing known analogs of natural nucleotides having binding properties similar to those of the reference nucleic acid. Unless otherwise indicated, a specific nucleic acid sequence also implicitly includes its conservedly modified variants (e.g., synonymous codon substitutions), alleles, heterohomologous variants, single nucleotide polymorphisms, and complementary sequences, as well as explicitly indicated sequences. Specifically, synonymous codon substitutions can be achieved by producing a sequence in which the third position of one or more selected (or all) codons is substituted with a mixed-base and / or deoxyinosine residue (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))."Nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. The nucleotides are joined together through the phosphate group. "Base" includes purines and pyrimidines, including natural compounds such as adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, including but not limited to modifications that place a new reactive group, including but not limited to amines, alcohols, thiols, carboxylates, and alkyl halides.

[0042] The term "gene" refers to a sequence of nucleic acid (e.g., DNA or RNA) containing a partial-length or full-length coding sequence required for the production of a polypeptide or a precursor polypeptide.

[0043] As used herein, "gene product" refers to a product of a gene, such as an RNA transcript or polypeptide.

[0044] The term "lipids" refers to a group of organic compounds including, but not limited to, esters of fatty acids, which are generally poorly soluble in water but soluble in many organic solvents. These are typically divided into at least three classes: (1) "simple lipids" including fats and oils as well as waxes; (2) "complex lipids" including phospholipids and glycolipids; and (3) "derived lipids" such as steroids.

[0045] "Steroids" are compounds containing the following carbon skeletons:

[0046] .

[0047] Non-limiting examples of steroids include cholesterol, etc.

[0048] "Cationic lipids" refer to lipids that can be positively charged. Exemplary cationic lipids contain one or more amine group(s) that have a positive charge. Desired cationic lipids can be ionized so that they exist in a positively charged or neutral form depending on the pH. The ionization of cationic lipids affects the surface charge of lipid nanoparticles under different pH conditions. These charge states can affect plasma protein uptake, blood removal, and tissue distribution (Semple, SC, et al., Adv. Drug Deliv Rev 32:3-17 (1998)), as well as the ability to form endosometically degradable non-bilayer structures important for intracellular delivery of nucleic acids (Hafez, IM, et al., Gene Ther 8:1188-1196 (2001)).

[0049] The term "polymer-conjugated lipid" refers to a molecule containing 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 containing 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), etc.

[0050] The term "neutral lipid" refers to any of the many lipid species that exist in an uncharged form or in a neutral zwitterion form at a selected pH. At physiological pH, these lipids include, but are not limited to, phosphotidylcholines such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM), ceramides, steroids such as sterols, and derivatives thereof. Neutral lipids may be synthetic or of natural origin.

[0051] The term “charged lipid” refers to any of a number of lipid species that exist in a positively or negatively charged form regardless of pH within a useful physiological range, e.g., pH ~3 to pH ~9. Charged lipids may be synthetic or of natural origin. Examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyl trimethylammonium-propane (e.g., DOTAP, DOTMA), dialkyl dimethylaminopropane, ethyl phosphocholine, and dimethylaminoethane carbamoyl sterol (e.g., DC-Chol).

[0052] The term “lipid nanoparticle” refers to a particle having at least one dimension of the nanometer scale (e.g., 1–1,000 nm) comprising one or more compounds of Formula I or other specified cationic lipids. In some embodiments, lipid nanoparticles comprising the disclosed cationic lipids (e.g., compounds of Formula I) are included in a formulation that can be used to deliver an activator or therapeutic agent, e.g., nucleic acid (e.g., mRNA), to a target site of interest (e.g., cells, tissues, organs, tumors, etc.). In some embodiments, the lipid nanoparticles comprise a compound of Formula I and a nucleic acid. These lipid nanoparticles typically comprise a compound of Formula I and one or more excipients selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. In some embodiments, an activator or therapeutic agent, such as a nucleic acid, may be encapsulated in an aqueous space surrounded by the lipid portion of the lipid nanoparticle or by part or all of the lipid portion of the lipid nanoparticle to be protected from enzymatic degradation or other undesirable effects induced by mechanisms of the host organism or cell, e.g., a reverse immune response.

[0053] In various embodiments, the lipid nanoparticles are about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or 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 They have an average diameter of nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the lipid nanoparticles are substantially non-toxic. In certain embodiments, nucleic acids, when present in the lipid nanoparticles, are resistant to degradation by nucleases in aqueous solution. Lipid nanoparticles containing nucleic acids and methods for preparing the same are disclosed, for example, in U.S. Patent Publications No. 2004 / 0142025, No. 2007 / 0042031 and PCT Publications No. WO 2013 / 016058 and No. WO 2013 / 086373, the full disclosure thereof is incorporated herein by reference in its entirety for all purposes.

[0054] As used herein, "encapsulated lipid" refers to lipid nanoparticles that provide total, partial, or both encapsulation to an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA). In an embodiment, the nucleic acid (e.g., mRNA) is completely encapsulated in the lipid nanoparticle.

[0055] As used herein, the term "aqueous solution" refers to a composition containing water.

[0056] With respect to nucleic acid-lipid nanoparticles, "serum stability" means that nucleotides are not significantly degraded after exposure to serum or nuclease assays that significantly degrade free DNA or RNA. Suitable assays include, for example, standard serum assays, DNAse assays, or RNAse assays.

[0057] As used herein, "systemic delivery" refers to the delivery of a therapeutic product that may result in extensive exposure of the active agent within an organism. Some administration techniques may induce systemic delivery of a specific formulation, while others may not. Systemic delivery means that an effective amount, preferably a therapeutic amount, of the formulation is exposed to most of the body. Systemic delivery of lipid nanoparticles may be achieved by any means known in the art, including, for example, intravenous, intra-arterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is achieved by intravenous delivery.

[0058] As used herein, "local delivery" refers to the direct delivery of an active agent to a target site within an organism. For example, a formulation may be delivered locally by direct injection to a diseased site, such as a tumor; other target sites, such as a site of inflammation; or target organs, such as the liver, heart, pancreas, or kidney. Local delivery may also include topical application or local injection techniques, such as intramuscular, subcutaneous, or intradermal injection. Local delivery does not exclude systemic pharmacological effects.

[0059] "Alkyl" is saturated or unsaturated (i.e., containing one or more double (alkenyl) and / or triple (alkynyl) bonds), for example, 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-C15 alkyl), 1 to 12 carbon atoms (C1-C 12 A straight-chain or branched-chain hydrocarbon radical having 1 to 8 carbon atoms (C1-C8 alkyl) or 1 to 6 carbon atoms (C1-C6 alkyl), composed only of carbon and hydrogen atoms attached to the rest of the molecule by single bonds, e.g., 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, ethinyl, propynyl, butynyl, pentinyl, hexinyl, etc. Unless specifically stated otherwise in the specification, the alkyl group is optionally substituted.

[0060] "Alkylene" or "alkylene chain" is saturated or unsaturated (i.e., containing one or more double (alkenylene) and / or triple (alkynylene) bonds), for example, 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 A straight-chain or side-chain divalent hydrocarbon chain having 1 to 8 carbon atoms (C1-C8 alkylene), 1 to 6 carbon atoms (C1-C6 alkylene), 2 to 4 carbon atoms (C2-C4 alkylene), or 1 to 2 carbon atoms (C1-C2 alkylene), composed only of carbon and hydrogen, connecting the remainder of the molecule to a radical group, e.g., methylene, ethylene, propylene, n -Butylene, Ethenylene, Propenylene, n - Butenylene, Propynylene, n- Refers to butylene, etc. The alkylene chain is attached to the rest of the molecule via single or double bonds and to the radical group via single or double bonds. The attachment points of the alkylene chain to the rest of the molecule and to the radical group may be through one carbon or any two carbons within the chain. Unless specifically stated otherwise in the specification, the alkylene chain may be optionally substituted.

[0061] "Cycloalkyl" or "carbocyclic ring" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical composed solely of carbon and hydrogen atoms that are saturated or unsaturated and attached to the rest of the molecule by single bonds, which may include a fused or bridging ring system having 3 to 15 carbon atoms, preferably 3 to 10 carbon atoms. Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycycline radicals include, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, etc. Unless specifically stated otherwise in the specification, the cycloalkyl group may be optionally substituted.

[0062] "Cycloalkylene" is a divalent cycloalkyl group. Unless specifically stated otherwise in the specification, the cycloalkylene group may be optionally substituted.

[0063] "Heterocyclil" or "heterocyclic ring" refers to a stable 3 to 18 (e.g., 5, 6, or 7) non-aromatic ring radical 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 heterocyclil radicals include, but are not limited to, azetidinyl, pyrrolidinyl, piperidinyl, imidazolidinyl, tetrahydropyrimidinyl, etc. Unless specifically stated otherwise in the specification, the heterocyclil group may be optionally substituted.

[0064] "Heteroaryl" refers to a 5- to 14-membered ring system radical comprising 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, etc. Unless specifically stated otherwise in the specification, the heteroaryl group may be optionally substituted.

[0065] As used herein, the term "substituted" refers to at least one hydrogen atom being a halogen atom such as F, Cl, Br, or I; an oxo group (=O); a hydroxyl group (-OH); or a C1-C 12 Alkyl group; cycloalkyl group; -(C=O)OR ' ; -O(C=O)R ' ; -C(=O)R ' ; -OR ' ; -S(O) x R ' ; -S-SR ' ; C(=O)SR ' ; -SC(=O)R ' ; -NR ' R ' ; -NR ' C(=O)R ' ; -C(=O)NR' R ' ; -NR ' C(=O)NR ' R ' ; -OC(=O)NR ' R ' ; -NR ' C(=O)OR ' ; -NR ' S(O) x NR ' R ' ; -NR ' S(O) x R ' ; and -S(O) x NR ' R ' (here, R ' is, at each occurrence, independently H, C1-C 15 It refers to any of the above groups (e.g., alkyl, alkylene, cycloalkyl, or cycloalkylene) that are replaced by bonding to a non-hydrogen atom that is the same as, but not limited to, alkyl, or cycloalkyl, where x is 0, 1, or 2. In some embodiments, the substituent is C1-C 12 It is an alkyl group. In another embodiment, the substituent is a cycloalkyl group. In another embodiment, the substituent is a halo group such as a 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 ' ) is. In another embodiment, the substituent is a carboxyl group. In another embodiment, the substituent is an amine group (-NR ' R ' )am.

[0066] "Any" or "any" (e.g., "any substituted") means that the event of the situation described below may or may not occur, and the description includes cases where said event or situation occurs and cases where it does not occur. For example, "any substituted alkyl" means that the alkyl radical may or may not be substituted, and the description includes both the substituted alkyl radical and the unsubstituted alkyl radical.

[0067] The disclosures disclosed herein are also intended to include all pharmaceutically acceptable compounds of isotope-labeled compounds of Formula I by replacing one or more atoms with atoms having different atomic masses or mass numbers. Examples of isotopes that may be included in the disclosed compounds are, respectively 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 It includes isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as I. These radiolabeled compounds can be useful in helping to determine or measure the efficacy of a compound by, for example, characterizing the site or mode of action, or binding affinity to pharmacologically important sites. Specific isotope-labeled compounds of chemical formulas I, IA, or IB, for example, compounds containing radioisotopes, are useful for studies on drug and / or substrate tissue distribution. The radioisotope tritium, i.e. 3 H, and carbon-14, i.e. 14It is particularly useful for this purpose in that C is easy to include and the means of detection are easy.

[0068] Deuterium, that is 2 Substitution with heavier isotopes such as H results in greater metabolic stability, for example, In vivo It may provide specific therapeutic benefits resulting from an increased half-life or reduced dosage requirements, which may be desirable in some situations.

[0069] 11 C, 18 F, 15 O and 13 Substitution with a positron-emitting isotope such as N can be useful in positron emission topography (PET) studies to investigate substrate acceptor occupancy. The isotope-labeled compound of Formula I can be prepared by using a suitable isotope-labeled reagent instead of a previously used unlabeled reagent by conventional techniques generally known to those skilled in the art or by a process similar to that described in the preparation and examples presented below.

[0070] The embodiments disclosed herein also include the disclosed compounds In vivo It is intended to include metabolic products. Such products may be, for example, the result of oxidation, reduction, hydrolysis, amidation, esterification, etc. of the administered compound due to primarily enzymatic processes. Accordingly, embodiments of the present disclosure include compounds produced by a method comprising administering the compound of the present disclosure to a mammal for a period sufficient to produce its metabolic products. Such products are typically identified by administering the radiolabeled compound of the present disclosure to an animal, e.g., a rat, mouse, guinea pig, monkey, or human, at a detectable dose, allowing sufficient time for metabolism to occur, and isolating the converted products from urine, blood, or other biological samples.

[0071] "Stable compound" and "stable structure" refer to a compound that is robust enough to withstand isolation from the reaction mixture to a useful degree of purity and formulation into an effective therapeutic agent.

[0072] "Mammals" include humans, domestic animals such as laboratory animals and domestic pets (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits), and non-domestic animals such as wild animals.

[0073] "Pharmaceuticalally acceptable carriers, diluents, or excipients" include, without limitation, any adjuvants, carriers, excipients, lubricants, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers approved by the U.S. Food and Drug Administration (FDA) as acceptable for use in humans or livestock.

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

[0075] "Pharmaceutically acceptable acid addition salts" are inorganic acids that are not biologically or otherwise undesirable, such as but not limited to hydrochloric acid, hydrobromide, sulfuric acid, nitric acid, and phosphoric acid, and acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphor acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactar acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, Glycerophosphate, glycolic acid, hipfuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucinous 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, parmoic acid, propionic acid, pyroglutamic acid, pyruvate, salicylic acid, 4-aminosalicylic acid, sebactic acid, stearic acid, succinic acid, tartaric acid, thiocyanate, p - Refers to a salt formed from an organic acid, such as but not limited to toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid, that possesses the biological effects and properties of a free base.

[0076] "Pharmaceuticalally acceptable base-added salts" refers to salts that retain the biological effects and properties of a free acid, which are biologically or otherwise undesirable. Such salts are prepared by adding an inorganic or organic base to a 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, etc. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, e.g., ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydravamin, choline, betaine, benetamine, benzathine, ethylenediamine, glucosamine, methylglucarmin, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N - It includes, but is not limited to, salts such as ethylpiperidine and polyamine resins. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0077] Often, crystallization produces a solvate of the compound of the present disclosure (i.e., the compound of Formula I). ​​As used herein, the term “solvate” refers to an aggregate comprising one or more molecules of the compound of the present disclosure and one or more molecules of the solvent. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compound of the present disclosure may exist in hydrates, including monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate, etc., as well as corresponding solvated forms. The solvate of the compound of the present disclosure may be a true solvate, whereas in other cases, the compound of the present disclosure may simply contain adventitious water or be a mixture of water and some adventitious solvent.

[0078] "Pharmaceutical composition" refers to a formulation of the compound of the present disclosure and a medium generally accepted in the art for delivering a biologically active compound to a mammal, e.g., a human. Such a medium comprises any pharmaceutically acceptable carrier, diluent, or excipient for this purpose.

[0079] "Effective dose" or "therapeutic effective dose" refers to an amount of the compound of the present disclosure sufficient to perform treatment in a mammal, preferably a human, when administered to a mammal, preferably a human. The amount of lipid nanoparticles of the present disclosure constituting the "therapeutic effective dose" will vary depending on the compound, the condition and severity thereof, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by a person skilled in the art in consideration of their own knowledge and the present disclosure.

[0080] As used herein, "treating" or "treatment" encompasses the treatment of the disease or condition of interest in mammals having the disease or condition of interest, preferably in humans, and includes the following:

[0081] (i) preventing a disease or pathological condition from occurring in mammals, in particular, where such mammals are susceptible to disease but have not yet been diagnosed with the disease;

[0082] (ii) suppressing a disease or pathological condition, that is, inhibiting its development;

[0083] (iii) alleviating a disease or condition, i.e., causing a regression of the disease or condition; or

[0084] (iv) Alleviating symptoms caused by a disease or condition, that is, relieving pain without addressing the underlying disease or condition. The terms “disease” and “condition” as used herein may be used interchangeably or may differ in that a specific disease or condition may not have known causative factors (so the etiology is not yet known) and is therefore not recognized as a disease but only as an undesirable condition or syndrome, in which a somewhat specific series of symptoms has been identified by a clinician.

[0085] The compounds of the present disclosure, or pharmaceutically acceptable salts thereof, may contain one or more stereocenters and thus, in terms of enantiomers, diastereomers, and absolute stereochemistry ( R )- or ( SThis may result in other stereoisomer forms that can be defined as (D)- or (L)- in the case of amino acids. The present disclosure implies that all such possible isomers, as well as their racemic and optically pure forms, are included. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers may be prepared using chiral synthones or chiral reagents, or may be decomposed using conventional techniques, e.g., chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from suitable optically pure precursors or, e.g., decomposition of racemics (or racemics of salts or derivatives) using chiral high-pressure liquid chromatography (HPLC). Where the compounds described herein contain olefin double bonds or other centers of geometric asymmetry, the compounds are intended to include both E and Z geometric isomers unless otherwise specified. Likewise, it is intended to include all tautomeric forms.

[0086] "Stereoisomer" refers to a compound having different three-dimensional structures composed of the same atoms bonded by the same bond but which are non-interchangeable. The present disclosure considers various stereoisomers and mixtures thereof, and includes "enantiomers," which refer to two stereoisomers that are mirror images of each other and whose molecules do not overlap.

[0087] "Tautomer" refers to the transfer of a proton from one atom of a molecule to another atom of the same molecule. The present disclosure includes tautomers of any of the said compounds.

[0088] compound

[0089] In one aspect, the present disclosure provides novel lipid compounds capable of forming oligonucleotides and lipid nanoparticles in combination with other lipid components, such as neutral lipids, charged lipids, steroids, and / or polymer-conjugated lipids. Without intending to be theoretical, these lipid nanoparticles protect oligonucleotides from degradation in serum and In vitro and In vivo It is thought to provide effective delivery of oligonucleotides into cells.

[0090] In one embodiment, the compound has the following formula I or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:

[0091] Chemical Formula I

[0092]

[0093] In the above chemical formula I,

[0094] R 1 is an arbitrarily substituted C1-C 24 alkyl or optionally substituted C2-C 24 It is alkenyl;

[0095] R 2 and R 3 Each is an independently and arbitrarily substituted C1-C 36 It is alkyl;

[0096] R 4 and R 5 are each independently and arbitrarily substituted C1-C6 alkyls, or R 4 and R 5 They combine with the attached N to form a heterocyclile or heteroaryl;

[0097] L 1 , L 2 , and L 3 Each is an independently and arbitrarily substituted C1-C 18 It is an alkylene;

[0098] G 1 is a direct bond, -(CH2) nO(C=O)-, -(CH2) n (C=O)O-, or -(C=O)- and;

[0099] G 2 and G 3 Each is independently -(C=O)O- or -O(C=O)- and;

[0100] n is an integer greater than or equal to 0.

[0101] In some embodiments, the compound has the following chemical formula IA:

[0102] [Chemical Formula IA]

[0103] .

[0104] In some embodiments, the compound has the following chemical formula IB:

[0105] [Chemical Formula IB]

[0106] .

[0107] In some embodiments, R 1 is an arbitrarily substituted C6-C 18 alkyl or C 14 -C 18 It is an alkenyl. In certain embodiments, R 1 C8alkyl, C9alkyl, C 10 Alkyl, C 12 Alkyl, C 14 alkyl, or C 16 It is an alkyl. In some more specific embodiments, R 1 C 16 It is an alkenyl. In certain more specific embodiments, R 1 is not branched. In some embodiments, R 1 is branched. In certain embodiments, R 1 is not substituted.

[0108] In some embodiments, G 1 is a direct bond, -(CH2) n O(C=O)-, or -(CH2) n (C=O)O- is. In certain embodiments, G 1is a direct bond. In some more specific embodiments, G 1 -(CH2) n (C=O)O- and n is 1 or greater. In some embodiments, n is 1-20. In some embodiments, n is 1-10. In some embodiments, n is 5-11. In some embodiments, n is 6-10. In certain more specific embodiments, n is 5, 6, 7, 8, 9, or 10. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In certain embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10.

[0109] In some embodiments, L 1 is a C1-C6 alkylene. In certain embodiments, L 1 is a C2-alkylene, C3-alkylene, or C4-alkylene. In some more specific embodiments, L 1 is not branched. In a specific, more specific embodiment, L 1 is not substituted.

[0110] In some embodiments, R 2 is C8-C 24 It is an alkyl. In some embodiments, R 3 C8-C 24 It is an alkyl. In some more specific embodiments, R 2 and R 3 Both are C8-C 24 It is an alkyl. In some embodiments, R 2 and R 3 C 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 It is an alkyl. In certain embodiments, R 2is branched. In a more specific embodiment, R 3 is branched. In some more specific embodiments, R 2 and R 3 Each independently has one of the following structures:

[0111] or

[0112] Here:

[0113] R 6 and R 7 C2-C each independently 12 It is an alkyl.

[0114] In some embodiments, R 2 and R 3 Each independently has one of the following structures:

[0115] ; ; ; ; or .

[0116] In some embodiments, L 2 and L 3 Each independently C4-C 10 It is an alkylene. In certain embodiments, L 2 and L 3 Both are C5 alkylenes. In some more specific embodiments, L 2 and L 3 Both are C6-alkylenes. In certain embodiments, L 2 and L 3 Both are C8-alkylenes. In some more specific embodiments, L 2 and L 3 Both are C9-alkylenes. In some embodiments, L 2 is not branched. In some embodiments, L 3 is not branched. In a more specific embodiment, L 2 is not substituted. In some embodiments, L 2is not substituted.

[0117] In some embodiments, R 4 and R 5 Each is independently a C1-C6 alkyl. In a more specific embodiment, R 4 and R 5 Both are methyl. In certain embodiments, R 4 and R 5 Both are ethyl. In certain embodiments, R 4 is methyl and R 5 is n-butyl. In some embodiments, R 4 and R 5 Both are n-butyl. In different embodiments, R 4 is methyl and R 5 is n-hexyl.

[0118] In some embodiments, R 4 and R 5 They combine with the N to which they are attached to form a heterocyclile. In certain embodiments, the heterocyclile is a pentagonal heterocyclile. In some embodiments, the heterocyclile has the following structure:

[0119] .

[0120] In various different embodiments, the compound has one of the structures presented in Table 1 below.

[0121] Table 1 . Compound included in the embodiment of the compound of Chemical Formula I

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129] It is understood that any embodiment of the compound of Formula I as presented above, and any specific substituent and / or variable in the compound of Formula I as presented above, may independently be combined with other embodiments of the compound of Formula I and / or substituents and / or variables to form embodiments of the present disclosure not specifically presented above. Furthermore, where a list of substituents and / or variables is listed for any specific R group, G group, L group, or variable n, it is understood that, in a specific embodiment and / or claim, each individual substituent and / or variable may be omitted from the specific embodiment and / or claim, and the remaining list of substituents and / or variables shall be deemed to be within the scope of the present disclosure.

[0130] In this description, it is understood that combinations of substituents and / or variables of the illustrated chemical formulas are permitted only if such contribution produces a stable compound.

[0131] In some embodiments, lipid nanoparticles comprising a compound of Formula I are provided. The lipid nanoparticles optionally comprise an excipient selected from neutral lipids, steroids, and polymer-conjugated lipids.

[0132] In some embodiments, a composition is provided comprising any one or more compounds of Formula I and a therapeutic agent. For example, in some embodiments, the composition comprises any compound of Formula I, a therapeutic agent, and one or more excipients selected from neutral lipids, steroids, and polymer-conjugated lipids. Other pharmaceutically acceptable excipients and / or carriers are also included in various embodiments of the composition.

[0133] 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.

[0134] In various embodiments, the composition further comprises a steroid or a steroid analog. In certain embodiments, the steroid or steroid analog is cholesterol. In some of these embodiments, the molar ratio of the compound to cholesterol is in the range of about 5:1 to 1:1.

[0135] In various embodiments, the polymer-conjugated lipid is a pegylated lipid. For example, some embodiments are pegylated diacylglycerol (PEG-DAG), e.g., 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), pegylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG), e.g., 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), pegylated ceramide (PEG-cer), or PEG dialkoxypropylcarbamate, e.g., ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or It includes 2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of the compound to the pegylated lipid is in the range of about 100:1 to about 20:1.

[0136] In some embodiments, the composition comprises a pegylated lipid having the following formula II or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:

[0137] [Chemical Formula II]

[0138]

[0139] In the above chemical formula II,

[0140] R 8 and R 9 Each is a straight or side chain saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms independently, wherein the alkyl chain is optionally blocked by one or more ester bonds;

[0141] w has an average value in the range of 30 to 60.

[0142] In some embodiments, R 8 and R 9 Each is a straight-chain saturated alkyl chain containing 12 to 16 carbon atoms independently. In another embodiment, the average w is in the range of about 42 to 55, for example, about 49.

[0143] In some embodiments of the above composition, the therapeutic agent comprises a nucleic acid. For example, in some embodiments, the nucleic acid is selected from antisense and messenger RNA. In some of the above embodiments, the composition comprises lipid nanoparticles.

[0144] Some related embodiments provide lipid nanoparticles comprising a compound of any one of the embodiments (e.g., a compound of Formula I). ​​In certain embodiments, the lipid nanoparticles further comprise a therapeutic agent (e.g., a nucleic acid such as antisense and messenger RNA).

[0145] In some embodiments, the lipid nanoparticles further comprise one or more excipients selected from neutral lipids, steroids, and polymer-conjugated lipids. In some embodiments, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In a more specific embodiment, the neutral lipid is DSPC.

[0146] In some more specific embodiments, the molar ratio of the compound to the neutral lipids is in the range of 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 is in the range of 5:1 to 1:1.

[0147] In certain embodiments, the polymer-conjugated lipid is a pegylated lipid. In certain more specific embodiments, the molar ratio of the compound to the pegylated lipid is about 100:1 to about 20:1.

[0148] In some embodiments, the pegylated lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, or PEG-dialkeopropylcarbamate. In another embodiment, the pegylated lipid has the following formula II or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:

[0149] Chemical Formula II

[0150]

[0151] In the above chemical formula II,

[0152] R 8 and R 9 Each is a straight or side chain saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms independently, wherein the alkyl chain is optionally blocked by one or more ester bonds;

[0153] w has an average value in the range of 30 to 60.

[0154] In some more specific embodiments of Chemical Formula II, R 8 and R 9 Each is a straight-chain saturated alkyl chain containing 12 to 16 carbon atoms independently. In a more specific embodiment, the average w is about 49.

[0155] In another different embodiment, the present disclosure relates to a method for administering a therapeutic agent to a patient in need thereof, comprising the steps of preparing or providing any of the above-mentioned compositions and administering the composition to a patient.

[0156] For the purpose of administration, embodiments of the compounds of the present disclosure (typically in the form of lipid nanoparticles combined with a therapeutic agent) may be administered as raw chemicals or formulated as pharmaceutical compositions. Pharmaceutical compositions of embodiments of the present disclosure comprise the compound of Formula I and one or more pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the compound of Formula I forms lipid nanoparticles and is present in the composition in an amount effective for delivering a therapeutic agent to treat, for example, a specific disease or condition of interest. Appropriate concentrations and dosages can be readily determined by a person skilled in the art.

[0157] The administration of the composition of the embodiments of the present disclosure may be carried out through any of the permitted modes of administration of formulations to provide similar utility. The pharmaceutical composition of the embodiments of the present disclosure may be formulated into solid, semi-solid, liquid, or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes of administration of such pharmaceutical compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. As used herein, the term parenteral includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection, or infusion techniques. The pharmaceutical composition of the embodiments of the present disclosure is formulated so that the active ingredient contained therein is bioavailable when the composition is administered to a patient. A composition to be administered to a subject or patient takes the form of one or more dosage units in some embodiments, wherein, for example, a tablet may be a single dosage unit, and a container of a compound of an embodiment of the present disclosure in an aerosol form may contain multiple dosage units. Actual methods for preparing such dosage forms will be known or obvious to those skilled in the art; for example, see literature; Remington : The Science and Practice of Pharmacy , 20th Edition (Philadelphia College of Pharmacy and Science, 2000). In some embodiments, the composition to be administered will, in any case, contain a therapeutically effective amount of the compound of the present disclosure or a pharmaceutically acceptable salt thereof for the treatment of a disease or condition of interest in accordance with the teachings of the present disclosure.

[0158] The pharmaceutical composition of an embodiment of the present disclosure may be in solid or liquid form. In one aspect, since the carrier(s) are particulates, the composition is, for example, in tablet or powder form. The carrier(s) may be liquid, and the composition is, for example, an oral syrup, an injectable liquid, or an aerosol, which is, for example, useful for inhalation administration.

[0159] When intended for oral administration, a pharmaceutical composition of a particular embodiment is preferably in a solid or liquid form, wherein semi-solid, semi-liquid, suspension, and gel forms are included within the forms considered as solid or liquid herein.

[0160] As a solid composition for oral administration, the pharmaceutical composition of some embodiments may be formulated in the form of powder, granules, compressed tablets, pills, capsules, chewing gum, wafers, etc. Such solid compositions will typically contain one or more inert diluents or edible carriers. Additionally, one or more of the following may be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch, lactose, or dextrin; disintegrants such as alginate, sodium alginate, Primogel, corn starch, etc.; lubricants such as magnesium stearate or Sterotex; lubricants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavoring agents such as peppermint, methyl salicylate, or orange flavor; and coloring agents.

[0161] In some embodiments, when the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain a liquid carrier such as polyethylene glycol or oil in addition to the material of the above type.

[0162] The pharmaceutical composition of some embodiments may be in the form of a liquid, e.g., an elixir, syrup, solution, emulsion, or suspension. The liquid may be for oral administration or delivery by injection, in two examples. For oral administration, the preferred composition contains, in addition to the compound of Formula I, one or more of a sweetener, a preservative, a colorant / coloring agent, and a flavor enhancer. For compositions intended for administration by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersant, a suspending agent, a buffer, a stabilizer, and an isotonic agent may be included.

[0163] A liquid pharmaceutical composition of an embodiment of the present disclosure, whether in a solution, suspension, or other similar form, may comprise one or more of the following adjuvants: water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, a fixative oil such as synthetic mono or diglycerides that can act as a solvent or suspension medium, a sterile diluent such as polyethylene glycol, glycerin, propylene glycol, or other solvents; an antimicrobial agent such as benzyl alcohol or methyl paraben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate and an adrenal regulator such as sodium chloride or dextrose; and an adrenal regulator such as sucrose or trehalose. Parenteral formulations may be enclosed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. The injectable pharmaceutical composition is preferably in a sterile state.

[0164] A liquid pharmaceutical composition of an embodiment of the present disclosure intended for parenteral or oral administration must contain an amount of the compound of the present disclosure such that an appropriate dosage is obtained.

[0165] The pharmaceutical composition of an embodiment of the present disclosure may be for topical administration, wherein the carrier may suitably comprise a solution, emulsion, ointment, or gel base. The base may comprise, for example, one or more of the following: diluents such as petroleum jelly, lanolin, polyethylene glycol, beeswax, mineral oil, water, and alcohol, and emulsifiers and stabilizers. Thickeners may be present in the pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may comprise a transdermal patch or an iontophoresis device.

[0166] Pharmaceutical compositions of embodiments of the present disclosure may be intended for rectal administration, for example, in the form of suppositories that melt in the rectum to release a drug. Compositions for rectal administration may contain an oily base as a suitable non-irritating excipient. Such bases include, without limitation, lanolin, cocoa butter, and polyethylene glycol.

[0167] Pharmaceutical compositions of embodiments of the present disclosure may include various materials that modify the physical form of solid or liquid dosage units. For example, the composition may include a material that forms a coating shell around an active ingredient. The material forming the coating shell is typically inert and may be selected from, for example, sugars, shellacs, and other enteric coating agents. Alternatively, the active ingredient may be packaged in a gelatin capsule.

[0168] A pharmaceutical composition of an embodiment of the present disclosure in solid or liquid form may include a formulation that binds to the compound of the present disclosure and assists in the delivery of the compound. Suitable formulations capable of acting with such ability include monoclonal or polyclonal antibodies or proteins.

[0169] The pharmaceutical composition of the embodiments of the present disclosure may consist of a dosing unit that can be administered as an aerosol. The term aerosol is used to denote various systems ranging from colloids to systems composed of pressurized packages. Delivery may be carried out by liquefied or compressed gas or by a suitable pump system that dispenses the active ingredient. The aerosol of the compound of the embodiments of the present disclosure may be delivered to a single-phase, two-phase, or three-phase system to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activator, valve, sub-container, etc., which can together form a kit. A person skilled in the art can determine the desired aerosol without excessive experimentation.

[0170] Pharmaceutical compositions of embodiments of the present disclosure may be prepared by methodologies widely known in the pharmaceutical field. For example, pharmaceutical compositions for administration by injection may be prepared by combining lipid nanoparticles of the present disclosure with sterile distilled water or other carriers to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. The surfactant is a compound that interacts non-covalently with the compound of the present disclosure to facilitate the dissolution or homogeneous suspension of the compound in an aqueous delivery system.

[0171] The composition of an embodiment of the present disclosure, or a pharmaceutically acceptable salt thereof, is administered in a therapeutically effective amount, which will depend on various factors including the activity of the specific therapeutic agent used; the metabolic stability and duration of action of the therapeutic agent; the age, weight, general health, gender, and diet of the patient; the manner and time of administration; the rate of elimination; the combination of drugs; the severity of the specific disorder or condition; and the subject being treated.

[0172] The composition of the embodiments of the present disclosure may 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 form of the composition of the embodiments of the present disclosure and one or more additional active agents, as well as the administration of the composition of the embodiments of the present disclosure and each active agent in its own separate pharmaceutical dosage form. For example, the composition of the embodiments of the present disclosure and other active agents may be administered together to a patient as a single oral dosage composition, such as a tablet or capsule, or each formulation may be administered in a separate oral dosage form. Where separate dosage forms are used, the compound of the embodiments of the present disclosure and one or more additional active agents may be administered essentially at the same time, i.e., simultaneously, or separately at a time apart, i.e., sequentially; it is understood that combination therapy includes all such administrations.

[0173] Methods for preparing the above compounds and compositions are described below in this document and / or are known in the art.

[0174] It will be recognized by those skilled in the art that in the method described herein, the active group of the intermediate compound may need to be protected by a suitable protective group. Such active groups include hydroxyl, amino, mercapto, and carboxylic acids. Suitable protective groups for hydroxyl groups are trialkylsilyl or diarylalkylsilyl (e.g., t -butyldimethylsilyl, t Suitable protection groups for amino, amidino, and guanidino include butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, etc. t - Includes butoxycarbonyl, benzyloxycarbonyl, etc. Suitable protection groups for mercapto are -C(O)-R" (where R" is alkyl, aryl, or arylalkyl), p- Includes methoxybenzyl, trityl, etc. Suitable protection groups for carboxylic acids include alkyl, aryl, or arylalkyl esters. Protection groups may be added or removed according to standard techniques known to those skilled in the art or as described herein. The use of protection groups [Green, TW and PGM Wutz, Protective Groups in Organic Synthesis (1999), 3 rd This is described in detail in Ed., Wiley. As is known to those skilled in the art, the protective group may also be a polymeric resin such as Wang resin, Rink resin, or 2-chlorotrityl-chloride resin.

[0175] Although such protected derivatives of the compounds of the present disclosure may not possess pharmacological activity in themselves, it will also be recognized by those skilled in the art that they may be metabolized in the body after administration to mammals to form pharmacologically active compounds of the present disclosure. Thus, such derivatives may be described as "prodrugs." All prodrugs of the compounds of the present disclosure are included within the scope of the present disclosure.

[0176] Additionally, compounds of embodiments of the present disclosure existing in the form of a free base or acid may be converted into pharmaceutically acceptable salts thereof by treating with a suitable inorganic or organic base or acid by a method known to those skilled in the art. Salts of compounds of embodiments of the present disclosure may be converted into their free base or acid forms by standard techniques.

[0177] The following general reaction scheme 1 illustrates an exemplary method for preparing the compounds of the present disclosure, namely the compound of formula I or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:

[0178] Chemical Formula I

[0179]

[0180] In the above chemical formula I, R 1 , R 2 , R 3 , R 4 , R 5 , L 1 , L 2 , L 3 , G 1 , G 2 , and G 3 ...as defined herein. It is understood that those skilled in the art may prepare these compounds by similar methods or by combining other methods known to those skilled in the art. Furthermore, it is understood that those skilled in the art may prepare other compounds of Formula I not specifically exemplified below in a manner similar to that described below by using appropriate starting ingredients and modifying the parameters of synthesis as necessary. In general, starting ingredients may be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, synthesized according to sources known to those skilled in the art (e.g., see Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)) or prepared as described in this disclosure.

[0181] General reaction equation 1

[0182]

[0183] General Reaction Scheme 1 provides an exemplary method for the preparation of a compound of Formula I. In General Reaction Scheme 1, R 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 2 is a reactive moiety (e.g., halo) selected to promote the desired reaction. A compound of structure A1 is purchased or prepared according to methods known in the art. Under appropriate reducing conditions (e.g., sodium triacetoxyborohydride), the reaction of A1 produces A3, a product of the reductive amination between A1 and A2. Subsequently, A3 is reacted with A4 under appropriate basic conditions (e.g., using triethylamine and DMAP) to obtain compound A5. Subsequently, A5 is reacted with amine A6 under appropriate conditions (e.g., heat) to produce a compound of formula I as shown.

[0184] It should be noted that various alternative strategies for the preparation of compounds of Formula I are available to those skilled in the art. For example, other compounds of Formula I that can be prepared according to similar methods using appropriate starting materials. The use of protection groups and other modifications to the general reaction scheme, as necessary, will be readily apparent to those skilled in the art.

[0185] The following examples are provided for illustrative purposes, not for limitation.

[0186] Example 1

[0187] In vivo evaluation of luciferase mRNA using lipid nanoparticle composition

[0188] Lipid nanoparticles were prepared and tested according to the general procedures described in PCT Publications WO 2015 / 199952 and WO 2017 / 004143, the full contents of which are incorporated herein by reference. Briefly, cationic lipids, DSPC, cholesterol, and PEG-lipids were dissolved in ethanol in molar ratios of about 50:10:38.5:1.5 or about 47.5:10:40.8:1.7. Lipid nanoparticles (LNPs) were prepared with a total lipid to mRNA weight ratio of approximately 10:1 to 30:1. mRNA was diluted to 0.2 mg / mL in 10 to 50 mM citrate or acetate buffer, pH 4. Using a syringe pump, the ethanolic lipid solution was mixed with the mRNA aqueous solution at a ratio of about 1:5 to 1:3 (vol / vol) with a total flow rate of at least 15 mL / min. Afterward, the ethanol was 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 lipid nanoparticle size was approximately 55–95 nm in diameter, as determined by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK), and in some cases, approximately 70–90 nm in diameter.

[0189] Studies were conducted on 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 Animal Care Committee (CCAC). Various doses of mRNA-lipid nanoparticles were administered systemically by tail vein injection, and the animals were euthanized at a specific time point (e.g., 4 hours) after administration. Livers and spleens were collected in pre-weighed tubes, weighed, immediately flash-frozen in liquid nitrogen, and stored at -80°C until processing for analysis.

[0190] For the liver, approximately 50 mg was dissected in a 2 mL FastPrep tube (MP Biomedicals, Solon OH) for analysis. A ¼" ceramic sphere (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 using a FastPrep24 instrument (MP Biomedicals) at 2 x 6.0 m / s for 15 seconds. The homogenate was incubated at room temperature for 5 minutes before diluting it 1:4 in GLB and evaluated using the SteadyGlo luciferase assay system (Promega). Specifically, 50 μL of the diluted tissue homogenate was reacted with 50 μL of SteadyGlo substrate, shaken for 10 seconds, incubated for 5 minutes, and then quantified using a CentroXS³ LB 960 luminometer (Berthold Technologies, Germany). The amount of analyzed protein was determined using a BCA protein assay kit (Pierce, Rockford IL). Subsequently, relative luminescence units (RLU) was normalized against the total μg protein analyzed. A standard curve was generated using QuantiLum recombinant luciferase (Promega) to convert RLU to ng luciferase.

[0191] FLuc mRNA (L-6107 or L-7202) from Trilink Biotechnologies will express the luciferase protein isolated from the firefly *Photinus pyralis*. FLuc is commonly used in mammalian cell cultures to measure gene expression and cell viability. It emits bioluminescence in the presence of the substrate luciferin. These capped and poly-adenylated mRNAs were completely substituted for uridine and / or cytidine nucleosides.

[0192] Example 2

[0193] pK of formulation lipids a decision

[0194] As described elsewhere, the pK of formulated cationic lipids a is correlated with the effect of LNPs for nucleic acid delivery (cf. Jayaraman et al, Angewandte Chemie , International Edition (2012), 51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176 (2010)). pK a The desirable range is ~5 to ~7. pK of each cationic lipid a TNS was determined 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%) in PBS with a total lipid concentration of 0.4 mM were prepared using an inline process as described in Example 1. TNS was prepared as a 100 μM stock solution in distilled water. The vesicles were diluted to 24 μM lipid in 2 mL of a buffer solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl, where the pH range is 2.5 to 11. Aliquots of TNS solution were added to provide a final concentration of 1 μM, and after vortex mixing, fluorescence intensity was measured at room temperature using an SLM Aminco Series 2 emission spectrophotometer with excitation and emission wavelengths of 321 nm and 445 nm. S-shaped optimal fit analysis was applied to the fluorescence data, and pK a It was measured as the pH that produces the half-maximum fluorescence intensity.

[0195] Example 3

[0196] Determination of the efficacy of lipid nanoparticle formulations containing various cationic lipids using an in vivo luciferase mRNA expression rodent model

[0197] The cationic lipids shown in Table 2 were previously tested as nucleic acids. For comparison purposes, these lipids were also used to formulate lipid nanoparticles containing FLuc mRNA (L-6107) using an inline mixing method as described in Example 1 and PCT / US10 / 22614, the full text of which is incorporated herein by reference. The lipid nanoparticles were formulated using the following molar ratios: 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid ("PEG-DMG", i.e., 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol, average PEG molecular weight 2000). In an alternative embodiment, the cationic lipid, DSPC, cholesterol, and PEG-lipid are formulated in 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 and 1.0 mg mRNA / kg and expressed as ng luciferase / g measured in the liver 4 hours after administration as described in Example 1.

[0198] Table 2. Comparative lipids showing mRNA and activity

[0199]

[0200] The representative compounds of the disclosures shown in Table 3 were formulated using the following molar ratios: 50% cationic lipids / 10% distearoylphosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipids ("PEG-DMA" 2-[2-(ω-methoxy(polyethylene glycol)). 2000)ethoxy]-N,N-ditetradecylacetamide) or 47.5% cationic lipids / 10% DSPC / 40.7% cholesterol / 1.8% PEG lipids. 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 measured in the liver 4 hours after administration as described in Example 1. The compound numbers in Table 3 correspond to the compound numbers in Table 1.

[0201] Table 3 Novel cationic lipids and related activities

[0202]

[0203]

[0204]

[0205] Example 4

[0206] Synthesis of bis(2-butyloctyl) 10-(N-decyl-5-(dimethylamino)pentanamido)nonadecandioate (Compound I-7)

[0207]

[0208] Synthesis of Compound 4-2

[0209] Ketones in DCE (10 mL) 4-1 A solution of (1.10 g, 1.62 mmol) and 1-decylamine (2.43 mmol, 382 mg, 0.486 mL) was stirred at room temperature for 15 minutes, and then sodium triacetoxyboron hydrogen (2.43 mmol, 515 mg) and acetic acid (2.43 mmol, 146 mg; 0.138 mL) was added. The mixture was stirred at room temperature for 2 days, after which the reaction mixture was concentrated. The residue was diluted with a mixture of nucleic acids and washed with dilute NaOH, saturated NaHCO3, and brine. The organic phase was separated, dried with 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 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).

[0210] Synthesis of Compound 4-3

[0211] A solution of thionyl chloride (3.36 mmol, 400 mg, 0.25 mL) in CH2Cl2 (5 mL) was added within 1 minute to a stirred solution of 5-bromovaleric acid (1.12 mmol, 204 mg) in CH2Cl2 (1 mL) at room temperature, followed by the addition of DMF (ca. 16 mg). The mixture was then heated to reflux for 2 hours. The reaction mixture was then concentrated under vacuum. The acid chloride was used immediately for the next step.

[0212] The solution of the above 5-bromopentanoyl chloride in benzene (5 mL) within 2 minutes at room temperature in benzine (5 mL) 4-2(230 mg, 0.28 mmol) and triethylamine (5.6 mmol, 565 mg, 0.780 mL) and DMAP (5 mg) were added dropwise to a solution of DMAP. After addition, the reaction mixture was stirred at room temperature for 1 hour. Methanol (1 mL) was added and the mixture was stirred for 2 hours. The reaction mixture was concentrated under reduced pressure. The crude product was purified by 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).

[0213] Synthesis of Compound I-7

[0214] 4-2 Dimethylamine (2 M in THF, 10 mL) was added to (250 mg, 0.25 mmol). The solution was stirred overnight at 64°C. The reaction mixture was concentrated. The residue was absorbed into a mixture of hexane, ethyl acetate, and Et3N (80:20:1), filtered through a silica gel pad, and washed with the same solvent mixture. The filtrate was concentrated to yield a brownish oil (ca 233 mg). The crude product (233 mg) was purified by scintillation-drying column chromatography on silica gel (0 to 6% methanol in chloroform). The desired product was obtained as (194 mg, colorless oil, 0.20 mmol, 82%). 1 ¹H NMR (400 MHz, CDCl3) δ: 4.60–4.20 (br, estimated 0.3H, due to slow isomerization for amide bonds), 3.97, 3.96 (two sets of doublets, 5.8 Hz, 4H), 3.60 (pentatonic-like, 7.0 Hz, 0.7H), 3.06–2.99 (m, 2H), 2.34–2.24 (m, 8H), 2.21 (monatonic, 6H), 1.72–1.56 (m, 8H), 1.56–1.37 (m, 8H), 1.37–1.10 (66H), 0.91–0.85 (m, 15H).

[0215] Example 5

[0216] Synthesis of bis(2-butyloctyl) 10-(N-decyl-4-(dimethylamino)butanamido)nonadecandioate (Compound I-18)

[0217]

[0218] Synthesis of Compound I-18, (Method A)

[0219] Oxalyl chloride (5.6 mmol, 722 mg, 0.496 mL) was added to a stirred solution of 4-(dimethylamino)butyric acid hydrochloride (1.12 mmol, 188 mg) and DMF (10-20 μL) in CH2Cl2 (10 mL) at room temperature. The resulting mixture was stirred overnight at room temperature. The reaction mixture (deep red) was concentrated under reduced pressure. The resulting acid chloride (red brick-colored solid) was used immediately for the next step.

[0220] The solution of the above acyl chloride in CH2Cl2 (10 mL) at room temperature in CH2Cl2 (5 mL) 4-2 (230 mg, 0.28 mmol) and triethylamine (5.6 mmol, 565 mg, 0.780 μL) and DMAP (5 mg) were added dropwise to a solution. The resulting mixture was stirred overnight at room temperature and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (hexane / EtOAc / Et3N, 80:20:0.1 to 75:25:1) and further purified by scintillation-drying column chromatography on silica gel (0 to 5% methanol in chloroform). The desired product was obtained as (92 mg, colorless oil, 0.10 mmol, 35%). 1¹H NMR (400 MHz, CDCl3) δ: 4.57-4.29 (br. 0.4H), 3.97, 3.96 (2 sets of doublets, 5.8 Hz, 5.8 Hz, 4H), 3.63 (quadruple-like, 6.8 Hz, 0.6H), 3.06-3.00 (m, 2H), 2.35-2.26 (m, 8H), 2.213, 2.211 (2 sets of singlets, 6H), 1.82 (quadruple-like, 7.6 Hz, 2H), 1.65-1.56 (m, 6H), 1.54-1.48 (m, 2H), 1.47-1.37 (m, 4H), 1.36-1.06 (66H), 0.91-0.86 (m, 15H).

[0221] Synthesis of Compound I-18, (Method B)

[0222] DCC (2.2 equiv, 3.34 mmol, 690 mg) was added 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), and the mixture was stirred at room temperature for 45 minutes. In CH2Cl2 (6 mL) 4-2 A solution of (1.25 g, 1.52 mmol) was added, and the resulting mixture was stirred overnight. The next day, more DCC (450 mg) was added, and the mixture was stirred for another day. Afterward, the mixture was concentrated under reduced pressure. The residue was absorbed into a mixture of hexane, ethyl acetate, and Et3N (80:20:1), filtered through a short column of silica gel, and washed with the same solvent mixture. The filtrate was concentrated to yield a colorless oil. The product was further purified by scintillation-drying column chromatography on silica gel (0 to 5% methanol in chloroform containing trace amounts of Et3N). The desired product was obtained as a colorless oil (1.14 g, 81%).

[0223] Example 6

[0224] Synthesis of bis(2-butyloctyl) 10-(N-decyl-5-(diethylamino)pentanamido)nonadecandioate (Compound I-8)

[0225]

[0226] Synthesis of Compound I-8

[0227] in acetonitrile (6 mL) 4-3 (179 mg, 0.18 mmol), diethylamine A mixture of (0.90 mmol, 66 mg, 0.093 mL) and N,N-diisopropylethylamine (0.36 mmol, 46 mg, 0.063 mL) was sealed and heated at 83°C for 24 hours. The reaction mixture was concentrated. The residue was absorbed into a mixture of hexane, ethyl acetate, and Et3N (80:20:1), filtered through a silica gel pad, and washed with the same solvent mixture. The filtrate was concentrated to yield a brownish oil (153 mg). The crude product (233 mg) was purified by scintillation-drying column chromatography on silica gel (0 to 6% MeOH in chloroform). The desired product was obtained as (136 mg, colorless oil, 0.14 mmol, 77%).

[0228] Example 7

[0229] Synthesis of bis(2-butyloctyl) 10-(N-decyl-5-(pyrrolidin-1-yl)pentanamido)nonadecandioate (Compound I-9)

[0230]

[0231] Synthesis of Compound I-9

[0232] in THF (10 mL) 4-3A mixture of (200 mg, 0.20 mmol) and pyrrolidine (50 eq. 0.83 mL, 10 mmol) was sealed and heated at 64°C for 24 hours. The reaction mixture was concentrated. The residue was absorbed into a mixture of hexane, ethyl acetate, and Et3N (80:20:1), filtered through a silica gel pad, and washed with the same solvent mixture. The filtrate was concentrated to yield a brownish oil. The crude product (233 mg) was purified by scintillation-drying column chromatography on silica gel (0 to 6% MeOH in chloroform). The desired product was obtained as (158 mg, colorless oil, 0.16 mmol, 80%).

[0233] Example 8

[0234] Synthesis of bis(2-hexyldecyl) 7-(N-decyl-4-(dimethylamino)butanamido)tridecanedioate (Compound I-16)

[0235]

[0236] Synthesis of 8-2

[0237] in DCE (10 mL) 8-1 (1 eq., 1.15 g , A solution of 1-decylamine (1.62 mmol) and 1-decylamine (1.5 eq., 2.43 mmol, 382 mg, 0.486 mL) was stirred at RT for about 15 minutes. Sodium triacetoxyborohydrogen (1.5 eq., 2.43 mmol, 515) was added to the solution. (mg) and AcOH (1.5 eq, 2.43 mmol, 146 mg, 0.14 mL) were added. The mixture was stirred at RT for 3 days. Afterward, the reaction mixture was concentrated. The residue was diluted with hexane / EtOAc (99:1) and washed with dilute NaOH solution, sat NaHCO3, and brine. The organic extract was dried with 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). 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-like, 5.5 Hz, 1H), 2.30 (t, 7.5 Hz, 4H), 1.68-1.57 (m, 6H), 1.49-1.40 (m, 2H), 1.40-1.08 (74H), 0.91-0.85 (m, 15H), 0.83-0.74 (br. 1H).

[0238] Synthesis of Compound I-16

[0239] Oxalyl chloride (3 eq., 6.3 mmol, 800 mg, 0.55 mL) was added under Ar to a stirred solution of 4-(dimethylamino)butyric acid hydrochloride (2.1 mmol, 352 mg) and DMF (ca. 13 mg) in CH2Cl2 (15 mL) at room temperature. The resulting mixture was stirred overnight at RT. The reaction mixture (bright orange solution) was concentrated under vacuum. The resulting acid chloride ( 8-3 , bright brown solid) was used immediately for the next reaction.

[0240] The solution of the above acyl chloride in CH2Cl2 (10 mL) at RT in CH2Cl2 (5 mL) 8-2(300 mg, 0.35 mmol) and triethylamine (10.5 mmol, 1.06 g, 1.5 mL) and DMAP (5 It was added to a solution of mg. After addition, the reaction mixture was stirred overnight at RT. After concentrating the mixture, the product was separated by column chromatography on silica gel (hexane, EtOAc and Et3N, 80:20:0.1 to 70:30:1) and the product was further purified by scintillation drying column chromatography on silica gel (0 to 5% MeOH in chloroform). The desired product was obtained as a slightly yellow oil (110 mg, 0.11 mmol, 32%). 1 HYNMR (CDCl3 at 400 MHz, 7.26 ppm) δ: 4.57-4.34 (br. 0.4H), 3.98-3.94 (m, 4H), 3.64 (quadruple-like, 6.8 Hz, 0.6H), 3.06-3.00 (m, 2H), 2.35-2.25 (m, 8H), 2.213, 2.210 (2 sets of monolinear, 6H), 1.82 (hexalinear-like, 7.4 Hz, 2H), 1.65-1.56 (m, 6H), 1.54-1.48 (m, 2H), 1.48-1.37 (m, 4H), 1.37-1.06 (70H), 0.91-0.86 (m, 15H).

[0241] Example 9

[0242] Synthesis of bis(2-butyloctyl) 10-(4-(dimethylamino)-N-(2-ethylhexyl)butanamido)nonadecandioate (Compound I-20)

[0243]

[0244] Synthesis of 9-2

[0245] in DCE (8 mL) 9-1A solution of (1 eq., 0.82 g, 1.21 mmol) and 2-ethyl-1-hexylamine (1.5 eq., 1.81 mmol, 234 mg) was stirred at RT for about 15 minutes. Sodium triacetoxyborohydride (1.5 eq., 1.81 mmol, 384 mg) was added to the solution. (mg) and AcOH (1.5 eq, 1.81 mmol, 109 mg) were added. The mixture was stirred at RT for 2 days. Afterward, the reaction mixture was concentrated. The residue was diluted with hexane and EtOAc (ca 99:5) and washed with dilute NaOH, sat 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 second wash was concentrated until dry. This yielded a 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 (pentangel-like, 5.5 Hz, 1H), 2.30 (t, 7.5 Hz, 4H), 1.68-1.57 (m, 6H), 1.41-1.08 (65H), 0.92-0.85 (m, 18H), 0.84-0.78 (br. 1H).

[0246] Synthesis of 9-4

[0247] A solution of thionyl chloride (3 eq. 6.72 mmol, 800 mg, 0.49 mL) in CH2Cl2 (5 mL) was slowly added within 1 minute to a stirred solution of 5-bromovaleric acid (2.24 mmol, 405 mg) in CH2Cl2 (2 mL) at room temperature. DMF (two small drops, ca 16 mg) was added to the reaction mixture. The mixture was then heated to reflux for 2 hours. The reaction mixture was concentrated under vacuum. The resulting acid chloride, 9- 3 It was used immediately for the next stage.

[0248] The solution of the above 5-bromopentanoyl chloride in benzene (8 mL) within 2 minutes at RT in benzene (5 mL) 9-2 (444 mg, 0.56 mmol) was added to a solution of triethylamine (1.56 mL) and DMAP (5 mg). After addition, the mixture was stirred overnight at RT. After evaporating the solvent under vacuum, the product was separated 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%).

[0249] Synthesis of Compound I-20

[0250] 9-4Dimethylamine (2 M in THF, 10 mL) was added to a pressure flask containing (260 mg, 0.27 mmol). The solution was stirred overnight at 64°C (oil bath temperature). Excess amine and solvent were evaporated. The residue was absorbed into a mixture of ethyl acetate and hexane (95:5) and filtered through a silica gel pad. The pad was washed with a mixture of hexane and EtOAc (95:5), followed by a mixture of hexane, EtOAc, and Et3N (80:20:1). The filtrate from the second wash was concentrated until dry. This yielded a crude product as a brown oil (233 mg). The crude product was further purified by scintillation-drying column chromatography on silica gel (0 to 5% MeOH in chloroform). The desired product was obtained as a colorless oil (204 It was obtained as mg, 0.22 mmol, 82%). 1 HNMR (CDCl3 at 400 MHz, 7.27 ppm) δ: 3.97 (d, 5.8 Hz, 4H), 3.66–3.57 (m, ca 1H), 3.19–2.99 (2 sets of peaks, 2H), 2.38–2.24 (m, 8H), 2.22 (singleline, 6H), 1.72–1.37 (m, 15H), 1.37–1.10 (60H), 0.91–0.85 (m, 18H).

[0251] Example 10

[0252] Synthesis of bis(2-butyloctyl) 10-(3-(dimethylamino)-N-nonylpropaneamido)nonadecandioate (Compound I-25)

[0253]

[0254] Synthesis of 10⁻¹

[0255] Oxalyl chloride (5.05 mmol, 641 mg, 0.44 mL) was added at RT to a solution of 3-bromopropionic acid (2.02 mmol, 311 mg) in CH2Cl2 (5 mL) and DMF (0.01 mL). The resulting mixture was stirred overnight at RT. Afterward, the mixture was concentrated under vacuum. The residual liquid / solid (yellow) was dissolved in 10 mL of CH2Cl2, and within 4 minutes at RT, the [substance] in CH2Cl2 (10 mL) 4-2 (833 mg, 1.02 mmol) and triethylamine (5.05 mmol, 0.7 mL) and DMAP (5 mg) were added to a solution. After addition, the mixture was stirred at RT for 2 hours. After evaporating the solvent under vacuum, the product was separated by column chromatography on silica gel (hexane / EtOAc, 99:1 to 90:10). The desired product was obtained as a colorless oil (794 mg, 0.83 mmol, 81%).

[0256] Synthesis of Compound I-25

[0257] 10-1 A mixture of (283 mg, 0.30 mmol) and dimethylamine (2 M in THF, 12 mL) was stirred overnight in a pressure flask at 68°C (oil bath temperature). The reaction mixture was concentrated. The residue was absorbed into a mixture of hexane, ethyl acetate, and Et3N (80:20:1), filtered through a silica gel pad, and washed with the same solvent mixture. The filtrate was concentrated to yield a brownish oil (302 mg). The crude product (302 mg) was purified by scintillation-drying column chromatography on silica gel (0 to 5% MeOH in chloroform containing trace amounts of Et3N). The desired product was obtained as a colorless oil (169 mg, 0.18 mmol, 61%). 1HYNMR (CDCl3 at 400 MHz, 7.26) δ: 4.50–4.31 (br, estimated 0.3H, due to slow isomerization for the amide bond), 3.97 (shouldered doublet, 5.8 Hz, 4H), 3.60 (pentatonic-like, 7.0 Hz, 0.7H), 3.07–3.00 (m, 2H), 2.65 (q-like, 7.6 Hz, 2H), 2.48 (q-like, 7.6 Hz, 2H), 2.29 (shouldered triplet, 7.6 Hz, 4H), 2.26, 2.25 (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).

[0258] Example 11

[0259] Synthesis of bis(2-hexyldecyl) 7-(N-decyl-4-(pyrrolidin-1-yl)butanamido)tridecandioate (Compound I-29)

[0260]

[0261] Synthesis of 11-1

[0262] Oxalyl chloride (3 eq. 2.91 mmol, 370 mg, 0.25 mL) was added at RT to a solution of 4-bromobutyric acid (0.97 mmol, 161 mg) in CH2Cl2 (3 mL) and DMF (0.01 mL). The mixture was stirred overnight at RT. Afterward, the reaction mixture was concentrated under vacuum. The residual liquid / solid (slightly yellow) was dissolved in 5 mL of CH2Cl2, and within 2 minutes at RT, in CH2Cl2 (20 mL) 8-2Solutions of (410 mg, 0.48 mmol), triethylamine (0.4 mL), and DMAP (2 mg) were added. After addition, the resulting mixture was stirred at RT for 2.5 hours. TLC (hexane / ethyl acetate = 9:1) showed two major spots. The reaction mixture was then concentrated under reduced pressure at RT. The residue was used for the next reaction without purification.

[0263] Synthesis of Compound I-29

[0264] 11-1 The residue containing [the substance] was absorbed into a mixture of pyrrolidine (2.10 mL, 25 mmol) and THF (15 mL). The mixture was transferred to a pressure flask and heated overnight at 68°C. The mixture was cooled and concentrated under reduced pressure. The residue was absorbed into a mixture of hexane, ethyl acetate, and Et3N (80:20:1), filtered through a short column of silica gel, and washed with the same solvent mixture. The filtrate was concentrated to yield 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% Et3N in CH2Cl2). The desired product was obtained as a yellow oil (215 mg). The product (215 mg) was further purified by scintillation-drying 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%). 1HNMR (CDCl3 at 400 MHz, 7.26 ppm) δ: 4.57-4.34 (br. 0.4H), 3.98-3.94 (m, 4H), 3.64 (quadruple-like, 6.8 Hz, 0.6H), 3.06-3.00 (m, 2H), 2.51-2.44 (m, 6H), 2.37-2.21 (m, 6H), 1.86 (hexauple-like, 7.6 Hz, 2H), 1.80-1.71 (m, 4H), 1.65-1.48 (m, 8H), 1.48-1.37 (m, 4H), 1.37-1.06 (70H), 0.91-0.86 (m, 15H).

[0265] Example 12

[0266] Synthesis of bis(2-butyloctyl) 10-(4-(dimethylamino)-N-(2-ethylhexyl)butanamido)nonadecandioate (Compound I-30)

[0267]

[0268] Synthesis of Compound I-30

[0269] DCC (1.1 mmol x 2,226 mg) was added 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), and the mixture was stirred at room temperature for 45 minutes. In CH2Cl2 (1 mL) 9-2A solution of (160 mg, 0.20 mmol) was added to the reaction mixture, and the resulting mixture was stirred over the weekend. More DCC (135 mg) was added, and the mixture was stirred for one more day. Based on TLC analysis, no progress was observed. The reaction mixture was concentrated under reduced pressure. The residue was absorbed into a mixture of hexane and EtOAc (approx. 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 Et3N (80:20:1). The filtrate from the second wash was concentrated until dry (133 mg). The crude product (133 mg) was further purified by scintillation-drying column chromatography on silica gel (0 to 5% MeOH in chloroform containing trace amounts of Et3N). The desired product was obtained as (48 mg, colorless oil, 0.053 mmol, 27%). 1 HNMR (CDCl3 at 400 MHz, 7.27 ppm) δ: 3.97 (d, 5.8 Hz, 4H), 3.70–3.60 (m, ca 1H), 3.19–2.99 (2 sets of peaks, 2H), 2.39–2.25 (m, 8H), 2.22 (singleline, 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).

[0270] Example 13

[0271] Synthesis of bis(2-butyloctyl) 10-(N-decyl-5-(dibutylamino)pentanamido)nonadecandioate (Compound I-31)

[0272]

[0273] Synthesis of Compound I-31

[0274] In a pressure flask 4-3A mixture of (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. The reaction mixture was concentrated. The residue was absorbed into a mixture of hexane, ethyl acetate, and Et3N (80:20:1), filtered through a silica gel pad, and washed with the same solvent mixture. The filtrate was concentrated to yield a brownish oil (product and dibutylamine). The oil was diluted with hexane, washed twice with a dilute aqueous HCl solution (0.5 M), washed with sat NaHCO3 and brine, and dried with sodium sulfate. The extract was concentrated under reduced pressure. The crude product (309 mg) was purified by scintillation-drying column chromatography on silica gel (0 to 5% MeOH in chloroform containing trace amounts of Et3N). The desired product was obtained as a colorless oil (216 mg, 0.21 mmol, 72%). 1 HNMR (400 MHz, CDCl3) δ: 4.50-4.35 (br, estimated 0.3H, due to slow isomerization for amide bonds), 3.97, 3.96 (two sets of doublets, 5.8 Hz, 4H), 3.61 (pentatonic-like, 7.0 Hz, 0.7H), 3.07-2.99 (m, 2H), 2.45-2.36 (m, 6H), 2.34-2.27 (m, 6H), 1.70-1.56 (m, 8H), 1.56-1.36 (m, 12H), 1.37-1.10 (70 H), 0.97-0.85 (m, 21H).

[0275] Example 14

[0276] Synthesis of 10-(4-(dimethylamino)-N-nonylbutanamido)nonadecane-1,19-diylbis(2-butyloctanoate) (Compound I-32)

[0277]

[0278] Synthesis of Compound I-32

[0279] DCC (2.2 equiv, 1.18 mmol, 245 mg) was added 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), and the mixture was stirred at room temperature for 45 minutes. Then, in CH2Cl2 (2 mL) 14-1 A solution of (442 mg, 0.54 mmol) was added. The resulting mixture was stirred overnight. More DCC (140 mg) was added, and the mixture was stirred for one more day. Afterward, the mixture was concentrated under reduced pressure. The residue was absorbed into a mixture of hexane, ethyl acetate, and Et3N (80:20:1), filtered through a short column of silica gel, and washed with the same solvent mixture. The filtrate was concentrated to yield a yellow oil (381 mg). The crude product (381 mg) was purified by scintillation-drying column chromatography on silica gel (0 to 5% MeOH in chloroform containing trace amounts of Et3N). The desired product was obtained as a colorless oil (345 mg, 0.38 mmol, 70%). 1 HYNMR (CDCl3 at 400 MHz, 7.26 ppm) δ: 5.30-4.34 (br., 0.3H), 4.061, 4.056 (2 sets of triplet, 6.7 Hz, 4H), 3.64 (quadruple-like, 6.8 Hz, 0.7H), 3.07-3.01 (m, 2H), 2.35-2.26 (m, 6H), 2.214, 2.211 (2 sets of singlet, 6H), 1.82 (quadruple-like, 7.6 Hz, 2H), 1.65-1.48 (m, 10H), 1.48-1.37 (m, 8H), 1.37-1.02 (60H), 0.90-0.85 (m, 15H).

[0280] Example 15

[0281] Synthesis of bis(2-butyloctyl) 10-(N-decyl-3-(pyrrolidin-1-yl)propaneamido)nonadecandioate (Compound I-33)

[0282]

[0283] Synthesis of Compound I-33

[0284] In the pressure tube 10-1 A mixture of (283 mg, 0.30 mmol), pyrrolidine (1.25 mL, 15 mmol), and THF (10 mL) was heated overnight at 64°C. The mixture was cooled and concentrated under reduced pressure. The residue was absorbed into a mixture of hexane, ethyl acetate, and Et3N (80:20:1), filtered through a short column of silica gel, and washed with the same solvent mixture. The filtrate was concentrated to yield a yellow oil. The crude product (314 mg) was further purified by scintillation-drying column chromatography on silica gel (0 to 5% MeOH in chloroform containing trace amounts of Et3N). The desired product was obtained as a colorless oil (113 mg, 0.12 mmol, 40%). 1 HYNMR (400 MHz, CDCl3at 7.26) δ: 4.55-4.30 (br, estimated 0.3H, due to slow isomerization for the amide bond), 3.97 (shouldered doublet, 5.8 Hz, 4H), 3.61 (pentatonic-like, 7.0 Hz, 0.7H), 3.06-3.00 (t-like, 2H), 2.81 (q-like, 7.6 Hz, 2H), 2.58-2.51 (m, 6H), 2.292, 2.285 (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).

[0285] Example 16

[0286] Synthesis of bis(2-butyloctyl) 10-(N-decyl-5-(hexyl(methyl)amino)pentanamido)nonadecandioate (Compound I-34)

[0287]

[0288] Synthesis of 16-1

[0289] in acetonitrile (6 mL) 4-3 A mixture of (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) was sealed and heated at 70°C for 24 hours. The reaction mixture was concentrated under reduced pressure at 75 to 85°C (ca 30 mmHg). TLC showed that most of the excess hexylamine was removed. The residue was absorbed into a mixture of hexane, ethyl acetate, and Et3N (80:20:1), filtered through a short column of silica gel, and washed with the same solvent mixture. The filtrate was concentrated to yield brown oil (192 mg), which was used for the next step without further purification.

[0290] Synthesis of Compound I-34

[0291] in THF (5 mL) 16-1A formaldehyde HCHO solution (500 mg, 37 wt.% solution in water) was added at RT to a solution of (192 mg, 0.19 mmol). The resulting mixture was stirred for 30 minutes before introducing sodium triacetoxyborohydride (1.2 mmol, 243 mg). The resulting mixture was stirred overnight at RT. The reaction mixture was concentrated. The residue was absorbed into a mixture of hexane and washed with dilute NaOH solution, saturated sodium bicarbonate solution, and brine. After drying with sodium sulfate, the solution was concentrated until dry (yellow oil). The residue was absorbed into a mixture of hexane, ethyl acetate, and Et3N (80:20:1), filtered through a short column of silica gel, and washed with the same solvent mixture. The filtrate was concentrated to yield yellow oil (220 mg). The crude product (220 mg) was further purified by scintillation-drying column chromatography on silica gel (0 to 5% MeOH in chloroform containing trace amounts of Et3N). The desired product was obtained as a colorless oil (113 It was obtained as mg, 0.13 mmol, 69%). 1 HYNMR (CDCl3 at 400 MHz, 7.26 ppm) δ: 4.50–4.35 (br, estimated 0.3H, due to slow isomerization for amide bonds), 3.97, 3.96 (2 sets of doublets, 5.8 Hz, 4H), 3.60 (pentatonic-like, 7.0 Hz, 0.7H), 3.06–2.98 (m, 2H), 2.36–2.26 (m, 10H), 2.191, 2.189 (2 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).

[0292] Example 17

[0293] Synthesis of bis(2-hexyldecyl) 7-(N-decyl-3-(dimethylamino)propaneamido)tridecanedioate (Compound I-35)

[0294]

[0295] Synthesis of Compound I-35

[0296] 3 in acetonitrile (10 mL) - DCC (2.2 equiv, 0.68 mmol, 141 mg) was added to a solution of dimethylaminopropionic acid hydrochloride (2 equiv, 0.62 mmol, 95 mg) and 4-dimethylaminopyridine (3 equiv, DMAP, 0.93 mmol, 114 mg), and the mixture was stirred at room temperature for 45 minutes. In CH2Cl2 (2 mL) 8-2 A solution of (262 mg, 0.31 mmol) was added, and the resulting mixture was stirred over the weekend. TLC (chloroform / MeOH, 9:1) showed a major spot on the solvent front that could be the elimination product, with no starting material and a small amount of the desired product. The reaction mixture was concentrated. The possible elimination product (107 mg colorless oil) was separated by column chromatography (hexane-EtOAc, 95:5) and treated with dimethylamine in THF (2 M, 9 mL) for 4 days at RT. The mixture was concentrated. The residue was absorbed into a mixture of hexane, ethyl acetate, and Et3N (80:20:1), filtered through a short column of silica gel, and washed with the same solvent mixture. The filtrate was concentrated to yield a yellow oil. The crude product was further purified by scintillation-drying column chromatography on silica gel (0 to 5% MeOH in chloroform containing trace amounts of Et3N). The desired product is a colorless oil (62 It was obtained as mg. 1HF NMR (CDCl3 at 400 MHz, 7.26) δ: 4.50–4.36 (br, estimated 0.3H, due to slow isomerization for amide bonds), 3.97, 3.96 (2 sets of doublets, 5.8 Hz, 4H), 3.61 (pentatonic-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 (2 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)

[0297] Example 18

[0298] Synthesis of bis(2-hexyldecyl) 7-(3-(dimethylamino)-N-(6-((2-ethylhexyl)oxy)-6-oxohexyl)propaneamido)tridecanedioate (Compound I-36)

[0299]

[0300] Synthesis of 18-2

[0301] CH2Cl2 ( Oxalyl chloride (0.86 mmol, 109 mg, 74 µL) was added at RT to a solution of 3-bromopropionylic acid (0.34 mmol, 52 mg) in 3 mL of CH2Cl2 and DMF (1 drop from a fine needle). The mixture was stirred overnight at RT. Subsequently, the mixture was concentrated at RT under reduced pressure for 60 minutes. The residual liquid / solid (yellow) was dissolved in 5 mL of CH2Cl2, and within 1 minute at RT, the [substance] in CH2Cl2 (5 mL) 18-1(160 mg, 0.17 mmol) and triethylamine (0.86 mmol, 0.12 mL) and DMAP (1 mg) were added to a solution of DMAP. After addition, the mixture was stirred at RT for 3 hours and then concentrated. The product was separated by column chromatography on silica gel (hexane, EtOAc, and Et3N, 95:5 to 85:15). The desired product was obtained as a colorless oil (99 mg, 0.09 mmol, 53%).

[0302] Synthesis of Compound I-36

[0303] 18-2 Dimethylamine (2 M in THF, 5 mL) was added to a pressure flask containing (99 mg, 0.09 mmol). The solution was stirred at 68°C (oil bath temperature) for 2 days. The mixture was cooled and concentrated under reduced pressure. The residue was absorbed into a mixture of hexane, ethyl acetate, and Et3N (80:20:1), filtered through a short column of silica gel, and washed with the same solvent mixture. The filtrate was concentrated to yield a brown oil (94 mg). The product (94 mg) was further purified by scintillation-drying column chromatography on silica gel (0 to 5% MeOH in chloroform containing trace amounts of Et3N). The desired product was obtained as a colorless oil (71 It was obtained as mg, 0.069 mmol, 76%). 1HF NMR (CDCl3 at 400 MHz, 7.26) δ: 4.53-4.35 (br, estimated 0.3H, due to slow isomerization for amide bonds), 4.02-3.93 (m, 6H), 3.62 (pentaline-like, 7.0 Hz, 0.7H), 3.07-3.01 (m, 2H), 2.65 (q-like, 7.6 Hz, 2H), 2.50-2.44 (m, 2H), 2.34-2.26 (m, 6H), 2.26, 2.25 (2 sets of singles, 6H), 1.69-1.56-1.48 (m, estimated 11H, overlapped with water peak), 1.49-1.37 (m, 4H), 1.37-1.10 (66H), 0.92-0.86 (m, 18H).

[0304] Example 19

[0305] Synthesis of di(tridecan-7-yl)10-(N-decyl-4-(dimethylamino)butanamido)nonadecandioate (Compound I-37)

[0306]

[0307] Synthesis of 19-2

[0308] in DCE (10 mL) 19-1A solution of sodium hydroxide (1 eq., 0.493 g, 0.70 mmol) and 1-decylamine (1.5 eq., 1.05 mmol, 165 mg, 0.21 mL) was stirred at RT for about 15 minutes. Sodium triacetoxyborohydride (1.5 eq., 1.05 mmol, 222 mg) and AcOH (1.5 eq., 1.05 mmol, 63 mg, 0.059 mL) were added to the solution. The mixture was stirred at RT for 2 days. Afterward, the reaction mixture was concentrated. The residue was diluted with hexane and washed with dilute NaOH, sat NaHCO3, and brine. The organic extract was dried with sodium sulfate, and the solvent was removed under reduced pressure. The residue was absorbed into a mixture of hexane, ethyl acetate, and Et3N (80:20:1), filtered through a short column of silica gel, and washed with the same solvent mixture. The filtrate was concentrated to provide the desired product as a colorless oil (582 mg, 0.69 mmol, 98%). The product was used for the next step without further purification.

[0309] Synthesis of Compound I-37

[0310] DCC (2.2 equiv, 1.52 mmol, 313 mg) was added 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), and the mixture was stirred at room temperature for 45 minutes. In CH2Cl2 (3 mL) 19-2A solution of (582 mg, 0.69 mmol) was added, and the resulting mixture was stirred overnight. The next day, more DCC (200 mg) was added, and stirring was continued over the weekend (4 days). Afterward, the mixture was concentrated under reduced pressure. The residue was absorbed into a mixture of hexane, ethyl acetate, and Et3N (80:20:1), filtered through a short column of silica gel, and washed with the same solvent mixture. The filtrate was concentrated to yield a yellow oil. The product was further purified by scintillation-drying column chromatography on silica gel (0 to 5% MeOH in chloroform containing trace amounts of Et3N). The desired product was obtained as a colorless oil.

[0311] Example 20

[0312] Synthesis of bis(2-hexyldecyl) 10-(N-decyl-4-(dimethylamino)butanamido)nonadecandioate (Compound I-38)

[0313]

[0314] 20-2 synthesis

[0315] Ketones in DCE (6 mL) 20-1 A solution of (0.92 g, 1.16 mmol) and 1-decylamine (2.03 mmol, 319 mg, 0.40 mL) was stirred at RT for 15 minutes, and then sodium triacetoxyborohydrogenated (2.03 mmol, 429 mg) and AcOH (2.03 mmol, 121 mg, 0.115 mL) were added. The mixture was stirred at RT for 2 days, after which 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 with 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).

[0316] Synthesis of I-38

[0317] DCC (2.2 equiv, 0.84 mmol, 172 mg) was added to a solution of 4-dimethylaminobutyric acid hydrochloride (2 equiv, 0.76 mmol, 127 mg) and 4-dimethylaminopyridine (3 eq, 1.14 mmol, 139 mg) in CH3CN (5 mL), and the mixture was stirred at room temperature for 45 minutes. In DCM (1 mL) 20-2 A solution of (350 mg, 0.38 mmol) was added, and the resulting mixture was stirred overnight. The next day, more DCC (50 mg) was added, and the mixture was stirred for another day. Afterward, the mixture was concentrated under reduced pressure. The residue was absorbed into a mixture of hexane, ethyl acetate, and Et3N (80:20:1), filtered through a short column of silica gel, and washed with the same solvent mixture. The filtrate was concentrated to yield a colorless oil. The product was further purified by scintillation-drying column chromatography on silica gel (0 to 5% MeOH in chloroform containing trace amounts of Et3N). The desired product was obtained as a colorless oil (260 mg).

[0318] Example 21

[0319] Synthesis of bis(2-hexyldecyl) 10-(N-decyl-4-(pyrrolidin-1-yl)butanamido)nonadecandioate (Compound I-39)

[0320]

[0321] Synthesis of 21-1

[0322] Oxalyl chloride (3 eq. 3.00 mmol, 381 mg, 0.26 mL) was added at RT to a solution of 4-bromobutyric acid (1.00 mmol, 167 mg) in DCM (3 mL) and DMF (a small drop). The mixture was stirred overnight at RT. Afterward, the reaction mixture was concentrated under vacuum. The residual liquid / solid (slightly yellow) was dissolved in 5 mL of DCM, and within 2 minutes at RT, the [substance] in DCM (5 mL) 20-2 Solutions of (464 mg, 0.50 mmol) and triethylamine (0.42 mL) and DMAP (2 mg) were added. After addition, the resulting mixture was stirred at RT for 2.5 hours. TLC (hexane / ethyl acetate = 9:1) showed two major spots. The reaction mixture was then concentrated under reduced pressure at RT. The residue was used for the next step without purification.

[0323]

[0324] Synthesis of I-39

[0325] The residue was absorbed into a mixture of pyrrolidine (2.20 mL, 26 mmol) and THF (15 mL). The mixture was transferred to a pressure flask and heated overnight at 68°C. The mixture was cooled and concentrated under reduced pressure. The residue was absorbed into a mixture of hexane, ethyl acetate, and Et3N (80:20:1), filtered through a short column of silica gel, and washed with the same solvent mixture. The filtrate was concentrated to yield a yellow oil / solid (359 mg). The crude product (359 mg) was further purified by scintillation-drying column chromatography on silica gel (0 to 5% MeOH in chloroform). The desired product was obtained as a colorless oil (165 mg).

[0326] ​The various embodiments described above may be combined to provide additional embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification and / or listed in application data sheets, including U.S. Nos. 62 / 791,566 and 62 / 890,469, are incorporated herein by reference in their entirety. Aspects of the embodiments may be modified as necessary to use concepts from various patents, applications, and publications to provide additional embodiments. These and other modifications may be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be interpreted as limiting the claims to the specific embodiments disclosed in the specification and claims, but should be interpreted as including all possible embodiments with the full scope of equivalents to which such claims are granted. Accordingly, the claims are not limited by the present disclosure.

Claims

Claim 1 A compound having the following structure (I) or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof: (I) Here, R 1 is substituted or unsubstituted C1-C 24 Alkyl or substituted or unsubstituted C2-C 24 It is alkenyl;R 2 and R 3 Each is independently substituted or unsubstituted C1-C 36 It is alkyl and;R 4 and R 5 are each independently substituted or unsubstituted C1-C6 alkyls, or R 4 and R 5 are connected together with the N to which they are attached to form a heterocyclile or heteroaryl; L 1 , L 2 , and L 3 Each is independently substituted or unsubstituted C1-C 18 It is an alkylene; G 1 is a direct bond, -(CH2) n O(C=O)-, -(CH2) n (C=O)O-, or -(C=O)- and;G 2 and G 3 are each independently -(C=O)O- or -O(C=O)-; n is an integer greater than 0; where each alkyl, alkenyl, and alkylene is substituted or not substituted with one or more halo, -C(=O)R', and -O(C=O)R' groups, and where R' is independently C1-C at each occurrence 15 It is an alkyl. Claim 2 In claim 1, a compound having the following structure (IA): (IA) . Claim 3 In claim 1, a compound having the following structure (IB): (IB) . Claim 4 In any one of paragraphs 1 to 3, R 1 This substituted or unsubstituted C6-C 18 alkyl or C 14 -C 18 It is an alkenyl, wherein each alkyl and alkenyl is substituted or not substituted with one or more halo, -C(=O)R', and -O(C=O)R' groups, where R' is independently C1-C at each occurrence 15 Alkyl compounds. Claim 5 In paragraph 4, R 1 This C8alkyl, C9alkyl, C 10 Alkyl, C 12 Alkyl, C 14 alkyl, or C 16 Alkyl compound. Claim 6 In paragraph 4, R 1 This C 16 Alkenyl compounds. Claim 7 In paragraph 4, R 1 This unbranched compound. Claim 8 In paragraph 4, R 1 This branched compound. Claim 9 In paragraph 4, R 1 This unsubstituted compound. Claim 10 In any one of paragraphs 1 to 3, G 1 This direct bond, -(CH2) n O(C=O)-, or -(CH2) n (C=O)O-compound. Claim 11 In Paragraph 10, G 1 A compound in which this is a direct bond. Claim 12 In Paragraph 10, G 1 This -(CH2) n A compound where (C=O)O- and n is greater than 1. Claim 13 In paragraph 12, a compound in which n is 5, 6, 7, 8, 9, or 10. Claim 14 In paragraph 13, a compound in which n is 7. Claim 15 In paragraph 13, a compound in which n is 8. Claim 16 In any one of paragraphs 1 to 3, L 1 This is a C1-C6 alkylene compound. Claim 17 In Paragraph 16, L 1 A compound that is a C2-alkylene, C3-alkylene, or C4-alkylene. Claim 18 In any one of paragraphs 1 to 3, L 1 This unbranched compound. Claim 19 In any one of paragraphs 1 to 3, L 1 This unsubstituted compound. Claim 20 In any one of paragraphs 1 to 3, R 2 C8-C 24 Alkyl compound. Claim 21 In any one of paragraphs 1 to 3, R 3 This C8-C 24 Alkyl compound. Claim 22 In any one of paragraphs 1 to 3, R 2 and R 3 Both of these are C8-C 24 Alkyl compound. Claim 23 In any one of paragraphs 1 to 3, R 2 and R 3 These 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 compound. Claim 24 In any one of paragraphs 1 to 3, R 2 A branched compound. Claim 25 In any one of paragraphs 1 to 3, R 3 This branched compound. Claim 26 In any one of paragraphs 1 to 3, R 2 and R 3 Compounds that each independently have one of the following structures: or Here:R 6 and R 7 C2-C each independently 12 It is an alkyl. Claim 27 In Paragraph 26, R 2 and R 3 Compounds that each independently have one of the following structures: ; ; ; ; or . Claim 28 In any one of paragraphs 1 to 3, L 2 and L 3 These each independently C4-C 10 Alkylene compound. Claim 29 In any one of paragraphs 1 to 3, L 2 and L 3 These are compounds that are both C5 alkylenes. Claim 30 In any one of paragraphs 1 to 3, L 2 and L 3 These are compounds in which both are C6-alkylenes. Claim 31 In any one of paragraphs 1 to 3, L 2 and L 3 These are both compounds that are C8-alkylenes. Claim 32 In any one of paragraphs 1 to 3, L 2 and L 3 These are both compounds that are C9 alkylenes. Claim 33 In any one of paragraphs 1 to 3, L 2 A compound that is not branched. Claim 34 In any one of paragraphs 1 to 3, L 3 This unbranched compound. Claim 35 In any one of paragraphs 1 to 3, L 2 A compound that is not substituted. Claim 36 In any one of paragraphs 1 to 3, L 2 is substituted with one or more halos, -C(=O)R' and -O(C=O)R' groups, where R' is independently C1-C at each occurrence 15 Alkyl compounds. Claim 37 In any one of paragraphs 1 to 3, R 4 and R 5 Compounds in which each is independently a C1-C6 alkyl. Claim 38 In Paragraph 37, R 4 and R 5 A compound in which both are methyl. Claim 39 In Paragraph 37, R 4 and R 5 A compound in which both are ethyl. Claim 40 In Paragraph 37, R 4 ga is methyl and R 5 a n-butyl compound. Claim 41 In any one of paragraphs 1 to 3, R 4 and R 5 a, compounds that are connected together with the attached N to form a heterocyclile. Claim 42 In paragraph 41, a compound in which the heterocyclile is a 5-membered heterocyclile. Claim 43 In paragraph 42, heterocyclil A compound having the structure of Claim 44 In claim 1, the compound having one of the following structures: ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; or . Claim 45 A composition comprising a compound of any one of claims 1 to 3 and a messenger RNA encoding a protein. Claim 46 A composition according to claim 45, further comprising one or more excipients selected from neutral lipids, steroids, and polymer-conjugated lipids. Claim 47 In paragraph 46, 1,2-distearoyl- sn -Glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl- sn -Glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl- sn -Glycero-3-phosphocholine (DMPC), 1,2-dioleoyl- sn -Glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl- sn -Glycero-3-phosphocholine (POPC), 1,2-dioleoyl- sn A composition comprising one or more neutral lipids selected from glycero-3-phosphoethanolamine (DOPE) and sphingomyelin (SM). Claim 48 In claim 47, a composition in which the neutral lipid is DSPC. Claim 49 A composition according to claim 46, wherein the molar ratio of the compound to the neutral lipid is in the range of 2:1 to 8:

1. Claim 50 In Clause 46, a composition in which the steroid is cholesterol. Claim 51 A composition according to claim 50, wherein the molar ratio of the compound to cholesterol is in the range of 5:1 to 1:

1. Claim 52 In claim 46, a composition in which the polymer-conjugated lipid is a pegylated lipid. Claim 53 A composition according to claim 52, wherein the molar ratio of the compound to the pegylated lipid is in the range of 100:1 to 20:

1. Claim 54 A composition according to claim 52, wherein the pegylated lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, or PEG diallyoxypropylcarbamate. Claim 55 In paragraph 52, a composition in which the pegylated lipid has the following structure (II) or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: (II) Here, R 8 and R 9 Each is a straight or branched saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms independently, wherein the alkyl chain is optionally blocked by one or more ester bonds; w has an average value in the range of 30 to 60. Claim 56 In paragraph 55, R 8 and R 9 A composition comprising a straight-chain saturated alkyl chain, each independently containing 12 to 16 carbon atoms. Claim 57 A composition according to claim 55, wherein the average w is 49. Claim 58 In paragraph 55, a composition comprising lipid nanoparticles. Claim 59 In paragraph 45, a composition administered to a patient for delivering messenger RNA to a patient who requires it. Claim 60 A lipid nanoparticle comprising a compound of any one of claims 1 to 3. Claim 61 In paragraph 60, lipid nanoparticles further comprising messenger RNA encoding a protein. Claim 62 Lipid nanoparticles according to claim 61, further comprising one or more excipients selected from neutral lipids, steroids, and polymer-conjugated lipids. Claim 63 In paragraph 62, the neutral lipid is 1,2-distearoyl- sn -Glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl- sn -Glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl- sn -Glycero-3-phosphocholine (DMPC), 1,2-dioleoyl- sn -Glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl- sn -Glycero-3-phosphocholine (POPC), 1,2-dioleoyl- sn Lipid nanoparticles selected from glycero-3-phosphoethanolamine (DOPE) and sphingomyelin (SM). Claim 64 In paragraph 63, lipid nanoparticles in which the neutral lipid is DSPC. Claim 65 In paragraph 62, lipid nanoparticles in which the molar ratio of the compound to the neutral lipid is in the range of 2:1 to 8:

1. Claim 66 In paragraph 62, lipid nanoparticles in which the steroid is cholesterol. Claim 67 In claim 66, lipid nanoparticles in which the molar ratio of the compound to cholesterol is in the range of 5:1 to 1:

1. Claim 68 In paragraph 62, lipid nanoparticles in which the polymer-conjugated lipid is a pegylated lipid. Claim 69 In claim 68, lipid nanoparticles in which the molar ratio of the compound to the pegylated lipid is in the range of 100:1 to 20:

1. Claim 70 In paragraph 68, lipid nanoparticles in which the pegylated lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, or PEG-dialkyoxypropylcarbamate. Claim 71 In paragraph 68, lipid nanoparticles having pegylated lipids having the following structure (II) or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: structure (II) Here, R 8 and R 9 Each is a straight or branched saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms independently, wherein the alkyl chain is optionally blocked by one or more ester bonds; w has an average value in the range of 30 to 60. Claim 72 In Paragraph 71, R 8 and R 9 Lipid nanoparticles that are straight-chain saturated alkyl chains, each independently containing 12 to 16 carbon atoms. Claim 73 In paragraph 71, lipid nanoparticles with an average w of 49. Claim 74 delete Claim 75 delete Claim 76 delete Claim 77 delete

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