Lipids for use in lipid nanoparticles

Abstract

The following formula (I): JPEG2026521723000074.jpg4535〔Wherein, R 1 , R 2 , R 3 , G 1 , G 2 , L 1 , and L 2 are as defined herein.〕 There is provided a compound having or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. Also provided are uses of the compound as a formulation of a lipid nanoparticle formulation for the delivery of therapeutic agents, compositions containing the compound and their uses and methods of manufacture.

Description

[Technical Field]

[0001] background Technical field The present invention relates to novel lipid compounds that can be used in combination with other lipid components such as neutral lipids, cholesterol, and polymer-conjugated lipids to form lipid nanoparticles with oligonucleotides to promote intracellular delivery of therapeutic nucleic acids (e.g., oligonucleotides, messenger RNA) both in vitro and in vivo. [Background technology]

[0002] Description of related technologies Many challenges exist in the delivery of nucleic acids to act on desired responses in biological systems. While nucleic acid-based therapeutics hold enormous potential, realizing this potential still requires more effective delivery of nucleic acids to the appropriate sites within cells or organisms. Therapeutic nucleic acids include, for example, messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, immunostimulatory nucleic acids, antagonists, antimyls, mimes, supermyls, and aptamers. Some nucleic acids, such as mRNA or plasmids, can be used to induce the expression of specific cellular products, for example, to be useful in treating diseases associated with protein or enzyme deficiencies. Therapeutic applications of translatable nucleotide delivery are extremely broad, as constructs can be synthesized for the production of any selected protein sequence, whether innate in the system or not. 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 into cells or organisms.

[0003] Certain nucleic acids, such as miRNA inhibitors, can be used to induce the expression of miRNA-regulated specific cellular products, for example, to treat diseases associated with protein or enzyme deficiencies. The therapeutic applications of miRNA inhibition are extremely broad, as constructs can be synthesized to inhibit one or more miRNAs, thereby controlling the expression of mRNA products. Inhibition of endogenous miRNAs can increase the expression of their downstream target endogenous proteins, restoring proper cellular or biological 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 the corresponding proteins through processes such as RNA interference (RNAi) or complementary binding of antisense RNA. The therapeutic applications of antisense oligonucleotides and RNAi are also extremely broad, as oligonucleotide constructs can be synthesized with any nucleotide sequence relative to the target mRNA. Targets can include mRNA from normal cells, mRNA associated with disease conditions 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. Furthermore, antisense oligonucleotide constructs are currently being evaluated in clinical trials.

[0005] However, there are currently two problems when using oligonucleotides in therapeutic settings. First, free RNA is sensitive to nuclease digestion in plasma. Second, free RNA has a limited ability to access intracellular compartments where the relevant translation mechanisms reside. Lipid nanoparticles formed from cationic lipids and other lipid components such as neutral lipids, cholesterol, PEG, PEGylated lipids, and oligonucleotides are being used to block RNA degradation in plasma and promote cellular uptake of oligonucleotides. [Overview of the project] [Problems that the invention aims to solve]

[0006] Improvements to cationic lipids and lipid nanoparticles remain necessary for oligonucleotide delivery. 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. Furthermore, these lipid-nucleic acid particles must be well-tolerated and provide an appropriate therapeutic index so that patient treatment with an effective dose of nucleic acid is not associated with unacceptable toxicity and / or risk to the patient. The present invention provides these and related advantages. [Means for solving the problem]

[0007] overview In short, the present invention provides lipid compounds (including their stereoisomers, pharmaceutically acceptable salts, or tautomers) that can be used alone or in combination with other lipid components such as neutral lipids, charged lipids, steroids (e.g., all sterols) and / or their analogues, and / or polymer-conjugated lipids, to form lipid nanoparticles for therapeutic agent delivery. In some cases, the lipid nanoparticles are used for the delivery of 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 agents and / or protein deficiencies, are also provided.

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

[0009] A pharmaceutical composition is also provided, comprising one or more compounds of formula (I) above and a therapeutic agent. In one embodiment, the pharmaceutical composition further comprises one or more components selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. Such a composition is useful for forming lipid nanoparticles for therapeutic agent delivery.

[0010] In another embodiment, the present invention provides a method for administering a therapeutic agent to a patient in need of treatment, comprising preparing a composition of lipid nanoparticles containing a compound of formula (I) and the therapeutic agent, and delivering the composition to the patient. Such a method is useful, for example, for inducing protein expression in a target to express an antigen or gene-editing protein for the purpose of vaccination.

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

[0012] Detailed description In the following description, certain specific details are provided to provide a complete understanding of various embodiments of the present invention. However, it will be understood by those skilled in the art that embodiments of the present invention can be carried out without these details.

[0013] The present invention is based in part on the discovery of novel cationic lipids that offer advantages when used in lipid nanoparticles for in vivo delivery of activators or therapeutic agents, such as nucleic acids, to mammalian cells. Embodiments of the present invention provide nucleic acid-lipid nanoparticle compositions comprising one or more of the novel cationic lipids described herein, which increase nucleic acid activity in vivo and improve the tolerability of the composition, resulting in a significantly increased therapeutic index compared to previously described nucleic acid-lipid nanoparticle compositions.

[0014] In one embodiment, the present invention provides novel cationic lipids that enable formulations of improved compositions for in vitro and in vivo delivery of mRNA and / or other oligonucleotides. In one embodiment, improvements to these lipid nanoparticle compositions are useful for the expression of mRNA-encoded proteins. In another embodiment, improvements to these lipid nanoparticle compositions are useful for upregulating endogenous protein expression by delivery of a single lipid nanoparticle composition or a group of miRNAs that control one or several target mRNAs. In yet another embodiment, improvements to these lipid nanoparticle compositions are useful for downregulation (e.g., silencing) at the protein level and / or the mRNA level of a target gene. In yet another embodiment, lipid nanoparticles are also useful for the delivery of mRNA and plasmids for transgene expression. 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 delivery of appropriate erythropoietin mRNA, or protection against infection via delivery of mRNA encoding an appropriate antibody.

[0015] The lipid nanoparticles and compositions of the present invention can be used for a variety of purposes, both in vitro and in vivo, including the delivery of encapsulated or bound (e.g., complexed) 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, comprising lipid nanoparticles that encapsulate or bind the subject and a suitable therapeutic agent, wherein the lipid nanoparticles comprise one or more of the novel cationic lipids described herein.

[0016] The embodiments of the lipid nanoparticles of the present invention described herein are particularly useful for the delivery of nucleic acids, including, for example, mRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomyl / antimyl), messenger RNA interference complementary RNA (micRNA), DNA, multivalent RNA, Dicer substrate RNA, and complementary DNA (cDNA). Accordingly, by using the lipid nanoparticles and compositions of the present invention, the expression of a desired protein can be induced both in vitro and in vivo by contacting cells with lipid nanoparticles containing one or more novel cationic lipids described herein, where the lipid nanoparticles encapsulate or bind nucleic acids to be expressed for the production of the desired protein (e.g., messenger RNA or plasmid encoding the desired protein). Alternatively, by using the lipid nanoparticles and compositions of the present invention to contact cells with lipid nanoparticles containing one or more novel cationic lipids described herein, the expression of target genes and proteins may be reduced both in vitro and in vivo, where the lipid nanoparticles encapsulate or bind nucleic acids (e.g., antisense oligonucleotides or small interfering RNA (siRNA)) that reduce target gene expression. The lipid nanoparticles and compositions of the present invention may also be used for the co-delivery of different nucleic acids (e.g., mRNA and plasmid DNA), either separately or in combination, which may be useful to provide effects requiring the co-localization of different nucleic acids (e.g., mRNA encoding a suitable gene-modifying enzyme and a DNA segment for integration into the host genome).

[0017] The nucleic acids used in this invention can be prepared according to any available technology. For mRNA, the primary method of production includes, but is not limited to, enzymatic synthesis (also known as in vitro transcription), which currently represents the most efficient method for producing long sequence-specific mRNA. In vitro transcription refers to the process of template-directed synthesis of RNA molecules from an engineered DNA template consisting of an upstream bacteriophage promoter sequence (including, but not limited to, those derived from T7, T3, and SP6 Escherichia coli phages) bound to a downstream sequence encoding the gene of interest. Template DNA can be prepared by in vitro transcription from several sources using appropriate techniques well known in the field, including but not limited to plasmid DNA and polymerase chain reaction amplification (see Linpinsel, JL and Conn, GL, General protocols for preparation of plasmid DNA template and Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LD in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941 Conn GL (ed), New York, NY Humana Press, 2012).

[0018] RNA transcription is performed 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 possibility of degradation of the resulting mRNA transcript. In vitro transcription can be performed using a variety of commercially available kits, including but not limited to the RiboMax Large Scale RNA Production System (Promega) and the MegaScript Transcription Kit (Life Technologies), and commercially available reagents containing RNA polymerase and rNTPs. Methods for in vitro transcription of mRNA are well known in this field (see, for example, Losick, R., 1972, In vitro transcription, Ann Rev Biochem v.41 409-46; Kamakaka, RT and Kraus, WL 2001. In Vitro Transcription. Current Protocols in Cell Biology. 2:11.6:11.6.1-11.6.17; Beckert, B. And Masquida, B., (2010) Synthesis of RNA by In Vitro Transcription in RNA in Methods in Molecular Biology v. 703 (Neilson, H. Ed), New York, NY Humana Press, 2010; Brunelle, JL and Green, R., 2013, Chapter Five - In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology v. 530, 101-114). All of these are incorporated herein by reference.

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

[0020] Furthermore, while reverse transcription can yield a large amount of mRNA, the product may contain several abnormal RNA impurities associated with unwanted polymerase activity, which may need to be removed from the full-length mRNA preparation. These include transcription initiation failure and RNA-dependent RNA polymerase activity, double-stranded RNA (dsRNA) produced by RNA priming transcription from RNA templates and self-complementary 3' extension. These impurities with dsRNA structures have been shown to result in unwanted 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. This, in turn, can dramatically reduce mRNA translation, leading to decreased protein synthesis during the innate cellular immune response. Therefore, further techniques for removing these dsRNA impurities have been developed, including, but are not limited to, HPLC purification, which are known in this field (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, v. 39 e142; Weissman, D., Pardi, N., Muramatsu, H., and Kariko, K., HPLC Purification of in vitro transcribed long RNA in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH Ed), 2013). HPLC-purified mRNA has been reported to be translated at much higher levels, particularly in primary cells and in vivo.

[0021] A considerable variety of modifications used to alter the specific properties of in vitro transcribed mRNA and improve its utility have been documented in the literature. These include, but are not limited to, modifications to the 5' and 3' ends of mRNA. Endogenous eukaryotic mRNA typically contains a cap structure at the 5' end of the mature molecule, which plays a crucial role in mediating the binding of mRNA cap-binding proteins (CBPs), thereby contributing to enhanced mRNA stability and the efficiency of mRNA translation in cells. Consequently, the highest levels of protein expression are achieved with capped mRNA transcripts. The 5' cap contains a 5'-5' triphosphate bond between the furthest 5' nucleotide and the guanine nucleotide. The conjugated guanine nucleotide is methylated at the N7 position. Further modifications include methylation of the 2'-hydroxyl group of the furthest 5' end and the second nucleotide from the end.

[0022] Multiple different cap structures can be used to produce the 5' cap of in vitro transcribed synthetic mRNA. 5' capping of synthetic mRNA can be carried out co-transcribed using chemical cap analogs (i.e., capping during in vitro transcription). For example, anti-reverse cap analog (ARCA) caps contain a 5'-5'-triphosphate guanine-guanine bond with an N7 methyl group and a 3'-O-methyl group on one side of the guanine. However, up to 20% of the transcript remains uncapped during this co-transcription process, and synthetic cap analogs are not identical to the 5' cap structure of true cellular mRNA, potentially reducing translatability and cellular stability. Alternatively, synthetic mRNA molecules can also be capped by post-transcriptional enzymes. These can produce more authentic 5' cap structures that more closely mimic the endogenous 5' cap structurally or functionally, enhancing the binding of cap-binding proteins, extending half-lives, reducing sensitivity to 5' endonucleases, and / or reducing 5' cap removal. Numerous synthetic 5' cap analogs have been developed and are known to enhance mRN stability and translatability in this field (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 v.969 (Rabinovich, PH Ed), 2013).

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

[0024] Poly(A) tailing of in vitro transcribed mRNA can be achieved using a variety of approaches, including, but not limited to, cloning of poly(T) tracts to a DNA template or post-transcriptional addition using poly(A) polymerase. The first example allows in vitro transcription of mRNA with a poly(A) tail of a predetermined length, depending on the size of the poly(T) tract, but requires further manipulation of the template. The latter example involves enzymatic addition of a poly(A) tail to in vitro transcribed mRNA using poly(A) polymerase, which catalyzes the incorporation of an adenine residue into the 3' end of the RNA, and does not require further manipulation of the DNA template, but results in mRNA with poly(A) tails of different lengths. 5' capping and 3'-poly(A) tailing can be performed using a variety of commercially available kits and reagents, including but not limited to the Poly(A) Polymerase Tailing kit (EpiCenter), mMESSAGE mMACHINE T7 Ultra kit, and Poly(A) Tailing kit (Life Technologies), as well as various ARCA caps and poly(A) polymerases.

[0025] In addition to 5' capping and 3' polyadenylation, other modifications of in vitro transcripts have been reported to offer benefits in terms of translation efficiency and stability. It is well known in this field that pathogenic DNA and RNA can be recognized by a variety of sensors within eukaryotes, potentially triggering a potent innate immune response. The ability to distinguish pathogenic DNA and RNA from self DNA and RNA is, at least in part, based on structural and nucleoside modifications, as most nucleic acids of natural sources contain modified nucleosides. In contrast, in vitro synthesized RNA lacks these modifications, making it immunostimulant, which in turn allows it to effectively inhibit mRNA translation, as outlined above.The introduction of modified nucleosides into in vitro transcribed mRNA can be used to inhibit the recognition and activation of RNA sensors, thereby 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, v.10 523-532; Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH Ed), 2013); Kariko, K., Muramatsu, H., Welsh, FA, Ludwig, J., Kato, H., Akira, S., (See Weissman, D., 2008, Incorporation of Pseudouridine Into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability, Mol Ther v.16, 1833-1840). Modified nucleosides and nucleotides used in the synthesis of modified RNA can be manufactured, monitored, and made available using general methods and procedures known in the art. A wide variety of nucleoside modifications are available that can be incorporated to some extent into in vitro transcribed mRNA, either alone or in combination with other modified nucleosides (see, e.g., U.S. Publication 2012 / 0251618).In vitro synthesis of nucleoside-modified mRNA has been reported to have the ability to activate immune sensors and to enhance associated translational activity.

[0026] Other components of mRNA that can be modified to provide benefits in terms of translatability and stability include the 5' and 3' untranslated regions (UTRs). Optimization of the UTR (advantageous 5' and 3' UTRs can be obtained from cellular or viral RNA) has been shown to increase the mRNA stability and translational efficiency of in vitro transcribed mRNA, both or independently (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 v.969 (Rabinovich, PH Ed), 2013).

[0027] In addition to mRNA, other nucleic acid payloads may be used in the present invention. Regarding oligonucleotides, preparation methods include, but are not limited to, the aforementioned chemical synthesis and enzymatic or chemical cleavage of long precursors, or in vitro transcription. Methods for synthesizing DNA and RNA nucleotides are widely used and well-known in this field (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, v. 288 (Clifton, NJ) Totowa, NJ: Humana Press, 2005; both are incorporated herein by reference).

[0028] Regarding plasmid DNA, the preparation for use in this invention generally involves, but is not limited to, in vitro expansion and isolation of plasmid DNA in a liquid culture of bacteria containing the plasmid of interest. The presence of genes encoding resistance to specific antibiotics (such as penicillin and kanamycin) in the plasmid of interest allows for the selective growth of bacteria containing the plasmid of interest in antibiotic-containing cultures. Methods for isolating plasmid DNA are widely used and well-known in this field (see, for example, Heilig, J., Elbing, KL and Brent, R (2001) Large-Scale Preparation of Plasmid DNA. Current Protocols in Molecular Biology. 41:II:1.7:1.7.1-1.7.16; Rozkov, A., Larsson, B., Gillstroem, S., Bjoernestedt, R. and Schmidt, SR (2008) Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture. Biotechnol. Bioeng., 99: 557-566; and U.S. Patents 6,197,553). Plasmid isolation can be performed using a variety of commercially available kits and reagents, including but not limited to Plasmid Plus (Qiagen), GenJET plasmid MaxiPrep (Thermo), and PureYield MaxiPrep (Promega) kits.

[0029] Various exemplary embodiments of the use of the compounds of the present invention, quality nanoparticles and compositions containing them, and active or therapeutic agents such as nucleic acids for regulating gene and protein expression are described in further detail below.

[0030] The following terms used herein, unless otherwise specified, have the meanings associated with them.

[0031] Unless the context necessitates a different interpretation, throughout this specification and the claims, the terms “including,” “inclusion,” and “containing,” and their variations thereof, should be interpreted in an open and inclusive sense, i.e., “including, but not limited to.”

[0032] Throughout this specification, the phrase "one embodiment" or "a certain embodiment" means that any specific characteristic, structure, or feature described in relation to that embodiment is included in at least one embodiment of the present invention. Therefore, the expressions "one embodiment" or "a certain embodiment" found in various places in this specification do not necessarily refer to the same embodiment. Furthermore, characteristics, structures, or features may be combined in any suitable manner in one or more embodiments.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention pertains. Singular expressions used herein and in the claims include plural subjects unless otherwise clearly indicated by the context.

[0034] The expression "induces the expression of a desired protein" refers to the ability of nucleic acids to increase the expression of a desired protein. To test 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 such as a human or rodent (e.g., mouse) or non-human primate (e.g., 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 such as a human or rodent (e.g., mouse) or non-human primate (e.g., monkey) model) that has not been in contact with or administered nucleic acids. When the desired protein is present in the control sample or control mammal, a value of 1.0 may be assigned to the expression of the desired protein in the control sample or control mammal. In one embodiment, induction of the expression of a desired protein is achieved when the ratio of the desired protein expression level in the test sample or test mammal to the desired protein expression level 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. When the desired protein is not present in the control sample or control mammal, induction of the expression of the desired protein is achieved when a measurable level of the desired protein is detected in the test sample or test mammal. Those skilled in the art will understand appropriate assays for determining the level of protein expression in a sample, such as dot blotting, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays, or assays based on reporter proteins that can produce fluorescence or luminescence under appropriate conditions.

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

[0036] The “effective dose” or “therapeutic effective dose” of an activator or therapeutic agent, such as a therapeutic nucleic acid, is an amount sufficient to increase or inhibit the expression of a target sequence compared to the normal expression level detected in the absence of the nucleic acid, for example. An increase in the expression of a target sequence is achieved when some measurable level is detected, in the case of an expression product that does not exist in the absence of the nucleic acid. If an expression product is present at a certain level before contact with the nucleic acid, an increase in expression is achieved when the increase factor of the value obtained with nucleic acids such as mRNA, compared to the control, is approximately 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000 or more. Inhibition of the expression of a target gene or target sequence is achieved when the value obtained by nucleic acids such as antisense oligonucleotides, relative to a control, is approximately 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring the expression of a target gene or target sequence include, for example, protein or RNA-level tests 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 a suitable reporter protein, and phenotypic assays known to those skilled in the art.

[0037] As used herein, the term “nucleic acid” refers to a polymer comprising at least two deoxyribonucleotides or ribonucleotides in single- or double-stranded form, and includes DNA, RNA, and hybrids thereof. DNA may be in the form of an antisense molecule, plasmid DNA, cDNA, PCR product, or vector. RNA may be in the form of small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA, dicer substrate RNA, or viral RNA (vRNA), and combinations thereof. Nucleic acids include those that are synthetic, naturally occurring, or not naturally occurring, and that contain known nucleotide analogs or modified backbone residues or bindings that have similar binding properties to a reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2'-O-methylribonucleotides, and peptide-nucleic acids (PNAs). Unless otherwise specified, this term encompasses nucleic acids that contain known analogs of naturally occurring nucleotides that have similar binding properties to a reference nucleic acid. Unless otherwise specified, a given nucleic acid sequence implicitly includes its conserved modification variants (e.g., degenerate codon substitutions), alleles, orthologues, single nucleotide polymorphisms, and complementary sequences, as well as sequences explicitly shown. In particular, degenerate codon substitutions can be achieved by producing sequences in which the third of a selected (or whole) codon is substituted with a mixed base and / or a deoxyinosine residue (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). A "nucleotide" consists of the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked to each other via phosphate groups."Bases" include purines and pyrimidines, and further include synthetic derivatives of purines and pyrimidines, including but not limited to modifications that insert novel reactive groups such as, but not limited to, natural compounds adenine, thymidine, guanine, cytosine, uracil, inosine, and natural analogs, as well as amines, alcohols, thiols, carboxylates, and alkyl halides.

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

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

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

[0041] "Steroids" are compounds containing the following carbon skeleton: [ka]

[0042] Non-limiting examples of steroids include cholesterol, etc.

[0043] "Cationic lipids" refer to lipids that can be positively charged. Examples of cationic lipids include those containing one or more positively charged amine groups. Preferred cationic lipids are ionizable so that they can exist in a positively charged or neutral form depending on the pH. Ionization of cationic lipids affects the surface charge of lipid nanoparticles under various pH conditions. This charge affects the ability to form endosomal destabilizing non-double-layer structures, which are important for plasma protein absorption, blood clearance and tissue distribution (Semple, SC, et al., Adv. Drug Deliv Rev 32:3-17 (1998)) and intracellular delivery of nucleic acids (Hafez, IM, et al., Gene Ther 8:1188-1196 (2001)).

[0044] The term “lipid nanoparticles” refers to particles having at least one dimension on the order of nanometers (e.g., 1 to 1,000 nm) and containing one or more compounds of formula (I) or other specific components. In some embodiments, lipid nanoparticles are included in formulations that can be used to deliver activators 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 contain nucleic acids. Such lipid nanoparticles typically contain a compound of formula (I) and one or more additives selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. In some embodiments, activators or therapeutic agents, such as nucleic acids, may be encapsulated in an aqueous space surrounded by the lipid portion of the lipid nanoparticle or some or all of the lipid molecules of the lipid nanoparticle, thereby protecting them from enzymatic degradation or other undesirable effects induced by host organism or cellular mechanisms, such as adverse immune responses.

[0045] In various embodiments, lipid nanoparticles have an average diameter of approximately 30 nm to 150 nm, approximately 40 nm to 150 nm, approximately 50 nm to 150 nm, approximately 60 nm to 130 nm, approximately 70 nm to 110 nm, approximately 70 nm to 100 nm, approximately 80 nm to 100 nm, approximately 90 nm to 100 nm, approximately 70 nm to 90 nm, approximately 80 nm to 90 nm, approximately 70 nm to 80 nm, or 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 nontoxic. In one embodiment, when present in lipid nanoparticles, nucleic acids are resistant to nuclease degradation in aqueous solution. 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, and these entire disclosures are incorporated herein by reference in their entirety for all purposes.

[0046] As used herein, “encapsulating lipids” refers to lipid nanoparticles that provide an active or therapeutic agent, such as nucleic acids (e.g., mRNA), that is fully encapsulated, partially encapsulated, or both. In one embodiment, the nucleic acid (e.g., mRNA) is fully encapsulated in the lipid nanoparticles.

[0047] The term "polymer-conjugated lipid" refers to a molecule containing both a lipid portion and a polymer portion. An example of a polymer-conjugated lipid is a PEGylated lipid. The term "PEGylated lipid" refers to a molecule containing both a lipid portion and a polyethylene glycol portion. PEGylated lipids are well known in this field and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), among others.

[0048] The term "neutral lipid" refers to any of several lipid species that exist in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, but are not limited to, phosphotidylcholine, 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), phophatidylethanolamine, e.g., 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM), ceramides, steroids, e.g., sterols and their derivatives. Neutral lipids may be synthetic or naturally occurring.

[0049] The term "charged lipid" refers to several lipid species that exist with a positive or negative charge, regardless of the useful physiological pH range, e.g., pH approximately 3 to pH approximately 9. Charged lipids can 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).

[0050] The term "aqueous solution" used here refers to a composition containing water.

[0051] In relation to nucleic acid-lipid nanoparticles, "serum-stable" means that the nucleotides are not significantly degraded after exposure to serum or nuclease assays that would significantly degrade free DNA or RNA. Suitable assays include, for example, standard serum assays, DNAe assays, or RNAe assays.

[0052] As used herein, "systemic delivery" refers to the delivery of a therapeutic product that can result in broad exposure of the active agent within an organism. Certain administration techniques can effect systemic delivery of a particular agent, while others cannot. Systemic delivery means that a majority of the body is exposed to a useful, preferably therapeutic amount of the agent. Systemic delivery of lipid nanoparticles can be achieved by any means known in the art, including, for example, intravenous, intraarterial, subcutaneous, and intraperitoneal delivery. In certain embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery.

[0053] As used herein, "local delivery" refers to the direct delivery of an active agent to a target site within an organism. For example, an agent can be locally delivered by direct injection to a diseased site such as a tumor, an inflamed site, or other target sites such as target organs like the liver, heart, pancreas, kidney, etc. Local delivery can also include local or localized injection techniques such as intramuscular, subcutaneous, or intradermal injection. Local delivery does not exclude systemic pharmacological effects.

[0054] "Alkyl" consists of only carbon and hydrogen atoms, is saturated (i.e., contains no double and / or triple bonds), and has from 1 to 24 carbon atoms (C1-C 24 alkyl), from 1 to 16 carbon atoms (C1-C 16 alkyl), from 1 to 12 carbon atoms (C1-C 12 alkyl), from 6 to 24 carbon atoms (C6-C 24 alkyl), from 1 to 8 carbon atoms (C1-C8 alkyl) or from 1 to 6 carbon atoms (C1-C6 alkyl), and is a straight-chain or branched hydrocarbon chain radical bonded by single bonds to the rest of the molecule, for example, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, etc. Unless specifically indicated otherwise herein, alkyl groups are optionally substituted.

[0055] "Alkenyl" consists of only carbon and hydrogen atoms, contains at least one carbon-carbon double bond, and has from 1 to 24 carbon atoms (C2-C 24 alkenyl), from 1 to 12 carbon atoms (C2-C12 Alkenyl), 6-24 carbon atoms (C6-C6) 24 Alkenyl), 2-16 carbon atoms (C2-C 16 Alkenyl), 4-12 carbon atoms (C4-C 12 Alkenyls are linear or branched hydrocarbon chain radicals having 1 to 8 carbon atoms (C2-C8 alkenyls) or 1 to 6 carbon atoms (C2-C6 alkenyls) bonded to the rest of the molecule by single bonds, such as ethenyl, n-propenyl, 1-methylethenyl, n-butenyl, n-pentenyl, 1,1-dimethylethenyl, 3-methylhexenyl, and 2-methylhexenyl. Unless otherwise specifically indicated herein, the alkenyl groups are optionally substituted.

[0056] "Alkynyl" consists only of carbon and hydrogen atoms, contains at least one carbon-carbon triple bond, and has 1 to 24 carbon atoms (C2-C 24 Alkynyl), 1 to 12 carbon atoms (C2-C 12 Alkynyl refers to a linear or branched hydrocarbon chain radical having 1 to 8 carbon atoms (C2-C8 alkynyl) or 1 to 6 carbon atoms (C2-C6 alkynyl) bonded to the rest of the molecule by a single bond, such as ethynyl, n-propynyl, 1-methylethynyl, n-butynyl, n-pentynyl, 1,1-dimethylethynyl, 3-methylhexynyl, 2-methylhexynyl, etc. Unless otherwise specifically indicated herein, the alkynyl group may be substituted as desired.

[0057] An alkylene or alkylene chain is a linear or branched divalent saturated hydrocarbon chain consisting only of carbon and hydrogen, with the rest of the molecule linked to radical groups. In one embodiment, an alkylene chain has 1 to 24 carbon atoms (C1-C1). 24 Alkylene), 1 to 15 carbon atoms (C1-C 15 Alkylene), 1 to 12 carbon atoms (C1-C 12Alkylenes have 1 to 8 carbon atoms (C1-C8 alkylenes), 1 to 6 carbon atoms (C1-C6 alkylenes), 4 to 6 carbon atoms (C4-C6 alkylenes), 2 to 4 carbon atoms (C2-C4 alkylenes), and 1 to 2 carbon atoms (C1-C2 alkylenes), such as methylene, ethylene, propylene, and n-butylene. Alkylene chains are bonded to the rest of the molecule by single bonds and to radical groups by single bonds. Bonding sites of alkylene chains to the rest of the molecule and to radical groups may be via one carbon or any two carbons in the chain. Unless otherwise specifically indicated herein, alkylene chains are optionally substituted.

[0058] An "alkenylene" or "alkenylene chain" is a linear or branched divalent hydrocarbon chain consisting only of carbon and hydrogen, containing at least one carbon-carbon double bond, with the rest of the molecule linked to a radical group. In one embodiment, an alkenylene chain has 2 to 24 carbon atoms (C2-C 24 Alkenylenes), 2 to 15 carbon atoms (C2-C 15 Alkenylenes), 2 to 12 carbon atoms (C2-C 12 Alkenylenes have 2-8 carbon atoms (C2-C8 alkenylenes), 2-6 carbon atoms (C2-C6 alkenylenes), 4-6 carbon atoms (C4-C6 alkenylenes), and 2-4 carbon atoms (C2-C4 alkenylenes), such as etenylene, propenylene, and n-butenylene. Alkenylene chains are bonded to the rest of the molecule via single bonds and to radical groups via single bonds. The bonding sites of the alkenylene chain to the rest of the molecule and to radical groups may be via one carbon or any two carbons in the chain. Unless otherwise specifically indicated herein, alkenylene chains are optionally substituted.

[0059] A "cycloalkyl" or "carbocyclic ring" consists only of carbon and hydrogen atoms and may include condensed or bridging ring systems, comprising 3 to 15 ring carbon atoms (C3-C3). 15 ), 3 to 10 ring carbon atoms (C3-C 10A cycloalkyl group is a stable, non-aromatic monocyclic or polycyclic hydrocarbon radical having 3 to 8 ring carbon atoms (C3-C8), saturated or unsaturated, and bonded to the rest of the molecule by a single bond. Examples of monocyclic radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of polycyclic radicals include adamantyl, norbolonyl, dekalinyl, and 7,7-dimethylbicyclo[2.2.1]heptanyl. Unless otherwise specifically stated herein, cycloalkyl groups are optionally substituted.

[0060] "Aryl" refers to a carbocyclic cyclic radical comprising hydrogen, 6 to 18 carbon atoms, and at least one aromatic ring. For the purposes of the present invention, aryl radicals are monocyclic, bicyclic, tricyclic, or tetracyclic and may include condensed or crosslinked cyclic systems. Aryl radicals include, but are not limited to, aryl radicals derived from acetantrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluorantene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene.

[0061] "Arylalkyl" is represented by the formula -R b -R c This refers to the radical of, where R b R is an alkylene or alkenylene as defined above, c arylalkyl groups are one or more aryl radicals as defined above, such as benzyl or diphenylmethyl. Unless otherwise specifically indicated herein, arylalkyl groups are optionally substituted.

[0062] A “heterocyclyl” or “heterocyclic ring” refers to a stable 3-18 membered non-aromatic ring radical having 1-12 ring carbon atoms (e.g., 2-12) and 1-6 ring heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Unless otherwise specifically indicated herein, heterocyclyl radicals are monocyclic, dicyclic, tricyclic, or tetracyclic, and may include condensed spirocyclic ("spiro-heterocyclyl") and / or bridging ring systems; and the nitrogen, carbon, or sulfur atoms of the heterocyclyl radical are optionally oxidized; the nitrogen atom is optionally quaternized; and the heterocyclyl radical is partially or completely saturated. Examples of such heterocyclyl radicals 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-piperidonyl, pyrrolidinil, pyrazolidinil, quinuclidinil, thiazolidinil, tetrahydrofuryl, trithianil, tetrahydropyranil, thiomorpholinil, thiamorpholinil, 1-oxo-thiomorpholinil, and 1,1-dioxo-thiomorpholinil. Unless otherwise specifically indicated herein, heterocyclyl groups are optionally substituted.

[0063] A "heteroaryl" refers to a 5-14 membered ring radical comprising a hydrogen atom, 1-13 carbon atoms, a heteroatom selected from the group consisting of 1-6 nitrogen, oxygen, and sulfur atoms, and at least one aromatic ring. For the purposes of the present invention, heteroaryl radicals may be monocyclic, bicyclic, tricyclic, or tetracyclic, and may include condensed or bridging ring systems; and the nitrogen, carbon, or sulfur atoms in the heteroaryl radical may be oxidized as desired; and the nitrogen atom may be quaternized as desired.Examples include azepinyl, acridinyl, benzimidazolyl, benzthiazolyl, benzindolyl, benzodioxolyl, benzofuranil, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanil, benzonaphthofuranil, benzoxazolyl, benzodioxolyl, benzodioxynil, benzopyranil, benzopyranonil, benzofuranil, benzothi Enyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, benzoxazolinonyl, benzimidazolethionyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indazolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolidinyl, isoxazolyl, Naphthilidinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxyranil, 1-oxidepyridinyl, 1-oxidepyrimidinyl, 1-oxidepyradinyl, 1-oxidepyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxadinyl, phthalazinyl, pteridinyl, pteridinonyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyridinonyl, pyridinyl, pyrimidinyl, pyrimidinyl, This includes, but is not limited to, pyridadinyl, pyrrolyl, pyrido[2,3-d]pyrimidinonyl, quinazolinyl, quinazolinonyl, quinoxalinyl, quinoxalinonyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, thieno[3,2-d]pyrimidine-4-onyl, thieno[2,3-d]pyrimidine-4-onyl, triazolyl, tetrazolyl, triazidinyl, and thiophenyl (i.e., thienyl). Unless otherwise specifically indicated herein, heteroaryl groups may be substituted as desired.

[0064] The term "substitution" used here refers to the substitution of at least one hydrogen atom with a halogen atom such as F, Cl, Br, and I; an oxo group (=O); a hydroxyl group (-OH); or an alkoxy group (-OR). a , here, Ra is C1-C 12 Alkyl or cycloalkyl group; carboxyl group (-OC(=O)R a Or -C (=O) OR a , here, R a H, C1-C 12 Alkyl or cycloalkyl group; amine group (-NR) a R b , here, R a and R b H and C1-C are independent of each other. 12 Alkyl or cycloalkyl); C1-C 12 Alkyl group; and cycloalkyl group; and any of the above groups (e.g., alkyl, alkenyl, alkynyl, alkylene, cycloalkyl, aryl, heteroaryl, or heterocyclyl) which are replaced by bonding to a non-hydrogen atom, but are not limited to these. In some embodiments, the substituent is 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 an 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.

[0065] "Optional" or "optionally" (e.g., optionally substituted) means that the event or situation described thereafter may or may not occur, and this description includes cases where such event or situation occurs and cases where it does not. For example, "optionally substituted alkyl" means that the alkyl radical may or may not be substituted, and this description includes both substituted alkyl radicals and unsubstituted alkyl radicals. In one embodiment, "optionally substituted" means that a particular radical is halo (e.g., F, Cl, Br, and I), oxo (=O), hydroxyl (-OH), alkoxy (-OR) a , here, R a is C1-C12 Alkyl), cycloalkoxy (-OR a , here, R a (C3-C8 cycloalkyl), carboxyl (-OC(=O)R a Or -C (=O) OR a , here, R a H, C1-C 12 Alkyl, or C3-C8 cycloalkyl, amine (-NR) a R b , here, R a and R b H and C1-C are independent of each other. 12 Alkyl, or C3-C8 cycloalkyl, C1-C 12 This means that the molecule is substituted with one or more substituents selected from the group consisting of alkyl and C3-C8 cycloalkyl groups.

[0066] In one embodiment, "optionally substituted" means substituted with one or more halo substituents. In one embodiment, "optionally substituted" means substituted with one or more oxo substituents. In one embodiment, "optionally substituted" means substituted with one or more hydroxyl substituents. In one embodiment, "optionally substituted" means substituted with one or more alkoxy substituents. In one embodiment, "optionally substituted" means substituted with one or more cycloalkoxy substituents. In one embodiment, "optionally substituted" means substituted with one or more carboxy substituents. In one embodiment, "optionally substituted" means substituted with one or more amine substituents. In one embodiment, "optionally substituted" means one or more C1-C 12 This means that it is substituted with an alkyl substituent. In one embodiment, "optionally substituted" means that it is substituted with one or more C3-C8 cycloalkyl substituents.

[0067] When a functional group is described as "optionally substituted," and then a substituent on that functional group is also described as "optionally substituted," for the purposes of the present invention, such repetitions are limited to five, preferably two. In one embodiment, such repetitions are limited to one. In another embodiment, such repetitions are limited to zero.

[0068] The present invention is also intended to encompass all pharmaceutically acceptable compounds of the compound of formula (I) that are isotope-labeled, in which atoms with different atomic masses or mass numbers are substituted. Examples of isotopes that may be incorporated into the compounds disclosed herein are 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 125 Contains I. These radiolabeled compounds are useful, for example, in determining or measuring the efficacy of compounds by characterizing the site of action or mechanism. Compounds of formula (I) that are isotope-labeled, for example, those incorporating radioactive isotopes, are useful in drug and / or substrate tissue distribution studies. Radioactive isotope tritium, i.e., 3 H, and carbon-14, that is, 14 C is particularly useful for this purpose due to its ease of integration and readiness for detection.

[0069] Deuterium, that is, 2 Substitution with heavier isotopes such as 1H may offer certain therapeutic benefits due to greater metabolic stability, such as an extended in vivo half-life or a reduced required dose, and therefore may be preferable in certain situations.

[0070] 11 C, 18 F, 15 O, and 13 Substitution with positron-emitting isotopes such as 1N may be useful in positron emission tomography (PET) tests for substrate-receptor selection. Compounds of formula (I) with isotope labeling can generally be prepared by using a suitable isotope-labeled reagent instead of the previously used unlabeled reagent, by the methods described in the production and examples shown in the conventional art known to those skilled in the art.

[0071] The present invention is also intended to encompass in vivo metabolites of the compounds of the present invention. Such products may be produced by enzymatic processes, such as oxidation, reduction, hydrolysis, amidation, or esterification of the administered compound. Accordingly, the present invention includes compounds produced by methods 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, allowing a sufficient period for metabolism to occur, and then isolating the converted products from urine, blood, or other biological samples.

[0072] The terms "stable compound" and "stable structure" are intended to indicate compounds that are robust enough to be isolated from a reaction mixture at a useful purity and survive to be formulated into an effective therapeutic agent.

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

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

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

[0076] "Pharmacologically acceptable acid addition salts" are those that retain the biological efficacy and properties of the free base and are not biologically or otherwise undesirable, and include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and 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, carbonate, 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, glucoheptonic acid, and gluconic acid. This refers to salts formed with organic acids such as, but not limited to, 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, mucinic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid.

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

[0078] Crystallization often produces solvates of the compounds of the present invention. The term "solvate" as used herein refers to an aggregate containing one or more molecules of the compound of the present invention and one or more molecules of solvent. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Therefore, the compounds of the present invention may exist as hydrates, including monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate, etc., and as corresponding solvated forms. The compounds of the present invention may be true solvates, or in other cases, they may simply hold undefined water or a mixture of water and a certain undefined solvent.

[0079] "Pharmaceutical composition" refers to a formulation of the compound of the present invention and a medium generally accepted in the art for the delivery of the biologically active compound to a mammal, such as a human. Such a medium includes all pharmaceutically acceptable carriers, diluents, or additives.

[0080] "Effective dose" or "therapeutic effective dose" means the amount of the compound of the present invention that, when administered to a mammal, preferably a human, is sufficient to perform treatment in said mammal, preferably a human. The amount of lipid nanoparticles of the present invention constituting the "therapeutic effective dose" varies depending on the compound, its state and severity, the method of administration, and the age of the mammal being treated, but can be routinely determined by a person skilled in the art in consideration of the knowledge of a person skilled in the art and the present disclosure.

[0081] As used herein, “to treat” or “to treat” encompasses the treatment of the disease or condition in a mammal, preferably a human, that has the disease or condition of interest, and includes the following: (i) In particular, to prevent the occurrence of the disease or condition in a mammal when the mammal is predisposed to the condition but has not yet been diagnosed as having it; (ii) To prevent a disease or condition, that is, to halt its progression; (iii) to alleviate a disease or condition, i.e., to cause recovery from a disease or condition; or (iv) Experiencing symptoms resulting from a disease or condition, i.e., pain relief without addressing the underlying disease or condition. The terms “disease” and “condition” as used herein may be interchangeable, or they may differ in that the disease or condition may not have known causative factors (and therefore its etiology is not yet understood), and therefore not yet recognized as a disease, but is recognized only as an undesirable condition or syndrome in which a more or less specific set of symptoms has been identified by clinicians.

[0082] The compounds of the present invention, or their pharmaceutically acceptable salts, may contain one or more chiral centers, and thus may give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined in terms of absolute stereochemistry as (R)- or (S)- or, for amino acids, (D)- or (L)-. The present invention is intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, e.g., chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include 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). When a compound described herein contains an olefinic double bond or other geometrically asymmetric center, and unless otherwise specified, it is intended that the compound includes both E and Z geometric isomers. Similarly, all tautomers are also intended to be included.

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

[0084] A "tautomer" is defined as a proton shift from one atom in a molecule to another atom in the same molecule. This invention includes tautomers of the compound.

[0085] compound In one aspect, the present invention provides novel lipid compounds that can form lipid nanoparticles in combination with other lipid components such as neutral lipids, charged lipids, steroids, and / or polymer-conjugated lipids. Without wishing to be bound by theory, these lipid nanoparticles are thought to shield the oligonucleotide from degradation in serum and provide for efficient delivery of the oligonucleotide to cells in vitro and in vivo.

[0086] One embodiment is of formula (I):

Chemical formula

[0087] One reason, R 1b and R 1c These combine with the nitrogen to which they are bound to form a 3-12 membered heterocycline. In one embodiment, R 1b and R 1c These molecules, along with the nitrogen atoms to which they are bound, form 3- to 10-membered heterocyclines.

[0088] In one embodiment, the compound is of the following formula (Ia): [ka] It is a compound or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

[0089] In one embodiment, the compound is of the following formula (Ib): [ka] It is a compound or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

[0090] One reason, R 1a C8-C is replaced with oxo as desired. 12 It is alkyl. A more specific example is R 1a The following structure: [ka] It has one of them.

[0091] One reason, R 1 The following structures are available upon request: -OH, oxo, fluoro, -N(CH3)2, and the following: [ka] It is substituted with one or two substituents selected from the group consisting of the following.

[0092] One reason, R 1 It is substituted with -OH. In one embodiment, R 1 is -CH3. In one other embodiment, R 1 The following structure: [ka] It has one of them.

[0093] One reason, R 1 The following structure: [ka] It has one of them.

[0094] One reason, R 2 is -OC(=O)OR 2a In one example, R 2a is C4-C 24 It is alkyl. In one example, R 2a is unsubstituted C4-C 24 It is alkyl. In one example, R 2a is non-branched C4-C 24 It is alkyl. In one example, R 2a Branch C4-C 24 It is alkyl.

[0095] One reason, R 2 -OC(=O)R 2b In one example, R 2 is -C(=O)OR 2c In one example, R 2b or R 2c The following structure: [ka] It holds.

[0096] One reason, R 2b or R 2c The following structure: [ka] It holds.

[0097] One reason, R 2d is an unbranched C4-C8 alkyl group. In one embodiment, R 2e is an unbranched C4-C8 alkyl group. In one embodiment, R 2dR is an unbranched C4 alkyl. In one embodiment, R 2e R is an unbranched C4 alkyl. In one embodiment, R 2d is an unbranched C6 alkyl. In one embodiment, R 2e is an unbranched C6 alkyl. In one embodiment, R 2d is an unbranched C8 alkyl. In one embodiment, R 2e It is an unbranched C8 alkyl group.

[0098] One reason, R 2 The following structure: [ka] It has one of them.

[0099] One reason, R 2 The following structure: [ka] [ka] It has one of them.

[0100] One reason, R 3 is -C(=O)N(R 3a )R 3b In one example, R 3 -NR 3a -C(=O)R 3b In one example, R 3a and R 3b Each is independently C6-C 24 It is alkyl. In one example, R 3a and R 3b Each is independently C6-C 12 It is alkyl. In one example, R 3a and R 3b Each is independently C8-C 10 It is alkyl. In one example, R 3a and R 3bBoth are C8 alkyl groups. In one example, R 3a and R 3b Both are C 10 It is alkyl. In one example, R 3a and R 3b Both are C 12 It is alkyl.

[0101] In one statement, L 1 and L 2 Each of these is independently a C4-C8 alkylene. In one embodiment, L 1 and L 2 Each is independently C4-C 10 It is an alkylene. 1 and L 2 Each of these is independently a C5, C7, or C8 alkylene. In one embodiment, L 1 and L 2 Each of these is independently a C4, C5, C6, C7, C8, or C9 alkylene. In one embodiment, L 1 and L 2 They are the same. In one example, L 1 and L 2 They are different. In one embodiment, L 1 and L 2 L is non-substitutable. In one embodiment, L 1 、 L 2 , or both are substituted with one or more fluoro substituents.

[0102] In various embodiments, the compound has one of the structures shown in Table 1 below (or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof).

[0103] [Table 1] [Table 2] [Table 3] [Table 4] [Table 5]

[0104] It is understood that any embodiment of the compound of formula (I) above, and any specific substituent and / or variable group in the compound of formula (I) above, can be independently combined with other embodiments and / or substituents and / or variable groups of the compound of formula (I) above to form embodiments of the present invention not specifically shown above. Furthermore, it is understood that if the list of substituents and / or variable groups mentions any specific R, G, or L group in a particular embodiment and / or claim, each individual substituent and / or variable group can be removed from that particular embodiment and / or claim, and the remainder of the list of substituents and / or variable groups is considered to be within the scope of embodiments of the present invention.

[0105] In this description, it is understood that combinations of substituents and / or variable groups in the formulas described are permissible only if such contributions result in a stable compound.

[0106] For administration purposes, the compound of the present invention (typically in the form of lipid nanoparticles combined with a therapeutic agent) may be administered as a raw compound or formulated as a pharmaceutical composition. The pharmaceutical composition of the present invention comprises the compound of formula (I) and one or more pharmaceutically acceptable carriers, diluents, or additives. The compound of formula (I) forms lipid nanoparticles and is present in the composition in an amount effective to deliver the therapeutic agent, for example, for the treatment of a particular disease or condition of interest. Appropriate concentrations and doses can be determined by those skilled in the art.

[0107] The embodiments provide compositions (e.g., lipid nanoparticles) comprising a compound of formula (I) and a therapeutic agent. In one embodiment, the composition (e.g., lipid nanoparticles) further comprises one or more additives selected from neutral lipids, steroids, and polymer-conjugated lipids.

[0108] In one embodiment, the therapeutic agent comprises nucleic acid. In one embodiment, the nucleic acid is selected from antisense and messenger RNA.

[0109] In one embodiment, the composition (e.g., lipid nanoparticles) comprises one or more neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In one embodiment, the neutral lipid is DSPC. In one embodiment, the molar ratio of the compound to the neutral lipid is in the range of about 2:1 to about 8:1. In one embodiment, the steroid is cholesterol. In one embodiment, the molar ratio of the compound to cholesterol is in the range of about 2:1 to 1:1. In one embodiment, the molar ratio of the compound to cholesterol is in the range of 5:1 to 1:1 or 2:1 to 1:1.

[0110] In one embodiment, the polymer conjugate lipid is a PEGylated lipid. In various embodiments, the polymer conjugate lipid is a PEGylated lipid. For example, one embodiment includes PEGylated diacylglycerols (PEG-DAG) such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidyl ethanolamines (PEG-PE), PEG succinate diacylglycerols (PEG-S-DAG) such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanediol (PEG-S-DMG), PEGylated ceramides (PEG-cer), or PEG dialkoxypropyl carbamates such as Ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecaneoxy)propyl) carbamate or 2,3-di(tetradecaneoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl) carbamate.

[0111] In one embodiment, the molar ratio of the compound to the PEGylated lipid is in the range of approximately 100:1 to approximately 10:1 or approximately 100:1 to approximately 25:1. In another embodiment, the molar ratio of the compound to the PEGylated lipid is in the range of approximately 100:1 to approximately 20:1 or approximately 100:1 to approximately 10:1.

[0112] In one embodiment, the PEGylated lipid is given by the following formula (II): [ka] [During the ceremony, R 10 and R 11 Each is independently a linear or branched, alkyl, alkenyl or alkynyl chain of 10 to 30 carbon atoms, where the alkyl, alkenyl or alkynyl is optionally interrupted by one or more ester bonds; and w has an average value in the range of 30 to 60. It is a compound or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

[0113] One reason, R 10 and R 11 Each is an independent linear alkyl chain containing 12 to 16 carbon atoms. In one embodiment, the average w is approximately 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55. In one embodiment, the average w is approximately 49. In one embodiment, w has a value in the range of 30 to 60. In one embodiment, w is in the range of 40 to 50. In one embodiment, w is 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0114] In one embodiment, the lipid nanoparticles or composition comprises a plurality of PEGylated lipids of formula (II), the average w for this plurality being in the range of 40 to 50. In one embodiment, the average w is 43, 44, 45, 46, 47, or 48.

[0115] The synthesis of PEGylated lipids can be found in U.S. Patent 9,738,593, the disclosure of which is incorporated herein by reference.

[0116] The compositions of the present invention may be administered via any acceptable method of administering a drug that provides similar utility. The pharmaceutical compositions of the present invention may be formulated into solid, semi-solid, liquid, or gaseous formulations such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, non-enteral, sublingual, buccal, rectal, vaginal, and intranasal. As used herein, non-enteral includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection, or infusion techniques. The pharmaceutical compositions of the present invention are formulated such that the active ingredients contained therein become bioavailable upon administration of the composition to a patient. The composition to be administered to a subject or patient may take the form of one or more dose units, where, for example, a tablet may be a single dose unit, and a container of the compound of the present invention in aerosol form may hold multiple dose units. The practical preparation methods for such dosage forms are known or obvious 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 administered composition in any case comprises a therapeutically effective amount of the compound of the present invention, or a pharmaceutically acceptable salt thereof, for the treatment of the disease or condition of interest as taught in this disclosure.

[0117] The pharmaceutical composition of the present invention may be in solid or liquid form. In one embodiment, the carrier is particles, and therefore the composition may be, for example, in tablet or powder form. The carrier may be liquid, and the composition may be, for example, an oral syrup, a liquid for injection, or an aerosol useful for inhalation administration.

[0118] When intended for oral administration, the pharmaceutical composition is preferably in solid or liquid form, where semi-solid, semi-liquid, suspension, and gel forms are included within the range of forms considered as solid or liquid.

[0119] As a solid composition for oral administration, pharmaceutical compositions can be formulated in the form of powders, granules, compressed tablets, pills, capsules, chewing gum, wafers, etc. Such solid compositions typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth gum, or gelatin; additives such as starch, lactose, or dextrin; disintegrants such as alginic acid, sodium alginate, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotex; flow enhancers such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavoring agents such as peppermint, methyl salicylate, or orange flavor; and colorants.

[0120] When the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain a liquid carrier such as polyethylene glycol or oil in addition to the above-mentioned type of substance.

[0121] Pharmaceutical compositions may be in the form of liquids, such as elixirs, syrups, solutions, emulsions, or suspensions. Liquids may be for oral administration or injection, as two examples. When intended for oral administration, preferred compositions include, in addition to the compound, one or more sweeteners, preservatives, colorants, and flavor enhancers. Compositions intended for injection may include one or more surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents.

[0122] Liquid The pharmaceutical composition of the present invention, whether in solution, suspension or other similar form, may contain one or more of the following adjuvants: water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixing oils such as synthetic mono or diglycerides that can serve as solvents or suspension media, sterile diluents such as polyethylene glycol, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetic acid, citric acid or phosphoric acid and tonic modifiers such as sodium chloride or dextrose; and agents that act as cryoprotective agents such as sucrose or trehalose. Non-enteral formulations may be sealed in glass or plastic ampoules, disposable syringes or multi-dose vials. Physiological saline is a preferred adjuvant. The pharmaceutical composition for injection is preferably sterile.

[0123] Liquid pharmaceutical compositions of the present invention intended for non-enteral or oral administration must contain an amount of the compound of the present invention such that an appropriate dose is obtained.

[0124] The pharmaceutical composition of the present invention may be intended for topical administration, in which case the carrier may appropriately comprise a solution, emulsion, ointment, or gel base. The base may comprise, for example, one or more of the following: diluents such as petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, water, and alcohol, and emulsifiers and stabilizers. Concentrating agents may be present in the topical pharmaceutical composition. If intended for transdermal administration, the composition may comprise a transdermal patch or an ion electrophoresis device.

[0125] 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 to release the drug. Compositions for rectal administration may include an oily base as a suitable non-irritating additive. Such bases include, but are not limited to, lanolin, cocoa butter, and polyethylene glycol.

[0126] The pharmaceutical compositions of the present invention may contain various substances that modify the physical form of solid or liquid dosage units. For example, the composition may contain a substance that forms a coating shell around the active ingredient. The substance forming the coating shell is typically inert and can be selected from, for example, sugars, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule.

[0127] The pharmaceutical compositions of the present invention, in solid or liquid form, may contain agents that bind to the compounds of the present invention, thereby assisting in the delivery of the compounds. Suitable agents that may be useful in this capacity include monoclonal or polyclonal antibodies or proteins.

[0128] The pharmaceutical compositions of the present invention can constitute dosage units that can be administered as aerosols. The term aerosol is used to describe a variety of forms, from colloidal substances to systems consisting of pressurized packages. Delivery is achieved 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 one-phase, two-phase, or three-phase systems for delivering the active ingredient. Aerosol delivery includes necessary containers, activators, valves, subcontainers, etc., which together may form a kit. Those skilled in the art can determine a preferred aerosol without excessive experimentation.

[0129] 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 mixing the lipid nanoparticles of the present invention with sterile, distilled water or other carriers to form a solution. A surfactant may be added to promote the formation of a homogeneous solution or suspension. The surfactant is a compound that interacts with the compounds of the present invention non-covalently and promotes the dissolution or homogeneous suspension of the compounds in an aqueous delivery system.

[0130] The compositions of the present invention or their pharmaceutically acceptable salts are administered in therapeutically effective doses that vary depending on a variety of factors, including the activity of the particular therapeutic agent used; the metabolic stability and duration of action of the therapeutic agent; the patient's age, weight, general health, sex, and dietary habits; the method and timing of administration; the rate of excretion; drug combinations; the severity of a particular disorder or condition; and the subject being treated.

[0131] The composition of the present invention may 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-dose formulation of the composition of the present invention and one or more additional activators, as well as the administration of each separate-dose formulation of the composition of the present invention and each activator. For example, the composition of the present invention and other activators may be administered to the patient together as a single oral-dose composition such as a tablet or capsule, or as separate oral-dose formulations of each agent. When using separate-dose formulations, the compound of the present invention and one or more additional activators may be administered essentially simultaneously, i.e., together, or separately, staggered by time, i.e., sequentially; it is understood that combination therapies include all of these regimens.

[0132] Methods for preparing the above compounds and compositions are described below and / or known in the art.

[0133] Those skilled in the art will recognize that in the methods described herein, it may be necessary to protect the functional groups of intermediate compounds with appropriate protecting groups. Such functional groups include hydroxy, amino, mercapto, and carboxylic acids. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl, or trimethylsilyl), tetrahydropyranyl, benzyl, etc. Suitable protecting groups for amino, amidino, and guanidino include t-butoxycarbonyl, benzyloxycarbonyl, etc. Suitable protecting groups for mercapto include -C(O)-R'' (where R'' is alkyl, aryl, or arylalkyl), p-methoxybenzyl, trityl, etc. Suitable protecting groups for carboxylic acids include alkyl, aryl, or arylalkyl esters. Protecting groups can be added or removed according to the standard techniques known to those skilled in the art and described herein. The use of protecting groups is described in Green, TW and PGM Wutz, Protective Groups in Organic Synthesis (1999), 3 rd As detailed in Ed., Wiley, the protecting group may also be a polymer resin such as Wang resin, Rink resin, or 2-chlorotrityl chloride resin, as will be understood by those skilled in the art.

[0134] Such protected derivatives of the compounds of the present invention may not possess pharmacological activity themselves, but it will also be recognized by those skilled in the art that, upon administration to mammals, they may be metabolized in the body to form pharmacologically active compounds of the present invention. Such derivatives may therefore be described as “prodrugs.” All prodrugs of the compounds of the present invention are encompassed within the scope of the present invention.

[0135] Furthermore, all compounds of the present invention, existing in free base or acid form, can be converted to pharmaceutically acceptable salts by treatment with a suitable inorganic or organic base or acid using methods known to those skilled in the art. Salts of the compounds of the present invention can be converted to free base or acid form by standard techniques.

[0136] The following reaction scheme depicts the compounds of the present invention, namely, those of formula (I):

Chemical formula

[0137] General Reaction Scheme 1

Chemical formula

[0138] Embodiments of the compounds of formula (I) (e.g., Compounds I-1 and I-2) can be prepared according to General Reaction Scheme 1, where the variable groups (e.g., R 1 , R 2 , R 3 , G 1 , G 2 , L 1 , L2 , R 2a , R 3a , and R 3b ) is defined here as follows.

[0139] For general reaction scheme 1, reagents and starting materials (e.g., compounds 1A, 1B, 1D, 1F, and 1H) can be purchased from commercial sources or prepared by methods familiar to those skilled in the art. A mixture of 1A and 1B is combined under appropriate reaction conditions to promote the coupling reaction (e.g., (COCl)2, DMF, triethylamine, DMAP). The resulting product (1C) is combined with amine 1D under appropriate reagents and reaction conditions (e.g., acetonitrile under reflux) to obtain the desired product (1E). In a parallel route, compound 1F is reacted with appropriate reagents (e.g., p-nitrophenyl chloroformate, DCM solution of pyridine) to obtain compound 1G. Compound 1G is then reacted with compound 1H using desired reaction conditions (e.g., DMAP, DCM solution of pyridine) to obtain compound 1I. Compound 1I is then reacted with compound 1E using appropriate conditions (e.g., DIPEA, acetonitrile under reflux) to obtain the compound of formula (I).

[0140] General reaction scheme 2 [ka]

[0141] Embodiments of the compound of formula (I) (e.g., compound I-8) can be prepared according to general reaction scheme 2, where a variable group (e.g., R) is used. 1 , R 2 , R 3 , G 1 , G 2 , L 1 , L 2 , R 2c , R 3a , R 1a , and R 3b ) is defined here as follows.

[0142] For general reaction scheme 2, the starting materials and other reagents (e.g., compounds 2A, 2B, 2D, 2F, and 2H) can be purchased from commercial sources or prepared by methods familiar to those skilled in the art. As the first step, a mixture of 2A and 2B is combined under appropriate reaction conditions to promote the coupling reaction (e.g., a DCM solution of DIPEA and HATU) to obtain the desired product as shown. Compound 2C is then reacted with compound 2D using appropriate conditions (e.g., a DCM solution of DCC and DMAP) to obtain compound 2E. The reaction product is then reacted with compound 2F under appropriate conditions (e.g., NaBH(AcO)3, acetic acid, and DCE) to obtain compound 2G. Compound 2G and compound 2H are then combined under appropriate conditions (e.g., a DCM solution of DIPEA and HATU) to obtain the compound of formula (I).

[0143] Those skilled in the art will understand that compounds of formula (I) can be prepared by similar methods or combinations of other methods known to those skilled in the art. Those skilled in the art will also understand that other compounds of formula (I) not specifically described below can be prepared by methods similar to those described below, using appropriate starting components as necessary and modifying the synthetic parameters. Generally, starting components can be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, or synthesized from sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)), or prepared as described in this disclosure.

[0144] Example 1 In vivo evaluation of luciferase mRNA using lipid nanoparticle compositions The lipids of formula (I), DSPC, cholesterol, and PEG-lipids of formula (II) are solubilized in ethanol at a molar ratio of 50:10:38.5:1.5 or 47.5:10:40.7:1.8. Lipid nanoparticles (LNPs) are prepared at a total lipid to mRNA weight ratio of approximately 10:1 to 40:1. Briefly, mRNA is diluted to 0.2 mg / mL in 10-50 mM citrate buffer, pH 4-6 or 10-25 mM acetate buffer, pH 4-6. Using a syringe pump, the ethanol-soluble lipid solution and the mRNA aqueous solution are mixed at a ratio of approximately 1:5 to 1:3 (vol / vol) at a total flow rate exceeding 15 mL / min. Then, the ethanol is removed and the external buffer is replaced with PBS by dialysis. Finally, the lipid nanoparticles are filtered through a 0.2 μm pore sterile filter.

[0145] The tests are conducted in 6-8 week old female C57BL / 6 mice (Charles River) or 8-10 week old CD-1 mice (Charles River or Inotiv) in accordance with guidelines established by the Institutional Animal Care Committee (ACC) and the Canadian Animal Care Association (CCAC). Various doses of mRNA-lipid nanoparticles are administered systemically by tail vein injection, and the mice are sacrificed at a specific time (e.g., 4 hours) after administration. The liver and spleen are collected in pre-weighed tubes, weighed, immediately rapid-frozen in liquid nitrogen, and stored at -80°C until processing for analysis.

[0146] For liver tissue, approximately 50 mg is cut into 2 mL FastPrep tubes (MP Biomedicals, Solon OH) for analysis. A 1 / 4” ceramic sphere (MP Biomedicals) is added to each tube, and 500–750 μL of Glo Lysis buffer-GLB (Promega, Madison WI), equilibrated to room temperature, is added to the liver tissue. The liver tissue is homogenized for 15 seconds at 2 × 6.0 m / s using a FastPrep24 instrument (MP Biomedicals). The homogenate is incubated at room temperature for 5 minutes, then diluted 1:4–1:6 with GLB and evaluated using the SteadyGlo Luciferase assay system (Promega). Specifically, 50 μL of diluted tissue homogenate is reacted with 50 μL of SteadyGlo substrate, shaken for 10 seconds, followed by incubation for 5 minutes, and then the luminescence is measured using CentroXS 3 Quantification was performed using an LB 960 luminometer (Berthold Technologies, Germany) or a Filter Max F5 Microplate Reader (Molecular Devices, USA). The amount of assayed protein was determined using the BCA protein assay kit (Pierce, Rockford, IL). Relative luminescence units (RLU) were then normalized to total protein μg or assayed tissue weight (g). A standard curve was created using QuantiLum recombinant luciferase (Promega) to convert RLU to luciferase ng.

[0147] FLuc mRNA (7202) 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. In the presence of the substrate, luciferin, it emits bioluminescence. This capped and polyadenylated mRNA is modified with 5-methiooxyuridine to optimize it for mammalian systems.

[0148] Example 2 In vivo evaluation of immunoglobulin G (IgG) mRNA using lipid nanoparticle compositions The lipids of formula (I), DSPC, cholesterol, and PEG-lipids of formula (II) are solubilized in ethanol at a molar ratio of 50:10:38.5:1.5 or 47.5:10:40.7:1.8. Lipid nanoparticles (LNPs) are prepared at a total lipid to mRNA weight ratio of approximately 10:1 to 40:1. Briefly, mRNA is diluted to 0.2 mg / mL in 10-50 mM citrate buffer, pH 4-6 or 10-25 mM acetate buffer, pH 4-6. Using a syringe pump, the ethanol-soluble lipid solution and the mRNA aqueous solution are mixed at a ratio of approximately 1:5 to 1:3 (vol / vol) at a total flow rate exceeding 15 mL / min. Then, the ethanol is removed and the external buffer is replaced with PBS by dialysis. Finally, the lipid nanoparticles are filtered through a 0.2 μm pore sterile filter.

[0149] The study will be conducted in 6-8 week old CD-1 / ICR mice (Charles River or Inotiv) in accordance with guidance established by the Animal Care Committee (ACC) and the Canadian Animal Care Association (CCAC). Various doses of mRNA-lipid nanoparticles will be administered systemically by tail vein injection, and the animals will be sacrificed at a specific time point (e.g., 24 hours) after administration. Whole blood will be collected, and serum will be separated by centrifugation of the whole blood tube at 2000 × g for 10 minutes at 4°C. The serum will be stored at -80°C until ready for analysis.

[0150] For the Immunoglobulin G (IgG) ELISA (Life Diagnostics Human IgG ELISA kit), serum samples are diluted 100 to 20,000 times with 1x diluent solution. 100 μL of diluted serum is duplicated and distributed with human IgG standard into an anti-human IgG coated 96-well plate, and incubated at 150 rpm in a plate shaker at 25°C for 45 minutes. The wells are washed five times using a plate washer with 1x washing solution (400 μL / well). 100 μL of HRP conjugate is added to each well, and the mixture is incubated in a plate shaker under the same conditions as above. The wells are washed five times using a plate washer with 1x washing solution (400 μL / well). 100 μL of TMB reagent is added to each well, and the mixture is incubated in a plate shaker under the same conditions as above. The reaction is stopped by adding 100 μL of stop solution to each well. The absorbance is read at 450 nm (A450) using a microplate reader. The amount of human IgG in mouse serum is determined by plotting the A450 value of the assay standard against the human IgG concentration.

[0151] Example 3 pK of formulated lipids a decision As described elsewhere, the pK of formulated lipids a This correlates with the effectiveness of LNPs for nucleic acid delivery (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176 (2010)). In one embodiment, pK a The preferred range is approximately 5 to 7. The pK of each lipid aThis can be determined using lipid nanoparticles with an assay based on 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). Lipid nanoparticles containing compound (I) / DSPC / cholesterol / PEG-lipid (50:10:38.5:1.5 or 47.5:10:40.7:1.8 mol%) in PBS at a total lipid concentration of 0.4 mM are prepared using the in-line process described in Example 1. TNS is prepared as a 100 μM stock solution in distilled water. The vesicles are diluted to 24 μM lipid in 2 mL with a buffer solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl in a pH range of 2.5–11. A fixed amount of TNS solution is added to achieve a final concentration of 1 μM. After vortex mixing, the fluorescence intensity is measured at room temperature using an SLM Aminco Series 2 Luminescence Spectrophotometer with excitation and emission wavelengths of 321 nm and 445 nm. Sigmoid optimality analysis is applied to the fluorescence data, and the pK a This is measured as the pH at which the fluorescence intensity reaches half of its maximum value.

[0152] Example 4 Determination of the efficacy of lipid nanoparticle formulations containing various cationic lipids using IgG mRNA-expressing rodent models. Representative compounds of the present invention shown in Table 2 were formulated using the following molar ratios: 50% cationic lipid / 10% distearoyl phosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid 2-[2-(ω-methoxy(polyethylene glycol)] 2000 (ethoxy)-N,N-ditetradecylacetamide) or 47.5% cationic lipid / 10% DSPC / 40.7% cholesterol / 1.8% PEG lipid. Relative activity was determined by measuring the amount of human IgG in mouse serum, as described in Example 1. Activity was compared at doses of 1.0 or 0.3 mg mRNA / kg and expressed as luciferase (ng) per 1 g of liver measured 4 hours after administration, as described in Example 1, or as IgG (μg) per 1 mL of serum measured 24 hours after administration, as described in Example 2. Compound numbers in Table 2 are referenced from compound numbers in Table 1.

[0153]

Table 6

[0154] Synthesis Example 1 Synthetic Routes of Compounds I-1 and I-2

Chem.

[0155] [ka] Synthesis of N,N-didecyl-8-(methylamino)octanamide A solution of 8-bromo-N,N-didecyloctanamide (1 equivalent) and methylamine (5 equivalents) in acetonitrile (10 mL / mmol) was heated overnight under reflux. The reaction mixture was concentrated, and the crude product was purified by column chromatography (Hex / Â 95:5~0:100, followed by DCM / 3%NH3 in MeOH solution 100:0~90:10). The product was obtained as a pale yellow oily substance (1.4 g, 3.09 mmol, 77%). ESI-MS: C 29 H 60 The calculated MW [M+H]+ value for N2O is 453.48; the measured value is 453.67.

[0156] [ka] Synthesis of N,N-didecyl-8-((5-hydroxypentyl)amino)octanamide N,N-didecyl-8-((5-hydroxypentyl)amino)octanamide was prepared from 8-bromo-N,N-didecyloctanamide (2 g, 3.99 mmol) and 5-amino-1-pentanol (2.05 g, 19.89 mmol) using a method similar to that used for N,N-didecyl-8-(methylamino)octanamide. The product was obtained as a pale yellow oily substance (1.2 g, 2.28 mmol, 57%). ESI-MS: C 33 H 68 N2O2 MW [M+H] + Calculated value: 525.54; Measured value: 526.64.

[0157] [ka] Synthesis of 8-bromooctyl(4-nitrophenyl) carbonate To a solution of 8-bromo-1-octanol (1.0 equivalent, 5 g, 23.91 mmol) in dichloromethane (3 mL / mmol), p-nitrophenyl chloroformate (1.2 equivalents, 5.78 g, 28.69 mmol) and pyridine (dropwise, 1.3 equivalents, 2.46 g, 31.08 mmol) were added under N2 conditions at room temperature. The resulting mixture was stirred overnight at room temperature. Subsequently, the reaction mixture was diluted with water and dichloromethane (DCM), and the aqueous and organic phases were separated. The aqueous phase was extracted twice with DCM, dried over Na2SO4, and concentrated under reduced pressure. The crude product was purified by column chromatography (Hex / siRNA, 100:0~80:20). The product was obtained as a colorless oil (7.5 g, 20 mmol, 84%). ESI-MS: C 15 H 20 m / z of BrNO5 [M+Na] + Calculated values: 396.04 and 398.04; measured values: 396.08 and 398.09.

[0158] [ka] Synthesis of 8-bromooctyldecyl carbonate To a solution of 8-bromooctyl(4-nitrophenyl) carbonate (1 equivalent, 5 g, 13.36 mmol) in DCM (3.5 mL / mmol), 1-decanol (4 equivalents, 8.45 g, 53.44 mmol), DMAP (0.2 equivalents, 0.33 g, 2.67 mmol), and pyridine (dropwise addition, 1.3 equivalents, 1.37 g, 17.37 mmol) were added at room temperature under N2. After stirring overnight at room temperature under N2, the reaction mixture was diluted with water and DCM. The resulting aqueous and organic phases were separated, the aqueous phase was extracted twice with DCM, dried over Na2SO4, and concentrated under reduced pressure. The crude product was purified by column chromatography (Hex / siRNA, 100:0~80:20). The product was obtained as a colorless oil (3.47 g, 8.85 mmol, 66%). 1H NMR (400 MHz, CDCl3) δ 4.12 (t, J = 6.7 Hz, 4H), 3.40 (t, J = 6.8 Hz, 2H), 1.89-1.78 (m, 2H), 1.66 (p, J = 6.7 Hz, 4H), 1.48-1.18 (m, 22H), 0.94-0.81 (m, 3H), ESI-MS: C 19 H 37 BrO3 m / z [M+H] + Calculated values: 393.20 and 395.20; measured values: 393.30 and 395.32.

[0159]

[0160] Synthesis of Decyl (8-((8-(didecylamino)-8-oxooctyl)(5-hydroxypentyl)amino)octyl) carbonate (compound I-1) To a solution of 8-bromooctyldecyl carbonate (1.2 equivalents, 0.36 g, 0.91 mmol) in acetonitrile (5.5 mL / mmol), DIPEA (4 equivalents, 0.39 g, 3.05 mmol) and N,N-didecyl-8-((5-hydroxypentyl)amino)octanamide (1.0 equivalent, 0.4 g, 0.76 mmol) were added. The reaction was carried out in a sealed tube at 80°C for 24 hours. The reaction mixture was then cooled to room temperature and concentrated under reduced pressure. The crude product was purified by column chromatography (Hex / 1% Et3N siRNA solution, 95:5~0:100). The product was obtained as a colorless oil (0.275 g, 0.328 mmol, 33%). 1 H NMR (400 MHz, CDCl3) δ 4.11 (t, J = 6.7 Hz, 4H), 3.64 (t, J = 6.5 Hz, 2H), 3.38 - 3.23 (m, 2H), 3.22 - 3.07 (m, 2H), 2.38 (m, 6H), 2.30 - 2.11 (m, 2H), 1.84 - 1.07 (m, 77H), 0.98 - 0.81 (m, 9H), ESI-MS: C 52 H 104 N2O5 m / z [M+H]+ Calculated value: 837.79, Measured value: 837.49.

[0161] [ka]

[0162] Synthesis of Decyl (8-((8-(didecylamino)-8-oxooctyl)(methyl)amino)octyl) carbonate (compound I-2) Compound I-2 was prepared using a method similar to that for compound I-1, from 8-bromooctyldecyl carbonate (0.417 g, 1.06 mmol), N,N-didecyl-8-(methylamino)octanamide (0.4 g, 0.88 mmol), and DIPEA (0.46 g, 3.53 mmol). The product was obtained as a colorless oil (0.262 g, 0.342 mmol, 34%). 1 H NMR (400 MHz, CDCl3) δ 4.11 (t, J = 6.7 Hz, 4H), 3.33 - 3.23 (m, 2H), 3.23 - 3.14 (m, 2H), 2.27 (m, 6H), 2.19 (s, 3H), 1.73 - 1.58 (m, 8H), 1.58 - 1.39 (m, 9H), 1.28 (d, J = 8.7 Hz, 57H), 0.88 (t, J = 6.6 Hz, 9H), ESI-MS: C 48 H 96 N2O4 m / z [M+H] + Calculated value: 765.75, Measured value: 765.40.

[0163] Synthesis Example 2 Synthetic routes of compounds I-3 and I-4 [ka] Synthesis of 4-nitrophenylpentadecane-8-yl carbonate 4-Nitrophenylpentadecane-8-yl carbonate was obtained from pentadecane-8-ol (2 g, 8.75 mmol), p-nitrophenyl chloroformate (2.12 g, 10.50 mmol), and pyridine (0.98 g, 11.38 mmol) according to the general procedure of Synthesis Example 1. The product was obtained as a colorless oil (2.75 g, 6.99 mmol, 80%). ESI-MS: C 22 H 35 NO5 m / z [M+H] + Calculated value: 394.53, Measured value: 394.90.

[0164] [ka] Synthesis of 8-bromooctylpentadecane-8-yl carbonate 8-Bromooctylpentadecane-8-yl carbonate was obtained from 4-nitrophenylpentadecane-8-yl carbonate (2.5 g, 6.35 mmol), 8-bromo-1-octanol (5.31 g, 25.41 mmol), DMAP (0.155 g, 1.27 mmol), and pyridine (0.98 g, 8.25 mmol) according to the general procedure of Synthesis Example 1. The product was obtained as a colorless solid (1.62 g, 3.49 mmol, 55%). 1 H NMR (400 MHz, CDCl3) δ 4.75-4.61 (m, 1H), 4.11 (t, J = 6.7 Hz, 2H), 3.40 (t, J = 6.8 Hz, 2H), 1.85 (m, 2H), 1.67 (m, 2H), 1.55 (m, 2H), 1.47-1.20 (m, 30H), 0.92-0.83 (m, 6H), ESI-MS: C 24 H 47 m / z of BrO3 [M+Na] + Calculated values: 485.26 and 487.26; measured values: 485.03 and 487.49.

[0165] [ka]

[0166] Synthesis of 8-((8-(didecylamino)-8-oxooctyl)(5-hydroxypentyl)amino)octylpentadecan-8-yl carbonate (compound I-3) Compound I-3 was obtained from 8-bromooctylpentadecane-8-yl carbonate (0.423 g, 0.914 mmol), N,N-didecyl-8-((5-hydroxypentyl)amino)octanamide (0.4 g, 0.76 mmol), and DIPEA (0.39 g, 3.05 mmol) according to the general procedure of Synthesis Example 1. The product was obtained as a colorless oil (0.274 g, 0.302 mmol, 35%). 1 H NMR (400 MHz, CDCl3) δ 4.76 - 4.63 (m, 1H), 4.11 (t, J = 6.7 Hz, 2H), 3.64 (t, J = 6.5 Hz, 2H), 3.32 - 3.23 (m, 2H), 3.22 - 3.14 (m, 2H), 2.38 (m, 6H), 2.32 - 2.21 (m, 2H), 1.86 - 1.14 (m, 83H), 0.99 - 0.76 (m, 12H), ESI-MS: C 57 H 114 N2O5 m / z [M+H] + Calculated value: 907.88, Measured value: 907.95.

[0167] [ka]

[0168] Synthesis of 8-((8-(didecylamino)-8-oxooctyl)(methyl)amino)octylpentadecane-8-yl carbonate (compound I-4) Compound I-4 was obtained from 8-bromooctylpentadecane-8-yl carbonate (0.491 g, 1.06 mmol), N,N-didecyl-8-(methylamino)octanamide (0.4 g, 0.88 mmol), and DIPEA (0.46 g, 3.53 mmol) according to the general procedure of Synthesis Example 1. The product was obtained as a colorless oil (0.253 g, 0.302 mmol, 32.6%). 1 H NMR (400 MHz, CDCl3) δ 4.68 (p, J = 7.1, 6.3 Hz, 1H), 4.11 (t, J = 6.7 Hz, 2H), 3.32 - 3.23 (m, 2H), 3.23 - 3.13 (m, 2H), 2.27 (m, 6H), 2.19 (s, 3H), 1.66 (m, 7H), 1.61 - 1.40 (m, 12H), 1.40 - 1.08 (m, 58H), 1.03 - 0.57 (m, 12H), ESI-MS: C 53 H 106 N2O4 m / z [M+H] + Calculated value: 835.83, Measured value: 835.72.

[0169] Synthesis Example 3 2-Butyloctyl 10-(N-decyl-3-(dimethylamino)propanamide)-19-(didecylamino)-19-oxononadecanoate (Compound I-8) [ka]

[0170] Synthesis of 19-(didecylamino)-10,19-dioxononadecanoic acid HATU (7.6 mmol, 2.9 g) was added to a solution of 10-oxononadecanedioic acid (5.8 mmol, 2.0 g), didecylamine (5.8 mmol, 1.7 g), and DIPEA (17.5 mmol, 3.05 mL) in DCM (29 mL), and the reaction mixture was stirred at room temperature for 50 minutes. The reaction mixture was concentrated, and the crude product was partitioned into siRNA and 1 M HCl. The organic layer was separated, dried over Na2SO4, and concentrated. The crude product was purified by automated flash chromatography (in a DCM solution of 1% to 10% MeOH). The isolated product was ground over hexane and filtered. The filtrate was further purified by automated flash chromatography (in a hexane solution of 10% to 70% siRNA) to obtain the desired product (500 mg, 14%).

[0171] [ka]

[0172] Synthesis of 2-butyloctyl 19-(didecylamino)-10,19-dioxononadecanoate A mixture of 19-(didecylamino)-10,19-dioxononadecanoic acid (0.8 mmol, 0.5 g), 2-butyloctan-1-ol (1.4 mmol, 262 mg), 4-dimethylaminopyridine (31.2 mmol, 147 mg), and DCC (1.6 mmol, 332 mg) in DCM (8 mL) was stirred overnight at room temperature. The reaction mixture was concentrated, the crude product was suspended in hexane, and filtered. The filtrate was purified by automated flash chromatography (1% to 15% siRNA solution in hexane) to obtain the desired product (396 mg, 62%).

[0173] [ka]

[0174] Synthesis of 2-butyloctyl 10-(decylamino)-19-(didecylamino)-19-oxononadecanoate Sodium triacetoxyborohydride (1.0 mmol, 215 mg) was added to a mixture of 2-butyloctyl 19-(didecylamino)-10,19-dioxononadecanoate (0.51 mmol, 400 mg), decylamine (0.76 mmol, 119 mg), and acetic acid (0.76 mmol, 0.043 mL) in dichloroethane (3 mL). The reaction mixture was stirred at room temperature for 22 hours. Another 1.0 mmol, 215 mg of sodium triacetoxyborohydride was added, and the reaction mixture was stirred for a further 24 hours. The reaction mixture was concentrated, and the crude product was partitioned into ethyl acetate and saturated NaHCO3. The organic layer was separated, dried over Na2SO4, and concentrated. Purification by automated flash chromatography (5% to 100% ethyl acetate in hexane solution) yielded the desired product (420 mg, 89%).

[0175] [ka]

[0176] Synthesis of 2-butyloctyl 10-(N-decyl-3-(dimethylamino)propanamide)-19-(didecylamino)-19-oxononadecanoate (compound I-8) HATU (0.16 mmol, 61 mg) was added to a mixture of 2-butyloctyl 10-(decylamino)-19-(didecylamino)-19-oxononadecanoate (0.11 mmol, 100 mg), 3-(dimethylamino)propanoate (0.14 mmol, 21 mg), and DIPEA (0.43 mmol, 0.075 mL) in DCM (1.1 mL). The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was partitioned into ethyl acetate and saturated NaHCO3. The organic layer was separated, dried over Na2SO4, and concentrated. Purification by automated flash chromatography (5%-65% ethyl acetate in hexane solution and 1% Et3N) yielded compound I-8 (69 mg, 62%). 1H NMR (400 MHz, CDCl3) δ 3.96 (m, 2H), 3.65 - 3.55 (m, 1H), 3.32 - 3.24 (m, 2H), 3.23 - 3.15 (m, 2H), 3.07 - 2.99 (m, 2H), 2.65 (m, m / z Formula: C 66 H 131 Calculated value of N3O4 = 1030.0. Measured value [M+H] + = 1031.3

[0177] Synthesis Example 4 8-((8-(didecylamino)-8-oxooctyl)(5-hydroxypentyl)amino)octyl(2-hexyldecyl) carbonate (compound I-19) [ka]

[0178] Synthesis of 2-hexyldecyl 1H-imidazole-1-carboxylate A solution of commercially available 2-hexyldecane-1-ol (7.9 mmol, 2.3 mL), CDI (7.9 mmol, 1.3 g), and potassium hydroxide (20 mg) in DCM (15 mL) was stirred in a sealed flask at 60°C for 6 hours. The reaction mixture was washed with water, dried over Na2SO4, and concentrated. The resulting crude product was purified by flash chromatography (in a hexane solution of 0% to 20% Â) to obtain 2-hexyldecyl 1H-imidazole-1-carboxylate (1.73 g, 46%).

[0179] Synthesis of 8-bromooctyl(2-hexyldecyl) carbonate A solution of 2-hexyldecyl 1H-imidazole-1-carboxylate (5.1 mmol, 1.73 g), commercially available 8-bromooctan-1-ol (5.1 mmol, 0.9 mL), and potassium hydroxide (10 mg) in DCM (15 mL) was stirred in a sealed flask at 60°C for 6 hours. The resulting crude material was purified by flash chromatography (in a hexane solution of 0% to 20% Â) to obtain 8-bromooctyl(2-hexyldecyl) carbonate (1.16 g, 48%).

[0180] Synthesis of 8-((8-(didecylamino)-8-oxooctyl)(5-hydroxypentyl)amino)octyl(2-hexyldecyl) carbonate A mixture of 8-bromooctyl(2-hexyldecyl) carbonate (0.71 mmol, 339 mg), N,N-didecyl-8-((5-hydroxypentyl)amino)octanamide (0.65 mmol, 339 mg), KI (1.9 mmol, 321 mg), and DIEA (2.6 mmol, 0.45 mL) in acetonitrile (2 mL) was heated in a microwave at 140 °C for 30 minutes. The reaction mixture was concentrated, and the resulting crude product was suspended in hexane and filtered. The filtered product was purified by flash chromatography to obtain the desired product (109 mg, 18%). 1 H NMR (400 MHz, MeOD) δ 4.15 (t, J = 6.5 Hz, 2H), 4.07 (d, J = 5.7 Hz, 2H), 3.59 (t, J = 6.6 Hz, 2H), 2.57 - 2.50 (m, 6H), 2.39 (t, 3H), 1.72 - 1.49 (m, 18H), 1.47 - 1.22 (m, 53H), 0.98 - 0.90 (m, 12H). ESI-MS: m / z C 58 H 116 Calculated value of N2O5 = 920.89, measured value [M+H] + = 922.17.

[0181] Synthesis Example 5 7-((8-(didecylamino)-8-oxooctyl)(5-hydroxypentyl)amino)heptylheptadecan-9-yl carbonate (compound I-18) [ka]

[0182] Synthesis of heptadecan-9-yl 1H-imidazole-1-carboxylate Heptadecan-9-yl 1H-imidazole-1-carboxylate was prepared from commercially available heptadecan-9-ol following the procedure outlined for 2-hexyldecyl 1H-imidazole-1-carboxylate. Yield (1.85 g, 70%).

[0183] Synthesis of 7-bromoheptylheptadecane-9-yl carbonate 7-Bromoheptylheptadecan-9-yl carbonate was prepared from commercially available 7-bromoheptan-1-ol following the procedure outlined for 8-bromooctyl(2-hexyldecyl) carbonate. Yield (1.91 g, 76%).

[0184] Synthesis of compound I-18 Compound I-18 was prepared according to the procedure outlined herein and the reaction scheme described above. Yield (119 mg, 23%). 1 H NMR (400 MHz, MeOD) δ 4.78 - 4.60 (m, 1H), 4.13 (t, J = 6.5 Hz, 2H), 3.57 (t, J = 6.6 Hz, 2H), 2.54 - 2.45 (m, 6H), 2.37 (t, J = 7.5 Hz, 2H), 1.72 - 1.46 (m, 9H), 1.44 - 1.30 (m, 72H), 0.96 - 0.88 (m, 12H). ESI-MS: m / z C 58 H 116 Calculated value of N2O5 = 920.89, measured value [M+H] + = 922.18.

[0185] Synthesis Example 6 9-((8-(didecylamino)-8-oxooctyl)(5-hydroxypentyl)amino)nonyltridecane-7-yl carbonate (compound I-17) [ka]

[0186] Synthesis of tridecane-7-yl 1H-imidazole-1-carboxylate Tridecane-7-yl 1H-imidazole-1-carboxylate was prepared from commercially available tridecane-7-ol according to the procedure outlined herein. Yield (559 mg, 76%).

[0187] Synthesis of 9-bromonoltridecane-7-yl carbonate The desired product was prepared from commercially available 9-bromononan-1-ol and tridecan-7-yl 1H-imidazole-1-carboxylate according to the procedure outlined herein. Yield (716 mg, 84%).

[0188] Synthesis of compound I-17 Compound I-17 was prepared according to the procedure outlined herein and the reaction scheme described above. Yield (156 mg, 28%). 1 H NMR (400 MHz, MeOD) δ 4.76 - 4.65 (m, 1H), 4.15 (t, J = 6.5 Hz, 2H), 3.59 (t, J = 6.6 Hz, 2H), 2.55 - 2.47 (m, 7H), 2.39 (t, J = 7.5 Hz, 2H), 1.77 - 1.22 (m, 82H), 0.99 - 0.91 (m, 12H). ESI-MS: m / z C 56 H 112 Calculated value of N2O5 = 892.86, measured value [M+H] + = 894.15.

[0189] Synthesis Example 7 5-((8-(didecylamino)-8-oxooctyl)(5-hydroxypentyl)amino)pentylnonan-5-yl carbonate (Compound I-16) [ka]

[0190] Synthesis of nonan-5-yl 1H-imidazole-1-carboxylate Nonan-5-yl 1H-imidazole-1-carboxylate was prepared from commercially available nonan-5-ol according to the procedure outlined herein. Yield (256 mg, 31%).

[0191] Synthesis of 5-bromopentylnonane-5-yl carbonate 5-Bromopentylnonan-5-yl carbonate was prepared from commercially available 5-bromopentan-1-ol and nonan-5-yl 1H-imidazole-1-carboxylate according to the procedure outlined herein. Yield (105 mg, 29%).

[0192] Synthesis of compound I-16 Compound I-16 was prepared according to the procedure outlined herein and the reaction scheme described above. Yield (105 mg, 33%). 1 H NMR (400 MHz, MeOD) δ 4.71 - 4.62 (m, 1H), 4.12 (t, J = 6.5 Hz, 2H), 3.55 (t, J = 6.6 Hz, 2H), 2.52 - 2.42 (m, 6H), 2.35 (t, J = 7.5 Hz, 2H), 1.77 - 1.22 (m, 57H), 0.97 - 0.83 (m, 12H). ESI-MS: m / z C 48 H 96 Calculated value of N2O5 = 780.73, measured value [M+H] + = 781.89.

[0193] Synthesis Example 8 5-((8-(didecylamino)-8-oxooctyl)(5-hydroxypentyl)amino)pentylhenicosan-11-yl carbonate (compound I-15) [ka]

[0194] Synthesis of henicosan-11-yl 1H-imidazole-1-carboxylate Henicosan-11-yl 1H-imidazole-1-carboxylate was prepared from commercially available henicosan-11-ol according to the procedure outlined herein. Yield (357 mg, 55%).

[0195] Synthesis of 5-bromopentylhenicosan-11-yl carbonate 5-Bromopentylhenicosan-11-yl carbonate was prepared from commercially available 5-bromopentan-1-ol and henicosan-11-yl 1H-imidazole-1-carboxylate according to the procedure outlined herein. Yield (198 mg, 45%).

[0196] Synthesis of compound I-15 Compound I-15 was prepared according to the procedure outlined herein and the reaction scheme described above. Yield (63.5 mg, 19%). 1 H NMR (400 MHz, MeOD) δ 4.73 - 4.62 (m, 1H), 4.13 (t, J = 6.4 Hz, 2H), 3.55 (t, J = 6.6 Hz, 2H), 2.53 - 2.41 (m, 6H), 2.35 (t, J = 7.5 Hz, 2H), 1.77 - 1.18 (m, 100H), 0.97 - 0.83 (m, 12H). ESI-MS: m / z C 60 H 120 Calculated value of N2O5 = 948.92, measured value [M+H] + = 950.22.

[0197] Synthesis Example 9 9-((8-(didecylamino)-8-oxooctyl)(5-hydroxypentyl)amino)nonylnonan-5-yl carbonate (Compound I-14) [ka]

[0198] Synthesis of nonan-5-yl 1H-imidazole-1-carboxylate Nonan-5-yl 1H-imidazole-1-carboxylate was prepared from commercially available nonan-5-ol according to the procedure outlined herein. Yield (268 mg, 73%).

[0199] Synthesis of 9-bromononylnonane-5-yl carbonate 9-Bromononylnonan-5-yl carbonate was prepared from commercially available 9-bromononan-1-ol and nonan-5-yl 1H-imidazole-1-carboxylate according to the procedure outlined herein. Yield (240 mg, 55%).

[0200] Synthesis of compound I-14 Compound I-14 was prepared according to the procedure outlined herein and the reaction scheme described above. Yield (89.3 mg, 19%). 1 H NMR (400 MHz, MeOD) δ 4.73 - 4.62 (m, 1H), 4.11 (t, J = 6.5 Hz, 2H), 3.55 (t, J = 6.6 Hz, 2H), 2.55 - 2.42 (m, 6H), 2.35 (t, J = 7.5 Hz, 2H), 1.72 - 1.19 (m, 68H), 0.97 - 0.84 (m, 12H). ESI-MS: m / z C 52 H 104 Calculated value of N2O5 = 836.79, measured value [M+H] + = 838.07.

[0201] Synthesis Example 10 9-((8-(didecylamino)-8-oxooctyl)(5-hydroxypentyl)amino)nonylhenicosan-11-yl carbonate (compound I-13) [ka]

[0202] Synthesis of 9-bromonolhenicosan-11-yl carbonate 9-Bromononylhenicosan-11-yl carbonate was prepared from commercially available 9-bromonan-1-ol and henicosan-11-yl 1H-imidazole-1-carboxylate according to the procedure outlined herein. Yield (269 mg, 40%).

[0203] Synthesis of compound I-13 Compound I-13 was prepared according to the procedure outlined herein and the reaction scheme described above. Yield (59.7 mg, 27%). 1 H NMR (400 MHz, MeOD) δ 4.73 - 4.62 (m, 1H), 4.12 (t, J = 6.4 Hz, 2H), 3.55 (t, J = 6.6 Hz, 2H), 2.54 - 2.43 (m, 6H), 2.35 (t, J = 7.5 Hz, 2H), 1.71 - 1.21 (m, 94H), 0.95 - 0.85 (m, 12H). ESI-MS: m / z C 64 H 128 Calculated value of N2O5 = 1004.98, measured value [M+H] + = 1006.20.

[0204] Synthesis Example 11 2-Butyloctyl(8-((8-(didecylamino)-8-oxooctyl)(5-hydroxypentyl)amino)octyl) carbonate (Compound I-12) [ka]

[0205] Synthesis of 2-butyloctyl 1H-imidazole-1-carboxylate 2-Butyloctyl 1H-imidazole-1-carboxylate was prepared from commercially available 2-butyloctan-1-ol according to the procedure outlined herein. Yield (743 mg, 43%).

[0206] Synthesis of 8-bromooctyl(2-butyloctyl) carbonate 8-Bromooctyl(2-butyloctyl) carbonate was prepared from commercially available 8-bromooctan-1-ol and 2-butyloctyl 1H-imidazole-1-carboxylate according to the procedure outlined herein. Yield (488 mg, 44%).

[0207] Synthesis of compound I-12 Compound I-12 was prepared according to the procedure outlined herein and the reaction scheme described above. Yield (72.7 mg, 25%). 1 H NMR (400 MHz, MeOD) δ 4.11 (t, J = 6.5 Hz, 2H), 4.03 (d, J = 5.7 Hz, 2H), 3.55 (t, J = 6.6 Hz, 2H), 2.53 - 2.43 (m, 6H), 2.35 (t, J = 7.5 Hz, 2H), 1.72 - 1.23 (m, 82H), 0.96 - 0.85 (m, 12H). ESI-MS: m / z C 54 H 108 Calculated value of N2O5 = 864.83, measured value [M+H] + = 866.11.

[0208] Synthesis Example 12 8-((8-(didecylamino)-8-oxooctyl)(5-hydroxypentyl)amino)octyl(2-ethylhexyl) carbonate (compound I-11) [ka]

[0209] Synthesis of 2-ethylhexyl 1H-imidazole-1-carboxylate 2-ethylhexyl 1H-imidazole-1-carboxylate was prepared from commercially available 2-ethylhexane-1-ol according to the procedure outlined herein. Yield (802 mg, 58%).

[0210] Synthesis of 8-bromooctyl(2-ethylhexyl) carbonate 8-Bromooctyl(2-ethylhexyl) carbonate was prepared from commercially available 8-bromooctan-1-ol and 2-ethylhexyl 1H-imidazole-1-carboxylate according to the procedure outlined herein. Yield (718 mg, 55%).

[0211] Synthesis of compound I-11 Compound I-11 was prepared according to the procedure outlined herein and the reaction scheme described above. Yield (79.7 mg, 24%). 1 H NMR (400 MHz, MeOD) δ 4.11 (t, J = 6.6 Hz, 2H), 4.04 (d, J = 5.7 Hz, 2H), 3.55 (t, J = 6.6 Hz, 2H), 2.52 - 2.43 (m, 7H), 2.35 (t, J = 7.5 Hz, 2H), 1.71 - 1.22 (m, 68H), 0.96 - 0.84 (m, 12H). ESI-MS: m / z C 50 H 100 Calculated value of N2O5 = 808.76, measured value [M+H] + = 809.99.

[0212] Synthesis Example 13 7-((8-(dioctylamino)-8-oxooctyl)(5-hydroxypentyl)amino)heptylheptadecan-9-yl carbonate (compound I-23) [ka]

[0213] Synthesis of 8-bromo-N,N-dioctyloctanamide 8-Bromo-N,N-dioctyloctanamide was prepared from commercially available dioctylamine and 8-bromooctanoyl chloride according to the procedure outlined herein. Yield (642 mg, 35%).

[0214] Synthesis of 8-((5-hydroxypentyl)amino)-N,N-dioctyloctanamide 8-((5-hydroxypentyl)amino)-N,N-dioctyloctanamide was prepared from commercially available 5-aminopentan-1-ol and 8-bromo-N,N-dioctyloctanamide according to the procedure outlined herein. Yield (294 mg, 88%).

[0215] Synthesis of compound I-23 Compound I-23 was prepared according to the reaction scheme outlined here. Yield (79.7 mg, 24%). 1 H NMR (400 MHz, MeOD) δ 4.72 - 4.62 (m, 1H), 4.11 (t, J = 6.5 Hz, 2H), 3.55 (t, J = 6.5 Hz, 2H), 2.53 - 2.42 (m, 6H), 2.35 (t, J = 7.5 Hz, 2H), 1.72 - 1.21 (m, 76H), 0.95 - 0.85 (m, 12H). ESI-MS: m / z C 54 H 108 Calculated value of N2O5 = 864.83, measured value [M+H] + = 866.09.

[0216] Synthesis Example 14 7-((8-(didodecylamino)-8-oxooctyl)(5-hydroxypentyl)amino)heptylheptadecan-9-yl carbonate (compound I-22) [ka]

[0217] Synthesis of 8-bromo-N,N-didodecyloctanamide 8-Bromo-N,N-didodecyloctanamide was prepared from commercially available didodecylamine and 8-bromooctanoyl chloride according to the procedure outlined herein. Yield (663 mg, 29%).

[0218] Synthesis of N,N-didodecyl-8-((5-hydroxypentyl)amino)octanamide N,N-didodecyl-8-((5-hydroxypentyl)amino)octanamide was prepared from commercially available 5-aminopentan-1-ol and 8-bromo-N,N-didodecyloctanamide according to the procedure outlined herein. Yield (220 mg, 64%).

[0219] Synthesis of compound I-22 Compound I-22 was prepared according to the procedure outlined herein and the reaction scheme described above. Yield (79.1 mg, 19%).

[0220] 1 H NMR (400 MHz, MeOD) δ 4.73 - 4.63 (m, 1H), 4.11 (t, J = 6.5 Hz, 2H), 3.55 (t, J = 6.5 Hz, 2H), 2.58 - 2.45 (m, 6H), 2.35 (t, J = 7.5 Hz, 2H), 1.71 - 1.20 (m, 101H), 0.96 - 0.85 (m, 12H). ESI-MS: m / z C 62 H 124 Calculated value of N2O5 = 976.95, measured value [M+H] + = 978.20.

[0221] Synthesis Example 15 7-((6-(didodecylamino)-6-oxohexyl)(5-hydroxypentyl)amino)heptylheptadecan-9-yl carbonate (compound I-21) [ka]

[0222] Synthesis of 6-bromohexanoyl chloride 6-bromohexanoyl chloride was prepared from commercially available 6-bromohexanoic acid according to the procedure outlined herein.

[0223] Synthesis of 6-bromo-N,N-didodecylhexaneamide 6-Bromo-N,N-didodecylhexanamide was prepared from commercially available didodecylamine and 6-bromohexanoyl chloride according to the procedure outlined herein. Yield (1.08 g, 44%).

[0224] Synthesis of N,N-didodecyl-6-((5-hydroxypentyl)amino)hexanamide N,N-didodecyl-6-((5-hydroxypentyl)amino)hexaneamide was prepared from commercially available 5-aminopentan-1-ol and 6-bromo-N,N-didodecylhexaneamide according to the procedure outlined herein. Yield (246 mg, 47%).

[0225] Synthesis of compound I-21 Compound I-21 was prepared according to the procedure outlined herein and the reaction scheme described above. Yield (53.8 mg, 14%). 1 H NMR (400 MHz, MeOD) δ 4.73 - 4.62 (m, 1H), 4.11 (t, J = 6.5 Hz, 3H), 3.55 (t, J = 6.5 Hz, 2H), 2.57 - 2.44 (m, 7H), 2.36 (t, J = 7.4 Hz, 3H), 1.71 - 1.19 (m, 106H), 0.96 - 0.85 (m, 12H). ESI-MS: m / z C 60 H 120 Calculated value of N2O5 = 948.92, measured value [M+H] + = 950.15.

[0226] Synthesis Example 16 7-((10-(dioctylamino)-10-oxodecyl)(5-hydroxypentyl)amino)heptylheptadecan-9-yl carbonate (compound I-20) [ka]

[0227] Synthesis of 10-bromodecanoyl chloride 10-Bromodecanoyl chloride was prepared from commercially available 10-bromodecanoic acid according to the procedure outlined herein.

[0228] Synthesis of 10-bromo-N,N-dioctyldecaneamide 10-Bromo-N,N-dioctyldecanamide was prepared from commercially available dioctylamine and 10-bromodecanoyl chloride according to the procedure outlined herein. Yield (1.01 g, 58%).

[0229] Synthesis of 10-((5-hydroxypentyl)amino)-N,N-dioctyldecaneamide 10-((5-hydroxypentyl)amino)-N,N-dioctyldecanamide was prepared from commercially available 5-aminopentan-1-ol and 10-bromo-N,N-dioctyldecanamide according to the procedure outlined herein. Yield (208 mg, 40%).

[0230] Synthesis of compound I-20 Compound I-20 was prepared according to the procedure outlined herein and the reaction scheme described above. Yield (21 mg, 5.6%). 1 H NMR (400 MHz, CDCl3) δ 4.74 - 4.61 (m, 1H), 4.11 (t, J = 6.8 Hz, 2H), 3.64 (t, J = 6.5 Hz, 2H), 3.33 - 3.13 (m, 4H), 2.46 - 2.32 (m, 6H), 2.30 - 2.22 (m, 2H), 1.72 - 1.17 (m, 79H), 0.93 - 0.79 (m, 12H). ESI-MS: m / z C56 H 112 Calculated value of N2O5 = 892.86, measured value [M+H] + = 894.13.

[0231] Synthesis Example 17 2-Butyloctyl 10-(N-decyl-4-(dimethylamino)butanamide)-19-(didecylamino)-19-oxononadecanoate (Compound I-25) [ka]

[0232] Synthesis of compound I-25 Compound I-25 was prepared from 2-butyloctyl 10-(decylamino)-19-(didecylamino)-19-oxononadecanoate and 4-(dimethylamino)butanoic acid by the procedure outlined in Synthesis Example 3. Yield (41 mg, 37%). 1 H NMR (400 MHz, CDCl3) δ 3.99 (dd, J = 5.8, 1.4 Hz, 2H), 3.70 - 3.60 (m, 1H), 3.34 - 3.26 (m, 2H), 3.25 - 3.17 (m, 2H), 3.10 - 3.01 (m, ESI-MS: m / z C 67 H 133 Calculated value of N3O4 = 1044.0, measured value [M+H] + = 1045.3.

[0233] Synthesis Example 18 2-Butyloctyl 19-(Didecylamino)-10-(N-Octyl-4-(Pyrrolidine-1-yl)butanamide)-19-Oxononadecanoate (Compound I-24) [ka]

[0234] Synthesis of 2-butyloctyl 19-(didecylamino)-10-(octylamino)-19-oxononadecanoate 2-Butyloctyl 19-(didecylamino)-10-(octylamino)-19-oxononadecanoate was prepared from 2-butyloctyl 19-(didecylamino)-10,19-dioxononadecanoate and octan-1-amine by the procedure outlined in Synthesis Example 3. Yield (440 mg, 85%).

[0235] Synthesis of 2-butyloctyl 10-(4-chloro-N-octylbutanamide)-19-(didecylamino)-19-oxononadecanoate A mixture of 2-butyloctyl 19-(didecylamino)-10-(octylamino)-19-oxononadecanoate (0.18 mmol, 160 mg), triethylamine (0.18 mmol, 0.050 mL), and 4-chlorobutanoyl chloride (0.18 mmol, 43 mg) in dichloromethane (1.8 mL) was stirred at room temperature for 1 hour. The reaction mixture was concentrated, and the resulting crude product was purified (in a hexane solution of 0% to 20% ethylbenzoate) to obtain 2-butyloctyl 10-(4-chloro-N-octylbutanamide)-19-(didecylamino)-19-oxononadecanoate (130 mg, 73%).

[0236] [ka]

[0237] Synthesis of compound I-24 A mixture of 2-butyloctyl 10-(4-chloro-N-octylbutanamide)-19-(didecylamino)-19-oxononadecanoate (0.13 mmol, 130 mg), pyrrolidine (0.77 mmol, 0.065 mL), DIEA (0.39 mmol, 0.067 mL), and potassium iodide (0.39 mmol, 65 mg) in acetonitrile (0.4 mL) was heated in a microwave at 140°C for 40 minutes. The reaction mixture was concentrated, and the resulting crude product was suspended in siRNA:hexane:Et3N (5:95:1) and filtered. The filtrate was purified by flash chromatography (5%-65% siRNA solution in hexane and 1% Et3N) to obtain compound I-24 (106 mg, 79%). 1 H NMR (400 MHz, CDCl3) δ 3.96 (dd, J = 5.8, 0.9 Hz, 2H), 3.62 - 3.45 (m, 1H), 3.31 - 3.23 (m, 2H), 3.23 - 3.14 (m, 2H), 3.12 - 2.66 (m, ESI-MS: m / z C 67 H 131 Calculated value of N3O4 = 1042.0, measured value [M+H] + = 1043.3.

[0238] Combining the various embodiments described above, further embodiments may be provided. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent literature referenced herein, including but not limited to U.S. Provisional Application 63 / 508,772 filed June 16, 2023, are incorporated herein by reference as a whole. The aspects of the embodiments may be modified as necessary to provide further embodiments using concepts from various patents, applications, and publications.

[0239] These and other modifications may be made to the embodiments in light of the detailed description above. In general, the terms used in the following claims should not be construed as limiting the claims to the specific embodiments disclosed in the specification and claims, but rather as encompassing the entire scope of equivalents to which such claims are given, along with all possible embodiments. Accordingly, the claims are not limited by this disclosure.

Claims

1. Equation (I): 【Chemistry 1】 [During the ceremony, G １ is N or CH; G ２ G １ When is N, it is either a direct bond or G ２ G １ When CH -NR １ａ - and; R １ａ is C ２ -C ４ -C １２ alkyl optionally substituted with one or more substituents selected from the group consisting of oxo, -OH, and NH R １ Oxo, -OH, -N(R) are available upon request. １ｂ )R １ｃ C is substituted with one or more substituents selected from the group consisting of cycloalkyl or heteroaryl. １ -C ８ It is alkyl; R １ｂ and R １ｃ Each is independently hydrogen or C １ -C ４ It is alkyl; or R １ｂ and R １ｃ They combine with the nitrogen to which they are bound to form a heterocycline; R ２ -OC(=O)OR ２ａ -OC(=O)R ２ｂ , or -C(=O)OR ２ｃ And; R ２ａ is C ４ -C ２４ Alkyl, C ４ -C ２４ Alkenyl, or C ４ -C ２４ It is alkinyl; R ２ｂ and R ２ｃ The structure is as follows: 【Chemistry 2】 Having; R ２ｄ and R ２ｅ Each is independently C ４ -C １２ Alkyl, C ４ -C １２ Alkenyl, or C ４ -C １２ It is alkinyl; R ３ Ha -C(=O)N(R ３ａ )R ３ｂ or -NR ３ａ -C(=O)R ３ｂ And; R ３ａ and R ３ｂ Each is independently C ６ -C ２４ Alkyl, C ６ -C ２４ Alkenyl, or C ６ -C ２４ It is Alkinnil; and L １ and L ２ Each is independently C ４ -C １２ It is alkylene, Here, each alkyl, alkenyl, alkynyl, alkylene, cycloalkyl, heterocyclyl, and heteroaryl is optionally substituted with one or more fluoropolymers. Compounds having the same property or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof.

2. The compound is given by the following formula (Ia): 【Transformation 3】 The compound according to claim 1, which has or is a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

3. The compound is represented by the following formula (Ib): 【Chemistry 4】 The compound according to claim 1, which has or is a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

4. R １ａ C is replaced with oxo as desired. ８ -C １２ A compound according to any one of claims 1 to 3, wherein the compound is alkyl.

5. R １ａ The structure is as follows: 【Transformation 5】 A compound according to any one of claims 1 to 4, having one of the above.

6. R １ However, if desired, -OH, oxo, fluoro, -N(CH ３ ) ２ , and the following structure: 【Transformation 6】 The compound according to any one of claims 1 to 5, which is substituted with one or two substituents selected from the group consisting of .

7. R １ The compound according to any one of claims 1 to 6, wherein the compound is substituted with -OH.

8. R １ ga-CH ３ The compound according to any one of claims 1 to 5.

9. R １ The structure is as follows: 【Transformation 7】 A compound according to any one of claims 1 to 8, having one of the above.

10. R ２ ga -OC(=O)OR ２ａ The compound according to any one of claims 1 to 9.

11. R ２ａ C ４ -C ２４ A compound according to any one of claims 1 to 10, wherein the compound is alkyl.

12. R ２ａ is non-substituted C ４ -C ２４ A compound according to any one of claims 1 to 11, wherein the compound is alkyl.

13. R ２ａ is non-branch C ４ -C ２４ A compound according to any one of claims 1 to 12, wherein the compound is alkyl.

14. R ２ａ Branch C ４ -C ２４ A compound according to any one of claims 1 to 12, wherein the compound is alkyl.

15. R ２ ga-OC(=O)R ２ｂ The compound according to any one of claims 1 to 9.

16. R ２ ga -C(=O)OR ２ｃ The compound according to any one of claims 1 to 9.

17. R ２ｂ or R ２ｃ has the following structure: 【Transformation 8】 A compound according to any one of claims 1 to 16, having the following characteristics.

18. R ２ｂ or R ２ｃ The structure is as follows: 【Chemistry 9】 A compound according to any one of claims 1 to 16, having the following characteristics.

19. R ２ The structure is as follows: 【Chemistry 10】 【Chemistry 11】 A compound according to any one of claims 1 to 18, having one of the above.

20. R ３ ga-C(=O)N(R ３ａ )R ３ｂ The compound according to any one of claims 1 to 19.

21. R ３ is -NR ３ａ -C(=O)R ３ｂ The compound according to any one of claims 1 to 19, wherein R is -NR-C(=O)R. ３ｂ

22. R ３ａ and R ３ｂ Each of them is independent of C ６ -C ２４ A compound according to any one of claims 1 to 21, wherein the compound is alkyl.

23. R ３ａ and R ３ｂ Each of them is independent of C ６ -C １２ A compound according to any one of claims 1 to 22, wherein it is alkyl.

24. R ３ａ and R ３ｂ Each of them is independent of C ８ -C １０ A compound according to any one of claims 1 to 23, wherein the compound is alkyl.

25. R ３ａ and R ３ｂ Both are C １２ A compound according to any one of claims 1 to 23, wherein the compound is alkyl.

26. R ３ａ and R ３ｂ Both are C ８ A compound according to any one of claims 1 to 23, wherein the compound is alkyl.

27. R ３ａ and R ３ｂ Both are C １０ A compound according to any one of claims 1 to 23, wherein the compound is alkyl.

28. L １ and L ２ Each of them is independent of C ４ -C １０ A compound according to any one of claims 1 to 27, which is an alkylene.

29. L １ and L ２ Each of them is independent of C ４ , C ５ , C ６ , C ７ , C ８ , or C ９ A compound according to any one of claims 1 to 28, which is an alkylene.

30. L １ and L ２ The compound according to any one of claims 1 to 29, wherein the same property is found.

31. L １ and L ２ The compound according to any one of claims 1 to 30, wherein is unsubstituted.

32. L １ 、 L ２ The compound according to any one of claims 1 to 30, wherein either or both are substituted with one or more fluoro substituents.

33. The compound has the following structure: 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】 【Chemistry 16】 A compound according to any one of claims 1 to 32, having one of the above, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

34. Lipid nanoparticles comprising any compound of claims 1 to 33 and a therapeutic agent.

35. A composition comprising any compound of claims 1 to 33 and a therapeutic agent.

36. Lipid nanoparticles or compositions according to claim 34 or 35, further comprising one or more additives selected from neutral lipids, steroids, and polymer-conjugated lipids.

37. Lipid nanoparticles or compositions comprising one or more neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPPC, DOPE, and SM.

38. Lipid nanoparticles or composition according to claim 36 or 37, wherein the neutral lipid is DSPC.

39. Lipid nanoparticles or composition according to any one of claims 36 to 38, wherein the molar ratio of the compound to neutral lipids is in the range of about 2:1 to about 8:

1.

40. Lipid nanoparticles or composition according to any one of claims 36 to 39, wherein the steroid is cholesterol.

41. The lipid nanoparticle or composition according to claim 40, wherein the molar ratio of the compound to cholesterol is in the range of 5:1 to 1:1 or 2:1 to 1:

1.

42. Lipid nanoparticles or composition according to any one of claims 36 to 41, wherein the polymer conjugate lipid is a PEG-modified lipid.

43. The lipid nanoparticle or composition according to claim 42, wherein the molar ratio of the compound to the PEGylated lipid is in the range of about 100:1 to about 20:1 or about 100:1 to about 10:

1.

44. The lipid nanoparticle or composition according to claim 43, wherein the PEGylated lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-Cer, or PEG-dialkyloxypropylcarbamate.

45. PEGylated lipids are given by the following formula (II): 【Chemistry 17】 [During the ceremony, R １０ and R １１ Each of these is independently a linear or branched, alkyl, alkenyl or alkynyl chain of 10 to 30 carbon atoms, where the alkyl, alkenyl or alkynyl is optionally interrupted by one or more ester bonds; and w has a value in the range of 30 to 60. Lipid nanoparticles or compositions according to claim 43, having or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

46. R １０ and R １１ The lipid nanoparticle or composition according to claim 45, wherein each is independently a linear alkyl chain containing 12 to 16 carbon atoms.

47. The lipid nanoparticle or composition according to claim 46, wherein w is in the range of 40 to 50.

48. Lipid nanoparticles or composition according to any one of claims 36 to 47, wherein the therapeutic agent comprises nucleic acid.

49. The lipid nanoparticle or composition according to claim 48, wherein the nucleic acid is selected from antisense and messenger RNA.

50. A method for inducing the expression of a desired protein in a subject requiring treatment, comprising administering to the subject a therapeutically effective amount of any lipid nanoparticles or pharmaceutical composition according to claims 34 to 49, wherein the therapeutic agent comprises a nucleic acid.

51. The method according to claim 50, wherein the nucleic acid is antisense RNA, mRNA, or Cas9 mRNA.

52. The method according to claim 51, wherein the mRNA encodes an antigen.

53. The method according to any one of claims 50 to 52, wherein the method is for vaccination against a viral pathogen.

54. The method according to claim 51, wherein the nucleic acid is Cas9 mRNA and the method is for editing a gene of interest.

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