Lipid and lipid nanoparticle formulations for nucleic acid delivery

Abstract

To provide a compound and a composition for use as a component of lipid nanoparticle formulations for delivery of a therapeutic agent.SOLUTION: A compound having the structure of Formula (I), or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof is provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention generally relates to novel cationic lipids that can be used in combination with other lipid components such as neutral lipids, cholesterol, and polymer conjugate lipids, and that can form oligonucleotide-containing lipid nanoparticles both in vitro and in vivo, thereby facilitating the intracellular delivery of therapeutic nucleic acids (e.g., oligonucleotides, messenger RNA). [Background technology]

[0002] Many challenges exist in relation to the delivery of nucleic acids to produce desired responses within biological systems. Nucleic acid-based therapeutics hold immense potential, but realizing this potential still requires more efficient 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 (antagomirs), antimirs, mimes, 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. Since constructs can be synthesized to produce any selected protein sequence, regardless of whether it is commensal 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 cellular products regulated by miRNAs, for example, to treat diseases associated with protein or enzyme deficiencies. Since constructs can be synthesized to inhibit one or more miRNAs, and this in turn modulates 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, thereby 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. Because 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 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 limited ability to acquire access to the intracellular compartments where the relevant translation mechanisms reside. Lipid nanoparticles formed from cationic lipids and oligonucleotides, which have other lipid components such as neutral lipids, cholesterol, PEG, and pegylated lipids, have been used to block RNA degradation in plasma and promote cellular uptake of oligonucleotides. [Overview of the project] [Means for solving the problem]

[0006] The need for improved cationic 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 delivery, and provide intracellular delivery of nucleic acids. In addition, these lipid-nucleic acid particles should be reasonably tolerable and provide a sufficient therapeutic index so that treatment of a patient with an effective dose of nucleic acid does not involve unacceptable toxicity and / or danger to the patient. The present invention provides these and related advantages.

[0007] In short, embodiments of the present invention provide lipid compounds, including stereoisomers, pharmaceutically acceptable salts, or tautomers of lipid compounds, 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-conjugate 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 formula (I):

Chemical formula

[0009] , R 1b , R 2a , R 2b , R 3a , R 3b , R 4a , R 4b , R 5 , R 6 , R 7 , R 8 , R 9 , L 1 , L 2 , G[[ID=4)), G 2 , G 3 , a, b, c and d are as defined herein) There is provided a compound having the above or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0009] There is also provided a pharmaceutical composition comprising one or more of the compounds of formula (I) described above and a therapeutic agent. In some embodiments, the pharmaceutical composition further comprises one or more components selected from neutral lipids, charged lipids, steroids and polymer-conjugated lipids. Such compositions are useful for the formation of 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 who needs administration of the therapeutic agent, the method comprising preparing a composition of lipid nanoparticles comprising a compound of formula (I) and a therapeutic agent and delivering the composition to the patient.

[0011] These and other aspects of the present invention will become apparent by reference to the following detailed description.

[0012] Within the diagram, the same reference number identifies similar elements. The size and relative position of elements in the diagram are not necessarily drawn to a fixed scale; some of these elements have been arbitrarily enlarged and positioned to improve the readability of the diagram. Furthermore, the specific shapes of the elements depicted are not intended to convey any information about the actual shape of those elements, but are merely selected to make the diagram easier to recognize. [Brief explanation of the drawing]

[0013] [Figure 1] Figure 1 shows the time course of luciferase expression in the liver of mice.

[0014] [Figure 2] Figure 2 illustrates the pKa calculation for MC3 as a representative example related to the disclosed lipids.

[0015] [Figure 3] Figure 3 provides comparative data on luciferase activity for different lipids. [Modes for carrying out the invention]

[0016] 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 the present invention can be carried out without these details.

[0017] The present invention is in part based on the discovery of novel cationic (amino) 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 cationic lipids described herein that provide increased nucleic acid activity and improved tolerance of the composition in vivo, resulting in a significant increase in therapeutic index compared to previously described nucleic acid-lipid nanoparticle compositions.

[0018] In certain embodiments, the present invention provides novel cationic 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 antibody.

[0019] 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 treatment or prevention of the disease or disorder, 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 cationic lipids described herein.

[0020] 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 (antago-mir / anti-mir), messenger-RNA-interfering complementary RNA (micRNA), DNA, multivalent RNA, Dicer substrate RNA, complementary DNA (cDNA), and the like. Accordingly, lipid nanoparticles and compositions of certain embodiments of the present invention can be used to induce the expression of a desired protein both in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more novel cationic lipids described herein, wherein the lipid nanoparticles encapsulate or accompany nucleic acids (e.g., messenger RNA or plasmid encoding the desired protein) to be expressed to produce the desired protein. Alternatively, lipid nanoparticles and compositions of certain embodiments of the present invention can be used both in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more novel cationic lipids described herein, 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, for example, 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(s) for integration into the host genome).

[0021] 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 colipages) 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).

[0022] 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 generated 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, See 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 (Neilson, H. ed.), 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).

[0023] 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.

[0024] 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 induce 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 greater levels, particularly in primary cells and in vivo.

[0025] 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.

[0026] Multiple 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 cap analog (i.e., capping during in vitro transcription). For example, anti-reverse cap analog (ARCA) caps contain 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 cap analog becomes non-identical to the 5'-cap structure of genuine cellular mRNA, potentially reducing translatability and cellular stability. Alternatively, synthetic mRNA molecules may also be enzymatically capped after transcription. These can produce more authentic 5'-cap structures that more closely mimic the endogenous 5'-cap structurally or functionally, with enhanced cap-binding protein binding, increased half-life, decreased sensitivity to 5' endonucleases, and / or reduced 5' decapping. Many synthetic 5'-cap analogs have been developed and are well known in the art to enhance mRNA stability and translatability (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).

[0027] 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, and in a process called polyadenylation, poly-A polymerase releases the 3' hydroxyl group that 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).

[0028] Poly(A) tailing of in vitro transcribed mRNA can be achieved using a variety of methods, including, but not limited to, cloning of the poly(T) region 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) region, but requires additional template manipulation. The latter case involves enzymatically adding the 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 length. 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.

[0029] 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.

[0030] 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).

[0031] 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).

[0032] 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.

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

[0034] As used herein, unless otherwise specified, the following terms have the meanings of these terms.

[0035] 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.”

[0036] 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.

[0037] 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.

[0038] 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 in the control sample or control mammal is greater than 1, for example, about 1.1, 1.5, 2.0, 5.0, or 10.0. If the desired protein is not present in the control sample or control mammal, induction of 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.

[0039] 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 approximately 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.

[0040] 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 exists at a certain level prior to contact with the nucleic acid, an increase in expression is achieved when the value obtained using a nucleic acid such as mRNA is doubled by 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.

[0041] 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 may be synthetic, natural, or unnatural, and include 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 “nucleic acid” encompasses nucleic acids containing known analogs of natural nucleotides having similar binding properties to a reference nucleic acid. Unless otherwise indicated, a given nucleic acid sequence also implicitly includes its variants modified in the traditional manner (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 3rd 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 their 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.

[0042] 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.

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

[0044] 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.

[0045] "Steroids" have the following carbon skeleton: [ka] It is a compound containing [a specific compound].

[0046] Non-limiting examples of steroids include cholesterol.

[0047] "Cationic lipids" refer to lipids that can be positively charged. Exemplary cationic lipids contain one or more amine groups that retain a positive charge. Exemplary 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)), as well as 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)).

[0048] 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 formula (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 a target site 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 formula (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 adverse effects.

[0049] In various embodiments, 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, and These nanoparticles 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 WO2013 / 016058 and WO2013 / 086373, the full disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0050] 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.

[0051] 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.

[0052] 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, etc., steroids, e.g., sterols, etc., and their derivatives. Neutral lipids may be synthetic or naturally occurring.

[0053] 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).

[0054] As used herein, the term "aqueous solution" refers to a composition containing water.

[0055] 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.

[0056] As used herein, "systemic delivery" refers to the delivery of a therapeutic product that can result in widespread exposure of an active drug within a living organism. Some administration techniques can result in systemic delivery of certain drugs, while others cannot. Systemic delivery means that a useful, preferably therapeutic, amount of the drug is exposed to a large portion 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.

[0057] As used herein, "local delivery" refers to the direct delivery of an active drug to a target site within a living organism. For example, a drug can be delivered locally by direct injection to a disease site, such as a tumor, another target site, such as an inflamed site, or a target organ, such as the liver, heart, pancreas, or kidney. Local delivery may also include techniques of local application or local injection, such as intramuscular, subcutaneous, or intradermal injection. Local delivery does not interfere with systemic pharmacological effects.

[0058] "Alkyl" means saturated or unsaturated (i.e., containing one or more double and / or triple bonds), for example, 1 to 24 carbon atoms (C1 to C24). 24 Alkyl), 4 to 20 carbon atoms (C4~C 20 Alkyl), 6-16 carbon atoms (C6-C 16 Alkyl), 6-9 carbon atoms (C6-C9 alkyl), 1-15 carbon atoms (C1-C 15 Alkyl), 1 to 12 carbon atoms (C1 to C 12Alkyl groups refer to linear or branched hydrocarbon chain groups consisting only of carbon and hydrogen atoms that have 1 to 8 carbon atoms (C1-C8 alkyl) or 1 to 6 carbon atoms (C1-C6 alkyl) and are bonded to the rest of the molecule by single bonds, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl, penta-1-enyl, penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc. Unless otherwise specifically stated in the specification, alkyl groups are optionally substituted.

[0059] "Alkylene" or "alkylene chain" is saturated or unsaturated (i.e., contains one or more double and / or triple bonds), for example, 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 Alkylenes are linear or branched divalent hydrocarbon chains consisting only of carbon and hydrogen, having 1 to 8 carbon atoms (C1-C8 alkylenes), 1 to 6 carbon atoms (C1-C6 alkylenes), 2 to 4 carbon atoms (C2-C4 alkylenes), and 1 to 2 carbon atoms (C1-C2 alkylenes), with the remaining molecules linked to radical groups. Examples include methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, etc. The alkylene chain is linked to the remaining molecules via single or double bonds and to radical groups via single or double bonds. The bonding points of the alkylene chain to the remaining molecules and radical groups can be via one carbon or any two carbons in the chain. Unless otherwise specifically stated in the specification, the alkylene chain may be optionally substituted.

[0060] "Heterocyclyl" or "heterocyclic" refers to a stable 3- to 18-membered (e.g., 5, 6, or 7-membered) non-aromatic ring group having 1 to 12 ring carbon atoms (e.g., 2 to 12) and 1 to 6 ring heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Unless otherwise specifically stated herein, heterocyclyl groups may be monocyclic, bicyclic, tricyclic, or tetracyclic ring systems, which may include fused or bridged ring systems, and the nitrogen, carbon, or sulfur atoms of the heterocyclyl group may be optionally oxidized, the nitrogen atom may be optionally quaternized, and the heterocyclyl group may be partially or fully saturated. Examples of such heterocyclyl groups include, but are not limited to, dioxolanil, thienyl[1,3]dithianil, decahydroisoquinolyl, imidazolinil, imidazolidinil, isothiazolidinil, isoxazolidinil, morpholinil, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinil, 2-oxopiperidinil, 2-oxopyrrolidinil, oxazolidinil, piperidinil, piperazinil, 4-piperidonil, pyrrolidinil, pyrazolidinil, quinuclidinil, thiazolidinil, tetrahydrofuryl, trithianil, tetrahydropyranil, thiomorpholinil, thiamorpholinil, 1-oxo-thiomorpholinil, and 1,1-dioxo-thiomorpholinil. Unless otherwise specifically stated herein, heterocyclyl groups may be optionally substituted.

[0061] As used herein, the term “substituted” means that at least one hydrogen atom (e.g., 1, 2, 3, or all hydrogen atoms) is replaced by a non-hydrogen atom, e.g., but not limited to halogen atoms, e.g., F, Cl, Br, or I; oxo group (=O); hydroxyl group (-OH); 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 ’ Each instance of appearance is independent of H, C1~C 15 This means any of the above groups (e.g., alkyl, alkylene, or heterocyclyl) that are replaced by a bond with an alkyl or cycloalkyl group (where x is 0, 1, or 2). In some embodiments, the substituents are C1-C 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. In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group.

[0062] "Optional" or "optionally substituted" (for example, optionally substituted) 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, "optionally substituted alkyl" means that the alkyl group may or may not be substituted, and the description means that it includes both substituted alkyl groups and unsubstituted alkyl groups.

[0063] The term "prodrug" is intended to refer to a compound that can be converted under physiological conditions or by solvolysis to the bioactive compound of the present invention. Therefore, the term "prodrug" refers to a pharmaceutically acceptable metabolite of the compound of the present invention. A prodrug may be inactive at the time of administration to a target requiring it, but is converted in vivo to the active compound of the present invention. Prodrugs are typically rapidly converted in vivo, for example, by hydrolysis in the blood, to produce the parent compound of the present invention. 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 Bioreversible Carriers in Drug Design, edited by Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

[0064] The term “prodrug” is also intended to include any covalent carrier that, when administered to a mammalian subject, releases the active compound of the present invention in vivo. Prodrugs of the compounds of the present invention (e.g., the compound of formula (I)) can be prepared by modifying a functional group present in the compound of the present invention such that the modification is cleaved either in the prescribed operation or in vivo to become the parent compound of the present invention. Prodrugs include compounds of the present invention in which a hydroxy, amino, or mercapto group is bonded to any group, and when the prodrug of the compound of the present invention 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 or amide derivatives of amine functional groups in the compounds of the present invention.

[0065] Embodiments of the present invention disclosed herein are also intended to encompass all pharmaceutically acceptable compounds of the compound of formula (I), wherein 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 having the structure of formula (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.

[0066] 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 requirements, resulting in certain therapeutic benefits.

[0067] 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. The isotope-labeled compound of formula (I) of (II) can generally be prepared by conventional techniques known to those skilled in the art, or by using a suitable isotope-labeled reagent instead of a previously used unlabeled reagent, by a process similar to that described in the preparations and examples below.

[0068] 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 the present invention in a detectable dose to animals such as rats, mice, guinea pigs, monkeys, or humans, allocating sufficient time for metabolism to occur, and then isolating the conversion products from urine, blood, or other biological samples.

[0069] "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.

[0070] "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.

[0071] "Pharmacologically acceptable carriers, diluents or excipients" include, but are not limited to, any adjuvants, carriers, excipients, flow enhancers, 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.

[0072] "Pharmacologically acceptable salts" include both acid addition salts and base addition salts.

[0073] "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 harmful, and include inorganic acids, for example, but not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and organic acids, for example, but not limited to acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, This refers to salts formed together with 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.

[0074] 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 harmful. 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, though not limited to these, include primary, secondary, and tertiary amines, substituted amines including naturally 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.

[0075] Crystallization often produces solvates of the compounds of the present invention. As used herein, the term “solvate” refers to an aggregate comprising one or more molecules of the compound of the present invention 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 monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate, etc., as well as the corresponding solvated forms. The compounds of the present invention may be true solvates, while in other cases, the compounds of the present invention may simply retain exogenous water or be a mixture of water and some exogenous solvent.

[0076] "Pharmaceutical composition" refers to a formulation of the compound of the present invention with 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.

[0077] "Effective dose" or "therapeutic effective dose" means the amount of the compound of the present invention, or lipid nanoparticles containing the compound, that is sufficient to perform a treatment in a mammal, preferably a human, when administered to a mammal, preferably a human. The amount of lipid nanoparticles of the present invention constituting the "therapeutic effective dose" will vary depending on the compound, its state and severity, the method of administration, and the age of the mammal to be treated, but can be determined as prescribed by those skilled in the art, taking into account their knowledge and this disclosure.

[0078] 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 susceptible 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 the cause has not yet been identified), 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.

[0079] The compounds of the present invention or pharmaceutically acceptable salts thereof may contain one or more chiral centers and thus may 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.

[0080] "Stereoisomers" refer to compounds composed of the same atoms bonded by the same bonds but having different three-dimensional structures that are not interchangeable. Embodiments of the present invention envision various stereoisomers and mixtures thereof, including "enantiomers", which refer to two stereoisomers whose molecules are mirror images that cannot be superimposed on each other.

[0081] "Tautomers" refer to the transfer of a proton from one atom of a molecule to another atom of the same molecule. Embodiments of the present invention include tautomers of any of the aforementioned compounds. Compound

[0082] In one aspect, 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, etc., to form lipid nanoparticles together with oligonucleotides. Without wishing to be bound 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. In one embodiment, the lipid compound has the formula (I): / / 这里的

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[0083] In some different embodiments, L 1 and L 2 These are -C(=O)-, -O-, and -S(O) respectively, independently. x -, -SS-, -C(=O)S-, -SC(=O)-, -NR a -, -NR aC(=O)-, -C(=O)NR a -, -NR a C(=O)NR a -OC(=O)NR a -, -NR a C(=O)O-, -NR a S(O) x NR a -, -NR a S(O) x - or -S(O) x NR a - is

[0084] In other embodiments of the aforementioned embodiments, the compound has the following structure (IA) or (IB): [ka] It has one of the following.

[0085] In some embodiments, the compound has structure (IA). In other embodiments, the compound has structure (IB).

[0086] In any of the embodiments described above, L 1 or L 2 One of them is -O(C=O)-. For example, in some embodiments, L 1 and L 2 Each of these is -O(C=O)-.

[0087] In some different embodiments of any of the embodiments described above, L 1 or L 2 One of them is -(C=O)O-. For example, in some embodiments, L 1 and L 2 Each of them is -(C=O)O-.

[0088] In a different embodiment, L 1 or L 2 One of them is a direct bond. As used herein, "direct bond" refers to a group (e.g., L 1 or L 2 This means that L is absent. For example, in some embodiments,1 and L 2 Each of these is a direct link.

[0089] In other different embodiments of the aforementioned embodiments, R 1a and R 1b For at least one occurrence of R 1a is H or C1~C 12 It is alkyl, R 1b And the carbon atom to which it is bonded is adjacent to R 1b And together with the carbon atom it is bonded to, it forms a carbon-carbon double bond.

[0090] In yet another different embodiment, R 4a and R 4b For at least one occurrence of R 4a is H or C1~C 12 It is alkyl, R 4b And the carbon atom to which it is bonded is adjacent to R 4b And together with the carbon atom it is bonded to, it forms a carbon-carbon double bond.

[0091] In further embodiments, R 2a and R 2b For at least one occurrence of R 2a is H or C1~C 12 It is alkyl, R 2b And the carbon atom to which it is bonded is adjacent to R 2b And together with the carbon atom it is bonded to, it forms a carbon-carbon double bond.

[0092] In any other different embodiment of the aforementioned embodiments, R 3a and R 3b For at least one occurrence of R 3a is H or C1~C 12 It is alkyl, R 3b And the carbon atom to which it is bonded is adjacent to R 3b And together with the carbon atom it is bonded to, it forms a carbon-carbon double bond.

[0093] The "carbon-carbon" double bond is understood to refer to one of the following structures: [ka] (In the formula, R c and R d (Each instance is independently either H or a substituent). For example, in some embodiments, R c and R d Each instance of appearance is independent of H, C1~C 12 Alkyl or cycloalkyl, e.g., H or C1-C 12 It is alkyl.

[0094] In various other embodiments, the compound has the following structure (IC) or (ID): [ka] (wherein e, f, g, and h are each independent integers between 1 and 12)

[0095] In some embodiments, the compound has a structure (IC). In other embodiments, the compound has a structure (ID).

[0096] In various embodiments of the compound of structure (IC) or (ID), e, f, g, and h are each independently integers from 4 to 10.

[0097] In some specific embodiments described above, a, b, c, and d are each independently integers between 2 and 12 or between 4 and 12. In other embodiments, a, b, c, and d are each independently integers between 8 and 12 or between 5 and 9. In some specific embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In yet another embodiment, a is 3. In yet another embodiment, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In yet another embodiment, a is 7. In yet another embodiment, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In yet another embodiment, a is 11. In yet another embodiment, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In yet another embodiment, a is 15. In yet another embodiment, a is 16.

[0098] In some embodiments, b is 1. In other embodiments, b is 2. In yet another embodiment, b is 3. In yet another embodiment, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In yet another embodiment, b is 7. In yet another embodiment, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In yet another embodiment, b is 11. In yet another embodiment, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In yet another embodiment, b is 15. In yet another embodiment, b is 16.

[0099] In some embodiments, c is 1. In other embodiments, c is 2. In yet another embodiment, c is 3. In yet another embodiment, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In yet another embodiment, c is 7. In yet another embodiment, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In yet another embodiment, c is 11. In yet another embodiment, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In yet another embodiment, c is 15. In yet another embodiment, c is 16.

[0100] In some specific embodiments, d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In yet another embodiment, d is 3. In yet another embodiment, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In yet another embodiment, d is 7. In yet another embodiment, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In yet another embodiment, d is 11. In yet another embodiment, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In yet another embodiment, d is 15. In yet another embodiment, d is 16.

[0101] In some embodiments, e is 1. In other embodiments, e is 2. In yet another embodiment, e is 3. In yet another embodiment, e is 4. In some embodiments, e is 5. In other embodiments, e is 6. In yet another embodiment, e is 7. In yet another embodiment, e is 8. In some embodiments, e is 9. In other embodiments, e is 10. In yet another embodiment, e is 11. In yet another embodiment, e is 12.

[0102] In some embodiments, f is 1. In other embodiments, f is 2. In yet another embodiment, f is 3. In yet another embodiment, f is 4. In some embodiments, f is 5. In other embodiments, f is 6. In yet another embodiment, f is 7. In yet another embodiment, f is 8. In some embodiments, f is 9. In other embodiments, f is 10. In yet another embodiment, f is 11. In yet another embodiment, f is 12.

[0103] In some embodiments, g is 1. In other embodiments, g is 2. In yet another embodiment, g is 3. In yet another embodiment, g is 4. In some embodiments, g is 5. In other embodiments, g is 6. In yet another embodiment, g is 7. In yet another embodiment, g is 8. In some embodiments, g is 9. In other embodiments, g is 10. In yet another embodiment, g is 11. In yet another embodiment, g is 12.

[0104] In some embodiments, h is 1. In other embodiments, e is 2. In yet another embodiment, h is 3. In yet another embodiment, h is 4. In some embodiments, e is 5. In other embodiments, h is 6. In yet another embodiment, h is 7. In yet another embodiment, h is 8. In some embodiments, h is 9. In other embodiments, h is 10. In yet another embodiment, h is 11. In yet another embodiment, h is 12.

[0105] In some other various embodiments, a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments, a and d are the same, and b and c are the same.

[0106] The sum of a and b, and the sum of c and d, are factors that can be varied to obtain lipids with desired properties. In one embodiment, a and b are selected such that their sum is an integer in the range of 14 to 24. In other embodiments, c and d are selected such that their sum is an integer in the range of 14 to 24. In further embodiments, the sum of a and b and the sum of c and d are the same. For example, in some embodiments, the sum of a and b and the sum of c and d are both the same integer, which may be in the range of 14 to 24. In yet another embodiment, a, b, c and d are selected such that the sum of a and b and the sum of c and d are 12 or greater.

[0107] R 1a , R 2a , R 3a and R 4a The substituents in are not particularly limited. In some embodiments, R 1a , R 2a , R 3a and R 4a At least one of them is H. In a particular embodiment, R 1a , R 2a , R 3a and R 4a H is present in each occurrence. In some other specific embodiments, R 1a , R 2a , R 3a and R 4a At least one of them is C1~C 12 It is alkyl. In certain other embodiments, R 1a , R 2a , R 3a and R 4a At least one of them is a C1-C8 alkyl group. In certain other embodiments, R 1a , R 2a , R 3a and R 4a At least one of them is a C1-C6 alkyl group. In some of the embodiments described above, the C1-C8 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0108] In a particular embodiment of the above, R 1a , R 1b , R 4a and R 4b Each time it appears, C1~C 12 It is alkyl.

[0109] In a further embodiment of the above, R 1b , R 2b , R 3b and R 4b At least one of them is H or R 1b , R 2b , R 3b and R 4b Each occurrence is H.

[0110] In a particular embodiment of the above, R 1b And the carbon atom to which it is bonded is adjacent to R 1b And together with the carbon atom to which it is bonded, it forms a carbon-carbon double bond. In other embodiments of the above, R 4b And the carbon atom to which it is bonded is adjacent to R 4b And together with the carbon atom it is bonded to, it forms a carbon-carbon double bond.

[0111] R 5 and R 6 The substituents in the above embodiments are not particularly limited. In a particular embodiment, R 5 or R 6 One of them is methyl. In other embodiments, R 5 or R 6 Each of them is methyl.

[0112] R 7 The substituents in the above embodiments are not particularly limited. In a particular embodiment, R 7 is C6~C 16 It is alkyl. In some other embodiments, R 7 R is a C6-C9 alkyl group. In some of these embodiments, R 7 is -(C=O)OR b-O(C=O)R b -C(=O)R b , -OR b , -S(O) x R b -S-SR b -C(=O)SR b -SC(=O)R b , -NR a R b , -NR a C(=O)R b -C(=O)NR a R b , -NR a C(=O)NR a R b -OC(=O)NR a R b , -NR a C(=O)OR b , -NR a S(O) x NR a R b , -NR a S(O) x R b or -S(O) x NR a R b It is replaced with, here, R a is H or C1~C 12 It is alkyl, R b is C1~C 15 It is alkyl, and x is 0, 1, or 2. For example, in some embodiments, R 7 is -(C=O)OR b Or -O(C=O)R b It has been replaced with.

[0113] In various embodiments of the above-described embodiments, R b is branched C1~C 15 It is alkyl. For example, in some embodiments, R b The structure is as follows: [ka] It has one of the following.

[0114] In certain other embodiments of the aforementioned embodiments, R 8 or R 9 One of them is methyl. In other embodiments, R 8 and R 9 Both are methyl.

[0115] In some different embodiments, R 8 and R 9 These, together with the nitrogen atoms to which they are bonded, form a 5, 6, or 7-membered heterocycle. In some embodiments of the above, R 8 and R 9 These, together with the nitrogen atoms to which they are bonded, form a five-membered heterocycle, such as a pyrrolidinyl ring. In some different embodiments of the embodiments described above, R 8 and R 9 These, together with the nitrogen atom to which they are bonded, form a six-membered heterocycle, such as a piperazinyl ring.

[0116] In yet another embodiment of the aforementioned compound, G 3 These are C2-C4 alkylenes, for example, C3 alkylenes.

[0117] In various different embodiments, the compound has one of the structures shown in Table 1 below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7]

[0118] It is understood that any embodiment of the compound of formula (I) as described above, and any particular substituent and / or variable in the compound of formula (I) as described above, can be independently combined with other embodiments and / or substituents and / or variables of the compound of formula (I) to form embodiments of the present invention not specifically described above. In addition, it is understood that 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-h or x, 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 present invention.

[0119] 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.

[0120] In some embodiments, compositions are provided comprising one or more compounds of formula (I) and a therapeutic agent. For example, in some embodiments, the composition comprises one of the compounds of formula (I), a therapeutic agent, and one or more excipients selected from neutral lipids, steroids, and polymer-conjugated lipids. Other pharmaceutically acceptable excipients and / or carriers are also included in various embodiments of the composition.

[0121] In some embodiments, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the neutral lipid is DSPC. In various embodiments, the molar ratio of the compound to the neutral lipid is in the range of about 2:1 to about 8:1.

[0122] In various embodiments, the composition further comprises a steroid or steroid analog. In certain embodiments, the steroid or steroid analog is cholesterol. In some of these embodiments, the molar ratio of the compound to cholesterol is in the range of about 2:1 to 1:1.

[0123] In various embodiments, the polymer conjugate lipid is a PEGylated lipid. For example, some embodiments use PEGylated diacylglycerol (PEG-DAG), e.g., 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG), e.g., 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(poly The compounds include ethoxy(ethyl)butanediolate (PEG-S-DMG), pegylated ceramide (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 molar ratio of the compound to the pegylated lipid is in the range of about 100:1 to about 25:1.

[0124] In some embodiments, the composition has the following structure (II): [ka] (In the formula, R 10 and R 11 Each of these is independently a linear or branched saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, and the alkyl chain is optionally interrupted by one or more ester bonds. z has an average value in the range of 30 to 60. This includes pegylated lipids having the same properties as pharmaceutically acceptable salts, tautomers, or stereoisomers thereof.

[0125] In some embodiments, R 10 and R 11 Each of these is an independent, linear, saturated alkyl chain containing 12 to 16 carbon atoms. In other embodiments, the average of z is approximately 45.

[0126] 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, plasmid DNA, and messenger RNA.

[0127] 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 preparing one of the aforementioned compositions and administering the composition to the patient.

[0128] For the purpose of administration, the compounds of the present invention (usually in the form of lipid nanoparticles combined with a therapeutic agent) may be administered as raw chemicals or formulated as pharmaceutical compositions. The pharmaceutical compositions of the present invention comprise the compound of formula (I) and one or more pharmaceutically acceptable carriers, diluents, or excipients. The compound of formula (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.

[0129] Administration of the compositions of the present invention can be effected via any of the recognized modes of administration of agents for achieving a similar utility. The pharmaceutical compositions of the present invention can be formulated into preparations in solid, semi-solid, liquid or gaseous form, for example, 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, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. As used herein, the term parenteral includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques. The pharmaceutical compositions of the present invention are formulated such that upon administration to a patient, the active ingredient contained therein is bioavailable. The composition administered to a subject or patient is in the form of one or more dosage units, in which case, for example, a tablet may be a single dosage unit, and a container of a compound of the present invention in aerosol form can hold a plurality of dosage units. The actual 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 event, contains a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof for the treatment of the targeted disease or condition, in accordance with the teachings of the present invention.

[0130] The pharmaceutical compositions of the present invention may be in solid or liquid form. In one aspect, the carrier(s) is / are microparticles, whereby the composition is in the form of, for example, a tablet or powder. The carrier(s) may be liquid and the composition may be, for example, an oral syrup, an injection solution or an aerosol useful for inhalation administration.

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

[0132] As a solid composition for oral administration, the pharmaceutical composition can be formulated in the form of powders, granules, compressed tablets, pills, capsules, chewing gums, cachets, etc. Such solid compositions usually contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, tragacanth gum or gelatin; excipients such as starch, lactose or dextrin; disintegrants such as alginic acid, sodium alginate, Primogel, corn starch; lubricants such as magnesium stearate or Sterotex; flow promoters such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavoring agents such as peppermint, methyl salicylate or orange flavor; and coloring agents.

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

[0134] The pharmaceutical composition may be in liquid form, such as elixirs, syrups, solutions, emulsions or suspensions. The liquid may, by way of two examples, be for oral administration or for delivery by injection. When oral administration is intended, the preferred composition contains, in addition to the compound, one or more of a sweetener, a preservative, a dye / coloring agent and a flavor enhancer. In compositions intended for administration by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer and an isotonic agent may be included.

[0135] The liquid pharmaceutical compositions 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., 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, etc.; buffering agents, e.g., acetates, citrates or phosphates, etc.; and tonicity modifiers, e.g., sodium chloride or dextrose, etc.; agents acting as antifreeze agents, e.g., sucrose or trehalose, etc. Parenteral preparations may be sealed in glass or plastic ampoules, disposable syringes or multi-dose vials. Physiological saline is a preferred adjuvant. The pharmaceutical compositions for injection are preferably sterile.

[0136] Liquid pharmaceutical compositions of the present invention intended for either parenteral or oral administration should contain a certain amount of the compound of the present invention so as to provide an appropriate dosage.

[0137] The pharmaceutical composition of the present invention may be intended for topical administration, in which case the carrier may appropriately include a liquid, emulsion, ointment, or gel base. 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. When intended for transdermal administration, the composition may include a transdermal patch or an iontophoresis device.

[0138] The pharmaceutical compositions 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. Compositions for rectal administration 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.

[0139] The pharmaceutical compositions of the present invention may include various materials that modify the physical form of solid or liquid dose units. For example, the composition may include a material that forms a coating shell around the active ingredient. The material forming the coating shell is usually 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.

[0140] The pharmaceutical compositions of the present invention, in solid or liquid form, may include agents that bind to the compound of the present invention, thereby assisting in the delivery of the compound. Suitable agents that can act with this ability include monoclonal or polyclonal antibodies or proteins.

[0141] The pharmaceutical compositions of the present invention may consist of dose units that can be administered as aerosols. The term aerosol is used to describe a variety of systems, ranging from colloidal forms to systems consisting of pressurized packages. Delivery may be by liquefaction or compressed gas, or by a suitable pump system for distributing the active ingredient. Aerosols of the compounds of the present invention can be delivered in a single-phase, two-phase, or three-phase system to deliver the active ingredient(s). Delivery of the aerosol may include necessary containers, active ingredients, valves, subcontainers, etc., which together may form a kit. Those skilled in the art can determine a preferred aerosol without excessive experimentation.

[0142] The pharmaceutical compositions 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 compounds of the present invention to promote the dissolution or homogeneous suspension of the compounds in an aqueous delivery system.

[0143] The compositions 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.

[0144] The compositions 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 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 which is itself a separate pharmaceutical formulation. For example, the composition of the present invention and other active agents may be administered together to the patient as a single oral formulation, such as a tablet or capsule, or each agent may be administered as a separate oral formulation. When separate formulations are used, the compounds of the present invention 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.

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

[0146] 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.

[0147] Such protected derivatives of the compounds of the present invention may not possess pharmacological activity in their own form, but it is also understood by those skilled in the art that they can be administered to mammals and subsequently metabolized in the body to form pharmacologically active compounds of the present invention. Therefore, such derivatives may be described as "prodrugs." All prodrugs of the compounds of the present invention are included within the scope of the present invention.

[0148] Furthermore, all compounds of the present invention that exist in the form of free bases or acids can be converted to pharmaceutically acceptable salts thereof by treatment with a suitable inorganic or organic base or inorganic or organic acid by methods known to those skilled in the art. Salts of the compounds of the present invention can be converted to the form of these free bases or free acids by standard techniques.

[0149] The following reaction scheme is for the compound of the present invention, i.e., the compound of formula (I): [ka] (In the formula, R 1a , R 1b , R 2a , R 2b , R 3a , R 3b , R 4a , R 4b , R 5 , R 6 , R 7 , R 8 , R 9 , L 1 , L 2 , G 1 , G 2 , G 3 (a, b, c, and d are as defined herein) Also exemplified is a method for preparing the compound or its pharmaceutically acceptable salt, tautomer or stereoisomer. It is understood by those skilled in the art that these compounds can be prepared in a similar manner or by combining other methods known to those skilled in the art. Those skilled in the art can prepare other compounds of formula (I) not specifically illustrated below in a similar manner as those described below by using appropriate starting components and modifying the synthetic parameters as necessary. Generally, the starting components may be obtained from suppliers such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI and Fluorochem USA, or may be synthesized according to information sources known to those skilled in the art (for example, see Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Edition (Wiley, December 2000)), or may be prepared as described in the present invention.

Chemical Formula

[0150] Embodiments of the compounds of structure (I) (e.g., Compound A-5 and A-7) generally follow reaction scheme 1 ("Method A") (wherein R 1a , R 1b , R 2a , R 2b , R 3a , R 3b , R <了 4a , R 4b , R 5 , R 6 , R 8 , R 9 , L 1 , L 2 , G 1 , G 2 , G 3 , a, b, c and d are as defined herein, and R 7’ is R 7 or C3-C 19It can be prepared according to alkyl. Referring to General Reaction Scheme 1, compounds of structures A-1 and A-2 can be purchased from commercial sources or prepared according to methods familiar to those skilled in the art. Solutions of A-1 and A-2 are treated with a reducing agent (e.g., sodium triacetoxyborohydride) to obtain A-3 after any necessary work-up. A solution of A-3 and a base (e.g., trimethylamine, DMAP) is treated with acyl chloride A-4 (or carboxylic acid and DCC) to obtain A-5 after any necessary work-up and / or purification. A-5 can be reduced with LiAlH4 A-6 to obtain A-7 after any necessary work-up and / or purification. [ka]

[0151] Embodiments of the compound of structure (I) (e.g., compound B-5) are shown in general reaction scheme 2 ("Method B") (wherein R 1a , R 1b , R 2a , R 2b , R 3a , R 3b , R 4a , R 4b , R 5 , R 6 , R 7 , R 8 , R 9 , L 1 , L 2 , G 3 , a, b, c and d are as defined herein) which can be prepared according to General Reaction Scheme 2. The compounds of structures B-1 and B-2 can be purchased from commercial sources or prepared according to methods familiar to those skilled in the art. A mixture of B-1 (in excess), B-2 and a base (e.g., potassium carbonate) is heated after any necessary work-up to obtain B-3. A solution of B-3 and a base (e.g., trimethylamine, DMAP) is treated with acyl B-4 chloride (or carboxylic acid and DCC) to obtain B-5 after any necessary work-up and / or purification. [ka]

[0152] Other embodiments of the compound of formula (I) (e.g., C-9) are prepared according to General Reaction Scheme 3. As illustrated in General Reaction Scheme 3, a well-protected ketone (C-1) is reacted under reductive amination conditions with amine C-2 to produce C-3. Acylation of C-3 with acid chloride C-4 produces the acylated product C-5. The alcohol protecting group on C-5 is removed and subsequently reacted with C-7 and / or C-8 and a suitable activating reagent (e.g., DCC) to produce the desired compound C-9.

[0153] The following examples are provided for illustrative purposes only and are not limiting. [Examples]

[0154] (Example 1) Synthesis of Compound 1 According to Method A, compound 1 was prepared from compound 5, yielding 240 mg of a colorless oily substance (0.32 mmol, 61%). ¹H NMR (400 MHz, CDCl₃) δ: 5.43–5.30 (m, 8H), 2.78 (t, 6.5 Hz, 4H), 2.39–2.25 (m, 7H), 2.22 (s, 6H), 2.06 (q, 6.8 Hz, 8H), 1.53 (quintuplet, 7.3 Hz, 2H), 1.41–1.11 (54H), 0.92–0.87 (m, 9H). (Example 2) Synthesis of Compound 2

[0155] Compound 2 was prepared according to Method A as follows: Compound 7 (0.84 g, 0.96 mmol) was dissolved in THF (15 mL), and LAH (2 equivalents, 1.92 mmol, 73 mg, MW 37.95) was added gradually at room temperature. The reaction mixture was heated overnight at 60 °C, and then sodium sulfate hydrate was added. The mixture was stirred for 2 hours and filtered through a layer of silica gel. The filtrate was concentrated to obtain a slightly yellowish oily substance (0.86 g). The crude product was purified by gravity column chromatography on silica gel (0-4% MeOH in chloroform). This yielded the desired product as a colorless oily substance (420 mg, 0.49 mmol, 51%). 1HNMR (400 MHz, CDCl3) δ: 5.43-5.30 (m, 12H), 2.78 (t, 6.4 Hz, 6H), 2.40-2.25 (m, 7H), 2.22 (s, 6H), 2.06 (q, 6.8 Hz, 12H), 1.53 (quintet, 7.3 Hz, 2H), 1.41-1.10 (58H), 0.90 (t, 6.8 Hz, 9H). (Example 3) Synthesis of Compound 3

[0156] According to Method A, compound 3 was prepared from compound 8 to produce 123 mg of a colorless oily substance (0.15 mmol, 41%). ¹H NMR (400 MHz, CDCl3) δ: 5.43–5.30 (m, 8H), 2.78 (t, 6.5 Hz, 4H), 2.35–2.24 (m, 5H), 2.22 (s, 6H), 2.15 (d, 5.5 Hz, 2H), 2.06 (q, 6.8 Hz, 8H), 1.52 (quintuplet, 7.3 Hz, 2H), 1.40–1.09 (65H), 0.92–0.87 (m, 12H). (Example 4) Synthesis of Compound 5 [ka] Compound 5 was prepared according to Method A as follows: Step 1

[0157] 3-Dimethylamine-1-propylamine (6 mmol, 612 mg) and ketone 5a (3.16 g, 6 mmol) were mixed in DCE (25 mL) and then treated with sodium triacetoxyborohydride (8.49 mmol, 1.8 g) and AcOH (6 mmol, 0.36 g, 0.340 mL). The mixture was stirred at room temperature under an Ar atmosphere for 2 days. The reaction mixture was quenched by adding 1N NaOH (approximately 20 mL), and the product was extracted with a mixture of hexane and ethyl acetate (approximately 5%). The organic extract was washed with water / brine (1:1), brine, and dried (Na2SO4). It was concentrated to obtain the desired product 5b as a yellow oily substance (3.55 g). The crude product was used in the next step without any further purification. Step 2

[0158] A solution of nonanoyl chloride (212 mg, 1.2 mmol) in benzene (10 mL) was added via syringe to a solution of compound 5b (600 mg, 0.978 mmol), triethylamine (5 mmol, 0.7 mL, 5 equivalents), and DMAP (20 mg) in benzene (10 mL) for 10 minutes at room temperature. After addition, the mixture was diluted with a mixture of hexane and ethyl acetate (approximately 5%), washed with water, washed with brine, dried over sodium sulfate, filtered, and concentrated. The crude product (0.77 g) was purified by gravity column chromatography on silica gel (230-400 mesh silica gel, 40 g, MeOH in chloroform, 0-4%). ​​This yielded the desired product 5 as a colorless oily substance (563 mg, 0.75 mmol, 76%). ¹H NMR (400 MHz, CDCl3) δ: 5.43-5.30 (m, 8H), 4.56-4.36 (br., 0.3H, due to slow isomerization around the amide bond), 3.64 (quintuplet, 7 Hz, 0.7H), 3.12-3.09 (m, 2H), 2.78 (t, 6.4 Hz, 4H), 2.33-2.25 (m, 4H), 2.23, 2.22 (two sets of monofilaments, 6H), 2.06 (q-like, 6.8 Hz, 8H), 1.76-1.66 (m, 4H), 1.50-1.40 (m, 4H), 1.40-1.15 (46H), 0.90 (t, 6.7 Hz, 6H), 0.88 (t, 6.8 Hz, 3H). (Example 5) Synthesis of Compound 6

[0159] Compound 6 was prepared according to general procedure A to produce 0.98 g of a slightly yellowish oily substance, 1.13 mmol, 58%. ¹H NMR (400 MHz, CDCl3) δ: 5.43–5.30 (m, 12H), 4.55–4.32 (br., 0.3H, due to slow isomerization around the amide bond), 3.63 (quintuplet-like, 7 Hz, 0.7H), 3.15–3.09 (m, 2H), 2.78 (t, 6.4 Hz, 6H), 2.33–2.25 (m, 4H), 2.22, 2.23 (two sets of singlelines, 6H), 2.06 (q-like, 6.8 Hz, 12H), 1.76–1.60 (m, 4H), 1.49–1.16 (54H), 0.90 (t-like, 6.8 Hz, 9H). (Example 6) Synthesis of Compound 7 Compound 7 was prepared according to Method A as follows:

[0160] To a solution of 2-ethylheptanoic acid (1.5 equivalents, 0.83 mmol, 130 mg) in benzene (6 mL) and DMF (5-10 μL), oxalyl chloride (5 equivalents, 2.8 mmol, 349 mg, 0.24 mL) was added at room temperature. The mixture was stirred at room temperature for 30 minutes, then heated under Ar at 60°C for 2 hours. The mixture was concentrated. The residue was dissolved in benzene (6 mL) and concentrated again to remove any oxalyl chloride. The remaining oily substance (pale yellow) was added to 4 mL of benzene and added via syringe to a solution of compound 5b (1 equivalent, 0.55 mmol, 337 mg), triethylamine (5 equivalents, 2.8 mmol, 283 mg, 390 μL), and DMAP (10 mg) in benzene (6 mL) for 10 minutes at room temperature. After addition, the resulting mixture was stirred overnight at room temperature. TLC showed little reaction. The reaction product was concentrated, thoroughly dried, and used as follows. The residue was dissolved in DCM (20 mL). DMAP (200 mg, 1.64 mmol) was added, followed by DCC (1.64 mmol, 338 mg). The mixture was stirred for 11 days and filtered. The filtrate was washed with 5% NaOH (100 mL). The organic phase was washed with brine and dried over sodium sulfate. A light brown oily substance was obtained by filtration and concentration (0.89 g). The crude product (0.89 g) was purified by column chromatography on silica gel (0-4% MeOH in chloroform). This yielded the desired product as a colorless oily substance (122 mg, 0.16 mmol, 29%). 11H NMR (400 MHz, CDCl3) δ: 5.43-5.30 (m, 8H), 4.69-4.51 (very broad, estimated 0.4H due to slow isomerization around the amide bond), 3.72 (quintuplet-like, 6.9 Hz, 0.6H), 3.19-3.09 (m, 2H), 2.78 (t, 6.4 Hz, 4H), 2.55 (quintuplet-like, 6.5 Hz, 0.5H), 2.42 (quintuplet-like, 6.5 Hz, 0.5H), 2.29 (q-like, but two overlapping triplets are also possible, 6.9 Hz, 2H), 2.24, 2.23 (two sets of monolines, integration ratio approximately 1:1, 6H) 2.09-2.02 (m, 8H), 1.77-1.58 (m, 4H), 1.55-1.15 (48H), 0.93-0.85 (m, 12H). (Example 7) Synthesis of compound 8

[0161] Compound 8 was prepared according to general procedure A to produce 0.39 g of a colorless oily substance, 0.46 mmol, 56%. ¹H NMR (400 MHz, CDCl3) δ: 5.43-5.30 (m, 8H), 4.55-4.32 (very broad, estimated 0.3H, due to slow isomerization around the amide bond), 3.71 (quintuplet-like, 7 Hz, 0.7H), 3.17-3.08 (m, 2H), 2.78 (t, 6.4 Hz, 4H), 2.59 (quintuplet-like, 6.5 Hz, 0.5H), 2.46 (quintuplet-like, 6.5 Hz, 0.5H), 2.40 (t, 7 Hz, 1H), 2.31 (t, 7 Hz, 1H), 2.28, 2.25 (two sets of singlelines, integration ratio approximately 1:1, 6H) 2.09-2.02 (m, 8H), 1.79-1.69 (m, 2H), 1.66-1.57 (m, 2H), 1.55-1.16 (62H), 0.92-0.86 (m, 12H). (Example 8) Synthesis of compound 9 [ka] Compound 9 was prepared according to Method A as follows: Step 1 3-dimethylamine-1-propylamine (1 equivalent, 1.3 mmol, 133 mg, 163 μL; MW102.18, d0.812) and ketone 9a (1 equivalent, 0.885 g, 1.3 mmol) were mixed in DCE (8 mL) and then treated with sodium triacetoxyborohydride (1.4 equivalents, 1.82 mmol, 386 mg; MW211.94) and AcOH (1 equivalent, 1.3 mmol, 78 mg, 74 μL; MW60.05, d1.06). The mixture was stirred at room temperature under an Ar atmosphere for 2 days. The reaction mixture was diluted with hexane-siRNA (9:1) and quenched by adding 0.1 N NaOH (20 mL). The organic phase was separated, washed with saturated NaHCO3 and brine, dried over sodium sulfate, decanted, and concentrated to obtain the desired product 9b as a slightly yellowish, cloudy oily substance (1.07 g, 1.398 mmol). Step 2

[0162] A solution of nonanoyl chloride (1.3 equivalents, 1.27 mmol, 225 mg) in benzene (10 mL) was added via syringe to a solution of compound 9b (0.75 g, 0.98 mmol), triethylamine (5 equivalents, 4.90 mmol, 0.68 mL), and DMAP (20 mg) in benzene (10 mL) from Step 1 for 10 minutes at room temperature. After addition, the mixture was stirred overnight at room temperature. Excess acyl chloride was removed by adding methanol (5.5 mL). After 3 hours, the mixture was filtered through a silica gel pad (1.2 cm). A colorless oily substance was obtained by concentration (0.70 g).

[0163] The crude product (0.70 g) was purified by flash-drying column chromatography on silica gel (0-4% MeOH in chloroform). This yielded 457 mg of a colorless oily substance, 0.50 mmol, 51%. ¹H NMR (400 MHz, CDCl3) δ: 4.54–4.36 (very broad, estimated 0.3H, due to slow isomerization around the amide bond), 3.977, 3.973 (two pairs of double lines, 5.8 Hz, 4H), 3.63 (similar to a quintuplet, 6.8 Hz, 0.7H), 3.14–3.09 (m, 2H), 2.33–2.25 (m, 8H), 2.23, 2.22 (two pairs of single lines, 6H), 1.76–1.56 (m, 10H), 1.49–1.39 (m, 4H), 1.37–1.11 (62H), 0.92–0.86 (m, 15H). (Example 9) Synthesis of compound 10

[0164] Compound 10 was prepared according to general procedure A to produce 245 mg of a colorless oily substance (0.27 mmol, 53% overall yield for 2 steps). 1 1H NMR (400 MHz, CDCl3) δ: 4.87 (quintuplet-like, 6.3 Hz, 2H), 4.54–4.36 (very broad, estimated 0.3H, due to slow isomerization around the amide bond), 3.63 (quintuplet-like, 6.8 Hz, 0.7H), 3.14–3.09 (m, 2H), 2.33–2.25 (m, 8H), 2.23, 2.22 (two sets of single lines, 6H), 1.76–1.56 (m, 8H), 1.55–1.39 (m, 12H), 1.37–1.11 (60H), 0.92–0.86 (m, 15H). (Example 10) Synthesis of compound 11

[0165] Compound 11 was prepared according to general procedure A to produce 239 mg of a colorless oily substance (0.26 mmol, 52% overall yield for 2 steps). 11H NMR (400 MHz, CDCl3) δ: 4.87 (quintuplet-like, 6.3 Hz, 2H), 4.54–4.36 (very broad, estimated 0.3H, due to slow isomerization around the amide bond), 3.63 (quintuplet-like, 6.8 Hz, 0.7H), 3.14–3.09 (m, 2H), 2.33–2.25 (m, 8H), 2.23, 2.22 (two sets of single lines, 6H), 1.76–1.56 (m, 8H), 1.55–1.39 (m, 12H), 1.37–1.11 (62H), 0.92–0.86 (m, 15H). (Example 11) Synthesis of compound 12

[0166] Compound 12 was prepared according to general procedure A to produce 198 mg of a colorless oily substance (0.20 mmol, 46% total yield for 2 steps). 1 1H NMR (400 MHz, CDCl3) δ: 4.54–4.36 (very broad, estimated 0.3H, due to slow isomerization around the amide bond), 3.974, 3.971 (two pairs of double lines, 5.8 Hz, 4H), 3.63 (similar to a quintuplet, 6.8 Hz, 0.7H), 3.14–3.09 (m, 2H), 2.33–2.25 (m, 8H), 2.23, 2.22 (two pairs of single lines, 6H), 1.76–1.56 (m, 10H), 1.49–1.39 (m, 4H), 1.37–1.11 (76H), 0.92–0.86 (m, 15H). (Example 12) Synthesis of compound 13

[0167] Compound 13 was prepared according to general procedure A to produce 217 mg of a colorless oily substance (0.21 mmol, 49% overall yield for 2 steps). 11H NMR (400 MHz, CDCl3) δ: 4.54–4.36 (very broad, estimated 0.3H, due to slow isomerization around the amide bond), 3.973, 3.970 (two pairs of double lines, 5.8 Hz, 4H), 3.63 (similar to a quintuplet, 6.8 Hz, 0.7H), 3.14–3.09 (m, 2H), 2.33–2.25 (m, 8H), 2.23, 2.22 (two pairs of single lines, 6H), 1.76–1.56 (m, 10H), 1.49–1.39 (m, 4H), 1.37–1.11 (78H), 0.92–0.86 (m, 15H). (Example 13) Synthesis of compound 14

[0168] Compound 14 was prepared according to general procedure A to produce 263 mg of a colorless oily substance (0.29 mmol, 39% total yield for 2 steps). 1 1H NMR (400 MHz, CDCl3) δ: 4.54-4.36 (br., estimated 0.3H, due to slow isomerization around the amide bond), 3.977, 3.973 (two pairs of double lines, 5.8 Hz, 4H), 3.63 (similar to a quintuplet line, 6.8 Hz, 0.7H), 3.17-3.10 (m, 2H), 2.53-2.43 (m, 6H), 2.34-2.26 (m, 6H), 1.83-1.71 (m, 6H), 1.70-1.57 (m, 8H), 1.49-1.38 (m, 4H), 1.37-1.11 (60H), 0.92-0.86 (m, 15H). (Example 14) Synthesis of compound 15

[0169] Compound 15 was prepared according to general procedure A to produce 234 mg of a colorless oily substance (0.25 mmol, 34% total yield for 2 steps). 11H NMR (400 MHz, CDCl3) δ: 4.54-4.36 (br., estimated 0.3H, due to slow isomerization around the amide bond), 3.977, 3.973 (two pairs of double lines, 5.8 Hz, 4H), 3.63 (similar to a quintuplet line, 6.8 Hz, 0.7H), 3.17-3.10 (m, 2H), 2.53-2.43 (m, 6H), 2.34-2.26 (m, 6H), 1.83-1.71 (m, 6H), 1.70-1.57 (m, 8H), 1.49-1.38 (m, 4H), 1.37-1.11 (62H), 0.92-0.86 (m, 15H). (Example 15) Synthesis of compound 16 [ka] Compound 16 was prepared according to Method B as follows:

[0170] To a solution of acid 018-19 (0.5 g, 0.90 mmol), N-hydroxysuccinimide (1.2 equivalents, 1.08 mmol, 124 mg), and DMAP (0.3 equivalents, 0.27 mmol, 33 mg) in DCM (20 mL), DCC (2 equivalents, 1.8 mmol, 371 mg) was added. The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was then filtered and added to a solution of amine 021-24 (1.26 mmol, 288 mg) in DCM (10 mL) and triethylamine (5 mmol, 696 μL). After 15 days, the mixture was concentrated. The residue was dissolved in hexane / ethyl acetate / Et3N (approximately 9:1:0.3), filtered through a small silica gel pad, and washed with a mixture of hexane / ethyl acetate / Et3N (approximately 9:1:0.3). The filtrate was concentrated to obtain a yellow oily substance (580 mg). The yellow oily substance was purified by column chromatography on silica gel (eluted with a gradient mixture of MeOH in chloroform, 0-4.2%). This yielded the desired product as a colorless oily substance (102 mg, 0.13 mmol, 14%). 1HNMR (400 MHz, CDCl3) δ: 5.43-5.30 (m, 8H), 3.38-3.29 (m, 3H), 3.28-3.23 (m, 1H), 2.78 (t, 6.4 Hz, 4H), 2.56-2.47 (m, 1H), 2.30-2.24 (m, 2H), 2.23, 2.22 (two pairs of singlets, 6H), 2.09-2.02 (m, 8H), 1.71 (quintet-like, 7.4 Hz, 2H), 1.66-1.48 (overlapping with water; estimated 4H), 1.47-1.18 (m, 50H), 0.92-0.86 (m, 9H). (Example 16) Synthesis of Compound 24

[0171] Compound 24 was prepared according to general procedure A to produce 279 mg of a slightly yellowish oily substance (0.29 mmol, 44% total yield for 2 steps). 1 HNMR (400 MHz, CDCl3) δ: 4.88 (quintet-like, 6.3 Hz, 3H), 3.62 (quintet-like, 6.8 Hz, 1H), 3.14-3.08 (m, 2H), 2.33-2.25 (m, 10H), 2.23, 2.22 (2 sets of singlets, 6H), 1.76-1.58 (m, 10H), 1.52 (q-like, 6.7 Hz, 12H), 1.49-1.39 (m, 4H), 1.38-1.14 (50H), 0.89 (t-like, 18H). (Example 17) Synthesis of Compound 35

[0172] Compound 35 was prepared according to general procedure A to produce 260 mg of a slightly yellowish oily substance (0.29 mmol, 33% total yield for 2 steps). 11H NMR (400 MHz, CDCl3) δ: 4.66–4.52 (very broad, estimated 0.3H, due to slow isomerization around the amide bond), 3.977, 3.973 (two sets of double lines, 5.8 Hz, 4H), 3.71 (quintuplet-like, 6.8 Hz, 0.7H), 3.19–3.09 (m, 2H), 2.54, 2.42 (two sets of quintuplet-like, 6.8 Hz, integral ratio approximately 1:1.2, 1H), 2.33–2.25 (m, 6H), 2.24, 2.22 (two sets of single lines, 6H), 1.77–1.11 (74H), 0.93–0.85 (m, 18H). (Example 18) Synthesis of Compound 17 [ka] Compound 17 was prepared according to Method C as follows: Step 1

[0173] 3-Dimethylamino-1-propylamine (4.14 mmol / 1 equivalent, 423 mg, 521 μL) and ketone 17a (2.0 g / 1 equivalent, 4.14 mmol) were mixed in DCE (30 mL), and then treated with sodium triacetoxyborohydride (1.4 equivalents, 5.80 mmol, 1.229 g) and AcOH (4.14 mmol / 1 equivalent, 249 mg, 235 μL). The mixture was stirred at room temperature under an Ar atmosphere for 2 days.

[0174] The reaction mixture was diluted with a mixture of hexane and siRNA (9:1, 200 mL) and quenched by adding a diluted solution of NaOH (0.1 N, 270 mL). The two phases were separated. The organic phase was washed with saturated NaHCO3 and brine, dried over sodium sulfate, and filtered through a silica gel pad. The pad was washed with 200 mL of a mixture of hexane and siRNA (9:1). The pad was then washed with 200 mL of a mixture of DCM / MeOH / Et3N (85:15:1). By concentrating the DCM / MeOH / Et3N washing solution, the desired product (17b) was obtained as a colorless oily substance (1.749 g, 3.07 mmol, 74%). Step 2

[0175] A solution of nonanoyl chloride (0.333 mL) in benzene (10 mL) was added at room temperature to a solution of compound 17b (0.75 g), triethylamine (0.92 mL), and DMAP (20 mg) in benzene (20 mL). The mixture was stirred overnight at room temperature. MeOH (1 mL) was added, and the mixture was stirred continuously for 2 hours. The reaction mixture was filtered through a silica gel pad. By concentration of the filtrate, the desired product (17c) was obtained as a yellow oily substance (0.945 g). Step 3

[0176] To a flask containing 17c (0.945 g, 1.33 mmol) and EtOH (25 mL), p-toluenesulfonic acid hydrate (1.33 mmol, 253 mg) was added at room temperature. The resulting mixture was stirred overnight at room temperature. The reaction mixture was heated at 85 °C for 2 hours. Further PTSA (160 mg) was added, and the reaction mixture was heated at 75 °C overnight. The mixture was concentrated. The residue was dissolved in DCM and washed with a diluted NH4OH solution. The organic phase was washed with a mixture of saturated sodium bicarbonate and brine and dried over sodium sulfate. Concentration yielded the desired product (17d) as a slightly yellowish viscous oily substance (0.799 g, 1.47 mmol). The crude product was purified by silica gel column chromatography (0-15% methanol and trace amounts of triethylamine in DCM). This yielded 17d as a colorless oily substance (647 mg, 1.20 mmol, 90%). Step 4

[0177] To a solution of 17d (216 mg, 0.40 mmol), 2-butyloctanoic acid (5 equivalents, 2 mmol, 401 mg), and 4-dimethylaminopyridine (DMAP) (5.5 equivalents, 2.2 mmol, 269 mg) in dichloromethane (20 mL), DCC (5.5 equivalents, 2.2 mmol, 454 mg) was added. After stirring for 4 days, 3 mL of MeOH was added. The mixture was stirred for another 16 hours. The mixture was filtered, and the filtrate was concentrated to dryness. The crude product was purified by gravity column chromatography on silica gel (MeOH in DCM, 0-6%). This yielded the desired compound (17) as a slightly yellowish oily substance (colorless oily substance, 175 mg, 0.19 mmol, 48%). 1 HNMR (400 MHz, CDCl3) δ: 4.07, 4.06 (2 sets of triplet, 6.7 Hz, 4H), 3.64 (quintet-like, 6.8 Hz, 1H), 3.21-3.09 (2 sets of multiplet, 2H), 3.00-2.37 (br. 6H), 2.36-2.20 (m, 6H), 2.05-1.85 (m, 2H), 1.79-1.53 ​​(m, 10H), 1.52-1.39 (m, 8H), 1.37-1.03 (58H), 0.91-0.86 (m, 15H). (Example 19) Synthesis of Compound 36

[0178] Compound 36 was prepared according to general procedure C to produce 156 mg of a colorless oily substance (0.15 mmol, 38% of the final step). 1 HNMR (400 MHz, CDCl3) δ: 4.07 (triplet, 6.7 Hz, 4H), 3.65 (quintet-like, 6.8 Hz, 1H), 3.21 (t-like, 6.8 Hz, 2H), 3.10-3.03 (br. 2H), 2.79, 2.78 (2 pairs of singlets, 6H), 2.35-2.28 (m, 4H), 2.09 (quintet-like, 7.5 Hz, 2H), 1.67-1.54 (m, 10H), 1.54-1.38 (m, 8H), 1.38-1.03 (74H), 0.91-0.86 (m, 15H). (Example 20) Synthesis of Compound 37

[0179] Compound 37 was prepared according to general procedure A to produce 397 mg of a colorless oily substance (0.49 mmol, 60% overall yield for 2 steps). 1 HNMR (400 MHz, CDCl3) δ: 5.43-5.30 (m, 8H), 4.13 (q, 7.1 Hz, 2H), 4.56-4.34 (br. 0.3H), 3.63 (quintet-like, 6.9 Hz, 0.7H), 3.15-3.08 (m, 2H), 2.78 (t similar, 6.4 Hz, 4H), 2.39-2.21 (m, 12H), 2.06 (q similar, 6.9 Hz, 8H), 1.79-1.55 (m, 6H), 1.50-1.40 (m, 4H), 1.40-1.15 (m, 45H), 0.90 (t similar, 6.8 Hz, 6H). (Example 21) Synthesis of compound 38 Compound 38 was prepared according to Method A as follows: [ka] Step 1

[0180] To a solution of 38a (1 equivalent, 1.266 g, 1.79 mmol) in DCE (15 mL), 3-dimethylamino-1-propylamine (1 equivalent, 1.79 mmol, 183 mg, 225 μL) was added, followed by sodium triacetoxyborohydride (1.4 equivalents, 2.51 mmol, 531 mg) and AcOH (1 equivalent, 1.79 mmol, 107 mg, 101 μL). The mixture was stirred at room temperature under an Ar atmosphere for 3 days.

[0181] The residue was diluted with hexane-siRNA (9:1, 150 mL), washed with diluted NaOH solution (0.12 N, 100 mL), saturated NaHCO3, and brine, and dried over sodium sulfate. The organic phase was filtered through a silica gel pad. The pad was washed with 200 mL of a mixture of hexane and siRNA (9:1). The pad was then washed with 200 mL of a mixture of DCM / MeOH / Et3N (85:15:1). The DCM / MeOH / Et3N washing solution was concentrated and dried in a high vacuum line to obtain the desired product (38b) as a colorless oily substance (1.1 g, 1.38 mmol, 77%). Step 2

[0182] A solution of nonanoyl chloride (1.5 equivalents, 0.68 mmol, 120 mg) in benzene (5 mL) was added to a solution of 38b (0.45 mmol, 360 mg), triethylamine (5 equivalents, 2.25 mmol, 228 mg, 314 μL), and DMAP (10 mg) in benzene (10 mL) under Ar for 2 minutes at room temperature. After addition, the mixture was stirred overnight at room temperature. MeOH (1 mL) was added, and the mixture was continuously stirred for 2 hours. The crude product was filtered through a silica gel pad. The filtrate was concentrated. The residue (457 mg) was purified by flash column chromatography on silica gel (230-400 mesh silica gel, 40 g, MeOH in chloroform, 0-4.6%). This yielded the desired product (38) as a colorless oily substance (410 mg, 0.44 mmol, 98%). 1 1H NMR (400 MHz, CDCl3) δ: 4.61–4.35 (br., estimated 0.4H, due to slow isomerization around the amide bond), 3.974, 3.964 (two pairs of double lines, 5.7 Hz, 4H), 3.64 (similar to a quintuplet line, 7.0 Hz, 0.6H), 3.14–3.08 (m, 2H), 2.34–2.25 (m, 8H), 2.23 (broad s, 6H), 1.77–1.58 (m, 10H), 1.53–1.39 (m, 4H), 1.37–1.15 (66H), 0.92–0.86 (m, 15H). (Example 22) Synthesis of compound 39

[0183] Compound 39 was prepared according to general procedure A to produce 370 mg of a colorless oily substance (0.40 mmol, 69% overall yield for 2 steps). 1 1H NMR (400 MHz, CDCl3) δ: 4.61–4.35 (br., estimated 0.4H, due to slow isomerization around the amide bond), 3.974, 3.964 (two pairs of double lines, 5.7 Hz, 4H), 3.64 (similar to a quintuplet, 7.0 Hz, 0.6H), 3.14–3.08 (m, 2H), 2.34–2.25 (m, 8H), 2.230, 2.221 (two pairs of single lines, 6H), 1.75–1.58 (m, 10H), 1.51–1.39 (m, 4H), 1.37–1.15 (64H), 0.92–0.86 (m, 15H). (Example 23) Synthesis of compound 40

[0184] Compound 40 was prepared according to general procedure A to produce 382 mg of a colorless oily substance (0.39 mmol, 68% overall yield for 2 steps). 1 1HNMR (400 MHz, CDCl3) δ: 4.60-4.35 (br., estimated 0.3H, due to slow isomerization around the amide bond), 4.13 (q, 7.2 Hz, 2H), 3.973, 3.964 (two pairs of double lines, 5.7 Hz, 4H), 3.63 (quintuplet-like, 7.0 Hz, 0.7H), 3.14-3.08 (m, 2H), 2.34-2.25 (m, 10H), 2.229, 2.220 (two pairs of single lines, 6H), 1.75-1.58 (m, 12H), 1.51-1.39 (m, 4H), 1.37-1.15 (64H), 0.89 (t-like, 7.8 Hz, 12H). (Example 24) Synthesis of compound 41

[0185] Compound 41 was prepared according to general procedure A to produce 309 mg of a colorless oily substance (0.30 mmol, 73% overall yield for 2 steps).1 1H NMR (400 MHz, CDCl3) δ: 4.60–4.35 (br., estimated 0.3H, due to slow isomerization around the amide bond), 3.972, 3.962 (two pairs of double lines, 5.7 Hz, 4H), 3.64 (similar to a quintuplet, 7.1 Hz, 0.7H), 3.14–3.08 (m, 2H), 2.34–2.25 (m, 8H), 2.23, 2.22 (two pairs of single lines, 6H), 1.75–1.58 (m, 10H), 1.51–1.39 (m, 4H), 1.35–1.21 (82H), 0.92–0.86 (m, 15H). (Example 25) Synthesis of compound 42

[0186] Compound 42 was prepared according to general procedure A to produce 235 mg of a colorless oily substance (0.23 mmol, 56% overall yield for 2 steps). 1 1H NMR (400 MHz, CDCl3) δ: 4.75-4.49 (br., estimated 0.4H, due to slow isomerization around the amide bond), 3.97, 3.96 (two pairs of double lines, 5.3 Hz, 4H), 3.72 (quintuplet-like, 7 Hz, 0.6H), 3.21-3.05 (m, 2H), 2.53, 2.42 (two pairs of quintuplet-like, 6.6 Hz, integral ratio approximately 1:1.7, 1H), 2.32-2.25 (m, 6H), 2.24, 2.22 (two pairs of single lines, 6H), 1.78-1.56 (m, 10H), 1.53-1.39 (m, 6H), 1.38-1.17 (76H), 0.93-0.85 (m, 18H). (Example 26) Synthesis of compound 43

[0187] Compound 43 was prepared according to general procedure C to produce 187 mg of a colorless oily substance (0.23 mmol, 57% of the final step). 1HNMR (400 MHz, CDCl3) δ: 4.077, 4.071 (2 sets of triplet, 6.7 Hz, 4H), 4.56-4.34 (br. 0.3H), 3.64 (quintet analog, 6.9 Hz, 0.7H), 3.15-3.09 (m, 2H), 2.34-2.24 (m, 6H), 2.234-2.224 (2 sets of singlets, 6H), 1.76-1.58 (m, 10H), 1.55-1.39 (m, 8H), 1.39-1.10 (48H), 0.92-0.86 (m, 15H). (Example 27) Synthesis of compound 44

[0188] Compound 44 was prepared according to general procedure A to produce 260 mg of a colorless oily substance (0.22 mmol, 53% overall yield for 2 steps). 1 1H NMR (400 MHz, CDCl3) δ: 4.59–4.35 (br., estimated 0.3H, due to slow isomerization around the amide bond), 4.03–3.95 (m, 6H), 3.63 (quintuplet-like, 6.9 Hz, 0.7H), 3.14–3.08 (m, 2H), 2.33–2.24 (m, 10H), 2.229, 2.221 (two sets of singlelines, 6H), 1.75–1.57 (m, 12H), 1.51–1.40 (m, 4H), 1.40–1.08 (87H), 0.92–0.86 (m, 18H). (Example 28) In vivo evaluation of luciferase mRNA using lipid nanoparticle compositions

[0189] Cationic lipids (MC3), DSPC, cholesterol, and PEG-lipids were solubilized in ethanol in a molar ratio of 50:10:38.5:1.5. Lipid nanoparticles (LNPs) were prepared with a weight ratio of total lipids to mRNA of approximately 10:1 to 30:1. Briefly, mRNA was diluted to 0.2 mg / mL in 10-50 mM citrate buffer at pH 4. Using a syringe pump, the ethanol-based lipid solution and the mRNA aqueous solution were mixed at a total flow rate of over 15 ml / min in a ratio of approximately 1:5 to 1:3 (vol / vol). The ethanol was then removed, and the external buffer was replaced with PBS by dialysis. Finally, the lipid nanoparticles were filtered through a sterile filter with a 0.2 μm pore size. The particle size of the lipid nanoparticles was determined to be 70-90 nm in diameter by quasi-elastic light scattering using a Nicomp 370 submicron particle size analyzer (Santa Barbara, CA).

[0190] The study was conducted in 6-8 week old female C57BL / 6 mice (Charles River) in accordance with 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 via tail vein injection, and the animals were euthanized at specific time points after administration (1, 2, 4, 8, and 24 hours). 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.

[0191] 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 solubilizer-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. The homogenate was incubated at room temperature for 5 minutes, then diluted 1:4 in GLB and evaluated using the SteadyGlo Luciferase assay system (Promega). Specifically, 50 μL of diluted tissue homogenate was reacted with 50 μL of SteadyGlo substrate, shaken for 10 seconds, followed by a 5-minute incubation, 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 (ug). A calibration curve was created using QuantiLum Recombinant Luciferase (Promega) to convert RLU to luciferase (ng). Based on the data provided in Figure 1, the 4-hour time point was selected for evaluating the efficacy of the lipid formulation (see Example 29).

[0192] FLuc mRNA (L-6107) 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. (Example 29) In vivo efficacy testing of lipid nanoparticle formulations containing various cationic lipids using a rodent model expressing luciferase mRNA.

[0193] The cationic lipids shown in Table 2 were previously tested using nucleic acids. For comparative purposes, lipid nanoparticles containing FLuc mRNA (L-6107) were also formulated using these lipids, employing a strain mixing method as described in Example 28 and PCT / US10 / 22614, the entirety of which is incorporated herein by reference. The lipid nanoparticles were 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, average PEG molecular weight 2000). Relative activity was determined by measuring luciferase expression in the liver 4 hours after administration via tail vein injection as described in Example 28. 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 28. [Table 2-1] [Table 2-2]

[0194] The novel lipids of the present invention and the selected comparative lipids shown in Table 3 are used in the following molar ratio: 50% cationic lipid / 10% distearoyl phosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid ("PEG-DMA") 2-[2-(ω-methoxy(polyethylene glycol) 2000The 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 28. Activity was compared at doses of 0.3 and 1.0 mg mRNA / kg and expressed as luciferase (ng) / liver (g) measured 4 hours after administration as described in Example 28. The plot of selected data is shown in Figure 3 (from top to bottom: circle = compound 10; triangle = compound 6; square = MC3).

[0195] [Table 3-1]

[0196] Table 3 continued [Table 3-2]

[0197] Table 3 continued [Table 3-3]

[0198] Table 8 continued [Table 3-4]

[0199] (Example 30) Determination of the pKa of formulated lipids

[0200] As described elsewhere, the pKa of formulated cationic lipids correlates with the efficacy of LNPs for nucleic acid delivery (see Jayaraman et al., Angewandte Chemie, International Edition (2012), Vol. 51 (No. 34), pp. 8529-8533; Semple et al., Nature Biotechnology, Vol. 28, pp. 172-176 (2010)). The preferred range for pKa is approximately 5 to 7. Using fluorescence-based assays of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS), the pKa of each cationic lipid is determined. a The properties were determined in lipid nanoparticles. Lipid nanoparticles containing cationic lipids / DSPC / cholesterol / PEG-lipids (50 / 10 / 38.5 / 1.5 mol%) at a total lipid concentration of 0.4 mM were prepared in PBS using an in-line process as described in Example 28. TNS was prepared as a 100 μM stock solution in distilled water. Vesicles were diluted to 24 μM lipids in 2 mL of buffer containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl (the pH range is 2.5 to 11). Aliquots of the TNS solution were 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. Sigmoid best-fit analysis was applied to the fluorescence data, and the pK was defined as the pH that elicits the maximum half of the fluorescence intensity. a The following measurements were taken (see Figure 2).

[0201] The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referenced herein and / or listed in the application datasheet, including U.S. Provisional Patent Application No. 62 / 186,210 filed June 29, 2015, are incorporated herein by reference in their entirety. The aspects of the embodiments can be modified as necessary to utilize the concepts of various patents, applications, and publications 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 rights. Thus, the claims are not limited by this disclosure.

Claims

1. Formula I: 【Chemistry 1】 (In the formula, L １ and L ２ These are independently -O(C=O)-, -(C=O)O-, or direct bonds, G １ and G ２ Each is independent and a direct connection, G ３ is C １ ~C ６ It is alkylene, R １ａ and R １ｂ each occur independently and are (a) H or C １ -C １２ alkyl or (b) R １ａ is H or C １ -C １２ alkyl, and the carbon atom to which R １ｂ is attached together with the carbon atom to which it is attached and the adjacent R １ｂ forms a carbon-carbon double bond, R ２ａ and R ２ｂ Each instance, independently, (a) H or C １ ~C １２ (b) Alkyl or R ２ａ is H or C １ ~C １２ It is alkyl, R ２ｂ And the carbon atom to which it is bonded is adjacent to R ２ｂ And together with the carbon atom it is bonded to, it forms a carbon-carbon double bond. R ３ａ and R ３ｂ Each instance, independently, (a) H or C １ ~C １２ (b) Alkyl or R ３ａ is H or C １ ~C １２ It is alkyl, R ３ｂ And the carbon atom to which it is bonded is adjacent to R ３ｂ And together with the carbon atom it is bonded to, it forms a carbon-carbon double bond. R ４ａ and R ４ｂ Each instance, independently, (a) H or C １ ~C １２ (b) Alkyl or R ４ａ is H or C １ ~C １２ It is alkyl, R ４ｂ And the carbon atom to which it is bonded is adjacent to R ４ｂ And together with the carbon atom it is bonded to, it forms a carbon-carbon double bond. R ５ and R ６ These are, independently, H or methyl, R ７ is C 9 to C ２０ It is alkyl, R ８ and R ９ Each of them is independent of C １ ~C １２ Alkyl or R ８ and R ９ These, together with the nitrogen atoms to which they are bonded, form a 5, 6, or 7-membered heterocycle. a, b, c, and d are each independent integers between 6 and 9. Each of the alkyl group, alkylene group, and heterocycle is unsubstituted. A compound having the structure of the above, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

2. L １ However, ~O(C=O)-, and here ~ is R １ａ and R １ｂ It is a covalent bond with a carbon atom having L ２ However, ~O(C=O)-, and here ~ is R ４ａ and R ４ｂ The compound according to claim 1, wherein it is covalently bonded with a carbon atom having [a specific characteristic].

3. L １ However, ~(C=O)O-, where ~ is R １ａ and R １ｂ It is a covalent bond with a carbon atom having L ２ However, ~(C=O)O-, where ~ is R ４ａ and R ４ｂ The compound according to claim 1, wherein it is covalently bonded with a carbon atom having [a specific characteristic].

4. L １ and L ２ The compound according to claim 1, wherein each of them is a direct bond.

5. R １ａ and R １ｂ For at least one occurrence of R １ａ is H or C １ ~C １２ It is alkyl, R １ｂ And the carbon atom to which it is bonded is adjacent to R １ｂ The compound according to any one of claims 1 to 4, wherein it and the carbon atom to which it is bonded form a carbon-carbon double bond.

6. For the occurrence of at least one of R 4a and R 4b, R ４ａ is H or C １ ~C １２ It is alkyl, R ４ｂ And the carbon atom to which it is bonded is adjacent to R ４ｂ The compound according to any one of claims 1 to 5, wherein it, together with the carbon atom to which it is bonded, forms a carbon-carbon double bond.

7. R １ａ , R ２ａ , R ３ａ and R ４ａ At least one of them is C １ ~C ８ A compound according to any one of claims 1 to 6, wherein it is alkyl.

8. C １ ~C ８ The compound according to claim 7, wherein the alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

9. R ５ and R ６ The compound according to any one of claims 1 to 8, wherein each of them is methyl.

10. R ８ and R ９ The compound according to any one of claims 1 to 9, wherein each of them is methyl.

11. R ８ and R ９ The compound according to any one of claims 1 to 9, wherein these combine with the nitrogen atom to which they are bonded to form a 5, 6, or 7-membered heterocycle.

12. The compound according to claim 11, wherein the heterocycle is pyrrolidinyl or piperazinyl.

13. G ３ C ２ ~C ４ The compound according to any one of claims 1 to 12, wherein it is an alkylene.

14. G ３ is C ３ The compound according to any one of claims 1 to 11, wherein the alkylene is C.

15. Below formula: 【Chemistry 2】 、 【Transformation 3】 ,or 【Chemistry 4】 A compound selected from the above, according to claim 1.

16. A composition comprising a compound according to any one of claims 1 to 15 and a therapeutic agent.

17. The composition according to claim 16, wherein the therapeutic agent comprises nucleic acid.

18. The composition according to claim 17, wherein the nucleic acid is selected from antisense and messenger RNA.

19. Use of a compound according to any one of claims 1 to 15, or a composition according to any one of claims 16 to 18, in the manufacture of a pharmaceutical product for administering a therapeutic agent to a patient requiring administration.

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