Lipids for use in lipid nanoparticle formulations

Abstract

To provide lipid compounds which can be used alone or in combination with other lipid components to form lipid nanoparticles for the delivery of therapeutic agents.SOLUTION: The present invention provides a compound having the structure (I) or a pharmaceutically acceptable salt, a prodrug or a stereoisomer thereof. (L1 represents -O(C=O) R1, -(C=O)OR1 or the like; L2 represents -O(C=O) R2, -(C=O)OR2 or the like; G1 and G2 independently represent C2-C12 alkylene or C2-C12 alkenylene; G3 represents C1-C24 alkylene, C2-C24 alkenylene or the like; R1 and R2 independently represent branched C6-C24 alkyl or branched C6-C24 alkenyl; R3 represents -N(R4) R5; R4 represents C1-C12 alkyl; R5 represents substituted C1-C12 alkyl; x represents 0, 1 or 2).SELECTED DRAWING: None

Description

[Technical Field]

[0001] Embodiments of the present invention generally relate to novel lipids that, when used in combination with other lipid components such as neutral lipids, cholesterol, and polymer-conjugated lipids, can form lipid nanoparticles for the delivery of therapeutic agents such as nucleic acids (e.g., oligonucleotides, messenger RNA) both in vitro and in vivo. [Background technology]

[0002] Many challenges exist in relation to the delivery of nucleic acids to influence desired responses within biological systems. Nucleic acid-based therapeutics hold immense potential, but realizing this potential still requires more effective delivery of nucleic acids to appropriate sites within cells or organisms. Examples of therapeutic nucleic acids include messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, immunostimulatory nucleic acids, antagonists, antimirs, mimics, supermirs, and aptamers. Some nucleic acids, such as mRNA or plasmids, can be used to induce the expression of specific cell products, for example, useful in treating diseases related to protein or enzyme deficiencies. Because any selected protein sequence can be generated by synthesizing constructs, regardless of whether they are constitutive in the system, the therapeutic applications of translatable nucleotide delivery are extremely broad. Nucleic acid expression products can increase existing levels of proteins, replace deficient or non-functional versions of proteins, or introduce novel proteins and associated functionalities within cells or organisms.

[0003] Certain nucleic acids, such as miRNA inhibitors, can be used to induce the expression of specific miRNA-regulated cell products, for example, to treat diseases related to protein or enzyme deficiencies. Since one or more miRNAs can be inhibited by synthesizing constructs, thereby regulating the expression of mRNA products, the therapeutic applications of miRNA inhibition are extremely broad. Inhibition of endogenous miRNAs can increase the expression of downstream target endogenous proteins within cells or organisms, restoring proper function, as a means of treating diseases associated with specific miRNAs or groups of miRNAs.

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

[0005] However, the use of oligonucleotides in therapeutic settings currently faces two problems. First, free RNA is susceptible to nuclease digestion in plasma. Second, free RNA has a limited ability to acquire access to the intracellular compartments where the relevant translation mechanisms reside. Lipid nanoparticles formed from lipids formulated with other lipid components such as neutral lipids, cholesterol, PEG, pegylated lipids, and oligonucleotides have been used to block RNA degradation in plasma and promote cellular uptake of oligonucleotides.

[0006] The need for improved lipids and lipid nanoparticles for oligonucleotide delivery remains. Preferably, these lipid nanoparticles provide an optimal drug-to-lipid ratio, protect nucleic acids from degradation and clearance in serum, are suitable for systemic or topical delivery, and provide intracellular delivery of nucleic acids. In addition, these lipid-nucleic acid particles should exhibit good tolerability and provide a sufficient therapeutic index so that treatment of patients with effective doses of nucleic acids does not pose unacceptable toxicity and / or risk to the patient. Embodiments of the present invention provide these and related advantages. [Overview of the project] [Means for solving the problem]

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

[0008] In one embodiment, the following structure (I): [ka] (In the formula, R 3 , L 1 , L 2 , G 1 , G 2 and G 3 (as defined herein) Compounds having the same property or pharmaceutically acceptable salts thereof, tautomers, prodrugs, or stereoisomers are provided.

[0009] Pharmaceutical compositions comprising one or more compounds of the aforementioned structure (I) and a therapeutic agent are also provided. Lipid nanoparticles (LNPs) comprising one or more compounds of structure (I) are also provided. In some embodiments, the pharmaceutical composition and / or LNP further comprises one or more components selected from neutral lipids, charged lipids, steroids and polymer-conjugated lipids. The disclosed compositions are useful for forming lipid nanoparticles for the delivery of therapeutic agents.

[0010] In other embodiments, the present invention provides a method for administering a therapeutic agent to a patient in need, comprising preparing a composition of lipid nanoparticles comprising a compound of structure (I) and a therapeutic agent, and delivering the composition to the patient. In some embodiments, a method for administering a therapeutic agent to a patient in need includes administering an LNP comprising one or more compounds of structure (I) and a therapeutic agent to the patient.

[0011] These and other aspects of the present invention will become apparent from the following detailed description. [Modes for carrying out the invention]

[0012] The following description includes certain details to allow for a thorough understanding of the various embodiments of the present invention. However, those skilled in the art will understand that embodiments of the present invention can be carried out without these details.

[0013] Embodiments of the present invention are in part based on the discovery of novel lipids that offer advantages when used in lipid nanoparticles for in vivo delivery of active agents or therapeutic agents, such as nucleic acids, to mammalian cells. In particular, embodiments of the present invention provide nucleic acid-lipid nanoparticle compositions comprising one or more of the novel lipids described herein that provide increased nucleic acid activity and improved compositional tolerance in vivo, resulting in a significant increase in therapeutic index compared to previously described nucleic acid-lipid nanoparticle compositions. For example, embodiments provide lipid nanoparticles comprising one or more compounds of structure (I).

[0014] In certain embodiments, the present invention provides novel lipids that enable the formulation of improved compositions for the in vitro and in vivo delivery of mRNA and / or other oligonucleotides. In some embodiments, these improved lipid nanoparticle compositions are useful for the expression of proteins encoded by mRNA. In other embodiments, these improved lipid nanoparticle compositions are useful for upregulating the expression of endogenous proteins by delivering miRNA inhibitors that target one specific miRNA or group of miRNAs that modulate one target mRNA or several mRNAs. In other embodiments, these improved lipid nanoparticle compositions are useful for downregulating (e.g., silencing) the protein and / or mRNA levels of a target gene. In some other embodiments, lipid nanoparticles are also useful for the delivery of mRNA and plasmids for the expression of transgenes. In yet another embodiment, lipid nanoparticle compositions are useful for inducing pharmacological effects resulting from protein expression, such as increased red blood cell production via the delivery of appropriate erythropoietin mRNA, or protection from infection via the delivery of mRNA encoding an appropriate antigen or antibody.

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

[0016] As described herein, embodiments of the lipid nanoparticles of the present invention are particularly useful for the delivery of nucleic acids, including, for example, mRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomyel / anti-mir), messenger-RNA-interfering complementary RNA (micRNA), DNA, multivalent RNA, Dicer substrate RNA, complementary DNA (cDNA), and the like. Accordingly, the lipid nanoparticles and compositions of embodiments of the present invention can be used to induce the expression of desired proteins both in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more novel lipids described herein, the lipid nanoparticles encapsulating or accompanying nucleic acids to be expressed to produce the desired protein (e.g., messenger RNA or plasmid encoding the desired protein) or to inhibit the process that terminates mRNA expression (e.g., a miRNA inhibitor). Alternatively, the lipid nanoparticles and compositions of embodiments of the present invention can be used to reduce the expression of target genes and proteins both in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more novel lipids described herein (e.g., compounds of structure (I)), wherein the lipid nanoparticles encapsulate or accompany nucleic acids (e.g., antisense oligonucleotides or small interfering RNA (siRNA)) that reduce the expression of the target gene. The lipid nanoparticles and compositions of embodiments of the present invention can also be used to co-deliver different nucleic acids (e.g., mRNA and plasmid DNA) separately or in combination, such as being useful to provide an action that requires the co-localization of different nucleic acids (e.g., mRNA encoding a suitable gene-modifying enzyme and DNA segments for integration into the host genome).

[0017] Nucleic acids for use in embodiments of the present invention can be prepared according to any available technique. With respect to mRNA, the primary method of preparation is, but is not limited to, enzymatic synthesis (also called in vitro transcription), which currently represents the most efficient method for generating long-sequence-specific mRNA. In vitro transcription describes the process of template instruction synthesis of an RNA molecule from an engineered DNA template consisting of an upstream bacteriophage promoter sequence (including, but not limited to, those from T7, T3, and SP6 E. coli phages) ligated to a downstream sequence encoding the gene of interest. Template DNA can be prepared for in vitro transcription from several sources using appropriate techniques known in the art, including plasmid DNA and polymerase chain reaction amplification, but is not limited to these (see Linpinsel, J. L and Conn, GL, General protocols for preparation of plasmid DNA template, and Bowman, JC, Azizi, B., Lenz, TK, Ray, P. and Williams, LD, RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods, Vol. 941, Conn GL (ed.), New York, NY Humana Press, 2012).

[0018] RNA transcription occurs in vitro using a linearized DNA template in the presence of the corresponding RNA polymerase and adenosine, guanosine, uridine, and cytidine ribonucleoside triphosphates (rNTPs), under conditions that support polymerase activity while minimizing the potential degradation of the resulting mRNA transcript. In vitro transcription can be carried out using various commercially available kits, including 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, but is not limited to these. Methods for in vitro transcription of mRNA are well known in the art. (For example, all of these are incorporated herein by reference: Losick, R., 1972, In vitro transcription, Ann Rev Biochem, Vol. 41, pp. 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, Vol. 703 (edited by Neilson, H.), New York, NY Humana Press, 2010; Brunelle, JL and Green, R., 2013, Chapter 5, In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology, Vol. 530, pp. 101–114).

[0019] Next, the desired, in vitro transcribed mRNA is purified from unwanted components of the transcription or related reactions (including unincorporated rNTPs, protein enzymes, salts, short RNA oligos, etc.). Techniques for isolating mRNA transcripts are well known in the art. Known procedures include phenol / chloroform extraction or precipitation with any alcohol (ethanol, isopropanol) in the presence of a monovalent cation or lithium chloride. Non-limiting examples of additional purification procedures that can be used include size exclusion chromatography (Lukavsky, PJ and Puglisi, JD, 2004, Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides, RNA, Vol. 10, pp. 889-893), silica-based affinity chromatography, and polyacrylamide gel electrophoresis (Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LD, RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods, Vol. 941, Conn GL (ed.), New York, NY Humana Press, 2012). Purification can be carried out using a variety of commercially available kits, including the SV Total Isolation System (Promega) and the in Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek), but is not limited to these.

[0020] Furthermore, while reverse transcription can produce large amounts of mRNA, its product may contain several abnormal RNA impurities associated with undesirable polymerase activity that may need to be removed from the full-length mRNA preparation. These include incomplete transcription initiation and RNA-dependent RNA polymerase activity, RNA-primed transcription from RNA templates, and short RNAs resulting from double-stranded RNA (dsRNA) produced by self-complementary 3' extension. It has been demonstrated that these impurities with dsRNA structures can lead to undesirable immunostimulatory activity through interaction with various innate immune sensors in eukaryotic cells that recognize specific nucleic acid structures and function to trigger a potent immune response. Consequently, this can dramatically reduce mRNA translation, as protein synthesis decreases during the cell's innate immune response. Therefore, additional techniques for removing these dsRNA contaminants, including, but not limited to, scalable HPLC purification, have been developed and are known in the art (see, for example, Kariko, K., Muramatsu, H., Ludwig, J., and Weissman, D., 2011, *Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA*, Nucl Acid Res, vol. 39, el42; Weissman, D., Pardi, N., Muramatsu, H., and Kariko, K., *HPLC Purification of in vitro transcribed long RNA in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology*, vol. 969 (Rabinovich, PH., ed.), 2013). HPLC-purified mRNA has been reported to be translated at much higher levels, particularly in primary cells and in vivo.

[0021] A fairly wide range of modifications used to alter specific properties of in vitro 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 mRNA. Endogenous eukaryotic mRNA typically contains a cap structure on the 5' end of the mature molecule, which plays a crucial role in mediating the binding of mRNA cap-binding proteins (CBPs) (which, in turn, contributes to enhancing intracellular RNA stability and mRNA translation efficiency). Therefore, the highest levels of protein expression are achieved using capped mRNA transcripts. The 5'-cap contains a 5'-5'-triphosphate bond between the furthest 5' nucleotide and a guanine nucleotide. The conjugated guanine nucleotide is methylated at the N7 position. Additional modifications include methylation of the furthest 5' nucleotide and the second-to-furthest 5' nucleotide on the 2'-hydroxyl group.

[0022] Several different cap structures can be used to generate the 5'-cap of synthetic mRNA transcribed in vitro. 5'-capping of synthetic mRNA can be carried out by co-transcription with a chemical capping analog (i.e., capping during in vitro transcription). For example, the Anti-Reverse Cap Analog (ARCA) cap contains a 5'-5'-triphosphate guanine-guanine bond in which one guanine contains both an N7 methyl group and a 3'-O-methyl group. However, up to 20% of the transcript remains uncapped during this co-transcription process, and the synthetic capping analog becomes non-identical to the 5'-cap structure of true cellular mRNA, potentially reducing translatability and cellular stability. Alternatively, the synthetic mRNA molecule may also be enzymatically capped after transcription. These can generate more authentic 5'-cap structures that more closely mimic endogenous 5'-caps structurally or functionally, with enhanced binding of cap-binding proteins, increased half-life, decreased sensitivity to 5'-endonucleases, and / or reduced 5'-cap removal. To enhance mRNA stability and translatability, many synthetic 5'-cap analogs have been developed and are known in the art (see, for example, 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, Vol. 969 (Rabinovich, PH. ed.), 2013).

[0023] At the 3' end, the long adenine nucleotide (poly-A tail) is usually attached to the mRNA molecule during RNA processing. Immediately after transcription, the 3' end of the transcript is cleaved, releasing the 3' hydroxyl group. In contrast, in a process called polyadenylation, poly-A polymerase attaches the adenine nucleotide chain to the RNA. Poly(A) tails have been widely shown to enhance both the translation efficiency and stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly(A), poly(A) binding protein and the regulation of mRNA stability, Trends Bio Sci, vol. 14, pp. 373-377; Guhaniyogi, J. and Brewer, G., 2001, Regulation of mRNA stability in mammalian cells, Gene, vol. 265, pp. 11-23; Dreyfus, M. and Regnier, P., 2002, The poly(A) tail of mRNAs: Bodyguard in eukaryotes, scavenger in bacteria, Cell, vol. 111, pp. 611-613).

[0024] Poly(A) tailing of in vitro transcribed mRNA can be achieved using a variety of methods, including, but not limited to, cloning of poly(T) tracts to a DNA template or post-transcriptional addition using poly(A) polymerase. The first case allows in vitro transcription of mRNA with a poly(A) tail of a defined length, depending on the size of the poly(T) tract, but requires additional template manipulation. The latter case involves enzymatically adding a poly(A) tail to in vitro transcribed mRNA using poly(A) polymerase that catalyzes the incorporation of adenine residues into the 3' end of the RNA, and does not require additional DNA template manipulation, but results in mRNA with poly(A) tails of non-uniform lengths. 5'-capping and 3'-poly(A) tailing can be performed using a variety of commercially available kits, including, but not limited to, poly(A) polymerase tailing kits (EpiCenter), mMESSAGE mMACHINE T7 Ultra kits and poly(A) tailing kits (Life Technologies), as well as using commercially available reagents, various ARCA caps, poly(A) polymerases, etc.

[0025] In addition to 5' capping and 3' polyadenylation, other modifications of in vitro transcripts have been reported to provide benefits related to translational efficiency and stability. It is well known in the art that pathogenic DNA and RNA can be recognized by various sensors in eukaryotic cells and trigger a potent innate immune response. Since most nucleic acids from natural sources contain modified nucleosides, the ability to distinguish pathogenic DNA and RNA from self DNA and RNA has been shown to be at least partially based on structural and nucleoside modifications. In contrast, RNA synthesized in vitro lacks these modifications and is therefore immunostimulant, which may, in turn, inhibit effective mRNA translation as outlined above.By introducing modified nucleosides into mRNA transcribed in vitro, the recognition and activation of RNA sensors can be prevented, thus mitigating this unwanted immunostimulatory activity and enhancing translational capacity (e.g., Kariko, K. and Weissman, D., 2007, Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: implication for therapeutic RNA development, Curr Opin Drug Discov Devel, Vol. 10, pp. 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, Vol. 969 (Rabinovich, PH. ed.), 2013; Kariko, K., Muramatsu, H., Welsh, FA, Ludwig, J., Kato, H., Akira, See S., Weissman, D., 2008, Incorporation of Pseudouridine Into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability, Mol Ther, Vol. 16, pp. 1833–1840. Modified nucleosides and nucleotides used in the synthesis of modified RNA can be prepared, monitored, and utilized using common methods and procedures known in the art. A wide variety of nucleoside modifications are available that can be incorporated to some extent into mRNA transcribed in vitro, either alone or in combination with other modified nucleosides (see, for example, US2012 / 0251618).In vitro synthesis of nucleoside-modified mRNA has been reported to simultaneously enhance translational capacity while reducing its ability to activate immune sensors.

[0026] Other components of mRNA that can be modified to provide benefits related to translatability and stability include the 5' and 3' untranslated regions (UTRs). Optimizing the UTRs, both or independently (preferred 5' and 3' UTRs can be obtained from cellular or viral RNA), has been shown to increase mRNA stability and translation efficiency of transcribed mRNA in vitro (see, for example, 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, Vol. 969 (Rabinovich, PH. ed.), 2013).

[0027] In addition to mRNA, other nucleic acid payloads can be used in embodiments of the present invention. Methods of preparation for oligonucleotides include, but are not limited to, the chemical synthesis and enzymatic or chemical cleavage of long precursors, and in vitro transcription as described above. Methods for synthesizing DNA and RNA nucleotides are widely used and well known in the art (see, for example, Gait, MJ (ed.), Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, DC: IRL Press, 1984; and Herdewijn, P. (ed.), Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, Vol. 288 (Clifton, NJ) Totowa, NJ: Humana Press, 2005).

[0028] With respect to plasmid DNA, preparation for use in embodiments of the present invention is not limited to these, but generally involves the in vitro amplification and isolation of plasmid DNA in a liquid culture of bacteria containing the plasmid of interest. The presence of a gene in the plasmid of interest that encodes resistance to a specific antibiotic (such as penicillin or kanamycin) makes it possible to selectively grow bacteria containing the plasmid of interest in a culture containing the antibiotic. Methods for isolating plasmid DNA are widely used and well-known in this art (see, for example, Heilig, J., Elbbing, KL, and Brent, R (2001) Large-Scale Preparation of Plasmid DNA. Current Protocols in Molecular Biology., Vol. 41: No. II: 1.7: 1.7.1-1.7.16; Rozkov, A., Larsson, B., Gillstrom, S., Bjornestedt, R., and Schmidt, SR (2008) Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture. Biotechnol. Bioeng., Vol. 99: 557-566; and US6197553B1). Plasmid isolation can be performed using a variety of commercially available kits, including, but not limited to, Plasmid Plus (Qiagen), GenJET Plasmid MaxiPrep (Thermo), and Pure Yield MaxiPrep (Promega) kits, as well as commercially available reagents.

[0029] Lipids, lipid nanoparticles and compositions containing the present invention, and various exemplary embodiments of their use for delivering active substances (e.g., therapeutic agents) such as nucleic acids that modulate gene and protein expression are described in further detail below.

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

[0031] Unless otherwise required by context, the word “comprises” and its variations, such as “comprises” and “to include,” shall be interpreted throughout this specification and the claims as having an open, comprehensive meaning, i.e., “including, but not limited to.”

[0032] Throughout this specification, any reference to “one embodiment” or “a certain embodiment” means that any particular feature, structure, or characteristic described in relation to this embodiment is included in at least one embodiment of the present invention. Therefore, occurrences of the phrase “in one embodiment” or “in a certain embodiment” in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, any particular feature, structure, or characteristic may be combined in any suitable manner in one or more embodiments.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the invention pertains. Where used in the specification and claims, the singular forms “a,” “an,” and “the” include plural references unless the context explicitly indicates otherwise.

[0034] The phrase "induces the expression of a desired protein" refers to the ability of nucleic acids to increase the expression of a desired protein. To investigate the degree of protein expression, a test sample (e.g., a cell sample in culture expressing the desired protein) or a test mammal (e.g., a mammal, e.g., a human, or an animal model, e.g., a rodent (e.g., a mouse), or a non-human primate (e.g., a monkey) model) is brought into contact with nucleic acids (e.g., nucleic acids combined with the lipids of the present invention). 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 cell sample in culture expressing the desired protein) or a control mammal (e.g., a mammal, e.g., a human, or an animal model, e.g., a rodent (e.g., a mouse), or a non-human primate (e.g., a monkey) model) that has not been brought into contact with nucleic acids or has not been administered nucleic acids. If the desired protein is present in the control sample or control mammal, a value of 1.0 can be assigned to the expression of the desired protein in the control sample or control mammal. In certain embodiments, induction of the expression of a 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 the desired protein expression in the control sample or control mammal exceeds 1, for example, about 1.1, 1.5, 2.0, 5.0, or 10.0. If the desired protein is not present in either the control sample or the control mammal, induction of the expression of the desired protein is achieved when any measurable level of the desired protein is detected in the test sample or test mammal. Those skilled in the art will understand appropriate assays for determining the level of protein expression in a sample, such as dot blotting, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays, or assays based on reporter proteins that can produce fluorescence or luminescence under appropriate conditions.

[0035] The phrase "inhibits the expression of a target gene" refers to the ability of a nucleic acid to silence, reduce, or inhibit the expression of a target gene. To investigate the degree of gene silencing, a test sample (e.g., a cell sample in culture expressing the target gene) or a test mammal (e.g., a mammal, e.g., a human, or an animal model, e.g., a rodent (e.g., a mouse), or a non-human primate (e.g., a monkey) model) is brought into contact with the nucleic acid that silences, reduces, or inhibits the expression of the target gene. The expression of the target gene in the test sample or test animal is compared to the expression of the target gene in a control sample (e.g., a cell sample in culture expressing the target gene) or a control mammal (e.g., a mammal, e.g., a human, or an animal model, e.g., a rodent (e.g., a mouse), or a non-human primate (e.g., a monkey) model) that has not been brought into contact with the nucleic acid or has not been administered the nucleic acid. The expression of the target gene in the control sample or control mammal can be assigned a value of 100%. In certain embodiments, silencing, inhibition, or reduction of target gene expression is achieved when the expression level of the target gene in the test sample or test mammal is approximately 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% relative to the level of target gene expression in the control sample or control mammal. In other words, nucleic acids can silence, reduce, or inhibit the expression of a target gene in a 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 expression level of the target gene in a control sample or control mammal that has not been in contact with or administered nucleic acids. Appropriate assays for determining the expression level of a target gene include, but are not limited to, techniques known to those skilled in the art, such as investigations of protein or mRNA levels using dot blotting, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those skilled in the art.

[0036] An "effective dose" or "therapeutic effective dose" of an active agent or therapeutic agent, such as a therapeutic nucleic acid, is an amount sufficient to produce the 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 a target sequence is achieved when any measurable level is detected, in the case of an expression product that does not exist in the absence of the nucleic acid. In the case of an expression product that is present at a certain level before contact with the nucleic acid, an increase in expression is achieved when the fold increase of the value obtained using a nucleic acid such as mRNA is approximately 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 times or more compared to the control. Inhibition of the expression of a target gene or target sequence is achieved when the value obtained using nucleic acids such as antisense oligonucleotides is approximately 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% compared to the control. Suitable assays for measuring the expression of a target gene or target sequence include, for example, protein or RNA level investigations using techniques known to those skilled in the art, such as dot blotting, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of appropriate reporter proteins, and phenotypic assays known to those skilled in the art.

[0037] As used herein, the term “nucleic acid” refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form, and includes DNA, RNA, and their hybrids. 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, multivalent RNA, dicer substrate RNA, or viral RNA (vRNA), and combinations thereof. Nucleic acids include synthetic, naturally occurring, and non-naturally occurring nucleic acids containing known nucleotide analogs or modified backbone residues or bindings having similar binding properties to a reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramides, methylphosphonates, chiral methylphosphonates, 2'-O-methylribonucleotides, and peptide-nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogs of naturally occurring nucleotides having similar binding properties to a reference nucleic acid. Unless otherwise indicated, a given nucleic acid sequence also implicitly includes its conservedly modified variants (e.g., degenerate codon substitutions), alleles, orthologues, single nucleotide polymorphisms, and complementary sequences, as well as explicitly indicated sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res., Vol. 19:p. 5081 (1991); Ohtsuka et al., J. Biol. Chem., Vol. 260:p. 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, Vol. 8:p. 91-98 (1994)). A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together via phosphate groups."Bases" include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogues, as well as synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications that introduce novel reactive groups such as amines, alcohols, thiols, carboxylates, and alkyl halides.

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

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

[0040] The term "lipids" generally refers to a group of organic compounds that include fatty acid esters, are poorly soluble in water but soluble in many organic solvents, and are not limited to these. Lipids are usually divided into at least three classes: (1) "simple lipids," which include fats and oils as well as waxes; (2) "complex lipids," which include phospholipids and glycolipids; and (3) "derived lipids," such as steroids.

[0041] "Steroids" have the following carbon skeleton: [ka] It is a compound containing [a certain substance]. Non-exclusive examples of steroids include cholesterol, etc.

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

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

[0044] The term "neutral lipid" refers to any of several lipid species that exist either uncharged or in a neutral zwitterionic form at a selected pH. Such lipids at physiological pH include, but are not limited to, phosphotidylcholines, e.g., 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), phophatidylethanolamines, e.g., 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM), ceramides, steroids, e.g., sterols, and their derivatives. Neutral lipids may be synthetic or naturally derived.

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

[0046] The term “lipid nanoparticles” refers to particles having a size of at least one nanometer (e.g., 1 to 1,000 nm) and comprising one or more compounds of structure (I) or other identified cationic lipids. In some embodiments, lipid nanoparticles are included in formulations that can be used to deliver active agents or therapeutic agents, such as nucleic acids (e.g., mRNA), to target sites of interest (e.g., cells, tissues, organs, tumors, etc.). In some embodiments, the lipid nanoparticles of the present invention include nucleic acids. Such lipid nanoparticles typically comprise a compound of structure (I) and one or more excipients selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. In some embodiments, active agents or therapeutic agents, such as nucleic acids, are encapsulated in an aqueous space surrounded by the lipid portion of the lipid nanoparticle or some or all of the lipid portion of the lipid nanoparticle, thereby protecting them from host organism or cellular mechanisms, such as enzymatic degradation induced by a harmful immune response or other undesirable effects.

[0047] In various embodiments, the lipid nanoparticles are approximately 30nm to 150nm, 40nm to 150nm, 50nm to 150nm, 60nm to 130nm, 70nm to 110nm, 70nm to 100nm, 80nm to 100nm, 90nm to 100nm, 70nm to 90nm, 80nm to 90nm, 70nm to 80nm, or They have an average diameter of approximately 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. In certain embodiments, when nucleic acids are present in lipid nanoparticles, they are resistant to degradation by nucleases in aqueous solutions. Lipid nanoparticles containing nucleic acids and methods for preparing them are disclosed, for example, in U.S. Patent Publications 2004 / 0142025, 2007 / 0042031 and PCT Publications WO2017 / 004143, WO2015 / 199952, WO2013 / 016058 and WO2013 / 086373, the complete disclosure thereof is incorporated herein by reference in its entirety for all purposes.

[0048] As used herein, “~encapsulated lipid” refers to lipid nanoparticles that provide an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), using complete encapsulation, partial encapsulation, or both. In some embodiments, the nucleic acid (e.g., mRNA) is completely encapsulated within the lipid nanoparticles.

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

[0050] In relation to nucleic acid lipid nanoparticles, "serum-stable" 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.

[0051] As used herein, "systemic delivery" refers to the delivery of a therapeutic product that can result in widespread exposure of an active agent within an organism. Some administration techniques can result in systemic delivery of a particular agent, while others cannot. Systemic delivery means that a useful, preferably therapeutic, amount of the agent is exposed to most of the body. Systemic delivery of lipid nanoparticles can be 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 by intravenous delivery.

[0052] As used herein, "local delivery" refers to the direct delivery of an active agent to a target site within an organism. For example, the agent can be delivered locally by direct injection into a diseased site, such as a tumor, another target site, such as an inflammatory site, or a target organ, such as the liver, heart, pancreas, kidney, etc. Local delivery can also include techniques of local application or localized injection, such as intramuscular, subcutaneous or intradermal injection. Local delivery does not interfere with systemic pharmacological effects.

[0053] "Alkyl" refers to, 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-C 15 alkyl), 1 to 12 carbon atoms (C1-C 12Alkyl) refers to saturated, linear or branched hydrocarbon chain radicals consisting only of carbon and hydrogen atoms, having 1 to 8 carbon atoms (C1-C8 alkyl) or 1 to 6 carbon atoms (C1-C6 alkyl) and being bonded 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, etc.). Unless otherwise specifically stated in the specification, alkyl groups are substituted as necessary.

[0054] "Alkenyl" refers to a group of atoms, for example, with 2 to 24 carbon atoms (C2 to C2). 24 Alkenyl), 4-20 carbon atoms (C4-C 20 Alkenyl), 6-16 carbon atoms (C6-C6) 16 Alkenyls), 6-9 carbon atoms (C6-C9 alkenyls), 2-15 carbon atoms (C2-C9 alkenyls), 15 Alkenyl), 2 to 12 carbon atoms (C2 to C2) 12 Alkenyls refer to linear or branched hydrocarbon chain radicals consisting only of carbon and hydrogen atoms, having 2 to 8 carbon atoms (C2-C8 alkenyls) or 2 to 6 carbon atoms (C2-C6 alkenyls), being bonded to the rest of the molecule by single bonds, and containing one or more carbon-carbon double bonds (e.g., ethenyl, propa-1-enyl, buta-1-enyl, penta-1-enyl, penta-1,4-dienyl, etc.). Unless otherwise specifically stated in the specification, alkenyl groups are substituted as necessary.

[0055] "Alkynyl" refers to, for example, a group of 2 to 24 carbon atoms (C2 to C2). 24 Alkynyl), 4 to 20 carbon atoms (C4~C 20 Alkynyl), 6-16 carbon atoms (C6-C6) 16 Alkynyl), 6-9 carbon atoms (C6-C9 alkynyl), 2-15 carbon atoms (C2-C 15 Alkynyl), 2 to 12 carbon atoms (C2 to C2) 12Alkynyl refers to a linear or branched hydrocarbon chain radical consisting only of carbon and hydrogen atoms, having 2 to 8 carbon atoms (C2-C8 alkynyl) or 2 to 6 carbon atoms (C2-C6 alkynyl), bonded to the rest of the molecule by single bonds, and containing one or more carbon-carbon triple bonds (e.g., ethynyl, propynyl, butynyl, pentynyl, etc.). Unless otherwise specifically stated in the specification, the alkynyl group may be substituted as needed.

[0056] "Alkylene" or "alkylene chain" refers to, for example, a chain of 1 to 24 carbon atoms (C1 to C24). 24 Alkylene), 1 to 15 carbon atoms (C1 to C) 15 Alkylene), 1 to 12 carbon atoms (C1 to C 12 Alkylene refers to a saturated, linear or branched divalent hydrocarbon chain (e.g., methylene, ethylene, propylene, n-butylene) consisting only of carbon and hydrogen, 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), and 1 to 2 carbon atoms (C1-C2 alkylene), with the rest of the molecule linked to a radical group. The alkylene chain is bonded to the rest of the molecule via single bonds and to the radical group via single bonds. The bonding sites of the alkylene chain to the rest of the molecule and to the radical group can be via one carbon or any two carbons in the chain. Unless otherwise specifically stated in the specification, the alkylene chain may be substituted as needed.

[0057] "Alkenylene" or "alkenylene chain" refers to, for example, a chain of 2 to 24 carbon atoms (C2 to C2). 24 Alkenylenes), 2 to 15 carbon atoms (C2 to C2) 15 Alkenylenes), 2 to 12 carbon atoms (C2 to C2) 12Alkenylenes refer to linear or branched divalent hydrocarbon chains (e.g., etenylene, propenylene, n-butenylene, etc.) having 2 to 8 carbon atoms (C2-C8 alkenylenes), 2 to 6 carbon atoms (C2-C6 alkenylenes), or 2 to 4 carbon atoms (C2-C4 alkenylenes), with the rest of the molecule linked to a radical group, consisting only of carbon and hydrogen, and containing one or more carbon-carbon double bonds. Alkenylene chains are linked to the rest of the molecule via single or double bonds and to the radical group via single or double bonds. The linkages of the alkenylene chain to the rest of the molecule and to the radical group can be via one carbon or any two carbons in the chain. Unless otherwise specifically stated in the specification, alkenylene chains may be substituted as needed.

[0058] "Aryl" refers to a carbocyclic radical comprising hydrogen, 6 to 18 carbon atoms, and at least one aromatic ring. For the purposes of the present invention, aryl radicals are monocyclic, bicyclic, tricyclic, or tetracyclic systems and may include fused or bridging ring systems. Examples of aryl radicals, but not limited to, include aryl radicals derived from acetantrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluorantene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene. Unless otherwise specifically stated in the specification, the term "aryl" or the prefix "ar-" (e.g., "aralkyl") is intended to include aryl radicals that are substituted as appropriate.

[0059] "Aralkill" is defined by formula -R b -R c (In the formula, R b R is an alkylene or alkenylene as defined above, cThe radicals refer to one or more aryl radicals as defined above, such as benzyl, diphenylmethyl, etc. Unless otherwise specifically stated in the specification, the aralkyl group is substituted as necessary.

[0060] "Cycloalkyl" refers to a stable, non-aromatic monocyclic or polycyclic hydrocarbon radical (which may include condensed or bridged ring systems) having 3 to 15 carbon atoms, 3 to 10 carbon atoms, or 3 to 8 carbon atoms, and being saturated, with the remainder of the molecule consisting only of carbon and hydrogen atoms bonded by single bonds. Monocyclic cycloalkyl radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example, adamantyl, norbornyl, dekalinyl, and 7,7-dimethyl-bicyclo[2.2.1]heptanyl. Unless otherwise specifically stated in the specification, cycloalkyl groups may be substituted as needed.

[0061] "Cycloalkylene" refers to a divalent cycloalkyl group. Unless otherwise specifically stated in the specification, the cycloalkylene group may be substituted as needed.

[0062] "Cycloalkenyl" refers to a stable, non-aromatic monocyclic or polycyclic hydrocarbon radical (which may include condensed or bridged ring systems) having 3 to 15 carbon atoms, 3 to 10 carbon atoms, or 3 to 8 carbon atoms, containing one or more carbon-carbon double bonds, and consisting only of carbon and hydrogen atoms bonded by single bonds to the rest of the molecule. Monocyclic cycloalkenyl radicals include, for example, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl. Unless otherwise specifically stated in the specification, the cycloalkenyl group may be substituted as necessary.

[0063] "Cycloalkenylene" refers to a divalent cycloalkenyl group. Unless otherwise specifically stated in the specification, the cycloalkenylene group may be substituted as needed.

[0064] As used herein, the term "substituted" means that at least one hydrogen atom is replaced by a non-hydrogen atom, such as, but not limited to, halogen atoms, such as F, Cl, Br, or I; an oxo group (=O); a hydroxyl group (-OH); or C1-C 12 Alkyl 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'(In the formula, R' is independent of H and C1~C each time it appears) 15 The substituents are C1-C12 12 In other embodiments, the substituent is an alkyl group. In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group, such as a fluoro group. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group (-OR'). In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group (-NR'R').

[0065] "As needed" or "as needed" (for example, substituted as needed) means that the event of the situation described below may or may not occur, and the description means both when the event or situation occurs and when it does not occur. For example, "alkyl as needed" means that the alkyl radical may or may not be substituted, and the description means that it includes both substituted alkyl radicals and unsubstituted alkyl radicals.

[0066] The term "prodrug" is intended to refer to a compound that can be converted under physiological conditions or by solvolysis to a bioactive compound of structure (I). Therefore, the term "prodrug" refers to a metabolite of a pharmaceutically acceptable compound of structure (I). A prodrug may be inactive when administered to a subject requiring it, but is converted in vivo to the active compound of structure (I). Prodrugs are typically rapidly converted in vivo, for example, by hydrolysis in the blood, to produce the parent compound of structure (I). Prodrug compounds often offer advantages in mammalian organisms, such as solubility, histocompatibility, or delayed release (see Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam)). Discussions of prodrugs are provided in Higuchi, T et al., ACS Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design, edited by Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

[0067] The term “prodrug” is also intended to include any covalent carrier that, when administered to a mammalian subject, releases the active compound of structure (I) in vivo. Prodrugs of compounds of structure (I) can be prepared by modifying a functional group present in the compound of structure (I) such that the modification is cleaved either by conventional manipulation or in vivo to become the parent compound of structure (I). Prodrugs include compounds of structure (I) in which a hydroxy, amino, or mercapto group is bonded to any group, and when the prodrug of the compound of structure (I) is administered to a mammalian subject, this any group is cleaved to form a free hydroxy, free amino, or free mercapto group, respectively. Examples of prodrugs, but not limited to, include acetate, formate, and benzoate derivatives of alcohols in the compound of structure (I) or amide derivatives of amine functional groups.

[0068] Embodiments of the present invention disclosed herein are also intended to encompass all pharmaceutically acceptable compounds of the compound of structure (I) in which one or more atoms are isotoped by being replaced with atoms having different atomic weights or mass numbers. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, for example, 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 125Examples include (I). These radiolabeled compounds may be useful in determining or measuring the efficacy of a compound by characterizing, for example, the site of action or mechanism of action, or the binding affinity to a pharmacologically important site of action. Compounds labeled with a specific isotope of structure (I) or (II), for example, those incorporating a radioactive isotope, are useful in drug and / or substrate tissue distribution studies. Radioactive isotope tritium, i.e., 3 H, and carbon 14, that is, 14 Given the ease of implementation and rapid detection means, C is particularly useful for this purpose.

[0069] Heavier isotopes, for example, deuterium, i.e., 2 Substitution with H, for example, can be preferable in some situations because it can lead to greater metabolic stability, such as increased in vivo half-life or reduced dose, resulting in certain therapeutic benefits.

[0070] Positron-emitting isotopes, for example, 11 C, 18 F, 15 O and 13 Substitution with N, etc., may be useful in positron emission topography (PET) studies to investigate substrate receptor occupancy. Isotope-labeled compounds of structure (I) can generally be prepared by conventional techniques known to those skilled in the art, or by using a suitable isotope-labeled reagent instead of previously used unlabeled reagents, as presented below, and by processes similar to those described in the preparations and examples.

[0071] Embodiments of the present invention disclosed herein are also intended to encompass in vivo metabolites of the disclosed compounds. Such products may arise primarily from enzymatic processes, such as oxidation, reduction, hydrolysis, amidation, or esterification of the administered compound. Accordingly, embodiments of the present invention include compounds produced by processes comprising administering the compounds of the present invention to mammals for a period of time sufficient to produce their metabolites. Such products are typically identified by administering a radiolabeled compound of structure (I) in a detectable dose to animals such as rats, mice, guinea pigs, monkeys, or humans, and isolating the converted products from urine, blood, or other biological samples after sufficient time for metabolism to occur.

[0072] "Stable compound" and "stable structure" refer to compounds that are robust enough to withstand isolation from a reaction mixture to a usable level of purity and formulation into an effective therapeutic agent.

[0073] "Mammals" include both humans and livestock, such as laboratory animals and household pets (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits), as well as non-domesticated animals, such as wild animals.

[0074] "Pharmacologically acceptable carriers, diluents or excipients" include, but are not limited to, any adjuvants, carriers, excipients, glidants, sweeteners, diluents, preservatives, colorants, flavor enhancers, surfactants, humectants, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers that are approved by the U.S. Food and Drug Administration as acceptable for use in humans or livestock.

[0075] "Pharmacologically acceptable salts" include both acid addition salts and base addition salts.

[0076] "Pharmacologically acceptable acid addition salts" are defined as those that retain the biological efficacy and properties of the free base and are not biologically or otherwise undesirable, and include inorganic acids, such as, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and organic acids, such as, but are not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, and gentian. This refers to salts formed together with sic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucoic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, etc.

[0077] A "pharmaceutically acceptable base addition salt" refers to a salt that retains the biological efficacy and properties of a free acid and is not biologically or otherwise undesirable. These salts are prepared by adding an inorganic or organic base to a free acid. Examples of salts derived from inorganic bases, but not limited to, include sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Examples of salts derived from organic bases, but not limited to these, include primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and salts of basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydravamin, choline, betaine, benetamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resins. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0078] Crystallization often produces solvates of compounds of structure (I). As used herein, the term “solvate” refers to an aggregate comprising one or more molecules of the compound of structure (I) and one or more molecules of a solvent. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of the present invention may exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, and the like, as well as the corresponding solvated forms. In some embodiments, the compound of structure (I) may exist as a true solvate, while in other cases, the compound of structure (I) may simply retain exogenous water or be a mixture of water and some exogenous solvent.

[0079] "Pharmaceutical composition" refers to a formulation of a compound of structure (I) and a medium commonly accepted in the art for delivering a bioactive compound to a mammal, such as a human. Such mediums include all pharmaceutically acceptable carriers, diluents, or excipients for this purpose.

[0080] "Effective dose" or "therapeutic effective dose" refers to the amount of the compound of structure (I) that, when administered to a mammal, preferably a human, is sufficient to perform a treatment in the mammal, preferably a human. The amount of lipid nanoparticles of the embodiments of the present invention constituting the "therapeutic effective dose" will vary depending on the compound, its state and its severity, the method of administration, and the age of the mammal to be treated, but can be conventionally determined by those skilled in the art, taking into account their knowledge and this disclosure.

[0081] As used herein, “to treat” or “treatment” encompasses the treatment of the disease or condition in a mammal, preferably a human, that has the disease or condition of interest. (i) In mammals, in particular, in cases where such mammals are predisposed to the condition but have not yet been diagnosed with it, to prevent the onset of the disease or condition. (ii) To inhibit a disease or condition, that is, to suppress its onset. (iii) to alleviate a disease or condition, that is, to cause regression of the disease or condition, (iv) Relieving symptoms caused by a disease or condition, that is, relieving pain without addressing the underlying disease or condition. This includes. As used herein, the terms “disease” and “condition” may be used interchangeably, or a particular disease or condition may not have a known causative agent (and therefore its etiology has not yet been resolved), and therefore not yet recognized as a disease, but only as an undesirable condition or syndrome, and may differ in that a particular set of symptoms, to varying degrees, has been identified by a clinician.

[0082] Compounds of structure (I) or pharmaceutically acceptable salts thereof may contain one or more chiral centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined with respect to an amino acid as (R)- or (S)-, or (D)- or (L)- in terms of absolute stereochemistry. Embodiments of the present invention are intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or they may be resolved using prior art, e.g., chromatography and fractional recrystallization. Prior art for the preparation / isolation of individual enantiomers includes chiral synthesis from a suitable optically pure precursor, or resolution of racemates (or racemates of salts or derivatives) using, for example, chiral high-performance liquid chromatography (HPLC). Where a compound described herein contains an olefinic double bond or other geometrically asymmetric center, unless otherwise specified, the compound is intended to include both E and Z geometric isomers. Similarly, all tautomer forms are also intended to be included.

[0083] A "stereoisomer" refers to a compound that is composed of the same atoms bonded together by the same bonds, but has different three-dimensional structures that are not interchangeable. This invention envisions various stereoisomers and mixtures thereof, and includes "enantiomers," which refer to two stereoisomers whose molecules are mirror images of each other and cannot be superimposed.

[0084] A "tautomer" refers to a proton shift from one atom of a molecule to another atom of the same molecule. This invention includes tautomers of any of the aforementioned compounds.

[0085] compound In some embodiments, the present invention provides novel lipid compounds that can be combined with other lipid components, such as neutral lipids, charged lipids, steroids, and / or polymer-conjugated lipids, to form lipid nanoparticles together with oligonucleotides. Without wishing to be constrained by theory, these lipid nanoparticles are thought to shield oligonucleotides from degradation in serum and provide effective delivery of oligonucleotides to cells in vitro and in vivo.

[0086] In one embodiment, the compound has the following structure (I): [ka] or having a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, in the formula, L 1 is -O(C=O)R 1 , -(C=O)OR 1 -C(=O)R 1 , -OR 1 , -S(O) x R 1 -S-SR 1 -C(=O)SR 1 -SC(=O)R 1 , -NR a C(=O)R 1 -C(=O)NR b R c , -NR aC(=O)NR b R c 、 -OC(=O)NR b R c or -NR a C(=O)OR 1 and is L 2 is -O(C=O)R 2 、 -(C=O)OR 2 、 -C(=O)R 2 、 -OR 2 、 -S(O) x R 2 、 -S - SR 2 、 -C(=O)SR 2 、 -SC(=O)R 2 、 -NR d C(=O)R 2 、 -C(=O)NR e R f 、 -NR d C(=O)NR e R f 、 -OC(=O)NR e R f ; -NR d C(=O)OR 2 or R 2 is a direct bond to, G 1 and G 2 are each independently C2 - C 12 alkylene or C2 - C 12 alkenylene and is G 3 is C1 - C 24 alkylene, C2 - C 24 alkenylene, C3 - C8 cycloalkylene or C3 - C8 cycloalkenylene and is R a 、R b 、R d and R e are each independently H or C1 - C 12 alkyl or C1 - C 12 alkenyl and is R c and R f are each independently C1 - C 12 alkyl or C2 - C 12It is alkenyl, R 1 and R 2 Each of them is independently branched, C6~C 24 Alkyl or branched C6-C 24 It is alkenyl, R 3 is -N(R 4 )R 5 And, R 4 is C1~C 12 It is alkyl, R 5 is substitution C1~C 12 It is alkyl, x is 0, 1, or 2. Alkyl, alkenyl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, aryl, and aralkyl are each independently substituted or unsubstituted unless otherwise specified.

[0087] In a particular embodiment, G 3 It is not replaced. In a more specific embodiment, G 3 is C2~C 12 It is an alkylene, and for example, in some embodiments, G 3 is a C3-C7 alkylene, or in other embodiments, G 3 is C3~C 12 It is an alkylene. 3 It is a C2 or C3 alkylene.

[0088] In some of the embodiments described above, the compound has the following structure (IA): [ka] The formula has the following characteristics, where y and z are each independently integers in the range of 2 to 12, for example, integers from 2 to 6, integers from 4 to 10, or for example, 4 or 5. In a particular embodiment, y and z are the same and selected from 4, 5, 6, 7, 8 and 9.

[0089] In some of the embodiments described above, L 1 is -O(C=O)R 1 , -(C=O)OR 1 Or -C(=O)NR b R c And L 2 is -O(C=O)R 2 , -(C=O)OR 2 Or -C(=O)NR e R f For example, in some embodiments, L 1 and L 2 These are -(C=O)OR respectively 1 and -(C=O)OR 2 In other embodiments, L 1 is -(C=O)OR 1 And L 2 is -C(=O)NR e R f In other embodiments, L 1 is -C(=O)NR b R c And L 2 is -C(=O)NR e R f That is the case.

[0090] In other embodiments of those described above, the compound has the following structure (IB), (IC), (ID), or (IE): [ka] It has one of the following.

[0091] In some of the embodiments described above, the compound has structure (IB), in other embodiments, the compound has structure (IC), and in yet another embodiment, the compound has structure (ID). In yet another embodiment, the compound has structure (IE).

[0092] In some different embodiments of those described above, the compound has the following structures: (IF), (IG), (IH), or (IJ): [ka] It has one of the following, where y and z are each independently integers in the range of 2 to 12, for example, integers from 2 to 6, for example, 4.

[0093] In some of the embodiments described above, y and z are integers in the ranges of 2 to 10, 2 to 8, 4 to 10, or 4 to 7, respectively. For example, in some embodiments, y is 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, z is 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, y and z are the same, while in other embodiments, y and z are different.

[0094] In some of the embodiments described above, R 1 Or R 2 Or both are branched C6~C 24 It is alkyl. For example, in some embodiments, R 1 and R 2 Each of these has the following independent structure: [ka] It has, in the formula, R 7a and R 7b Each occurrence is independent of H or C1~C 12 It is alkyl, a is an integer between 2 and 12. R 7a , R 7b and a are R 1 and R 2 Each of these is independently selected to contain 6 to 20 carbon atoms. For example, in some embodiments, a is an integer in the range of 5 to 9 or 8 to 12.

[0095] In some of the embodiments described above, R 7a At least one occurrence of is H. For example, in some embodiments, R 7aH is present in each occurrence. In other different embodiments of the aforementioned, R 7b At least one of the occurrences is a C1-C8 alkyl group. For example, in some embodiments, the C1-C8 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0096] In a different embodiment, R 1 Or R 2 Or both have the following structure: [ka] It has one of the following.

[0097] In some of the embodiments described above, R b , R c , R e and R f These are, independently, C3~C 12 It is alkyl. For example, in some embodiments, R b , R c , R e and R f is n-hexyl, and in other embodiments, R b , R c , R e and R f It is n-octyl.

[0098] In any of the embodiments described above, R 4 R is substituted or unsubstituted methyl, ethyl, propyl, n-butyl, n-hexyl, n-octyl, or n-nonyl. For example, in some embodiments, R 4 It is not substituted. For others, R 4 is -OR g , -NR g C(=O)R h -C(=O)NR g R h -C(=O)R h -OC(=O)R h, -C(=O)OR h and -OR i OH (in the formula, R g Each instance is independently either H or a C1-C6 alkyl group. R h Each instance is independently a C1-C6 alkyl group. R i (Each instance is independently a C1-C6 alkylene.) It is substituted with one or more substituents selected from the group consisting of the following:

[0099] In other embodiments described above, R 5 R is substituted with methyl, ethyl, propyl, n-butyl, n-hexyl, n-octyl, or n-nonyl. In some embodiments, R 5 R is substituted ethyl or substituted propyl. In other different embodiments, R 5 R is substituted with hydroxyl. In further embodiments, R 5 is -OR g , -NR g C(=O)R h -C(=O)NR g R h -C(=O)R h -OC(=O)R h , -C(=O)OR h and -OR i OH (in the formula, R g Each instance is independently either H or a C1-C6 alkyl group. R h Each instance is independently a C1-C6 alkyl group. R i (Each instance is independently a C1-C6 alkylene.) It is substituted with one or more substituents selected from the group consisting of the following:

[0100] In other embodiments, R 4 R is an unsubstituted methyl group, 5R is substituted with methyl, ethyl, propyl, n-butyl, n-hexyl, n-octyl, or n-nonyl. In some of these embodiments, R 5 It is substituted with a hydroxyl group.

[0101] In some other specific embodiments, R 3 The structure is as follows: [ka] It has one of the following.

[0102] In various different embodiments, the compound has one of the structures shown in Table 1 below. [Table 1-1] [Table 1-2]

[0103] The compounds in Table 1 were prepared and tested according to methods known in the art, for example, the general methods described below herein.

[0104] Any embodiment of the compound of structure (I) described above, and any specific substituents and / or variables in the compound of structure (I) described above, can be independently combined with other embodiments and / or substituents and / or variables of the compound of structure (I) to form embodiments of the present invention not specifically described above. It is understood that this is possible. In addition, if in a particular embodiment and / or claim a list of substituents and / or variables is enumerated for any particular R group, L group, G group or variable a, x, y, or z, each individual substituent and / or variable may be removed from the particular embodiment and / or claim, and the remaining list of substituents and / or variables is considered to be within the scope of the embodiments of the present invention.

[0105] It is understood in this document that combinations of substituents and / or variables in the given formulas are acceptable only if such contributions result in a stable compound.

[0106] In some embodiments, a composition is provided comprising one or more compounds of structure (I) and a therapeutic agent. In some embodiments, lipid nanoparticles comprising one or more compounds of structure (I) are provided. For example, in some embodiments, the composition comprises one of the compounds of structure (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.

[0107] 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 compound to neutral lipid is in the range of about 2:1 to about 8:1.

[0108] In various embodiments, the composition further comprises a steroid or steroid analog. In certain embodiments, the steroid or steroid analog is cholesterol. In some of these embodiments, the molar ratio of compound to cholesterol is in the range of about 5:1 to 1:1.

[0109] In various embodiments, the polymer conjugate lipid is a pegylated lipid. For example, some embodiments include pegylated diacylglycerols (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerols (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanediolate (PEG-S-DMG), pegylated ceramides (PEG-cer), or PEG dialkoxypropyl carbamates, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecaneoxy)propyl)carbamate or 2,3-di(tetradecaneoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the compound is used in pairs. The molar ratio of PEGylated lipids is in the range of approximately 100:1 to 20:1.

[0110] In some embodiments, the composition has the following structure (II): [ka] (In the formula, R 8 and R 9 Each of these is independently a linear or branched alkyl, alkenyl, or alkynyl molecule containing 10 to 30 carbon atoms, and the alkyl, alkenyl, or alkynyl molecule is optionally cleaved by one or more ester bonds. w has an average value in the range of 30-60. This includes pegylated lipids having the same properties as pharmaceutically acceptable salts, tautomers, or stereoisomers thereof.

[0111] In some embodiments, R 8 and R 9 Each of these is independently a linear alkyl group containing 12 to 16 carbon atoms. In some embodiments, w has an average value in the range of 43 to 53. In other embodiments, the average value of w is approximately 45. In yet another different embodiment, the average value of w is approximately 49.

[0112] In some embodiments, lipid nanoparticles (LNPs) are provided comprising one or more compounds of structure (I) and a therapeutic agent. For example, in some embodiments, the LNP comprises one of the compounds of structure (I), a therapeutic agent, and one or more excipients selected from neutral lipids, steroids, and polymer-conjugated lipids.

[0113] In some embodiments of LNP, 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 compound to neutral lipid ranges from about 2:1 to about 8:1.

[0114] In various embodiments of LNP, the composition further comprises a steroid or steroid analogue. In certain embodiments, the steroid or steroid analogue is cholesterol. In some of these embodiments, the molar ratio of compound to cholesterol is in the range of about 5:1 to 1:1.

[0115] In various embodiments of LNPs, the polymer conjugate lipid is a pegylated lipid. For example, some embodiments include pegylated diacylglycerols (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerols (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanediolate (PEG-S-DMG), pegylated ceramides (PEG-cer), or PEG dialkoxypropyl carbamates, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecaneoxy)propyl)carbamate or 2,3-di(tetradecaneoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the compound is used in pairs. The molar ratio of PEGylated lipids is in the range of approximately 100:1 to 20:1.

[0116] In some embodiments, the LNP has the following structure (II): [ka] (In the formula, R 8 and R 9 Each of these is independently a linear or branched alkyl, alkenyl, or alkynyl containing 10 to 30 carbon atoms, and the alkyl, alkenyl, or alkynyl is optionally cleaved by one or more ester bonds. w has an average value in the range of 30-60. This includes pegylated lipids having the same properties as pharmaceutically acceptable salts, tautomers, or stereoisomers thereof.

[0117] In some embodiments, R 8 and R 9 Each of these is independently a linear alkyl group containing 12 to 16 carbon atoms. In some embodiments, w has an average value in the range of 43 to 53. In other embodiments, the average value of w is approximately 45. In yet another different embodiment, the average value of w is approximately 49.

[0118] Methods for preparing the above-mentioned lipids, lipid nanoparticles, and compositions are described herein below and / or are known in the art in PCT Publications WO2015 / 199952, WO2017 / 004143, and WO 2017 / 075531, respectively, which are incorporated herein by reference in their entirety.

[0119] In some embodiments of the compositions described above, the therapeutic agent comprises nucleic acid. For example, in some embodiments, the nucleic acid is selected from antisense and messenger RNA.

[0120] In other different embodiments, the present invention relates to a method for administering a therapeutic agent to a patient in need thereof, comprising the steps of preparing or providing one of the aforementioned compositions, and administering the composition to the patient.

[0121] For the purpose of administration, the compound of structure (I) (usually in the form of lipid nanoparticles combined with a therapeutic agent) may be administered as a raw chemical or formulated as a pharmaceutical composition. The pharmaceutical compositions of embodiments of the present invention comprise the compound of structure (I) (e.g., as a component in LNPs) and one or more pharmaceutically acceptable carriers, diluents, or excipients. The compound of structure (I) is present in the composition in an amount effective to form lipid nanoparticles and deliver the therapeutic agent, for example, to treat a specific disease or condition of interest. Appropriate concentrations and dosages can be readily determined by those skilled in the art.

[0122] The compositions and / or LNPs of the embodiments of the present invention may be administered via any of the accepted modes of administration of the agent for similar utility. The pharmaceutical compositions of the embodiments of the present invention can be formulated into preparations in the form of solids, semi-solids, liquids, or gases, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes for administering such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, intraperitoneal, sublingual, buccal, rectal, vaginal, and nasal. As used herein, the term intraperitoneal includes subcutaneous, intravenous, intramuscular, intradermal, intrasternal, intrasternal injection, or infusion techniques. The pharmaceutical compositions of the present invention are formulated so that when the composition is administered to a patient, the active ingredients contained therein become bioavailable. The composition administered to the subject or patient takes the form of one or more dosing units, in which case, for example, a tablet may be a single dosing unit, and a container of the compound of structure (I) in aerosol form may hold multiple dosing units. Practical methods for preparing such dosage forms are known or will be apparent to those skilled in the art. See, for example, Remington: The Science and Practice of Pharmacy, 20th edition (Philadelphia College of Pharmacy and Science, 2000). The composition administered in any case contains a therapeutically effective amount of the compound of structure (I), or a pharmaceutically acceptable salt thereof, for the treatment of the disease or condition of interest, in accordance with the teachings of the embodiments of the present invention.

[0123] The pharmaceutical compositions of embodiments of the present invention may be in solid or liquid form. In one embodiment, the carrier(s) are fine particles, thereby the composition is in the form of, for example, tablets or powders. The carrier(s) may be liquid, and the composition is, for example, an oral syrup, an injection solution, or an aerosol useful for, for example, inhalation administration.

[0124] When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, and semi-solid, semi-liquid, suspension, and gel forms are included herein as either solid or liquid forms.

[0125] As solid compositions for oral administration, pharmaceutical compositions can be formulated in the form of powders, granules, compressed tablets, pills, capsules, chewing gum, cachets, etc. Such solid compositions typically contain one or more inert diluents or food carriers. In addition, one or more of the following may be present: binders, e.g., carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth gum, or gelatin; excipients, e.g., starch, lactose, or dextrin; disintegrants, e.g., alginic acid, sodium alginate, Primogel, corn starch, etc.; lubricants, e.g., magnesium stearate, or sterotex; flow enhancers, e.g., colloidal silicon dioxide, etc.; sweeteners, e.g., sucrose, or saccharin; flavoring agents, e.g., peppermint, methyl salicylate, or orange flavorings, etc.; and colorants.

[0126] If the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, the pharmaceutical composition may also contain a liquid carrier, such as polyethylene glycol or oil, in addition to the above-mentioned materials.

[0127] The pharmaceutical composition may be in liquid form, such as an elixir, syrup, solution, emulsion, or suspension. The liquid may be, as two examples, for oral administration or for delivery by injection. When intended for oral administration, a preferred composition contains, in addition to the compound or LNP, one or more sweeteners, preservatives, dyes / colorants, and flavor enhancers. Compositions intended for administration by injection may contain one or more surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents.

[0128] The liquid pharmaceutical compositions of embodiments of the present invention, whether they are liquids, suspensions or other similar forms, may contain one or more of the following adjuvants: sterile diluents, e.g., distilled water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, etc.; non-volatile oils, e.g., synthetic mono or diglycerides, polyethylene glycol, glycerin, propylene glycol, or other solvents that can act as solvents or suspension media; antimicrobial agents, e.g., benzyl alcohol or methylparaben; antioxidants, e.g., ascorbic acid or sodium bisulfite; chelating agents, e.g., ethylenediaminetetraacetic acid; buffering agents, e.g., acetates, citrates or phosphates, etc.; and tonicity modifiers, e.g., sodium chloride or dextrose, etc.; agents acting as antifreezes, e.g., sucrose or trehalose, etc. The peritoneal preparations may be sealed in glass or plastic ampoules, disposable syringes or multi-dose vials. Physiological saline is a preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

[0129] Liquid pharmaceutical compositions of embodiments of the present invention intended for either intraperitoneal or oral administration should contain a certain amount of the compound of structure (I) so as to yield a suitable LNP.

[0130] The pharmaceutical compositions of embodiments of the present invention may be intended for topical administration, in which case the carrier may appropriately include a base for a liquid, emulsion, ointment, or gel. The base may include, for example, one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, as well as emulsifiers and stabilizers. Thickeners may be present in the pharmaceutical composition for topical administration. If transdermal administration is intended, the composition may include a transdermal patch or an iontophoresis device.

[0131] The pharmaceutical compositions of embodiments of the present invention may be intended for rectal administration, for example, in the form of suppositories that dissolve in the rectum and release the drug. The rectal administration compositions may contain an oily base as a suitable non-irritating excipient. Examples of such bases, but not limited to, include lanolin, cocoa butter, and polyethylene glycol.

[0132] The pharmaceutical compositions of embodiments of the present invention may include a variety of materials that modify the physical form of a solid or liquid dosing unit. For example, the composition may include a material that forms a coating shell around the active ingredient. The material forming the coating shell is typically inert and can be selected from, for example, sugars, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule.

[0133] The pharmaceutical compositions of the embodiments of the present invention, in solid or liquid form, may include agents that bind to a compound of structure (I) and thereby assist in the delivery of the compound. Suitable agents that can act with this ability include monoclonal or polyclonal antibodies or proteins.

[0134] The pharmaceutical compositions of embodiments of the present invention may consist of dosing units that can be administered as aerosols. The term aerosol is used to describe a variety of systems, ranging from colloidal in nature to systems consisting of pressurized packages. Delivery may be by liquefied gas or pressurized gas, or by a suitable pump system for distributing the active ingredient. Aerosols of compounds of structure (I) can be delivered in a single-phase, two-phase, or three-phase system to deliver the active ingredient(s). Aerosol delivery may include necessary containers, activators, valves, sub-containers, etc., which together may form a kit. Those skilled in the art can determine a preferred aerosol without excessive experimentation.

[0135] Pharmaceutical compositions of embodiments of the present invention can be prepared by methods well known in the pharmaceutical field. For example, a pharmaceutical composition intended for administration by injection can be prepared by combining the lipid nanoparticles of the present invention with sterile, distilled water or other carriers to form a solution. Surfactants can be added to promote the formation of a homogeneous solution or suspension. Surfactants are compounds that interact non-covalently with the compound of structure (I) to promote the dissolution or homogeneous suspension of the compound in an aqueous delivery system.

[0136] The compositions of the embodiments of the present invention or their pharmaceutically acceptable salts are administered in therapeutically effective doses, which will vary depending on a variety of factors, including the activity of the particular therapeutic agent used, the metabolic stability and duration of action of the therapeutic agent, the patient's age, weight, overall health, sex, and diet, mode and timing of administration, elimination rate, drug combinations, the severity of a particular disorder or condition, and the subject being treated.

[0137] The compositions of the embodiments of the present invention may also be administered simultaneously with, before, or after the administration of one or more other therapeutic agents. Such combination therapies include the administration of a single pharmaceutical formulation of the composition of the embodiments of the present invention and one or more additional active agents, as well as the administration of the composition of the present invention and each active agent in separate pharmaceutical formulations. For example, the composition of the embodiments of the present invention and other active agents may be administered together to a patient as a single oral formulation, such as a tablet or capsule, or each agent may be administered as a separate oral formulation. When using separate formulations, the compound of structure (I) and one or more additional active agents may be administered at essentially the same time, i.e., simultaneously, or separately with a time stagger, i.e., sequentially, and combination therapy is understood to include all of these regimens.

[0138] Methods for preparing the above compounds and compositions are described below herein and / or are known in the art.

[0139] Those skilled in the art will understand that in the processes described herein, it may be necessary to protect the functional groups of intermediate compounds with appropriate protecting groups. Examples of such functional groups include hydroxyl, amino, mercapto, and carboxylic acids. Suitable protecting groups for hydroxyl groups include trialkylsilyl or diarylalkylsilyl (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl, or trimethylsilyl), tetrahydropyranyl, and benzyl. Suitable protecting groups for amino, amidino, and guanidino groups include t-butoxycarbonyl and benzyloxycarbonyl. Suitable protecting groups for mercapto groups include -C(O)-R'' (wherein R'' is alkyl, aryl, or arylalkyl), p-methoxybenzyl, and trityl. Suitable protecting groups for carboxylic acids include alkyl, aryl, or arylalkyl esters. These protecting groups are known to those skilled in the art and can be added or removed according to standard techniques such as those described herein. The use of protecting groups is described in detail in Green, TW, and PGM Wutz, Protective Groups in Organic Synthesis (1999), 3rd edition, Wiley. As those skilled in the art will understand, the protecting group may also be a polymer resin, such as Wang resin, Rink resin, or 2-chlorotrityl chloride resin.

[0140] Those skilled in the art will understand that such protected derivatives of the compounds of the present invention may not possess pharmacological activity on their own, but may, upon administration to a mammal, be metabolized in the body to form a pharmacologically active compound of structure (I). Therefore, such derivatives may be described as “prodrugs.” All prodrugs of the compound of structure (I) are included within the scope of embodiments of the present invention.

[0141] Furthermore, all compounds of structure (I) existing in free base or acid form can be converted to pharmaceutically acceptable salts thereof by treatment with a suitable inorganic or organic base or inorganic or organic acid using methods known to those skilled in the art. Salts of compounds of structure (I) can be converted to their free base or free acid forms by standard techniques.

[0142] Compounds of structure (I) and lipid nanoparticles containing the same can be prepared by methods known to those skilled in the art or derived by those skilled in the art, for example, by methods disclosed in PCT Publications WO2015 / 199952, WO2017 / 004143 and WO2017 / 075531 (each of which is incorporated herein by reference in whole).

[0143] The following general reaction scheme applies to compounds of structure (I): [ka] (In the formula, R 3 , L 1 , L 2 , G 1 , G 2 and G 3 (This is as defined herein.) Or, exemplary methods for preparing pharmaceutically acceptable salts, tautomers, or stereoisomers thereof are illustrated. Those skilled in the art will understand that these compounds may be prepared in a similar manner or in combination with other methods known to those skilled in the art. Those skilled in the art will also understand that other compounds of structure (I), not specifically shown below, may be prepared in a similar manner to those described below by using appropriate starting components and, as necessary, modifying the parameters of the synthesis. Generally, starting components may be obtained from suppliers such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, synthesized according to information known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)), or prepared as described in the present invention.

[0144] [ka] Embodiments of the compound of structure (I) (e.g., compound A-5) can be prepared according to general reaction scheme 1 ("Method A"), where R is independently present in each instance. 1 or R 2 The expression represents m, where m is an integer from 0 to 23, and each n is an integer from 2 to 12 independently. Referring to general reaction scheme 1, the compound of structure A-1 may be purchased from a commercial source or prepared according to methods well known to those skilled in the art. A mixture of A-1, A-2 and DMAP is treated in DCC to obtain bromide A-3. A mixture of bromide A-3, a base (e.g., N,N-diisopropylethylamine) and N,N-dimethyldiamine A-4 is heated at a temperature and time sufficient to produce A-5 after any necessary post-treatment and / or purification steps.

[0145] [ka] Embodiments of the compound of structure (I) (e.g., compound B-5) can be prepared according to general reaction scheme 2 ("Method B"), where R is independently present in each instance. 1 or R 2 The expression represents m, where m is an integer from 0 to 23, and each n is an integer from 2 to 12 independently. As shown in General Reaction Scheme 2, the compound of structure B-1 may be purchased from a commercial source or prepared according to methods well known to those skilled in the art. A solution of B-1 (1 equivalent) is treated with an acid chloride B-2 (1 equivalent) and a base (e.g., triethylamine). The crude product is treated with an oxidizing agent (e.g., pyridinium chlorochromate) to recover the intermediate product B-3. Then, a solution of crude B-3, an acid (e.g., acetic acid) and N,N-dimethylaminoamine B-4 is treated with a reducing agent (e.g., sodium triacetoxyborohydride) to obtain B-5 after any necessary post-treatment and / or purification.

[0146] Although starting materials A-1 and B-1 are shown above as containing only saturated methylene carbon, it should be noted that starting materials containing carbon-carbon double bonds can also be used to prepare compounds containing carbon-carbon double bonds.

[0147] [ka] Embodiments of the compound of structure (I) (e.g., compound C-7) can be prepared according to general reaction scheme 3 ("Method C"), where R is independently present in each instance. 1 or R 2where \(m\) represents an integer from 0 to 23, and each \(n\) independently represents an integer from 2 to 12. Referring to General Reaction Scheme 3, the compound of Structure C-1 may be purchased from a commercial source or prepared according to methods well known to those skilled in the art. Reaction of C-1 with a suitable hydroxylamine (e.g., C-2), followed by chlorination, yields chloride C-5, which can be treated with a suitable secondary amine (e.g., C-6) to produce the desired compound after any necessary post-treatment and / or purification.

[0148] It should be noted that various alternative strategies for the preparation of compounds of Structure (I) are available to those skilled in the art. For example, the R 5 moiety may contain substituents, such as hydroxyl, and an appropriate protecting group may be required to mask the substituent, or the substituent may be added after R 5 has been added to the rest of the molecule. The use of protecting groups when necessary, and other modifications to General Reaction Schemes 1 - 3, will be readily apparent to those skilled in the art. The following examples are provided for purposes of illustration, not limitation.

Example

[0149] (Example 1) In Vivo Evaluation of Luciferase mRNA Using Lipid Nanoparticle Compositions Lipids of Structure (I), DSPC, cholesterol, and PEG-lipid were solubilized in ethanol at a molar ratio of 50:10:38.5:1.5 or 47.5:10:40.8:1.7. Lipid nanoparticles (LNP) were prepared at a weight ratio of total lipid to mRNA of approximately 10:1 to 30:1. Briefly, mRNA was diluted to 0.2 mg / mL in 10 - 50 mM citrate buffer, pH 4. Using a syringe pump, the ethanolic lipid solution and the aqueous mRNA solution were mixed at a ratio of about 1:5 to 1:3 (vol / vol) at a total flow rate exceeding 15 mL / min. Ethanol was then removed, and the external buffer was replaced with PBS by dialysis. Finally, the lipid nanoparticles were filtered through a sterile 0.2-μm pore filter.

[0150] The study was conducted in 6-8 week old female C57BL / 6 mice (Charles River) and 8-10 week old CD-1 (Harlan) mice (Charles River), following guidelines established by the Institutional Animal Care Committee (ACC) and the Canadian Council on Animal Care (CCAC). Different doses of mRNA-lipid nanoparticles were administered systemically by tail vein injection, and the animals were euthanized at a specific time point (e.g., 4 hours) after administration. The liver and spleen were collected in pre-weighed tubes, weighed, immediately flash-frozen in liquid nitrogen, and stored at -80°C until processed for analysis.

[0151] For the liver, approximately 50 mg was dissected for analysis and placed in 2 mL FastPrep tubes (MP Biomedicals, Solon OH). A 1 / 4-inch 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 × 6.0 m / s for 15 seconds. After incubating the homogenate at room temperature for 5 minutes, it was diluted 1:4 with GLB and evaluated using the SteadyGlo Luciferase assay system (Promega). Specifically, 50 μL of diluted tissue homogenate was reacted with 50 μL of SteadyGlo substrate, shaken for 10 seconds, followed by incubation for 5 minutes, and then CentroXS 3 Quantification was performed using an LB 960 illuminometer (Berthold Technologies, Germany). The amount of assayed protein was determined using the BCA protein assay kit (Pierce, Rockford, IL). Then, relative luminescence units (RLU) were normalized to the total assayed protein (μg). A calibration curve was created using QuantiLum Recombinant Luciferase (Promega) to convert RLU to luciferase (ng).

[0152] FLuc mRNA (L-6107 or L-7602) from Trilink Biotechnologies expresses the luciferase protein originally isolated from the firefly, *Photinus pyralis*. FLuc is commonly used in mammalian cell cultures to measure both gene expression and cell viability. It emits bioluminescence in the presence of the substrate, luciferin. This capped and polyadenylated mRNA is completely substituted with 5-methylcytidine and pseudouridine.

[0153] Example 2 pK of formulated lipids a decision As described elsewhere, the pKa of formulated lipids correlates with the efficacy of LNPs for nucleic acid delivery (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176 (2010)). In some embodiments, pK a The preferred range is approximately 5 to approximately 7. Using a fluorescence-based assay of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS), the pK of a representative compound of structure (I) was determined. aThe properties were determined in lipid nanoparticles. Lipid nanoparticles containing compound (I) / DSPC / cholesterol / PEG-lipid (50 / 10 / 38.5 / 1.5 or 47.5:10:40.8:1.7 mol%) in PBS at a total lipid concentration of 0.4 mM were prepared using an in-line 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 buffered solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl (the pH of which is in the range of 2.5 to 11). A fixed volume of TNS solution was added to a final concentration of 1 μM, and after vortex mixing, the fluorescence intensity was measured at room temperature using an SLM Aminco Series 2 Luminescence Spectrophotometer with excitation and emission wavelengths of 321 nm and 445 nm. The best-fit analysis (PK) was applied to the fluorescence data, and the pK value was used as the pH that elicits the maximum half of the fluorescence intensity. a We measured it.

[0154] The particle size of the lipid nanoparticles was approximately 55–95 nm in diameter, and in one case, determined by quasi-elastic light scattering using Malvern Zetasizer Nano ZS (Malvern, UK), the diameter was approximately 70–90 nm. The diameters shown are intensity-weighted averages. Mounting was determined using a fluorescence intercalate dye-based assay (Ribogreen).

[0155] The compound in structure (I) has the following molar ratio: 47.5% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 40.8% cholesterol / 1.7% PEG lipid ("PEG-DMA") 2-[2-(ω-methoxy(polyethylene glycol) 2000 The formulation was prepared using [ethoxy]-N,N-ditetradecylacetamide. Relative activity was determined by measuring luciferase expression in the liver 4 hours after administration via tail vein injection as described in Example 1.

[0156] Example 3 Determination of the efficacy of lipid nanoparticle formulations containing various cationic lipids using an in vivo luciferase mRNA expression rodent model. The cationic lipids shown in Table 2 have been previously tested with nucleic acids. For comparison, these lipids were also used to formulate lipid nanoparticles containing FLuc mRNA (L-6107) using a strain mixing method, as described in Example 1 and in PCT / US10 / 22614 (which is thus incorporated herein by reference in its entirety). The lipid nanoparticles may be formulated using the following molar ratio: 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid ("PEG-DMG", i.e., 1-(monomethoxy-polyethylene glycol)-2,3-dimiristoylglycerol, with an average PEG molecular weight of 2000). In alternative embodiments, 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. The activity at doses of 0.3 and 1.0 mg mRNA / kg was compared and expressed as luciferase (ng) / liver (g) measured 4 hours after administration, as described in Example 1. [Table 2]

[0157] The representative compounds of the present invention shown in Table 3 are in the following molar ratios: 50% cationic lipid / 10% distearoyl phosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid ("PEG-DMA") 2-[2-(ω-methoxy(polyethylene glycol) 2000The formulations were prepared using either ethoxy-N,N-ditetradecylacetamide or 47.5% cationic lipids / 10% DSPC / 40.8% cholesterol / 1.7% PEG lipids. Unless otherwise stated, activity was compared at a dose of 0.5 mg mRNA / kg and expressed as luciferase (ng) / liver (g) measured 4 hours after administration, as described in Example 1. Compound numbers in Table 3 refer to compound numbers in Table 1. [Table 3]

[0158] Example 4 [ka]

[0159] 4-1 synthesis To a 50 mL solution of 6-bromohexanoic acid (16 mmol, 3.12 g), 2-hexyl-1-decanol (22.4 mmol, 5.43 g), and DMAP (8 mmol, 976 mg) in DCM, DCC (17.6 mmol, 3.62 g) was added. The resulting mixture was stirred at room temperature for 16 hours. The precipitate (DCU) was removed by filtration. The filtrate was concentrated, and the resulting residual oily substance / solid was purified by column chromatography on silica gel (0-5% ethyl acetate in hexane). This yielded the desired product as a colorless oily substance (5.79 g, colorless oily substance, 13.8 mmol, 86%).

[0160] 4-2 synthesis A 35 mL anhydrous THF solution of 2-aminoethanol (333 mg, 5.46 mmol) was added with 4-1 (4.37 g, 10.4 mmol), potassium carbonate (1.44 g, 10.4 mmol), cesium carbonate (534 mg, 1.64 mmol) and sodium iodide (30 mg). The resulting mixture was heated at 70 °C for 6 days in a sealed pressure flask. The solvent was evaporated under reduced pressure, and the residue was put into a mixture of hexane and ethyl acetate (94:4) and washed with water and brine. The organic layer was separated and dried over anhydrous sodium sulfate. The dried extract (320 mL) was loaded onto a silica gel column. The column was eluted with a mixture of hexane, EtOAc and triethylamine (95:5:0 to 80:20:1). Thereby, the desired product was obtained as a colorless oily substance (2.68 g, 3.63 mmol, 70%). 1 HNMR (400 MHz, CDCl3) δ: 3.97 (d, 5.8 Hz, 4H), 3.53 (t, 5.3 Hz, 2H), 3.08 - 2.79 (br. 1H), 2.57 (t, 5.3 Hz, 2H), 2.45 (t-like, 7.4 Hz, 4H), 2.31 (t, 7.5 Hz, 4H), 1.67 - 1.59 (m, 6H), 1.51 - 1.41 (m, 4H), 1.38 - 1.10 (52H), 0.89 (t-like, 6.8 Hz, 12H).

[0161] Synthesis of 4-3 A solution of thionyl chloride (1.23 mmol, 146 mg) in 5 mL of chloroform was added dropwise to an ice-cooled solution of 4-2 (300 mg, 0.41 mmol) in 1 mL of CHC13 under an Ar atmosphere. After the addition of SOCl2 (1 - 2 minutes) was completed, the ice bath was removed and the reaction mixture was stirred at room temperature (20 °C) for 16 hours. Removal of chloroform and SOCl2 under reduced pressure gave a thick dark red oily substance. The crude product was purified by flash column chromatography on silica gel (0 - 1% MeOH in chloroform containing a trace amount of Et3N). The desired product was obtained as a brown oily substance (190 mg, 0.25 mmol, 61%).

[0162] Synthesis of Compound 1 4-3 (190 mg, 0.25 mmol) was dissolved in THF (5 mL). N,N-diisopropylethylamine (0.217 mL) and 4-4 (0.75 mmol, 140 mg; prepared from 1-bromononane and aminoethanol) were added to the solution. The sealed mixture was heated overnight at 69°C. The following day, sodium iodide (10 mg) was added to the mixture and heating (at 65°C) was resumed. After 3 days, the mixture was cooled and concentrated. The crude product was purified by column chromatography on silica gel and eluted with a mixture of hexane, ethylamine and triethylamine (95:5:0~80:20:1). This yielded the desired product as a colorless oily substance (150 mg, 0.17 mmol, 66%). 1 HNMR (400 MHz, CDCl3) δ: 4.90-4.20 (br. 1H), 3.97 (d, 5.8 Hz, 4H), 3.52 (t, 5.0 Hz, 2H), 2.61-2.53 (m, 4H), 2.52-2.45 (m, 4H), 2.45-2.40 (m, 4H), 2.31 (t, 7.5 Hz, 4H), 1.69-1.60 (m, 6H), 1.52-1.40 (m, 6H), 1.36-1.18 (64H), 0.89 (t-like, 6.8 Hz, 15H).

[0163] Example 5 [ka] Compound 2 was synthesized using a method similar to that of Compound 1 (50 mg, colorless oily substance). 1 HNMR (400 MHz, CDCl3) δ: 5.65-5.43 (br. 1H), 3.97 (d, 5.8 Hz, 4H), 3.77 (t, 5.1 Hz, 2H), 2.62 (t-sama, 5.6 Hz, 2H), 2.57-2.46 (m, 4H), 2.44-2.38 (m, 6H), 2.31 (t, 7.5 Hz, 4H), 1.69-1.60 (m, 8H), 1.51-1.40 (m, 6H), 1.36-1.18 (62H), 0.89 (t-like, 6.8 Hz, 15H).

[0164] Example 6 [ka] Compound 3 was synthesized using a method similar to that of Compound 1 (62 mg, colorless oily substance). 1 1H NMR (400 MHz, CDCl3) d: 4.43-4.07 (br. 2H), 3.97 (d, 5.8 Hz, 4H), 3.58 (t, 5.0 Hz, 4H), 2.71 (t, 5.3 Hz, 4H), 2.68-2.63 (br, 2H), 2.57-2.35 (m, 6H), 2.30 (t, 7.5 Hz, 4H), 1.67-1.56 (m, 8H), 1.52-1.40 (br., 4H), 1.39-1.18 (60H), 0.89 (t, 6.8 Hz, 12H). Using the method described in Example 2, the pKa of this compound was determined to be 7.18.

[0165] Example 7 [ka] Compound 4 was synthesized using a method similar to that of Compound 1 (colorless oily substance, 251 mg, 0.31 mmol, 51% obtained in two steps from alcohol 4-2). 1 HNMR (400 MHz, CDCl3) d: 3.97 (d, 5.8 Hz, 4H), 3.57 (t-like, 5.5 Hz, 2H), 2.62-2.38 (m, 10H), 2.32 (s, 3H). 2.31 (t, 7.4 Hz, 4H), 1.91-1.64 (br. Estimated 2H, OH), 1.69-1.59 (m, 6H), 1.54-1.40 (m, 4H), 1.37-1.19 (m, 52H), 0.89 (t-like, 6.8 Hz, 12H).

[0166] Example 8 [ka] Step 1. To an ice-cold solution of 4-2 (2.16 g, 2.93 mmol) in 8 mL of CHC13, a solution of thionyl chloride (8.79 mmol, 1.05 g, 0.641 mmol) in 35 mL of chloroform was added dropwise under an Ar atmosphere. After the addition of SOCl2 (1-2 minutes) was completed, the ice bath was removed and the reaction mixture was stirred at room temperature (20°C) for 16 hours. Removal of CHC13 and SOCl2 under reduced pressure yielded a concentrated dark red oily substance. The crude product was purified by flash column chromatography on silica gel (0-1% MeOH in chloroform containing a small amount of Et3N). The desired product was obtained as a brown oily substance (1.786 g, 2.36 mmol, 80%).

[0167] Step 2. The mixture in a pressure flask containing the above chloride (190 mg, 0.25 mmol), 8-1 (3 equivalents, 0.75 mmol, 130 mg, prepared from 9-bromo-1-nonanol and methylamine), N,N-diisopropylethylamine (0.217 mL), sodium iodide (10 mg), and THF (6 mL) was heated at 63°C for 3 days.

[0168] The mixture was cooled and concentrated. The residue was purified twice by flash-dry column chromatography on silica gel (hexane-Â1-Et3N, 95:5:0~80:20:1, and MeOH in chloroform, 0~5%). The desired product was obtained as a colorless oily substance (140 mg, 0.16 mmol, 63%). 1 HNMR (400 MHz, CDCl3) δ:3.97 (d, 5.8 Hz, 4H), 3.65 (t, 6.6 Hz, 2H), 2.57-2.51 (m, 2H), 2.44-2.39 (m, 6H), 2.35-2.28 (m, 6H), 2.23 (s, 3H), 1.68-1.53 ​​(m, 9H), 1.50-1.41 (m, 6H), 1.39-1.10 (62H), 0.89 (t-like, 6.8 Hz, 12H).

[0169] Example 9 [ka] Compound 6 was synthesized using a method similar to that of Compound 1 (colorless oily substance, 115 mg, 0.13 mmol, 52%). 1 HNMR (400 MHz, CDCl3) δ: 5.63 (br. s, 1H), 3.97 (d, 5.8 Hz, 4H), 3.77 (t, 5.1 Hz, 2H), 2.63 (t-sama, 5.6 Hz, 2H), 2.57-2.48 (m, 6H), 2.43-2.38 (m, 4H), 2.30 (t, 7.5 Hz, 4H), 1.69-1.58 (m, 8H), 1.47-1.39 (m, 4H), 1.37-1.18 (60H), 1.05 (t, 7.1 Hz, 3H), 0.89 (Mr. t, 6.8 Hz, 12H). Using the method described in Example 2, the pKa of this compound was determined to be 6.82.

[0170] Example 10 [ka] Compound 7 was synthesized using a method similar to that of Compound 1 (colorless oily substance, 166 mg, 0.19 mmol, 65%). 1 HNMR (400 MHz, CDCl3) δ:3.97 (d, 5.8 Hz, 4H), 3.65 (t, 6.6 Hz, 2H), 2.58-2.28 (m, 14H), 2.23 (s, 3H), 1.68-1.53 ​​(m, 9H), 1.50-1.41 (m, 6H), 1.39-1.10 (56H), 0.89 (t-sama, 6.8 Hz, 12H).

[0171] Example 11 [ka]

[0172] 11-1 synthesis To a solution of 2-butyloctanoic acid (26.9 mmol, 5.388 g), 9-bromo-1-nonanol (4 g, 18 mmol), and DMAP (9 mmol, 1.10 g) in DCM (40 mL), DCC (19.8 mmol, 4.08 g) was added. The resulting mixture was stirred at room temperature for 16 hours. The precipitate (DCU) was removed by filtration. The filtrate was concentrated, and the crude product was purified by flash-dry column chromatography on silica gel (0-3% ethyl acetate in hexane). The desired compound was obtained as a colorless oily substance (6.42 g, 15.8 mmol, 88%).

[0173] 11-2 synthesis A mixture of 11-1 (2.41 g, 5.94 mmol), 2-aminoethanol (185 mg, 3.03 mmol), N,N-diisopropylethylamine (1.32 mL), and anhydrous acetonitrile (20 mL) in a pressure flask was heated at 80°C for 16 hours. The solvent was evaporated under reduced pressure, and the crude product was purified by flash-dry column chromatography on silica gel (hexane-siRNA-Et3N, 99:1:0~80:20:1). The desired compound was obtained as a colorless oily substance (1.441 g, colorless oily substance, 2.03 mmol, 68%).

[0174] 11-3 synthesis To an ice-cold solution of 11-2 (1.441 g, 2.03 mmol) in 8 mL of CHC13, a solution of thionyl chloride (6.09 mmol, 725 mg) in chloroform (25 mL) was added dropwise under an Ar atmosphere. After the addition of SOCl2 was complete, the ice bath was removed and the reaction mixture was stirred at room temperature (20°C) for 16 hours. Removal of CHC13 and SOCl2 under reduced pressure yielded 1.730 g of a concentrated brown oily substance. The crude product (1.730 g) was purified by flash-dry column chromatography on silica gel (silica gel 230-400 mesh grade, 1% MeOH in chloroform containing trace amounts of Et3N). The desired compound was obtained as a brown oily substance (1.35 g, 1.8 mmol, 91%).

[0175] Synthesis of compound 8 A mixture of 11-3 (268 mg, 0.37 mmol), 8-1 (0.75 mmol, 130 mg), N,N-diisopropylethylamine (0.22 mL), and sodium iodide (10 mg) in THF (6 mL) was sealed and heated at 70°C for 3 days. The mixture was cooled and concentrated. The crude product was purified by flash-dry column chromatography on silica gel (0-5% MeOH in chloroform containing trace amounts of Et3N). The desired compound was obtained as a colorless oily substance (135 mg, 0.16 mmol, 43%). 1 HNMR (400 MHz, CDCl3) δ:4.06 (t, 6.6 Hz, 4H), 3.64 (t, 6.6 Hz, 2H), 2.57-2.51 (m, 2H), 2.45-2.38 (m, 6H), 2.35-2.27 (m, 4H), 2.22 (s, 3H), 1.66-1.52 (m, estimated 11H), 1.50-1.38 (m, 10H), 1.38-1.10 (54H), 0.90-0.85 (m, 12H).

[0176] Example 12 [ka] Compound 9 was synthesized using a method similar to that of Compound 1 (colorless oily substance, 154 mg; colorless oily substance, 0.17 mmol, 56%). 1 1H NMR (400 MHz, CDCl3) δ: 3.96 (d, 5.8 Hz, 4H), 3.64 (t, 6.6 Hz, 2H), 2.56-2.51 (m, 2H), 2.44-2.37 (m, 6H), 2.36-2.31 (m, 2H), 2.29 (t, 7.5 Hz, 4H), 2.22 (s, 3H), 1.66-1.52 (m, 9H, estimated, overlapping with water peak), 1.52-1.37 (m, 8H), 1.37-1.08 (62H), 0.88 (t-like, 6.8 Hz, 12H).

[0177] Example 13 [ka] Compound 10 was synthesized in the same manner as compound 8, according to the scheme described above.

[0178] The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to herein and / or listed in the application datasheet, including U.S. Provisional Patent Application No. 62 / 546,346 filed August 16, 2017, are incorporated herein by reference in their entirety. The aspects of the embodiments can be modified as necessary to utilize various patent, application, and publication concepts and to provide further embodiments. These and other modifications can be made to the embodiments in consideration of the descriptions detailed above. In general, the terms used in the following claims should not be construed as limiting the claims to the specific embodiments disclosed herein and herein, but rather as encompassing all possible embodiments, together with the entire scope of equivalents to which such claims are granted. Thus, the claims are not limited by this disclosure.

Claims

1. The following structure (IA): 【Chemistry 1】 During the ceremony, L １ is -O(C=O)R １ , or -(C=O) OR １ And, L ２ is -O(C=O)R ２ , or -(C=O) OR ２ And, G ３ is C ２ or C ３ alkylene, and R １ and R ２ Each of them is independently branched C ６ ~C ２４ Alkyl or branched C ６ ~C ２４ It is alkenyl, R ３ ha-N(R ４ ) R ５ And, R ４ is C １ ~C １２ It is alkyl, R ５ -NR ｇ C(=O)R ｈ , and -C(=O)NR ｇ C is substituted with one or more substituents selected from the group consisting of R and h. １ ~C １２ Alkyl R ｇ Each instance is independently either H or C １ ~C ６ It is alkyl, R ｈ Each instance is independent of C １ ~C ６ It is alkyl, y and z are independent integers in the range of 4 to 12. x is 0, 1, or 2. Here, alkyl, alkenyl, alkylene, alkenylene, cycloalkylene, and cycloalkenylene are each independently substituted or unsubstituted, unless otherwise specified. A compound having, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

2. L １ -O(C=O)R １ And, L ２ -O(C=O)R ２ L １ - (C=O) OR １ And, L ２ -O(C=O)R ２ And the compound has the following structure (IF) or (IG): 【Chemistry 2】 The compound according to claim 1, having one of the following.

3. The compound according to any one of claims 1 to 2, wherein y and z are each independently integers in the range of 4 to 10.

4. R １ and R ２ However, each is independent, branched C ６ ~C ２４ A compound according to any one of claims 1 to 3, wherein it is alkyl.

5. R １ and R ２ However, each operates independently, with the following structure: 【Transformation 3】 It has, in the formula, R ７ａ and R ７ｂ Each instance is independently either H or C １ ~C １２ It is an alkyl group, and a is an integer between 2 and 12. R ７ａ , R ７ｂ and a are R １ and R ２ Each of these is independently branched and selected to independently contain 6 to 20 carbon atoms. The compound according to claim 4.

6. The compound according to claim 5, wherein a is an integer from 8 to 12.

7. R １ Or R ２ Or both, independently, have the following structure: 【Chemistry 4】 A compound according to any one of claims 1 to 6, having one of the above.

8. R ４ The compound according to any one of claims 1 to 7, wherein the compound is substituted or unsubstituted methyl, ethyl, propyl, n-butyl, n-hexyl, n-octyl, or n-nonyl.

9. R ５ The compound according to any one of claims 1 to 7, wherein the compound is substituted with methyl, ethyl, propyl, n-butyl, n-hexyl, n-octyl, or n-nonyl.

10. R ５ The compound according to claim 9, wherein the atom is substituted with ethyl or substituted with propyl.

11. R ４ The compound according to any one of claims 9 or 10, wherein the methyl group is unsubstituted.

12. A composition comprising a compound according to any one of claims 1 to 11 and a therapeutic agent.

13. The composition according to claim 12, wherein the therapeutic agent comprises nucleic acid.

14. The composition according to claim 13, wherein the nucleic acid is selected from antisense and messenger RNA.

15. Use of the composition according to any one of claims 12 to 14 for the manufacture of a pharmaceutical composition for administering a therapeutic agent to a patient in need thereof.

16. Lipid nanoparticles comprising the compound according to any one of claims 1 to 11.

17. A pharmaceutical composition comprising lipid nanoparticles according to claim 16 and a pharmaceutically acceptable diluent or excipient.

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