Cationic lipids for use in lipid nanoparticles - Patent Application 20070122999

Abstract

The following structure: 1 , G 2 , L 1 , L 2 , R 1a , R 1b , R 2a , R 2b , R 3a , R 3b , R 4a , R 4b , R 5 , R 6 , R 7 , R 8 and X is as defined herein, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. Use of the compound as a component of a lipid nanoparticle formulation for delivery of a therapeutic agent, nanoparticles comprising the compound, and methods of use and production thereof are also provided. TIFF2023535365000032.tif4868

Description

[Technical Field]

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

[0002] Numerous challenges exist for the delivery of nucleic acids to affect desired responses in biological systems. Nucleic acid-based therapeutics hold great promise, but to realize this potential, more effective delivery of nucleic acids to appropriate sites within cells or organisms is needed. Therapeutic nucleic acids include, for example, messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, immunostimulatory nucleic acids, antagomirs, antimirs, mimics, supermirs, and aptamers. Some nucleic acids, such as mRNA or plasmids, can be used to direct the expression of specific cellular products, useful, for example, for treating diseases associated with protein or enzyme deficiencies. The therapeutic applications of translatable nucleotide delivery are quite broad, as constructs can be synthesized to generate any selected protein sequence, whether native to the system or not. The expression product of a nucleic acid can enhance existing levels of a protein, replace a missing or dysfunctional protein, or introduce a new protein and associated function into a cell or organism.

[0003] Some nucleic acids, such as miRNA inhibitors, can be used to affect the expression of specific cellular products regulated by miRNAs, making them useful, for example, for treating diseases associated with protein or enzyme deficiencies. The therapeutic applications of miRNA inhibition are quite broad, as constructs can be synthesized to inhibit one or more miRNAs and control the expression of their mRNA products. Inhibition of endogenous miRNAs can enhance the expression of their downstream target endogenous proteins and restore proper cell or organism function as a means of treating diseases associated with specific miRNAs or groups of miRNAs.

[0004] Other nucleic acids 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 very broad, since oligonucleotide constructs can be synthesized with any nucleotide sequence directed against a target mRNA. Targets can include mRNAs from normal cells, mRNAs associated with disease states such as cancer, and mRNAs of infectious agents such as viruses. To date, antisense oligonucleotide constructs have demonstrated the ability to specifically downregulate target proteins through the degradation of cognate mRNAs in both in vitro and in vivo models. Furthermore, antisense oligonucleotide constructs are currently being evaluated in clinical studies.

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

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

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

[0008] In one embodiment, the following structure (I): [ka] [In the formula, a, b, c, d, G 1 , G 2 , L 1 , L 2 , R 1a , R 1b , R 2a , R 2b , R 3a , R 3b , R 4a , R4b , R 5 , R 6 , R 7 , R 8 and X is as defined herein. or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

[0009] Also provided are lipid nanoparticles (LNPs) and pharmaceutical compositions comprising the same, comprising one or more compounds of structure (I) and a therapeutic agent. In some embodiments, the nanoparticles further comprise one or more components selected from neutral lipids, charged lipids, steroids, and polymer-bound lipids. Such LNPs are useful, for example, for delivering therapeutic agents for the treatment of disease or vaccination against viral pathogens.

[0010] These and other aspects of the present disclosure will become evident upon reference to the following detailed description. DETAILED DESCRIPTION OF THE INVENTION

[0011] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the present disclosure. However, it will be understood by those skilled in the art that the present disclosure may be practiced without these details.

[0012] The present disclosure is based, in part, on the discovery of novel cationic (amino) lipids that offer advantages when used in lipid nanoparticles for the in vivo delivery of active or therapeutic agents, such as nucleic acids, to mammalian cells. In particular, embodiments of the present disclosure provide nucleic acid-lipid nanoparticle compositions comprising one or more of the novel cationic lipids described herein, which provide increased in vivo nucleic acid activity and improved tolerability of the composition, resulting in a significantly increased therapeutic index compared to previously described nucleic acid-lipid nanoparticle compositions. In other embodiments, the disclosed lipids, and lipid nanoparticles comprising them, have improved safety and / or tolerability when used to deliver active agents, such as nucleic acids.

[0013] In certain embodiments, the present disclosure provides novel cationic lipids that enable the formulation of improved compositions for in vitro and in vivo delivery of mRNA and / or other oligonucleotides. In some embodiments, these improved lipid nanoparticle compositions are useful for the expression of proteins encoded by mRNA. In other embodiments, these improved lipid nanoparticle compositions are useful for upregulating endogenous protein expression by delivering miRNA inhibitors that target a specific miRNA or a group of miRNAs that regulate a single target mRNA or several mRNAs. In other embodiments, these improved lipid nanoparticle compositions are useful for downregulating (e.g., silencing) the protein and / or mRNA levels of target genes. In some other embodiments, lipid nanoparticles are also useful for delivering mRNA and plasmids for transgene expression. In still other embodiments, lipid nanoparticle compositions are useful for inducing pharmacological effects resulting from protein expression, such as increased red blood cell production through delivery of appropriate erythropoietin mRNA, or protection against infection through delivery of mRNA encoding appropriate antigens or antibodies.

[0014] The lipid nanoparticles and compositions of the present disclosure can be used for a variety of purposes, including the delivery of encapsulated or associated (e.g., complexed) therapeutic agents, such as nucleic acids, to cells, both in vitro and in vivo. Accordingly, embodiments of the present disclosure provide methods for treating or preventing a disease or disorder in a subject in need thereof by contacting the subject with lipid nanoparticles having a suitable therapeutic agent encapsulated or associated therewith, the lipid nanoparticles comprising one or more of the novel cationic lipids described herein.

[0015] As described herein, lipid nanoparticle embodiments of the present disclosure are particularly useful for the delivery of nucleic acids, such as, for example, mRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomirs / antimirs), messenger RNA interference complementary RNA (micRNA), DNA, multivalent RNA, Dicer substrate RNA, and complementary DNA (cDNA). Thus, certain embodiments of lipid nanoparticles and compositions of the present disclosure can be used to induce expression of a desired protein both in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more of the novel cationic lipids described herein, where the lipid nanoparticles encapsulate or associate with a nucleic acid to be expressed to produce the desired protein (e.g., a plasmid or messenger RNA encoding the desired protein) or inhibit a process that terminates mRNA expression (e.g., an miRNA inhibitor). In certain embodiments, the protein expressed by the nucleic acid is an antigen, and thus the LNP induces an immune response (e.g., vaccination). Alternatively, the lipid nanoparticles and compositions of the present disclosure can be used to reduce target gene and protein expression both in vitro and in vivo by contacting cells with lipid nanoparticles containing one or more of the novel cationic lipids described herein, wherein the lipid nanoparticles encapsulate or associate with nucleic acids (e.g., antisense oligonucleotides or small interfering RNA (siRNA)) that reduce target gene expression. The lipid nanoparticles and compositions of the present disclosure can also be used, separately or in combination, for the simultaneous delivery of different nucleic acids (e.g., mRNA and plasmid DNA), which can be useful, for example, to provide effects that require the co-localization of different nucleic acids (e.g., mRNA encoding appropriate gene-modifying enzymes and DNA segments for integration into the host genome).

[0016] Nucleic acids for use in the embodiments of the present disclosure can be prepared according to any available technique. In the case of mRNA, the main method of preparation is, but is not limited to, enzymatic synthesis (also called in vitro transcription), which is currently the most efficient method for producing specific mRNAs of long sequences. In vitro transcription is described as the process of template-directed synthesis of RNA molecules from an engineered DNA template composed of an upstream bacteriophage promoter sequence (e.g., including but not limited to those derived from T7, T3, and SP6 coliphages) linked to a downstream sequence encoding a gene of interest. Template DNA can be prepared for in vitro transcription from many sources using suitable techniques well known in the art, including, but not limited to, plasmid DNA and polymerase chain reaction amplification (see Linpinsel, JL and Conn, GL, General protocols for preparation of plasmid DNA template and Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LD in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941 Conn GL (ed), New York, NY Humana Press, 2012).

[0017] RNA transcription is carried out 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 potential degradation of the resulting mRNA transcript. In vitro transcription can be carried out using a variety of commercially available kits and commercially available reagents containing RNA polymerase and rNTPs, including, but not limited to, RiboMax Large Scale RNA Production System (Promega) and MegaScript Transcription kits (Life Technologies). Methodologies for in vitro transcription of mRNA are well known in the art. (See, e.g., Losick, R., 1972, In vitro transcription, Ann Rev Biochem v.41 409-46; Kamakaka, RT and Kraus, WL 2001. In Vitro Transcription. Current Protocols in Cell Biology. 2:11.6:11.6.1-11.6.17; Beckert, B. And Masquida, B., (2010) Synthesis of RNA by In Vitro Transcription in RNA in Methods in Molecular Biology v. 703 (Neilson, H. Ed), New York, NY Humana Press, 2010; Brunelle, JL and Green, R., 2013, Chapter Five - In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology v. 530, 101-114; all of which are incorporated herein by reference).

[0018] The in vitro transcribed mRNA of interest is purified from undesired components of the transcription or related reaction, including unincorporated rNTPs, protein enzymes, salts, short RNA oligos, etc. Techniques for isolating mRNA transcripts are well known in the art. Well-known techniques include precipitation with alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride, or phenol / chloroform extraction. Further examples of purification techniques that can be used include, but are not limited to, size exclusion chromatography (Lukavsky, PJ and Puglisi, JD, 2004, Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides, RNA v.10, 889-893), silica-based affinity chromatography, and polyacrylamide gel electrophoresis (Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LD in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA synthesis Methods v. 941 Conn GL (ed), New York, NY Humana Press, 2012). Purification can be performed using various commercially available kits, including, but not limited to, the SV Total Isolation System (Promega) and the In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).

[0019] Furthermore, while reverse transcription can generate large amounts of mRNA, the product may contain many aberrant RNA impurities associated with undesired polymerase activity that may need to be removed from full-length mRNA preparations. These include short RNAs resulting from premature transcription initiation, double-stranded RNA (dsRNA) generated by RNA-dependent RNA polymerase activity, RNA-primed transcription from RNA templates, and self-complementary 3' extensions. It has been demonstrated that these contaminants, with their dsRNA structure, can result in unwanted immunostimulatory activity through interactions with various innate immune sensors in eukaryotic cells that recognize specific nucleic acid structures and function to induce potent immune responses. This can dramatically reduce mRNA translation due to reduced protein synthesis during the innate cellular immune response. Accordingly, additional techniques for removing these dsRNA contaminants have been developed and are known in the art, including, but not limited to, scalable HPLC purification (see, e.g., Kariko, K., Muramatsu, H., Ludwig, J. And Weissman, D., 2011, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA, Nucl Acid Res, v. 39 e142; Weissman, D., Pardi, N., Muramatsu, H., and Kariko, K., HPLC Purification of in vitro transcribed long RNA in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH Ed), 2013).HPLC-purified mRNA has been reported to be translated at much higher levels, especially in primary cells and in vivo.

[0020] A wide variety of modifications have been described in the art to alter specific properties of in vitro transcribed mRNA and improve its utility. These include, but are not limited to, modifications to the 5' and 3' ends of mRNA. Endogenous eukaryotic mRNAs typically contain a cap structure at the 5' end of the mature molecule, which plays an important role in mediating mRNA cap-binding protein (CBP) binding and thus enhances intracellular mRNA stability and the efficiency of mRNA translation. Therefore, the highest levels of protein expression are achieved with capped mRNA transcripts. The 5'-cap contains a 5'-5'-triphosphate linkage between the 5'-most nucleotide and a guanine nucleotide. The conjugated guanine nucleotide is methylated at the N7 position. Additional modifications include methylation of the 5'-last and penultimate 5'-nucleotides on the 2'-hydroxyl group.

[0021] Several different cap structures can be used to create 5'-caps for in vitro transcribed synthetic mRNAs. 5'-capping of synthetic mRNAs can be performed co-transcriptionally (i.e., capping during in vitro transcription) using chemical cap analogs. For example, the anti-reverse cap analog (ARCA) cap contains a 5'-5'-triphosphate guanine-guanine linkage, where one guanine contains an N7 methyl group and a 3'-O-methyl group. However, up to 20% of transcripts remain uncapped during this co-transcription process, and synthetic cap analogs are not identical to the 5'-cap structure of authentic cellular mRNAs, potentially reducing translatability and cellular stability. Alternatively, synthetic mRNA molecules can also be enzymatically capped post-transcriptionally. These may generate more authentic 5'-cap structures that structurally or functionally mimic endogenous 5'-caps, enhancing cap-binding protein binding, increasing half-life, reducing susceptibility to 5' endonucleases, and / or reducing 5' decapping. A number of synthetic 5'-cap analogs have been developed and are known in the art to enhance mRNA stability and translatability (see, e.g., Grudzien-Nogalska, E., Kowalska, J., Su, W., Kuhn, AN, Slepenkov, SV, Darynkiewicz, E., Sahin, U., Jemielity, J., and Rhoads, RE, Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH Ed), 2013).

[0022] At the 3' end, a long chain of adenine nucleotides (poly-A tail) is usually added to mRNA molecules during RNA processing. Shortly after transcription, the 3' end of the transcript is cleaved, freeing a 3' hydroxyl where poly-A polymerase adds a chain of adenine nucleotides to the RNA in a process called polyadenylation. The poly(A) tail has been widely shown to enhance both the translation efficiency and stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly(A), poly(A) binding protein and the regulation of mRNA stability, Trends Bio Sci, v. 14, 373-377; Guhaniyogi, J. and Brewer, G., 2001, Regulation of mRNA stability in mammalian cells, Gene, v. 265, 11-23; Dreyfus, M. and Regnier, P., 2002, The poly(A) tail of mRNAs: Bodyguard in eukaryotes, scavenger in bacteria, Cell, v. 111, 611-613).

[0023] Poly(A) tailing of in vitro transcribed mRNA can be achieved using a variety of techniques, including, but not limited to, cloning a poly(T) tract into a DNA template or post-transcriptional addition using poly(A) polymerase. In the first case, in vitro transcription of mRNA with a poly(A) tail of a predetermined length is possible, depending on the size of the poly(T) tract, but additional template manipulation is required. In the latter case, enzymatic addition of a poly(A) tail to in vitro transcribed mRNA using poly(A) polymerase, which catalyzes the incorporation of adenine residues into the 3' end of the RNA, does not require additional DNA template manipulation but can result in mRNAs with poly(A) tails of heterogeneous lengths. 5'-capping and 3'-poly(A) tailing can be performed using a variety of commercially available kits and reagents, including, but not limited to, the Poly(A) Polymerase Tailing Kit (EpiCenter), the mMESSAGE mMACHINE T7 Ultra Kit, and the Poly(A) Tailing Kit (Life Technologies), various ARCA caps, and poly(A) polymerases.

[0024] In addition to 5' caps and 3' polyadenylation, other modifications of in vitro transcripts have been reported to provide benefits related to translation efficiency and stability. It is well known in the art that pathogenic DNA and RNA can be recognized by various sensors in eukaryotes and trigger strong innate immune responses. Because most naturally occurring nucleic acids contain modified nucleosides, the ability to distinguish pathogenic from self DNA and RNA has been shown to be based, at least in part, on structural and nucleoside modifications. In contrast, in vitro-synthesized RNA lacks these modifications, making it immunostimulatory, potentially inhibiting effective mRNA translation, as outlined above.Introduction of modified nucleosides into in vitro transcribed mRNA can be used to prevent recognition and activation of RNA sensors, thus reducing this undesired immunostimulatory activity and enhancing translational competence (e.g., Kariko, K. And Weissman, D. 2007, Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: Implication for therapeutic RNA development, Curr Opin Drug Discov Devel, v.10 523-532; Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH Ed), 2013; Kariko, K., Muramatsu, H., Welsh, F.A., Ludwig, J., Kato, H., Akira, S., Weissman, (See, e.g., D., 2008, Incorporation of Pseudouridine Into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability, Mol Ther v.16, 1833-1840.) The modified nucleosides and nucleotides used in the synthesis of modified RNAs can be prepared, monitored, and utilized using common methods and procedures known in the art.A wide variety of nucleoside modifications are available that can be incorporated into in vitro transcribed mRNA to some degree, either alone or in combination with other modified nucleosides (see, e.g., US2012 / 0251618). In vitro synthesis of nucleoside-modified mRNA has been reported to reduce its ability to activate immune sensors while simultaneously increasing translational competence.

[0025] Other portions of mRNA that can be modified to provide advantages in terms of translatability and stability include the 5' and 3' untranslated regions (UTRs). Optimizing UTRs (preferred 5' and 3' UTRs can be derived from cellular or viral RNA) has been shown to increase mRNA stability and translation efficiency of in vitro transcribed mRNAs, both together or independently (see, e.g., Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH Ed), 2013).

[0026] In addition to mRNA, other nucleic acid payloads can be used for the present disclosure.For oligonucleotides, preparation methods include, but are not limited to, chemical synthesis, enzymatic methods, chemical cleavage of longer precursors, in vitro transcription as described above, etc.The method of synthesizing DNA and RNA nucleotides is widely used and well known in the art (for example, Gait, MJ (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, DC: IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, NJ) Totowa, NJ: Humana Press, 2005; both of which are incorporated herein by reference).

[0027] For plasmid DNA, preparation for use in embodiments of the present disclosure generally utilizes, but is not limited to, in vitro expansion and isolation of the plasmid DNA in liquid cultures of bacteria containing the plasmid of interest. The presence of a gene in the plasmid of interest that encodes resistance to a particular antibiotic (penicillin, kanamycin, etc.) allows bacteria containing the plasmid of interest to selectively grow in antibiotic-containing culture. Methods for isolating plasmid DNA are widely used and well known in the art (see, e.g., Heilig, J., Elbing, KL and Brent, R., (2001), Large-Scale Preparation of Plasmid DNA, Current Protocols in Molecular Biology, 41:11:1.7:1.7.1-1.7.16; Rozkov, A., Larsson, B., Gillstrom, S., Bjornestedt, R. and Schmidt, SR, (2008), Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture, Biotechnol. Bioeng., 99: 557-566; and U.S. Pat. No. 6,197,553B1). 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.

[0028] Various exemplary embodiments of the cationic lipids, lipid nanoparticles and compositions comprising the same of the present disclosure, and their use to deliver active substances (e.g., therapeutic agents), such as nucleic acids, to modulate gene and protein expression, are described in further detail below.

[0029] As used herein, the following terms have the meanings set forth therein unless otherwise specified.

[0030] Unless the context requires otherwise, throughout this specification and claims, the words "comprises" and variations thereof, such as "included" and "comprises," are to be interpreted in their open and inclusive sense, i.e., "including but not limited to."

[0031] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used in the specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0033] The phrase "induce the expression of a desired protein" refers to the ability of a nucleic acid to increase the expression of a desired protein.To determine the level of protein expression, a test sample (e.g., a sample of cultured cells expressing the desired protein) or a test mammalian (e.g., a mammalian, such as a human or animal) model, such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model, is contacted with a nucleic acid (e.g., a nucleic acid combined with a lipid of the present disclosure).The expression of the desired protein in the test sample or test animal is compared with the expression of the desired protein in a control sample (e.g., a sample of cultured cells expressing the desired protein) or a control mammalian (e.g., a mammalian, such as a human or animal) model, such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model, that has not been contacted or administered with the nucleic acid.If the desired protein is present in the control sample or control mammal, the expression of the desired protein in the control sample or control mammal can be assigned a value of 1.0. In certain embodiments, induction of expression of a desired protein is achieved when the ratio of the level of desired protein expression in a test sample or test mammal to the level of desired protein expression in a control sample or control mammal is greater than 1, for example, about 1.1, 1.5, 2.0, 5.0, or 10.0. Induction of expression of a desired protein is achieved when measurable levels of the desired protein are detected in a test sample or test mammal in the absence of the desired protein in the control sample or control mammal. Those skilled in the art will recognize appropriate assays for determining the level of protein expression in a sample, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays, or assays based on reporter proteins capable of generating fluorescence or luminescence under appropriate conditions.

[0034] The phrase "inhibiting expression of a target gene" refers to the ability of a nucleic acid to silence, reduce, or inhibit expression of a target gene. To determine the extent of gene silencing, a test sample (e.g., a sample of cultured cells expressing the target gene) or a test mammalian (e.g., a mammalian, such as a human or animal) model, e.g., a rodent (e.g., a mouse) or non-human primate (e.g., a monkey) model, is contacted with a nucleic acid that silences, reduces, or inhibits expression of the target gene. The expression of the target gene in the test sample or test animal is compared to the expression of the target gene in a control sample (e.g., a sample of cultured cells expressing the target gene) or a control mammalian (e.g., a mammalian, such as a human or animal, model, e.g., a rodent (e.g., a mouse) or non-human primate (e.g., a monkey) model, that has not been contacted or administered with the nucleic acid. The expression of the target gene in the control sample or control mammal can be assigned a value of 100%. In certain embodiments, silencing, inhibition, or reduction of expression of a target gene is measured by the expression level of the target gene in the test sample or test mammal compared to the expression level of the target gene in the control sample or control mammal. Achieved when the level of gene expression is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. In other words, the nucleic acid is capable of silencing, reducing, or inhibiting expression of the 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% relative to the level of gene expression in a control sample or control mammal not contacted or administered with the nucleic acid.Suitable assays for determining the expression level of a target gene include, but are not limited to, measuring protein or mRNA levels using techniques known to those of skill in the art, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzymatic function, and phenotypic assays known to those of skill in the art.

[0035] An "effective amount" or "therapeutically effective amount" of a therapeutic agent, such as an active agent or therapeutic nucleic acid, is an amount sufficient to produce a desired effect, such as increased or inhibited expression of a target sequence compared to the normal expression level detected in the absence of the nucleic acid. Increased expression of a target sequence is achieved when a measurable level of expression product not present in the absence of the nucleic acid is detected. When an expression product is present at a certain level prior to contact with the nucleic acid, increased expression is achieved when the fold increase over the value obtained with the nucleic acid, such as mRNA, is about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000, or more compared to a control. Inhibition of expression of a target gene or target sequence is achieved when the value obtained with a nucleic acid, such as an antisense oligonucleotide, is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% compared to a control. Suitable assays for measuring expression of a target gene or target sequence include measuring protein or RNA levels using techniques known to those skilled in the art, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of appropriate reporter proteins, and phenotypic assays known to those skilled in the art.

[0036] As used herein, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single- or double-stranded form, including DNA, RNA, and hybrids thereof. DNA can be in the form of an antisense molecule, plasmid DNA, cDNA, PCR product, or vector. RNA can be in the form of small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, polyvalent RNA, Dicer substrate RNA, or viral RNA (vRNA), and combinations thereof. Nucleic acids include synthetic, natural, and non-natural nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that have similar binding properties to the reference nucleic acid. Examples of such analogs (analogs) include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid. Unless otherwise specified, a particular nucleic acid sequence implicitly encompasses not only the explicitly indicated sequence, but also its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences. Specifically, degenerate codon substitutions can be made by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). A "nucleotide" comprises the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked via a phosphate group."Bases" include purines and pyrimidines, as well as the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and naturally occurring analogues, and synthetic derivatives of purines and pyrimidines, including modifications that place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0037] The term "gene" refers to a nucleic acid (eg, DNA or RNA) sequence that comprises partial or full-length coding sequences necessary for the production of a polypeptide or polypeptide precursor.

[0038] As used herein, "gene product" means the product of a gene, such as an RNA transcript or a polypeptide.

[0039] The term "lipid" refers to a group of organic compounds, including but not limited to esters of fatty acids, that are generally poorly soluble in water but soluble in many organic solvents. They are typically divided into at least three classes: (1) "simple lipids" (e.g., fats and oils and waxes); (2) "complex lipids" (e.g., phospholipids and glycolipids); and (3) "derived lipids" (e.g., steroids).

[0040] "Steroids" are compounds with the following carbon skeleton: [ka] Examples of steroids include, but are not limited to, cholesterol.

[0041] "Cationic lipid" refers to a lipid that can be positively charged. For example, cationic lipids contain one or more amine groups that are positively charged. Preferred cationic lipids are ionizable, so that they can exist in a positively charged or neutral form depending on pH. The ionization of cationic lipids affects the surface charge of lipid nanoparticles under various pH conditions. This charge state can affect the absorption of plasma proteins, blood clearance, and tissue distribution (Semple, SC, et al., Adv. Drug Deliv Rev 32:3-17 (1998)), and the ability to form endosomolytic non-bilayer structures that are important for intracellular delivery of nucleic acids (Hafez, IM, et al., Gene Ther 8:1188-1196 (2001)).

[0042] The term "polymer-conjugated lipid" refers to a molecule that contains both a lipid portion and a polymer portion. An example of a polymer-conjugated lipid is a PEGylated lipid. The term "PEGylated lipid" refers to a molecule that contains both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG).

[0043] The term "neutral lipid" refers to any of several lipid species that exist in either uncharged or neutral zwitterionic form at selected pH levels. At physiological pH, such lipids include, but are not limited to, phosphotidylcholines such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), steroids such as sphingomyelin (SM), ceramides, sterols, and derivatives thereof. Neutral lipids can be synthetic or naturally occurring.

[0044] The term "charged lipid" refers to any of several lipid species that exist in either a positively or negatively charged form, regardless of the useful physiological pH range, e.g., pH 3 to pH 9. Charged lipids can be synthetic or naturally derived. Examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, hemisuccinate sterols, dialkyltrimethylammonium-propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, and dimethylaminoethanecarbamoylsterol (e.g., DC-Chol).

[0045] The term "lipid nanoparticle" refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) that comprises one or more of the compounds of structure (I) or other specific cationic lipids. In some embodiments, lipid nanoparticles comprising the disclosed cationic lipids (e.g., compounds of structure (I)) are included in formulations that can be used to deliver active or therapeutic agents, such as nucleic acids (e.g., mRNA), to a desired target site (e.g., a cell, tissue, organ, tumor, etc.). In some embodiments, the lipid nanoparticle comprises a compound of structure (I) and a nucleic acid. Such lipid nanoparticles typically comprise a compound of structure (I) and one or more excipients selected from neutral lipids, charged lipids, steroids, and polymer-bound lipids. In some embodiments, the active or therapeutic agent, such as a nucleic acid, can be encapsulated in the lipid portion of the lipid nanoparticle or in the aqueous space enclosed by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects elicited by mechanisms of the host organism or cells, such as a harmful immune response.

[0046] In various embodiments, the lipid nanoparticles have an average particle size of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 7 ... The diameter of the lipid nanoparticles is about 30 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the lipid nanoparticles are substantially non-toxic. In certain embodiments, the nucleic acid, when present in the lipid nanoparticles, is resistant to degradation by nucleases in aqueous solution. Lipid nanoparticles containing nucleic acids and methods for their production are disclosed, for example, in U.S. Patent Publications 2004 / 0142025, 2007 / 0042031, and PCT Publications WO2013 / 016058 and WO2013 / 086373, the disclosures of which are all incorporated by reference in their entirety for all purposes.

[0047] As used herein, "lipid encapsulation" refers to lipid nanoparticles that provide an active or therapeutic agent, such as a nucleic acid (e.g., mRNA), with complete encapsulation, partial encapsulation, or both. In one embodiment, the nucleic acid (e.g., mRNA) is completely encapsulated in the lipid nanoparticle.

[0048] As used herein, the term "aqueous solution" refers to a composition that includes water.

[0049] "Serum stability" in reference to nucleic acid-lipid nanoparticles means that the nucleotides are not significantly degraded after exposure to serum or nuclease assays that significantly degrade free DNA or RNA. Suitable assays include, for example, standard serum assays, DNAse assays, or RNAse assays.

[0050] As used herein, "systemic delivery" refers to the delivery of a therapeutic product that allows widespread exposure of an active agent within an organism. Some techniques of administration can allow certain agents to be delivered systemically while others cannot. Systemic delivery means that a useful, preferably therapeutic, amount of the agent is exposed to most parts of the body. Systemic delivery of lipid nanoparticles can be by any means known in the art, including, for example, intravenous, intraarterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery.

[0051] As used herein, "local delivery" refers to the direct delivery of an active agent to a target site within an organism. For example, a drug can be delivered locally by direct injection into a disease site such as a tumor, other target site such as an inflammation site, or target organ such as the liver, heart, pancreas, or kidney. Local delivery can also include topical application or local injection techniques such as intramuscular, subcutaneous, or intradermal injection. Local delivery does not exclude systemic pharmacological effects.

[0052] "Alkyl" means a saturated, straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, e.g., 1 to 24 carbon atoms (C1-C 24 alkyl), 4 to 20 carbon atoms (C4-C 20 alkyl), 6 to 16 carbon atoms (C6-C 16 alkyl), 6 to 9 carbon atoms (C6-C9 alkyl), 1 to 15 carbon atoms (C1-C 15 alkyl), 1 to 12 carbon atoms (C1-C 12alkyl), 1 to 8 carbon atoms (C1-C8 alkyl), or 1 to 6 carbon atoms (C1-C6 alkyl), which is attached to the rest of the molecule by a single bond, for example, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, etc. Unless specified otherwise in the specification, an alkyl group is substituted or unsubstituted.

[0053] "Alkylene" means a saturated, straight or branched divalent hydrocarbon chain, consisting only of carbon and hydrogen, that links the remainder of the molecule to another group, e.g., 1 to 24 carbon atoms (C1-C 24 Alkylene, 1 to 15 carbon atoms (C1-C 15 Alkylene, 1 to 12 carbon atoms (C1-C 12 Alkylene chains have 1 to 8 carbon atoms (C1-C8 alkylene), 1 to 6 carbon atoms (C1-C6 alkylene), 2 to 4 carbon atoms (C2-C4 alkylene), and 1 to 2 carbon atoms (C1-C2 alkylene), such as methylene, ethylene, propylene, and n-butylene. The alkylene chain is attached to the rest of the molecule through a single bond and to other groups through a single bond. The points of attachment of the alkylene chain to the rest of the molecule and to other groups can be through one carbon or any two carbons within the chain. Unless specified otherwise in the specification, the alkylene chain is substituted or unsubstituted.

[0054] "Alkene" and "alkenylene" refer to alkyl and alkylene, respectively, containing at least one carbon-carbon double bond. Alkenes and alkenylenes contain the same number of carbon atoms as alkyl and alkylene, as defined above, except that alkenes and alkenylenes must contain at least two carbons. Unless otherwise specified in the specification, alkenes and alkenylenes are substituted or unsubstituted.

[0055] As used herein, the term "substituted" means any of the above groups (e.g., alkyl or alkylene) in which at least one hydrogen atom has been replaced by a bond to a non-hydrogen atom, including, but not limited to, substitutions such as a halogen atom, e.g., F, Cl, Br, or I; an oxo group (=O); a hydroxyl group (-OH); a carboxyl group (-COH); a C1-C2 12 Alkyl group; -(C=O)OR'; -O(C=O)R'; -C(=O)R'; -OR'; -S(O) x R';-S-SR';-C(=O)SR';-SC(=O)R';-NR'R';-NR'C(=O)R';-C(=O)NR'R';-NR'C(=O)NR'R';-OC(=O)NR'R';-NR'C(=O)OR';-NR'S(O) x NR'R';-NR'S(O) x R'; and -S(O) x NR'R', where R' at each occurrence is independently H, C-C 15 alkyl and x is 0, 1, or 2. In some embodiments, the substituent is C-C 12 In another embodiment, the substituent is an alkyl group. In another embodiment, the substituent is a halo group, such as fluoro. In another embodiment, the substituent is an oxo group. In another embodiment, the substituent is a hydroxyl group. In another embodiment, the substituent is an alkoxy group (-OR'). In another embodiment, the substituent is a carboxyl group. In another embodiment, the substituent is an amine group (-NR'R').

[0056] "Optional" or "optionally" (e.g., optionally substituted) means that the subsequently described circumstance event may or may not occur, and the description includes cases where said event or circumstance occurs and cases where it does not occur. For example, "optionally substituted alkyl" means that the alkyl group may be substituted or unsubstituted, and the description includes both substituted alkyl groups and alkyl groups that have no substituents.

[0057] The disclosure herein is also intended to encompass all pharmaceutically acceptable compounds of the compound of structure (I) that are isotopically labeled by replacing one or more atoms with atoms of different atomic mass or mass number. Isotopes that can be incorporated into the disclosed compounds include, for example, isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, for example, 2 H, 3 H, 11 C. 13 C. 14 C. 13 N, 15 N, 15 O. 17 O. 18 O. 31 P, 32 P, 35 S, 18 F, 36 Cl, 123 I, and 125 I. These radiolabeled compounds may be useful to help determine or measure the efficacy of compounds, for example, by characterizing the site or mechanism of action, or binding affinity to a pharmacologically important site of action. Certain isotopically labeled compounds of structure (I), (IA), or (IB), e.g., compounds incorporating a radioisotope, are useful in drug and / or substrate tissue distribution studies. The radioisotope tritium, i.e., 3 H, and carbon-l4, i.e. 14 C is particularly useful for this purpose in view of its ease of incorporation and ready means for detection.

[0058] Deuterium i.e. 2 Substitution with heavier isotopes, such as H, may confer certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced required dosage, and therefore may be preferable in some circumstances.

[0059] 11 C. 18 F, 15 O. 13Substitution with positron emitting isotopes, such as N, can be useful in positron emission tomography (PET) studies to examine substrate receptor occupancy. Isotopically labeled compounds of structure (I) can generally be prepared by conventional techniques known to those skilled in the art, or by processes analogous to those described in the preparations and examples below, by substituting a suitable isotopically labeled reagent for a previously employed unlabeled reagent.

[0060] "Stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.

[0061] "Mammal" includes both humans and domestic animals, such as laboratory animals and household pets (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits), and non-domestic animals, such as wildlife.

[0062] A "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, an adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier, which is approved by the U.S. Food and Drug Administration as acceptable for use in humans or domestic animals.

[0063] "Pharmaceutically acceptable salt" includes both acid and base addition salts.

[0064] "Pharmaceutically acceptable acid addition salts" means salts formed with inorganic and organic acids, including, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, which retain the biological effectiveness and properties of the free bases and are not biologically or otherwise undesirable, and organic acids, including, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, and the like. Acids that can be used include carboxylic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid.

[0065] "Pharmaceutically acceptable base addition salt" means a salt that retains the biological effectiveness and properties of the free acid and is not biologically or otherwise undesirable. These salts are prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0066] Crystallization often produces solvates of the compounds of the present disclosure (i.e., compounds of structure (I)). As used herein, the term "solvate" refers to an aggregate comprising one or more molecules of a compound of the present disclosure and one or more solvent molecules. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of the present disclosure may exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc., and their corresponding solvate forms. The solvates of the compounds of the present disclosure may be true solvates, while in other cases, the compounds of the present disclosure may simply retain incidental water or may be a mixture of water and some incidental solvents.

[0067] "Pharmaceutical composition" means a formulation of a compound of the present disclosure and a vehicle generally accepted in the art for delivering the biologically active compound to a mammal (e.g., a human). Such a vehicle includes any pharmaceutically acceptable carrier, diluent, or excipient therefor.

[0068] "Effective amount" or "therapeutically effective amount" refers to the amount of a compound of the present disclosure that, when administered to a mammal, preferably a human, is sufficient to effect treatment in the mammal, preferably a human. The amount of lipid nanoparticles of the present disclosure that constitutes a "therapeutically effective amount" varies depending on the compound, the disease and its severity, the method of administration, and the age of the mammal being treated, but can be determined by one skilled in the art in the course of routine, given their knowledge and this disclosure.

[0069] As used herein, "treating" or "treatment" encompasses the treatment of a disease or condition of interest in a mammal (preferably a human) having the disease or condition of interest, and includes: (i) To prevent a disease or illness from occurring in a mammal (especially if the mammal is susceptible to the disease but has not yet been diagnosed as having it); (ii) inhibiting a disease or illness (i.e., arresting its development); (iii) alleviating the disease or illness (i.e., causing regression of the disease or illness); or (iv) Relieving symptoms caused by a disease or illness (i.e., relieving pain without addressing the underlying disease or illness). As used herein, the terms "disease" and "illness" may be used interchangeably or may differ in that a particular illness or condition may not have a known causative agent (and thus the etiology has not yet been elucidated), and therefore is not yet recognized as a disease, but only as an undesirable illness or syndrome in which more or less specific symptoms have been identified by a clinician.

[0070] The compounds of the present disclosure, or pharmaceutically acceptable salts thereof, may contain one or more stereocenters and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms, which may be defined with respect to absolute stereochemistry as (R)- or (S)-, or, in the case of amino acids, as (D)- or (L)-. The present disclosure is intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+)- and (-), (R)- and (S)-, or (D)- and (L)-isomers can be prepared using chiral synthons or chiral reagents or separated using conventional techniques, such as chromatography and fractional crystallization. Conventional techniques for preparing / isolating individual enantiomers include chiral synthesis from appropriate optically pure precursors or separation of the racemate (or racemate of a salt or derivative) using, for example, chiral high-performance liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, unless specified otherwise, the compounds are intended to include both E and Z geometric isomers, as well as all tautomeric forms.

[0071] "Stereoisomers" refer to compounds consisting of the same atoms joined by the same bonds, but having different, incompatible three-dimensional structures. The present disclosure contemplates various stereoisomers and mixtures thereof, and includes "enantiomers," which refer to two stereoisomers whose molecules are non-superimposable mirror images of one another.

[0072] "Tautomer" means a shift of a proton from one atom of a molecule to another atom of the same molecule. The present disclosure includes tautomers of any of the aforementioned compounds.

[0073] compound In one aspect, the present disclosure provides novel lipid compounds that can be combined with other lipid components, such as neutral lipids, charged lipids, steroids, and / or polymer-bound lipids, to form lipid nanoparticles carrying therapeutic agents, such as oligonucleotides. Without being bound by theory, it is believed that these lipid nanoparticles protect the therapeutic agent from degradation in serum and provide effective cellular delivery of oligonucleotides in vitro and in vivo.

[0074] In one embodiment, the compound has the following structure (I): [ka] [In the formula, G 1 and G 2 are each independently C1-C6 alkylene; L 1 and L 2 are each independently —O(C═O)— or —(C═O)O—; R 1a and R 1b is, independently at each occurrence, (a) H, or C1-C 12 alkyl; or (b) R 1a is H, or C1-C 12 alkyl, and R 1b together with the carbon atom to which it is attached, form an adjacent R 1band together with the carbon atom to which it is attached, R 1b is taken to form a carbon-carbon double bond; R 2a and R 2b is, independently at each occurrence, (a) H, or C1-C 12 alkyl; or (b) R 2a is H, or C1-C 12 alkyl, and R 2b together with the carbon atom to which it is attached, form an adjacent R 2b and together with the carbon atom to which it is attached, R 2b is taken to form a carbon-carbon double bond; R 3a and R 3b is, independently at each occurrence, (a) H, or C1-C 12 alkyl; or (b) R 3a is H, or C1-C 12 alkyl, and R 3b together with the carbon atom to which it is attached, form an adjacent R 3b and together with the carbon atom to which it is attached, R 3b is taken to form a carbon-carbon double bond; R 4a and R 4b is, independently at each occurrence, (a) H, or C1-C 12 alkyl; or (b) R 4a is H, or C1-C 12 alkyl, and R 4b together with the carbon atom to which it is attached, form an adjacent R 4b and together with the carbon atom to which it is attached, R 4b is taken to form a carbon-carbon double bond; R 5 and R 6 are each independently H or methyl; R 7 is -O(C=O)R 10 , -(C=O)OR 10 , -NR 9 (C=O)R10 , or -(C=O)NR 9 R 10 and; R 8 is OH, -N(R 11 )(C=O)R 12 , -(C=O)NR 11 R 12 , -NR 11 R 12 , -(C=O)OR 12 , or -O(C=O)R 12 and; R 9 is H, or C1-C 15 is alkyl; R 10 is C1-C 15 is alkyl; R 11 is H, or C1-C6 alkyl; R 12 is C1-C6 alkyl; X is —(C═O)— or a direct bond; and a, b, c, and d are each independently an integer from 1 to 24; wherein each alkyl and alkylene is independently optionally substituted. or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

[0075] In other embodiments, the compound has the following structure (IA) or (IB): [ka] or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof,

[0076] In certain embodiments, G 1 is C2-C3 alkylene. In a different embodiment, G 1 is C4-C6 alkylene. For example, in various embodiments, G 1is a C2 alkylene, a C3 alkylene, a C4 alkylene, a C5 alkylene, or a C6 alkylene.

[0077] In other embodiments, G 2 is C2-C4 alkylene, for example, C2-C3 alkylene, or C3-C4 alkylene. In some embodiments, G 2 is a C2 alkylene, a C3 alkylene, or a C4 alkylene.

[0078] In various different embodiments, X is —(C═O)—, while in different embodiments, X is a direct bond.

[0079] In any of the above embodiments, R 7 is -O(C=O)R 10 , or -(C=O)OR 10 In certain of these embodiments, R 10 is a linear C1-C 15 alkyl, e.g., straight chain C-C 10 In other such embodiments, R 10 is methyl or R 10 is branch C2-C 15 alkyl, e.g., branched C 10 -C 15 It is alkyl.

[0080] In still other embodiments of the above embodiments, R 7 is -NR 9 (C=O) or -(C=O)NR 9 R 10 In some of these embodiments, R 9 is H. In other of these embodiments, R 9 and R 10 are independently C6-C 10 It is alkyl.

[0081] In other embodiments, R 1a and R 1bFor at least one occurrence of R 1a is H, or C1-C 12 alkyl, and R 1b together with the carbon atom to which it is attached, form an adjacent R 1b and together with the carbon atom to which it is attached, R 1b is taken to form a carbon-carbon double bond.

[0082] In a further embodiment, R 4a and R 4b For at least one occurrence of R 4a is H, or C1-C 12 alkyl, and R 4b together with the carbon atom to which it is attached, form an adjacent R 4b and together with the carbon atom to which it is attached, R 4b is taken to form a carbon-carbon double bond.

[0083] In yet other embodiments, R 2a and R 2b For at least one occurrence of R 2a is H, or C1-C 12 alkyl, and R 2b together with the carbon atom to which it is attached, form an adjacent R 2b and together with the carbon atom to which it is attached, R 2b is taken to form a carbon-carbon double bond.

[0084] In other embodiments, R 3a and R 3b For at least one occurrence of R 3a is H, or C1-C 12 alkyl, and R 3b together with the carbon atom to which it is attached, form an adjacent R 3b and together with the carbon atom to which it is attached, R 3b is taken to form a carbon-carbon double bond.

[0085] In various of the above embodiments, R 1a , R 1b , R 2a , R 2b , R 3a , R 3b , R 4a , and R 4b Each occurrence of is independently H or C1-C 12 In other embodiments, R 2a , R 2b , R 3a , and R 3b Each occurrence of R is H. For example, in certain embodiments, R 1a and R 4a Each occurrence of R is H. In a different embodiment, R 1b and R 4b At least one of is C1-C8 alkyl. For example, in some embodiments, the C1-C8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0086] In other more particular embodiments, [ka] or both, independently, the following: [ka] This is one of the structures shown below.

[0087] In further embodiments, a, b, c, and d are each independently an integer from 2 to 12. In further embodiments, a, b, c, and d are each independently an integer from 4 to 10, 5 to 10, 6 to 10, 4 to 9, 5 to 9, or 6 to 9. In yet other embodiments, b and c are independently 5, 6, 7, 8, 9, or 10.

[0088] In some embodiments, R 5 or R6 In another embodiment, one of R 5 and R 6 Each of is methyl.

[0089] In some embodiments, R 8 is OH.

[0090] In other embodiments, R 8 is -N(R 11 )(C=O)R 12 In a different embodiment, R 8 is -(C=O)NR 11 R 12 In yet another embodiment, R 8 is -NR 11 R 12 In some of these above embodiments, R 11 and R 12 are each independently H, or C1-C8 alkyl. In these other embodiments, R 11 and R 12 are each independently H or C1-C3 alkyl. For example, in some embodiments, the C1-C8 alkyl or C1-C3 alkyl is unsubstituted or substituted with hydroxyl. In other such embodiments, R 11 and R 12 are methyl, respectively.

[0091] In other embodiments of the compounds of structure (I), R 8 is -(C=O)OR 12 However, in a different embodiment, R 8 is -O(C=O)R 12 is.

[0092] In certain embodiments of any of the above compounds, R 8 is as follows: -OH, or [ka] This is one of the structures shown below.

[0093] In various different embodiments, the present disclosure provides compounds having one of the structures set forth in Table 1 below, or a pharmaceutically acceptable salt, or tautomer thereof.

[0094] Table 1. Representative compounds [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0095] It is understood that any embodiment of the compounds of structure (I) above, and any particular substituents and / or variables in the compounds of structure (I) above, can be independently combined with other embodiments of the compounds of structure (I) and / or substituents and / or variables to form embodiments of the present disclosure not specifically set forth above. Furthermore, when a list of substituents and / or variables is recited in a particular embodiment and / or claim for a particular R group, G group, L group, or variable a, b, c, d, or n, it is understood that the individual substituents and / or variables can be deleted from the particular embodiment and / or claim, respectively, and the remaining list of substituents and / or variables is considered to be within the scope of the present disclosure.

[0096] It is understood that combinations of substituents and / or variables within the depicted formulae herein are permissible only if such combinations result in stable compounds.

[0097] In some embodiments, lipid nanoparticles are provided comprising a compound of structure (I). The lipid nanoparticles may optionally include an excipient selected from a neutral lipid, a steroid, and a polymer-bound lipid.

[0098] In some embodiments, compositions are provided that include any one or more of the compounds of structure (I) and a therapeutic agent. For example, in some embodiments, the compositions include any one of the compounds of structure (I), a therapeutic agent, and one or more excipients selected from neutral lipids, steroids, and polymer-bound lipids. Other pharmaceutically acceptable excipients and / or carriers are also included in various embodiments of the compositions.

[0099] In some embodiments, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the neutral lipid is DSPC. In various embodiments, the molar ratio of the compound to the neutral lipid is in the range of about 2:1 to about 8:1.

[0100] In various embodiments, the composition further comprises a steroid or steroid analog. In certain embodiments, the steroid or steroid analog is cholesterol. In some of these embodiments, the molar ratio of the compound to cholesterol ranges from about 5:1 to 1:1.

[0101] In various embodiments, the polymer-bound lipid is a PEGylated lipid. For example, some embodiments include PEGylated diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG diacylglycerol succinate (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanediol. PEG-S-DMG), PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamate, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of the compound to the PEGylated lipid ranges from about 100:1 to about 20:1.

[0102] In some embodiments, the composition has the following structure (II): [ka] [In the formula, R 8 and R 9 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain may optionally be interrupted by one or more ester linkages; and The average value of w is in the range of 30 to 60. or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

[0103] In some embodiments, R 8 and R 9 are each independently a linear saturated alkyl chain containing 12 to 16 carbon atoms. In other embodiments, the average value of w is in the range of about 42 to 55, e.g., about 49.

[0104] In some embodiments of the above compositions, the therapeutic agent comprises a nucleic acid. For example, in some embodiments, the nucleic acid is selected from antisense RNA and messenger RNA. In some of the above embodiments, the composition comprises lipid nanoparticles.

[0105] In some related embodiments, a lipid nanoparticle is provided that comprises a compound of any one of the above embodiments (e.g., a compound of structure (I)). In some embodiments, the lipid nanoparticle further comprises a therapeutic agent (e.g., a nucleic acid such as an antisense RNA or a messenger RNA).

[0106] In some embodiments, the lipid nanoparticle further comprises one or more excipients selected from neutral lipids, steroids, and polymer-bound lipids.In some embodiments, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.In more specific embodiments, the neutral lipid is DSPC.

[0107] In some more specific embodiments, the molar ratio of the compound to the neutral lipid ranges from about 2:1 to about 8:1. In some embodiments, the steroid is cholesterol. In some embodiments, the molar ratio of the compound to the cholesterol ranges from 5:1 to 1:1.

[0108] In certain embodiments, the polymer-bound lipid is a pegylated lipid. In more specific embodiments, the molar ratio of the compound to the pegylated lipid ranges from about 100:1 to about 20:1.

[0109] In some embodiments, the pegylated lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, or PEG dialkyloxypropylcarbamate. In other embodiments, the pegylated lipid has the following structure (II): [ka] [In the formula, R 8 and R 9 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain may optionally be interrupted by one or more ester linkages; and The average value of w is in the range of 30 to 60. or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

[0110] In some more specific embodiments of structure (II), R 8 and R 9 are each independently a linear saturated alkyl chain containing 12 to 16 carbon atoms. In a more specific embodiment, the average value of w is about 49.

[0111] In other different embodiments, the present disclosure relates to a method of administering a therapeutic agent to a patient in need thereof, comprising preparing or providing any of the compositions described above and administering the composition to the patient.

[0112] For purposes of administration, embodiments of the compounds of the present disclosure (typically in the form of lipid nanoparticles combined with a therapeutic agent) can be administered as raw chemicals or formulated as pharmaceutical compositions. Pharmaceutical compositions of embodiments of the present disclosure include a compound of structure (I) and one or more pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the compound of structure (I) is present in the composition in an amount effective to form lipid nanoparticles and deliver, for example, a therapeutic agent for treating a particular disease or condition of interest. Appropriate concentrations and dosages can be readily determined by those skilled in the art.

[0113] Administration of the compositions of the presently disclosed embodiments can be carried out via any of the accepted methods of administering pharmaceuticals to provide similar benefits. Pharmaceutical compositions of the presently disclosed embodiments can be formulated into solid, semisolid, liquid, or gaseous formulations, including, for example, tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and nasal. As used herein, the term parenteral includes subcutaneous, intravenous, intramuscular, intradermal, and intrasternal injection or infusion techniques. Pharmaceutical compositions of the presently disclosed embodiments are formulated so that the active ingredient contained therein is bioavailable upon administration of the composition to a patient. In some embodiments, the composition administered to a subject or patient takes the form of one or more unit dosage forms; for example, a tablet may be one unit dosage form, and a container of the compound of the presently disclosed embodiments in aerosol form may hold multiple unit dosage forms. Actual methods for preparing such dosage forms are known or apparent to those skilled in the art. See, e.g., Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). In some embodiments, the composition administered comprises a therapeutically effective amount of a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, in each case for treating the disease or condition of interest according to the teachings of the present disclosure.

[0114] The pharmaceutical compositions of the present disclosure may be in solid or liquid form. In one aspect, the carrier is particulate, with the composition being, for example, in tablet or powder form. Alternatively, the carrier may be liquid, with the composition being, for example, an oral syrup, an injectable liquid, or an aerosol useful, for example, in inhalation administration.

[0115] When intended for oral administration, pharmaceutical compositions of certain embodiments are preferably in either solid or liquid form, with semi-solid, semi-liquid, suspension, and gel forms being included within the scope of forms considered herein as either solid or liquid.

[0116] As a solid composition for oral administration, some embodiments of the pharmaceutical composition can be formulated into the form of powder, granules, compressed tablets, pills, capsules, chewing gum, wafers, etc. Such solid compositions typically contain one or more inert diluents or edible carriers. Additionally, one or more of the following may be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch, lactose, or dextrin; disintegrants such as alginic acid, sodium alginate, Primogel, corn starch, etc.; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavorings such as peppermint, methyl salicylate, or orange flavor; and coloring agents.

[0117] When the pharmaceutical composition of some embodiments is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.

[0118] The pharmaceutical composition of some embodiments can be in the form of a liquid, such as an elixir, syrup, solution, emulsion, or suspension. The liquid can be for oral administration or for delivery by injection, as two examples. For oral administration, preferred compositions contain, in addition to the compound of structure (I), one or more of a sweetener, a preservative, a dye / colorant, and a flavoring agent. For compositions intended for injection administration, one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, and an isotonic agent can be included.

[0119] Liquid pharmaceutical compositions of the present disclosure, whether in solution, suspension, or other form, may contain one or more of the following adjuvants: sterile diluents, such as water for injection, saline, preferably saline, Ringer's solution, or isotonic sodium chloride; fixed oils, such as synthetic monoglycerides or diglycerides, polyethylene glycol, glycerin, propylene glycol, or other solvents that can serve as solvents or suspending media; antibacterial agents, such as benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates, and tonicity adjusters, such as sodium chloride or dextrose; and agents that act as cryoprotectants, such as sucrose or trehalose. Parenteral formulations can be placed in glass or plastic ampoules, disposable syringes, or multiple-dose vials. Saline is a preferred adjuvant. Pharmaceutical compositions for injection are preferably sterile.

[0120] The pharmaceutical composition of the present disclosure can be intended for topical administration, and in this case, the carrier can comprise a solution, emulsion, ointment, or gel base as appropriate.The base can comprise, for example, one or more of petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers.A thickener can also be present in the pharmaceutical composition for topical administration.When intended for transdermal administration, the composition can comprise a transdermal patch or iontophoresis device.

[0121] The pharmaceutical compositions of the present disclosure may contain various materials that modify the physical form of the solid or liquid dosage unit. For example, the compositions may contain materials that form a coating shell around the active ingredient. The coating shell material is usually inert and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredient can be encapsulated in a gelatin capsule.

[0122] Pharmaceutical compositions of embodiments of the present disclosure, in solid or liquid form, may include an agent that binds to the compounds of the present disclosure and thereby aids in the delivery of the LNP. Suitable agents that may act in this capacity include monoclonal or polyclonal antibodies, or proteins.

[0123] The pharmaceutical compositions of the present disclosure may be comprised of dosage units that can be administered as an aerosol. The term aerosol is used to describe a variety of systems, ranging from colloidal to pressurized packaging. Delivery can be achieved by a liquefied or compressed gas that disperses the active ingredient, or by a suitable pump system. The aerosols of the LNPs of the present disclosure may be delivered in single-phase, two-phase, or three-phase systems to deliver the active ingredient. Aerosol delivery includes the necessary containers, activators, valves, subcontainers, etc., which may be combined to form a kit. Those skilled in the art will be able to obtain a suitable aerosol without undue experimentation.

[0124] The pharmaceutical composition of the present disclosure can be prepared by methods well known in the pharmaceutical field.For example, pharmaceutical compositions intended for administration by injection can be prepared by combining the liquid lipid nanoparticles of the present disclosure with sterile distilled water or other carriers to form a solution.Surfactants may be added to promote the formation of a uniform solution or suspension.Surfactants are compounds that interact non-covalently with the compounds of the present disclosure to promote the dissolution or uniform suspension of the compound in aqueous delivery systems.

[0125] The compositions of the presently disclosed embodiments, or pharmaceutically acceptable salts thereof, are administered in a therapeutically effective amount, which will vary depending on a variety of factors, including the activity of the particular therapeutic agent used: the metabolic stability and duration of action of the therapeutic agent; the patient's age, weight, general health, sex, and diet; the mode and time of administration; excretion rate; drug combinations; the severity of the particular disorder or disease; and the subject being treated.

[0126] The composition of the present disclosure's embodiments can also be administered simultaneously with, before, or after the administration of one or more other therapeutic agents.Such combination therapy includes the administration of a single pharmaceutical dosage formulation of the composition of the present disclosure's embodiments and one or more additional active agents, as well as the administration of a composition of the present disclosure's embodiments and each active agent in a separate pharmaceutical dosage formulation.For example, the composition of the present disclosure's embodiments and other active agents can be administered to a patient together in a single oral dosage composition such as a tablet or capsule, or each agent can be administered in a separate oral dosage formulation.When separate dosage formulations are used, the compound of the present disclosure's embodiments and one or more additional active agents can be administered essentially simultaneously, i.e., together, or at different times, i.e., consecutively, and combination therapy is understood to include all of these regimens.

[0127] Methods for preparing the above compounds and compositions are described herein below and / or known in the art.

[0128] It will be understood by those skilled in the art that in the processes described herein, functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxy, amino, mercapto, and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl, or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino, and guanidino include t-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include -C(0)-R" (where R" is alkyl, aryl, or arylalkyl), p-methoxybenzyl, trityl, and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl, or arylalkyl esters. Protecting groups can be added or removed according to standard techniques known to those skilled in the art and described herein. The use of protecting groups is described in Green, TW and PGM Wutz, Protective Groups in Organic Synthesis (1999), 3 rd Ed., Wiley, As one skilled in the art will appreciate, the protecting group may also be a polymer resin such as a Wang resin, a Rink resin, or a 2-chlorotrityl chloride resin.

[0129] Such protected derivatives of the compounds of the present disclosure may not themselves have pharmacological activity, but they may be administered to a mammal and then metabolized in the body to form a pharmacologically active compound of the present disclosure. Such derivatives may therefore be described as "prodrugs." All prodrugs of the compounds of the present disclosure are included within the scope of the present disclosure.

[0130] Additionally, compounds of the disclosed embodiments that exist in a free base or acid form can be converted to their pharmaceutically acceptable salts by treatment with an appropriate inorganic or organic base or acid by methods known in the art. Salts of compounds of the disclosed embodiments can be converted to their free base or acid forms by standard techniques.

[0131] The following General Reaction Scheme 1 illustrates the synthesis of compounds of the present disclosure, i.e., structure (I): [ka] [In the formula, a, b, c, d, G 1 , G 2 , L 1 , L 2 , R 1a , R 1b , R 2a , R 2b , R 3a , R 3b , R 4a , R 4b , R 5 , R 6 , R 7 , R 8 and X is as defined herein. The following provides exemplary methods for preparing a compound of the formula (I), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. It will be understood that one skilled in the art can prepare these compounds by similar methods or by combining other methods known to those skilled in the art. It will also be understood that one skilled in the art can prepare other compounds of structure (I) not specifically shown below by using the appropriate starting components and modifying the synthetic parameters as needed in a manner similar to that described below. In general, the starting components can be obtained from commercial sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, or can be synthesized according to sources known to those skilled in the art (see, e.g., Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)), or can be prepared as described in this disclosure.

[0132] General Reaction Scheme 1 [ka]

[0133] General Reaction Scheme 1 provides an exemplary method for preparing a compound of structure (I) (i.e., A5). In General Reaction Scheme 1, a, b, c, d, and G 2 , L 1 , L 2 , R 1a , R 1b , R 2a , R 2b , R 3a , R 3b , R 4a , R 4b , R 5 , R 6 , R 8 , R 9 , and R 10is as defined herein, and Z is a G sufficient to form a bond with the NH group of A3. 1 (e.g., alkylene or alkene terminated with an aldehyde, acid halide, acrylate, etc.). The intermediates and reagents (e.g., A1 and A2) required to prepare compounds according to General Reaction Scheme 1 can be purchased or prepared according to the Examples below or methods known to those skilled in the art.

[0134] It should be noted that various alternative strategies for preparing compounds of structure (I) are available to those skilled in the art. For example, other compounds of structure (I) can be prepared according to similar methods using appropriate starting materials. The use of protecting groups as necessary and other modifications to the above general reaction scheme will be readily apparent to those skilled in the art. [Example]

[0135] The following examples are offered by way of illustration and not by way of limitation.

[0136] Example 1 In vivo assessment of luciferase mRNA using lipid nanoparticle compositions Lipid nanoparticles were prepared and tested according to the general methods described in PCT Publication Nos. WO2015 / 199952 and WO2017 / 004143, the disclosures of which are incorporated herein by reference in their entirety. Briefly, cationic lipid, DSPC, cholesterol, and PEG lipids were solubilized in ethanol at a molar ratio of approximately 50:10:38.5:1.5 or approximately 47.5:10:40.8:1.7. Lipid nanoparticles (LNPs) were prepared at a total lipid to mRNA weight ratio of approximately 10:1 to 30:1. The mRNA was diluted to 0.2 mg / mL with 10-50 mM citrate or acetate buffer (pH 4). Using a syringe pump, the ethanolic lipid solution was mixed with the aqueous mRNA solution at a ratio of approximately 1:5 to 1:3 (vol / vol) at a total flow rate of 15 mL / min or greater. The ethanol was then removed, and the external buffer was replaced with PBS by dialysis. Finally, the lipid nanoparticles were filtered through a 0.2 μm pore sterile filter. The particle size of the lipid nanoparticles was approximately 55–95 nm, and in some cases, approximately 70–90 nm, as determined by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK).

[0137] Studies were conducted in 6-8 week-old female C57BL / 6 mice (Charles River) or 8-10 week-old CD-1 (Harlan) mice (Charles River) in accordance with guidelines established by the Institutional Animal Care Committee (ACC) and the Canadian Council on Animal Care (CCAC). Various doses of mRNA-lipid nanoparticles were administered systemically via tail vein injection, and animals were euthanized at specific time points (e.g., 4 hours) post-administration. Livers and spleens were collected in pre-weighed tubes, weighed, immediately flash-frozen in liquid nitrogen, and stored at -80°C until further analysis.

[0138] Approximately 50 mg of liver was processed for analysis in a 2 mL FastPrep tube (MP Biomedicals, Solon, OH). A 1 / 4-inch ceramic ball (MP Biomedicals) was added to each tube, and 500 μL of Glo Lysis Buffer-GLB (Promega, Madison, WI), equilibrated to room temperature, was added to the liver tissue. The liver tissue was homogenized twice in a FastPrep24 instrument (MP Biomedicals) at 6.0 m / s for 15 seconds. The homogenate was incubated for 5 minutes at room temperature, then diluted 1:4 with GLB and measured using the SteadyGlo Luciferase Assay System (Promega). Specifically, 50 μL of the homogenate-diluted tissue was reacted with 50 μL of SteadyGlo substrate, shaken for 10 seconds, incubated for 5 minutes, and then assayed using a CentroXS. 3 Quantification was performed using an LB960 luminometer (Berthold Technologies, Germany). The amount of protein assayed was measured using a BCA protein assay kit (Pierce, Rockford, IL). Relative luminescence units (RLU) were then normalized to μg of total protein assayed. To convert RLU to ng of luciferase, a standard curve was generated using QuantiLum Recombinant Luciferase (Promega).

[0139] Trilink Biotechnologies' FLuc mRNA (L-6107 or L-7202) expresses the luciferase protein originally isolated from the firefly (Photinus pyralis). FLuc is commonly used in mammalian cell culture to measure both gene expression and cell viability. It emits bioluminescence in the presence of the substrate luciferin. This capped, polyadenylated mRNA is fully substituted for uridine and / or cytidine nucleosides.

[0140] Example 2 PK of formulated lipids A Measurement of As described elsewhere, the pK of the formulated cationic lipids a pK correlates with the efficacy of LNPs for nucleic acid delivery (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176 (2010)). a The preferred range of pK of each cationic lipid is from about 5 to about 7. a was measured in lipid nanoparticles using a fluorescence-based assay of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). Lipid nanoparticles containing cationic lipid / DSPC / cholesterol / PEG lipid (50 / 10 / 38.5 / 1.5 mol%) at a concentration of 0.4 mM total lipid in PBS were prepared using the in-line process described in Example 1. TNS was prepared as a 100 mM stock solution in distilled water. The vehicle was diluted to 24 μM lipid in 2 mL of buffer containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl, with a pH ranging from 2.5 to 11. An aliquot of TNS solution was added to a final concentration of 1 μM, followed by vortex mixing, and fluorescence intensity was measured at room temperature using an SLM Aminco Series 2 Luminescence Spectrophotometer at excitation and emission wavelengths of 321 nm and 445 nm. A sigmoidal best-fit analysis was applied to the fluorescence data and pKa was determined as the pH that gave rise to half the maximum fluorescence intensity.

[0141] Example 3 Determining the efficacy of lipid nanoparticle formulations containing various cationic lipids using an in vivo luciferase mRNA-expressing rodent model Representative compounds of the present disclosure shown in Table 2 were prepared in the following molar ratio: 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid 2-[2-(ω-methoxy(polyethylene glycol)] 2000The compounds were formulated with 47.5% cationic lipid / 10% DSPC / 40.7% cholesterol / 1.8% PEG lipid. Relative activity was determined by measuring luciferase expression in the liver 4 hours after administration via tail vein injection, as described in Example 1. Activity was compared at doses of 1.0 or 0.5 mg mRNA / kg and expressed as ng luciferase / g liver measured 4 hours after administration, as described in Example 1. Compound numbers in Table 2 refer to compound numbers in Table 1.

[0142] Table 2. Novel cationic lipids and their activities [Table 2-1] [Table 2-2] [Table 2-3]

[0143] Example 4 Synthesis of bis(2-butyloctyl) 7-((3-(dimethylamino)propyl)(3-(dioctylamino)-3-oxopropyl)amino) tridecanedioate (Compound I-7) [ka]

[0144] Synthesis of acryloyl chloride Acrylic acid (1.20 g, 16.65 mmol) was dissolved in 20 mL of anhydrous dichloromethane. Thionyl chloride (1.98 g, 16.65 mmol) was added dropwise with stirring under N2, and the reaction mixture was heated to reflux for 4 h. After completion of the reaction, the crude product was concentrated to give a pale yellow liquid, which was used in the next step without further purification.

[0145] Synthesis of N,N-dioctylacrylamide (intermediate A) Acryloyl chloride (1.12 g, 12.37 mmol) was added to a cooled solution (0 °C) of dioctylamine in dichloromethane containing triethylamine (1 equivalent) as a base. The reaction mixture was stirred at 0 °C for 1 h and at room temperature for an additional 1 h. The reaction mixture was filtered, and the resulting solution was washed with hydrochloric acid (1 N HCl), followed by saturated NaHCO3 solution and brine. The solvent was evaporated under reduced pressure to give the crude product (colorless liquid), which was used in the next step without further purification.

[0146] Synthesis of bis(2-butyloctyl) 7-oxotridecanedioate To a solution of 2-butyloctan-1-ol (3.85 g, 20.66 mmol), 7-oxotridecanedioic acid (1.34 g, 5.17 mmol), and 4-dimethylaminopyridine (DMAP) (1.9 g, 15.55 mmol) in anhydrous DCM was added DCC (4.27 g, 20.69 mmol). The resulting mixture was stirred at room temperature overnight. The solid (DCU) was then filtered and washed with DCM. The filtrate was concentrated. The residue (oil / solid) was purified by column chromatography on silica gel (0–5% ethyl acetate in hexane). The desired product was obtained as a colorless oil (2.55 g, 42.86 mmol, 83%).

[0147] Synthesis of bis(2-butyloctyl) 7-((3-(dimethylamino)propyl)amino) tridecanedioate A solution of 3-(dimethylamino)-1-propylamine (0.09 g, 0.88 mmol) and bis(2-butyloctyl) 7-oxotridecanedioate (0.37 g, 0.63 mmol) in DCE was treated overnight with sodium triacetoxyborohydride (0.20 g, 0.94 mmol) and AcOH (55 μL, 0.98 mmol). The solution was washed with dilute aqueous sodium hydroxide (1 N NaOH). The organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered, and the solvent removed. The residue was passed through a small pad of silica gel and washed with a mixture of DCM / MeOH / EtN (85:15:1). The filtrate was concentrated to give the desired product as a slightly yellow oil (240 mg, 0.35 mmol, 56%).

[0148] Synthesis of I-7 A solution of bis(2-butyloctyl) 7-((3-(dimethylamino)propyl)amino) tridecanedioate (210 mg, 0.30 mmol) and N,N-dioctylacrylamide (1.5 equiv., 136 mg, 0.46 mmol) in EtOH (10 mL) was stirred at room temperature overnight. The reaction mixture was heated to reflux for 7 days. After the reaction was complete, the solvent was removed. The residue was dissolved in a mixture of hexane and EtOAc (19:1) and washed with saturated sodium bicarbonate solution and brine. The extract was dried over sodium sulfate. The dried extract was filtered through a pad of silica gel. The pad was washed with a mixture of hexane / ethyl acetate / triethylamine (80:20:1). The washings were concentrated to give the desired crude product.

[0149] The crude product was further purified by flash dry column chromatography on silica gel (0–5% MeOH in chloroform), which afforded the desired product as a colorless oil (30 mg, 0.03 mmol, 10%). 1 HNMR (400 MHz, CDCl3) δ: 3.96 (d, 5.8 Hz, 4H), 3.30-3.16 (m, 4H), 2.74 (t, 7.2 Hz, 2H), 2.65-2.24 (m, 17H), 1.80-1.44 (m, 12H), 1.43-1.15 (64H), 0.93-0.82 (m, 18H).

[0150] Example 5 Synthesis of bis(2-hexyldecyl) 7-((4-(dihexylamino)-4-oxobutyl)(2-(dimethylamino)ethyl)amino) tridecanedioate (Compound I-19) [ka]

[0151] Synthesis of N,N-dihexyl-4-oxobutanamide (Intermediate B) Butyrolactone (2.51 g, 29.15 mmol) and dihexylamine (5.40 g, 29.13 mmol) were heated in a pressure flask at 61° C. for 4 days. The reaction mixture was cooled to room temperature. The crude product was purified by column chromatography on silica gel (0% to 5% MeOH in DCM) to give N,N-dihexyl-4-hydroxybutanamide as a slightly yellow oil (6.30 g, 79%).

[0152] N,N-Dihexyl-4-hydroxybutanamide (3.00 g, 11.05 mmol) was dissolved in DCM and treated with pyridinium chlorochromate (2.38 g, 11.05 mmol) for 2 hours. Diethyl ether was added, and the supernatant was filtered through a silica gel bed. The solvent was removed from the filtrate, and the resulting oil was dissolved in hexane. The suspension was filtered through a silica gel bed, and the solvent was removed. The crude product (colorless liquid) was used in the next step without further purification.

[0153] Synthesis of I-19 A solution of N,N-dihexyl-4-oxobutanamide (0.56 g, 1.97 mmol) and bis(2-hexyldecyl) 7-((2-(dimethylamino)ethyl)amino) tridecanedioate (0.44 g, 0.56 mmol, prepared by the procedure of Example 4) in 1,2-dichloroethane (10 mL) was stirred for 15 minutes, after which sodium triacetoxyborohydride (0.41 g, 1.97 mmol) was added in one portion and stirred at room temperature for an additional 16 hours. The mixture was concentrated. The residue was taken up in a mixture of hexane and ethyl acetate (96:4) and washed with saturated aqueous NaHCO3 and brine. The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to give a colorless oil. The crude product was purified by flash column chromatography on silica gel (0-5% MeOH in chloroform) to give the desired product as a colorless oil (260 mg, 0.25 mmol, 45%). 1HNMR (400 MHz, CDCl3) δ: 3.96 (d, 5.8 Hz, 4H), 3.28 (t-like, 7.7 Hz, 2H), 3.20 (t-like, 7.7 Hz, 2H), 2.56-2.47 (m, 2H), 2.44 (t, 6.8 Hz, 2H), 2.39-2.20 (m, 15H), 1.74-1.45 (m, 12H), 1.42-1.15 (72H), 0.93-0.84 (m, 18H).

[0154] Example 6 Synthesis of bis(2-butyloctyl) 10-((4-(dihexylamino)-4-oxobutyl)(3-(dimethylamino)propyl)amino)nonadecanedioate (Compound I-21) Compound I-21 was prepared according to the general procedure of Example 5 and obtained as 0.05 g of a colorless oil, 0.03 mmol, 32%. 1 HNMR (400 MHz, CDCl3) δ: 3.97 (d, 5.8 Hz, 4H), 3.28 (t-like, 7.6 Hz, 2H), 3.20 (t-like, 7.6 Hz, 2H), 2.43-2.23 (m, 13H), 2.20 (s, 6H), 1.75-1.45 (m, 14H), 1.40-1.12 (m, 68H), 0.93-0.84 (m, 18H).

[0155] Example 7 Synthesis of bis(2-butyloctyl) 7-((4-(dihexylamino)-4-oxobutyl)(3-(dimethylamino)propyl)amino) tridecanedioate (Compound I-20) Compound I-20 was prepared according to the general procedure of Example 5 and obtained as 0.06 g of a colorless oil, 0.06 mmol, 41%. 1HNMR (400 MHz, CDCl3) δ: 3.96 (d, 5.8 Hz, 4H), 3.27 (t-like, 7.6 Hz, 2H), 3.19 (t-like, 7.6 Hz, 2H), 2.62-2.17 (m, 19H), 1.79-1.43 (m, 14H), 1.42-1.10 (m, 56H), 0.95-0.81 (m, 18H).

[0156] Example 8 Synthesis of bis(2-butyloctyl) 10-(N-(3-(dimethylamino)propyl)-6-methoxy-6-oxohexanamido) nonadecanedioate (compound I-14) [ka]

[0157] A solution of adipic acid chloride (0.12 g, 0.68 mmol) in anhydrous benzene (5 mL) was added via syringe to a solution of bis(2-butyloctyl) 10-((3-(dimethylamino)propyl)amino)nonadecanedioate (0.26 g, 0.34 mmol, prepared according to Example 4), triethylamine (0.3 mL, 2.5 mmol), and DMAP (5 mg) in benzene (10 mL) over 5 min at room temperature. The mixture was stirred for 2 h, and then methanol (0.5 mL) was added to remove excess acyl chloride. The resulting mixture was stirred for an additional 1 h, then filtered through a pad of silica gel, washed with a mixture of hexane / EtOAc / EtN (70:30:1), and concentrated. The residue was passed through a silica gel column (0–4% MeOH gradient in DCM) to give compound I-14 (0.28 g, 0.30 mmol, 89%) as a colorless oil. 1HNMR (400 MHz, CDCl3) δ: 4.52-4.29 (br., estimated 0.3H, due to slow amide bond isomerization), 3.96 (d, 5.8 Hz, 4H), 3.65 (s, 3H), 3.59 (quintet-like, 7.0 Hz, 0.7H), 3.14-3.05 (m, 2H), 2.37-2.24 (m, 10H), 2.23-2.18 (m, 6H), 1.73-1.54 (m, 12H), 1.48-1.37 (m, 4H), 1.34-1.14 (m, 52H), 0.93-0.83 (m, 12H).

[0158] Example 9 Synthesis of bis(2-butyloctyl) 10-(N-(2-(dimethylamino)ethyl)-6-methoxy-6-oxohexanamido) nonadecanedioate (compound I-15) Compound I-15 was prepared according to the general procedure of Example 8 and obtained as 0.18 g of a colorless oil, 0.20 mmol, 85%. 1 HNMR (400 MHz, CDCl3) δ: 4.53-4.30 (br., 0.3H, due to slow amide bond isomerization), 3.96 (d, 5.8 Hz, 4H), 3.65 (s, 3H), 3.58 (quintet-like, 7 Hz, 0.7H), 3.27-3.15 (m, 2H), 2.46-2.22 (m, 16H), 1.75-1.54 (m, 10H), 1.50-1.36 (m, 4H), 1.35-1.09 (m, 52H), 0.94-0.82 (m, 12H).

[0159] Example 10 Synthesis of bis(2-hexyldecyl) 7-(N-(2-(dimethylamino)ethyl)-6-methoxy-6-oxohexanamido) tridecanedioate (Compound I-16) Compound I-16 was prepared according to the general procedure of Example 8 and obtained as 0.27 g of a colorless oil, 0.29 mmol, 81%. 1HNMR (400 MHz, CDCl3) δ: 4.53-4.30 (br., 0.3H, due to slow amide bond isomerization), 3.99-3.92 (m, 4H), 3.66 (s, 3H), 3.59 (quintet-like, 7.0 Hz, 0.7H), 3.28-3.14 (m, 2H), 2.46-2.20 (m, 16H), 1.75-1.53 ​​(m, 10H), 1.51-1.36 (m, 4H), 1.35-1.09 (m, 56H), 0.94-0.81 (m, 12H).

[0160] Example 11 Synthesis of bis(2-hexyldecyl) 7-(N-(3-(dimethylamino)propyl)-8-methoxy-8-oxooctanamido) tridecanedioate (Compound I-17) Compound I-17 was prepared according to the general procedure of Example 8 and obtained as 0.08 g of a colorless oil, 0.08 mmol, 75%. 1 HNMR (400 MHz, CDCl) δ: 4.53-4.30 (br., 0.3H, due to slow amide bond isomerization), 3.99-3.91 (m, 4H), 3.66 (s, 3H), 3.61 (quintet-like, 7.0 Hz, 0.7H), 3.15-3.06 (m, 2H), 2.34-2.23 (m, 10H), 2.22 (s, 6H), 1.75-1.53 ​​(m, 10H), 1.51-1.38 (m, 4H), 1.37-1.15 (m, 62H), 0.93-0.82 (m, 12H).

[0161] Example 12 Synthesis of bis(2-hexyldecyl) 7-(N-(2-(dimethylamino)ethyl)-8-methoxy-8-oxooctanamido) tridecanedioate (Compound I-18) Compound I-18 was prepared according to the general procedure of Example 8 and obtained as 0.15 g of a colorless oil, 0.16 mmol, 79%. 1HNMR (400 MHz, CDCl3) δ: 4.53-4.30 (br., 0.3H, due to slow amide bond isomerization), 3.99-3.92 (m, 4H), 3.66 (s, 3H), 3.61 (quintet-like, 7.0 Hz, 0.7H), 3.26-3.14 (m, 2H), 2.47-2.35 (m, 2H), 2.34-2.20 (m, 14H), 1.73-1.53 ​​(m, 8H), 1.51-1.39 (m, 4H), 1.38-1.14 (m, 62H), 0.93-0.82 (m, 12H).

[0162] Example 13 Synthesis of bis(2-butyloctyl) 10-(N-(3-(dimethylamino)propyl)-6-methoxy-6-oxohexanamido) nonadecanedioate (Compound I-1) [ka]

[0163] Synthesis of intermediate C To a solution of acrylic acid (1.1 equiv., 8.25 mmol, 594 mg), octanol (1 equiv., 975 mg, 7.5 mmol), and DMAP (0.4 equiv., 3 mmol, 366 mg) in DCM (15 mL) was added DCC (1.4 equiv., 10.5 mmol, 2.16 g). The resulting mixture was stirred at room temperature for 16 h. The reaction mixture was filtered, and the filtrate was concentrated. The residue was taken up in hexane (50 mL) and loaded onto a silica gel column. The column was washed with hexane (40 mL). The combined fractions were loaded onto the column again and eluted with a mixture of hexane and ethyl acetate (approximately 99:1 or 98:2, 200 mL). A colorless oil (986 mg, 71%) was obtained.

[0164] Synthesis of Compound I-1 A solution of bis(2-butyloctyl) 10-((4-(dimethylamino)butyl)amino)nonadecanedioate (1 equiv., 220 mg, 0.28 mmol, prepared according to the general procedure above) and Intermediate C (2.75 equiv., 0.77 mmol, 140 mg) in EtOH (10 mL) was placed in a sealed pressure flask and stirred under Ar at room temperature for 4 days. The reaction mixture was concentrated. The residue was purified twice by flash column chromatography on silica gel (hexane-EtOAc-EtN, 95:5:0 to 80:20:1, and 0 to 5% MeOH in chloroform). The desired product was obtained as a colorless oil (68 mg, 0.07 mmol, 25%). 1 HNMR (400 MHz, CDCl3) δ: 4.03 (t, 6.9 Hz, 2H), 3.97 (d, 5.8 Hz, 4H), 2.69 (t, 7.2 Hz, 2H), 2.38-2.33 (m, 4H), 2.33-2.26 (m, 1H), 2.29 (t, 7.5 Hz, 4H), 2.26-2.22 (m, 2H), 2.21 (s, 6H), 1.61 (quintet-like, 7.0 Hz, 8H), 1.48-1.08 (70H), 0.92-0.86 (m, 15H).

[0165] Example 14 Synthesis of bis(2-butyloctyl) 10-((5-(dimethylamino)pentyl)(3-(octyloxy)-3-oxopropyl)amino)nonadecanedioate (Compound I-2) Compound I-2 was prepared according to the general procedure of Example 13 and obtained as 0.05 g of a colorless oil, 0.05 mmol, 10%. 1 HNMR (400 MHz, CDCl3) δ: 4.03 (t, 6.8 Hz, 2H), 3.96 (d, 5.8 Hz, 4H), 2.69 (t, 7.2 Hz, 2H), 2.38-2.21 (m, 17H), 1.61 (quintet-like, 7.0 Hz, 8H), 1.51-1.10 (m, 72H), 0.93-0.84 (m, 15H).

[0166] Example 15 Synthesis of bis(2-butyloctyl) 7-((4-(dimethylamino)butyl)(3-(octyloxy)-3-oxopropyl)amino) tridecanedioate (Compound I-3) Compound I-3 was prepared according to the general procedure of Example 13 and obtained as 0.01 g of a colorless oil, 0.01 mmol, 11%. 1 HNMR (400 MHz, CDCl3) δ: 4.03 (t, 6.9 Hz, 2H), 3.96 (d, 5.6 Hz, 4H), 2.69 (t, 7.1 Hz, 2H), 2.38-2.20 (m, 17H), 1.69-1.56 (m, 10H), 1.48-1.09 (m, 56H), 0.92-0.84 (m, 15H).

[0167] Example 16 Synthesis of bis(2-hexyldecyl) 7-((4-(dimethylamino)butyl)(3-(octyloxy)-3-oxopropyl)amino) tridecanedioate (compound I-4) Compound I-4 was prepared according to the general procedure of Example 13 and obtained as 0.05 g of a colorless oil, 0.05 mmol, 13%. 1 HNMR (400 MHz, CDCl3) δ: 4.03 (t, 6.8 Hz, 2H), 3.96 (d, 5.8 Hz, 4H), 2.69 (t, 7.1 Hz, 2H), 2.39-2.21 (m, 17H), 1.66-1.09 (m, 82H), 0.88 (t, 7.0 Hz, 15H).

[0168] Example 17 Synthesis of bis(2-butyloctyl) 10-((4-(dimethylamino)butyl)(octyl)amino)nonadecanedioate (Compound I-23) [ka]

[0169] Synthesis of compound I-23 A solution of octanal (3.5 equiv., 0.90 mmol, 115 mg, 0.141 mL) and bis(2-butyloctyl) 10-((4-(dimethylamino)butyl)amino)nonadecanedioate (200 mg, 0.26 mmol, prepared according to the general procedure above) in 1,2-dichloroethane (5 mL) was stirred for 15 min, after which sodium triacetoxyborohydride (3.5 equiv., 0.9 mmol, 190 mg) was added in one portion. Stirring was continued at room temperature for 16 h. The reaction mixture was concentrated. The residue was purified twice by flash column chromatography on silica gel (hexane-EtOAc-Et3N, 95:5:0 to 80:20:1, and 0 to 5% MeOH in chloroform). The desired product was obtained as a colorless oil (203 mg, 0.23 mmol, 88%). 1 HNMR (400 MHz, CDCl3) δ: 3.97 (d, 5.8 Hz, 4H), 2.40-2.18 (m, 17H), 1.69-1.56 (m, 6H), 1.52-1.10 (m, 72H), 0.92-0.86 (m, 15H).

[0170] Example 18 Synthesis of bis(2-ethylhexyl) 10-((4-(dimethylamino)butyl)(6-((2-hexyldecanoyl)oxy)hexyl)amino)nonadecanedioate (Compound I-22) Compound I-22 was prepared according to the general procedure of Example 17 and obtained as 0.19 g of a colorless oil, 0.19 mmol, 80%. 1 HNMR (400 MHz, CDCl3) δ: 4.06 (t, 6.7 Hz, 2H), 3.97 (d, 5.6 Hz, 4H), 2.39-2.26 (m, 11H), 2.23 (s, 6H), 1.68-1.10 (m, 83H), 0.94-0.82 (m, 18H).

[0171] Example 19 Synthesis of bis(2-butyloctyl) 10-((4-(dimethylamino)butyl)(6-((2-hexyldecanoyl)oxy)hexyl)amino)nonadecanedioate (Compound I-5) Compound I-5 was prepared according to the general procedure of Example 17 and obtained as 0.04 g of a colorless oil, 0.04 mmol, 73%. 1 HNMR (400 MHz, CDCl3) δ: 4.05 (t, 7.0 Hz, 2H), 3.96 (d, 5., 8 Hz, 4H), 2.38-2.26 (m, 11H), 2.23 (s, 6H), 1.71-1.09 (m, 99H), 0.95-0.82 (m, 18H).

[0172] The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent literature referenced herein (including, but not limited to, U.S. Provisional Patent Application No. 63 / 052,815, filed July 16, 2020, and U.S. Provisional Patent Application No. 63 / 188,996, filed May 14, 2021) are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified, if necessary, to employ concepts from various patents, applications, and literature to provide further embodiments. These and other changes can be made to the embodiments in light of the above detailed description. Generally, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments, along with the full range of equivalents to which such claims are entitled. Accordingly, the scope of the claims is not limited by the present disclosure.

Claims

1. Formula (I): 【Chemistry 1】 [In the formula, G １ and G ２ are each independently C １ -C ６ alkylene; L １ and L ２ are each independently —O(C═O)— or —(C═O)O—; R １ａ and R １ｂ is independently at each occurrence H, or C １ -C １２ is alkyl; R ２ａ and R ２ｂ is independently at each occurrence H, or C １ -C １２ is alkyl; R ３ａ and R ３ｂ is independently at each occurrence H, or C １ -C １２ is alkyl; R ４ａ and R ４ｂ is independently at each occurrence H, or C １ -C １２ is alkyl; R ５ and R ６ are each independently H or methyl; R ７ is -O(C=O)R １０ , -(C=O)OR １０ , -NR ９ (C=O)R １０ or —(C═O)NR ９ R １０ and R ８ is -N(R １１ ) (C=O)R １２ , —(C═O)NR １１ R １２ , -NR １１ R １２ , -(C=O)OR １２ or —O(C═O)R １２ and R ９ is H or C １ -C １５ is alkyl; R １０ is C １ -C １５ is alkyl; R １１ is H or C １ -C ６ is alkyl; R １２ is C １ -C ６ is alkyl; X is —(C═O)— or a direct bond; and a, b, c, and d are each independently an integer from 4 to 10; wherein each alkyl and alkylene is independently optionally substituted with one or more fluoro. or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

2. The following structure (IA) or (IB): 【Chemistry 2】 10. The compound of claim 1, wherein:

3. G １ But C ２ -C ３ Alkylene or C ４ -C ６ 3. The compound of claim 1 or 2, which is alkylene.

4. G ２ But C ２ -C ４ Alkylene, C ２ -C ３ Alkylene, or C ３ -C ４ The compound according to any one of claims 1 to 3, which is alkylene.

5. R ７ is -O(C=O)R １０ or -(C=O)OR １０ and R １０ But linear C １ -C １５ alkyl, and R １０ But linear C ６ -C １０ alkyl, and R １０ is methyl, and R １０ But branch C ２ -C １５ alkyl, or R １０ But branch C １０ -C １５ The compound according to any one of claims 1 to 4, which is alkyl.

6. R ７ But, -NR ９ (C═O), or —(C═O)NR ９ R １０ and R ９ is H and R ９ and R １０ However, each independently, C ６ -C １０ The compound according to any one of claims 1 to 4, which is alkyl.

7. R １ａ , R １ｂ , R ２ａ , R ２ｂ , R ３ａ , R ３ｂ , R ４ａ , and R ４ｂ each occurrence independently represents H, or C １ -C １２ i) R is alkyl; ２ａ , R ２ｂ , R ３ａ , and R ３ｂ is H for each occurrence, and ii) R １ａ and R ４ａ is H for each occurrence, and iii) R １ｂ and R ４ｂ At least one of １ -C ８ alkyl, and iv) C １ -C ８ alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl; and v) 【Transformation 3】 or both, independently: 【Chemistry 4】 7. The compound of claim 1, wherein the compound has one of the structures:

8. The compound according to any one of claims 1 to 7, wherein a, b, c, and d are each independently an integer from 5 to 9.

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

10. R ８ But the following: 【Transformation 5】 The compound according to any one of claims 1 to 9, which has the structure:

11. The following structure: 【Transformation 6】 【Transformation 7】 【Transformation 8】 A compound represented by any one of the following:

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

13. The lipid nanoparticle of claim 12, wherein the therapeutic agent comprises a nucleic acid, preferably the nucleic acid is selected from antisense RNA and messenger RNA.

14. A pharmaceutical composition comprising the lipid nanoparticles of claim 12 or 13 and a pharmaceutically acceptable diluent or excipient.

15. Lipid nanoparticles described in either claim 12 or 13, or a pharmaceutical composition described in claim 14, for use in a method for treating or preventing a disease, preferably for use in a method for vaccinating a patient in need thereof against a viral pathogen, wherein the therapeutic agent is a viral antigen or a nucleic acid capable of transcribing a viral antigen.

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