Cationic lipids for use in lipid nanoparticles

Abstract

The present invention provides novel cationic lipids, the use of the compound as a component of lipid nanoparticle formulations for the delivery of therapeutic agents, nanoparticles containing the compound, and their uses. [Solution] A compound represented by the following structure, or a pharmaceutically acceptable salt thereof, a tautomer, or a stereoisomer thereof, is provided. JPEG2026062827000031.jpg4868

Description

[Technical Field]

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

[0002] There are many challenges associated with the delivery of nucleic acids that influence desired responses in biological systems. Nucleic acid-based therapies hold great potential, but realizing this potential requires more effective delivery of nucleic acids to the appropriate sites within cells or organisms. Examples of therapeutic nucleic acids include messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, immunostimulatory nucleic acids, antagonists, antimirs, mimics, supermirs, and aptamers. Some nucleic acids, such as mRNA or plasmids, can be used to induce the expression of specific cell products, for example, to be useful in treating diseases associated with protein or enzyme deficiencies. The therapeutic applications of translatable nucleotide delivery are very broad, as constructs can be synthesized to generate any selected protein sequence, whether system-specific or not. Nucleic acid expression products can enhance existing levels of proteins, replace deficient or dysfunctional proteins, or introduce novel proteins and associated functions into cells or organisms.

[0003] Some nucleic acids, such as miRNA inhibitors, can be used to influence the expression of specific cell products regulated by miRNAs, for example, to be useful in treating diseases associated with protein or enzyme deficiencies. The therapeutic applications of miRNA inhibition are very broad, as constructs can be synthesized to inhibit one or more miRNAs and control the expression of mRNA products. Inhibition of endogenous miRNAs can enhance the expression of their downstream target endogenous proteins and restore proper function of cells or organisms 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, and consequently downregulate the synthesis of the corresponding protein 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, because oligonucleotide constructs can be synthesized with any nucleotide sequence directed toward target mRNA. Targets can include mRNA from normal cells, mRNA associated with disease conditions such as cancer, and mRNA from infectious agents such as viruses. To date, antisense oligonucleotide constructs have demonstrated the ability to specifically downregulate target proteins through the degradation of homogeneous mRNA in both in vitro and in vivo models. Furthermore, antisense oligonucleotide constructs are currently being evaluated in clinical studies.

[0005] However, the use of oligonucleotides in therapeutic settings currently faces two challenges. First, free RNA is susceptible to nuclease digestion in plasma. Second, free RNA has a limited ability to access the intracellular regions where the relevant translation mechanisms reside. Lipid nanoparticles formed from cationic lipids, including other lipid components such as neutral lipids, cholesterol, PEG, PEGylated lipids, and oligonucleotides, are being used to block the degradation of RNA in plasma and promote the intracellular 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 nucleic acids from degradation and clearance in serum, are suitable for systemic or local delivery, and provide intracellular delivery of nucleic acids. Further, these lipid-nucleic acid particles need to be sufficiently tolerable and provide an appropriate therapeutic index such that patient treatment with an effective dose of the nucleic acid is not associated with unacceptable toxicity and / or risk to the patient. The present disclosure provides these and related advantages. SUMMARY OF THE INVENTION

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

[0008] In one embodiment, the following structure (I):

Chemical formula

[0009] Lipid nanoparticles (LNPs) comprising one or more compounds of structure (I) and a therapeutic agent, and pharmaceutical compositions comprising the same are also provided. 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 the delivery of therapeutic agents for the treatment of diseases or for vaccination against viral pathogens.

[0010] These and other aspects of this disclosure will become apparent by referring to the detailed description below. [Modes for carrying out the invention]

[0011] The following description includes certain details in order to provide a thorough understanding of the various embodiments of this disclosure. However, those skilled in the art will understand that this disclosure can be implemented without these details.

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

[0013] In certain embodiments, this disclosure provides novel cationic lipids that enable the formulation of improved compositions for the in vitro and in vivo delivery of mRNA and / or other oligonucleotides. In some embodiments, these improved lipid nanoparticle compositions are useful for the expression of mRNA-encoded proteins. In other embodiments, these improved lipid nanoparticle compositions are useful for the upregulation of endogenous protein expression by delivering miRNA inhibitors that target one specific miRNA or one target mRNA or a group of miRNAs that modulate several mRNAs. In other embodiments, these improved lipid nanoparticle compositions are useful for downregulating (e.g., silencing) the protein and / or mRNA levels of a target gene. In some other embodiments, lipid nanoparticles are also useful for the delivery of mRNA and plasmids for the expression of transgenes. In yet another embodiment, lipid nanoparticle compositions are useful for inducing pharmacological effects resulting from protein expression, e.g., increased red blood cell production by delivery of appropriate erythropoietin mRNA, or protection against infection by delivery of mRNA encoding an appropriate antigen or antibody.

[0014] The lipid nanoparticles and compositions of the embodiments of this disclosure may 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 this disclosure provide a method for treating or preventing a disease or disorder in a subject requiring such treatment by contacting the subject with lipid nanoparticles, which contain one or more novel cationic lipids described herein, and which encapsulate or associate a suitable therapeutic agent.

[0015] As described herein, embodiments of lipid nanoparticles of this disclosure are particularly useful for the delivery of nucleic acids, such as mRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomir / antimir), messenger RNA interference complementary RNA (micRNA), DNA, multivalent RNA, Dicer substrate RNA, and complementary DNA (cDNA). Therefore, using the lipid nanoparticles and compositions of specific embodiments of this disclosure, the expression of a desired protein can be induced both in vitro and in vivo by contacting cells with lipid nanoparticles containing one or more novel cationic lipids described herein, where the lipid nanoparticles encapsulate or associate the nucleic acid to be expressed to produce the desired protein (e.g., a plasmid or messenger RNA encoding the desired protein) or to inhibit the process that terminates mRNA expression (e.g., a miRNA inhibitor). In specific embodiments, the protein expressed by the nucleic acid is an antigen, and therefore the LNP induces an immune response (e.g., vaccination). Alternatively, the lipid nanoparticles and compositions of the embodiments of this disclosure can be used to reduce the expression of targeted genes and proteins both in vitro and in vivo by contacting cells with lipid nanoparticles containing one or more novel cationic lipids described herein, wherein the lipid nanoparticles encapsulate or associate nucleic acids (e.g., antisense oligonucleotides or small interfering RNAs (siRNAs)) that reduce the expression of the target gene. The lipid nanoparticles and compositions of the embodiments of this disclosure can also be used separately or in combination for the simultaneous delivery of different nucleic acids (e.g., mRNA and plasmid DNA), and may be useful, for example, to provide effects requiring the co-localization of different nucleic acids (e.g., mRNA encoding a suitable gene-modifying enzyme and a DNA segment for integration into the host genome).

[0016] Nucleic acids for use in embodiments of this disclosure can be prepared according to any available technique. In the case of mRNA, the primary 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 long-sequence specific mRNA. In vitro transcription is described as a template-directed synthesis process of an RNA molecule derived from a prepared DNA template, consisting of an upstream bacteriophage promoter sequence (including, but not limited to, those derived from T7, T3, and SP6 colipage) ligated to a downstream sequence encoding the gene of interest. Template DNA can be prepared for in vitro transcription from many sources using appropriate 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 performed in vitro using a linearized DNA template in the presence of the corresponding RNA polymerase and ribonucleoside triphosphates (rNTPs) of adenosine, guanosine, uridine, and cytidine, under conditions that support polymerase activity while minimizing potential degradation of the resulting mRNA transcript. In vitro transcription can be performed using a variety of commercially available kits and commercially available reagents including RNA polymerase and rNTPs, including, but not limited to, the 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, for example, Losick, R., 1972, In vitro transcription, Ann Rev Biochem v.41 409-46; Kamakaka, RT and Kraus, WL 2001. In Vitro Transcription. Current Protocols in Cell Biology. 2:11.6:11.6.1-11.6.17; Beckert, B. And Masquida, B., (2010) Synthesis of RNA by In Vitro Transcription in RNA in Methods in Molecular Biology v. 703 (Neilson, H. Ed), New York, NY Humana Press, 2010; Brunelle, JL and Green, R., 2013, Chapter Five - In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology v. 530, 101-114; all of these are incorporated herein by reference).

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

[0019] Furthermore, while reverse transcription can produce large amounts of mRNA, the product may contain numerous abnormal RNA impurities associated with undesirable polymerase activity that may need to be removed from the full-length mRNA preparation. These include short RNAs resulting from underdeveloped transcription initiations, double-stranded RNAs (dsRNAs) generated by RNA-dependent RNA polymerase activity, RNA priming transcription from RNA templates, and self-complementary 3' extensions. It has been demonstrated that these impurities with dsRNA structures can lead to undesirable immunostimulatory activity through interaction with various innate immune sensors in eukaryotic cells that function to recognize specific nucleic acid structures and induce potent immune responses. This can dramatically reduce mRNA translation, leading to decreased protein synthesis during innate cellular immune responses. Therefore, further techniques for removing these dsRNA contaminants have been developed, including, but are not limited to, scalable HPLC purification, which is known in the art (see, for example, Kariko, K., Muramatsu, H., Ludwig, J. And Weissman, D., 2011, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA, Nucl Acid Res, 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).mRNA purified by HPLC has been reported to be translated at much higher levels, particularly in primary cells and in vivo.

[0020] A fairly diverse range of modifications used to alter specific properties of in vitro transcribed mRNA and improve its utility have been described in the art. These include, but are not limited to, modifications to the 5' and 3' ends of mRNA. Endogenous eukaryotic mRNA typically contains a cap structure at the 5' end of the mature molecule, which plays a crucial role in mediating the binding of cap-binding proteins (CBPs) to mRNA and enhancing the stability of mRNA within the cell 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 furthest 5' nucleotide and the guanine nucleotide. The conjugated guanine nucleotide is methylated at the N7 position. Additional modifications include methylation of the last and second-to-last 5' nucleotides on the 5' end for a 2'-hydroxyl group.

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

[0022] At the 3' end, a long chain of adenine nucleotides (poly-A tail) is usually attached to the mRNA molecule during RNA processing. Immediately after transcription, the 3' end of the transcript is cleaved, releasing the 3' hydroxyl group, and poly-A polymerase then attaches the adenine nucleotide chain to the RNA in a process called polyadenylation. Poly(A) tails have been widely shown to enhance both the translation efficiency and stability of mRNA (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 performed using a variety of methods, including, but not limited to, cloning of poly(T) tracts to a DNA template or post-transcriptional addition using poly(A) polymerase. 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, the poly(A) tail is enzymatically added to in vitro transcribed mRNA using poly(A) polymerase that catalyzes the incorporation of an adenine residue into the 3' end of the RNA, and no additional DNA template manipulation is required, but mRNA with poly(A) tails of non-uniform lengths is obtained. 5'-capping and 3'-poly(A) tailing can be performed using a variety of commercially available kits, including, but not limited to, the poly(A) polymerase tailing kit (EpiCenter), mMESSAGE mMACHINE T7 Ultra Kit, and poly(A) tailing kit (Life Technologies), as well as commercially available reagents, various ARCA caps, poly(A) polymerases, etc.

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

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

[0026] In addition to mRNA, other nucleic acid payloads may be used for the purposes of this disclosure. For oligonucleotides, preparation methods include, but are not limited to, chemical synthesis, enzymatic methods, chemical cleavage of longer precursors, and in vitro transcription as described above. Methods for synthesizing DNA and RNA nucleotides are widely used and well known in the art (e.g., 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] With respect to plasmid DNA, the preparation for use in embodiments of this disclosure generally involves, but is not limited to, the in vitro expansion and isolation of plasmid DNA in a liquid culture of bacteria containing the plasmid of interest. The presence of genes within the plasmid of interest that encode resistance to specific antibiotics (such as penicillin or kanamycin) allows bacteria containing the plasmid of interest to grow selectively in antibiotic-containing cultures. Methods for isolating plasmid DNA are widely used and well known in the art (see, for example, Heilig, J., Elbing, KL and Brent, R., (2001), Large-Scale Preparation of Plasmid DNA, Current Protocols in Molecular Biology, 41:II:1.7:1.7.1-1.7.16; Rozkov, A., Larsson, B., 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 US6,197,553B1). Plasmid isolation can be performed using a variety of commercially available kits, including but not limited to Plasmid Plus (Qiagen), GenJET plasmid MaxiPrep (Thermo), and PureYield MaxiPrep (Promega) kits, as well as commercially available reagents.

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

[0029] When used in this specification, the following terms have the meanings set forth herein unless otherwise specified.

[0030] Unless otherwise specified in the context, throughout this specification and the claims, variations thereof such as “includes,” “includes,” and “contains” should be interpreted in an open and comprehensive sense, that is, “includes, but not limited to.”

[0031] Throughout this specification, any reference to “one embodiment” or “embodiment” means that a particular feature, structure, or characteristic described in relation to that embodiment is included in at least one embodiment of this disclosure. Therefore, occurrences of the phrase “in one embodiment” or “in an embodiment” in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, a particular feature, structure, or characteristic can be combined in any suitable manner in one or more embodiments.

[0032] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this disclosure belongs. When used in the specification and claims, the singular forms “a,” “an,” and “the” include plural references unless the context explicitly indicates otherwise.

[0033] The phrase "induces the expression of a desired protein" means the ability of nucleic acids to increase the expression of a desired protein. To examine the degree of protein expression, a test sample (e.g., a sample of cultured cells expressing the protein of interest) or a test mammalian model (e.g., a mammal such as a human or animal), such as a rodent (e.g., a mouse) or a non-human primate model (e.g., a monkey), is brought into contact with nucleic acids (e.g., nucleic acids combined with the lipids of this disclosure). The expression of the desired protein in the test sample or test animal is compared to the expression of the desired protein in a control sample (e.g., a sample of cultured cells expressing the desired protein) or a control mammalian model (e.g., a mammal such as a human or animal), such as a rodent (e.g., a mouse) or a non-human primate model (e.g., a monkey), which has not been brought into contact with or administered nucleic acids. If the desired protein is present in the control sample or control mammal, a value of 1.0 can be assigned to the expression of the desired protein in the control sample or control mammal. In certain embodiments, induction of the desired protein expression is achieved when the ratio of the desired protein expression level in the test sample or test mammal to the desired protein expression level in the control sample or control mammal is greater than 1, for example, about 1.1, 1.5, 2.0, 5.0, or 10.0. If the desired protein is not present in the control sample or control mammal, induction of the desired protein expression is achieved when a measurable level of the desired protein is detected in the test sample or test mammal. Those skilled in the art will understand appropriate assays for determining the level of protein expression in a sample, such as dot blotting, Northern blotting, in-situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays, or assays based on reporter proteins that can produce fluorescence or luminescence under appropriate conditions.

[0034] The phrase "inhibits the expression of a target gene" refers to the ability of a nucleic acid to silence, reduce, or inhibit the expression of a target gene. To investigate the degree of gene silencing, a test sample (e.g., a sample of cultured cells expressing the target gene) or a test mammalian model (e.g., a mammalian model such as a human or animal), such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey), is brought into contact with the nucleic acid that silences, reduces, or inhibits the expression of the target gene. The expression of the target gene in the test sample or test animal is compared to the expression of the target gene in a control sample (e.g., a sample of cultured cells expressing the target gene) or a control mammal (a mammalian model such as a human or animal, such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model) that has not been in contact with or administered the nucleic acid. The expression of the target gene in the control sample or control mammal may be assigned a value of 100%. In a particular embodiment, silencing, inhibition, or reduction of the expression of the target gene is compared to 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. This is achieved when the gene expression level is approximately 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. In other words, nucleic acids can silence, reduce, or inhibit the expression of a target gene in a test sample or test mammal by at least approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the gene expression level in a control sample or control mammal that has not been in contact with or administered nucleic acids.Suitable assays for determining the expression level of a target gene include, but are not limited to, measurements of protein or mRNA levels using techniques known to those skilled in the art, such as dot blotting, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those skilled in the art.

[0035] The “effective dose” or “therapeutic effective dose” of a therapeutic agent, such as an activator or therapeutic nucleic acid, is an amount sufficient to produce the desired effect, such as an increase or inhibition of the expression of a target sequence compared to the normal expression level detected in the absence of the nucleic acid. An increase in the expression of a target sequence is achieved when a measurable level is detected, in the case of an expression product that does not exist in the absence of the nucleic acid. If the expression product exists at a level present before contact with the nucleic acid, an increase in expression is achieved when the multiplier of the value obtained with the nucleic acid, such as mRNA, is approximately 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000 or more compared to the control. Inhibition of the expression of a target gene or target sequence is achieved when the value obtained with a nucleic acid such as an antisense oligonucleotide is approximately 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% compared to the control. Suitable assays for measuring the expression of a target gene or target sequence include protein or RNA level measurements using techniques known to those skilled in the art, such as dot blotting, Northern blotting, in-situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of a suitable reporter protein, and phenotypic assays known to those skilled in the art.

[0036] In this specification, the term “nucleic acid” means a polymer comprising at least two deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form, and includes DNA, RNA, and hybrids thereof. DNA may be in the form of an antisense molecule, plasmid DNA, cDNA, PCR product, or vector. RNA may be in the form of small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA, dicer substrate RNA, or viral RNA (vRNA), and combinations thereof. Nucleic acids include synthetic, natural, and unnatural nucleic acids that contain known nucleotide analogs or modified backbone residues or linkages having similar binding properties to the reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramides, methylphosphonates, chiral methylphosphonates, 2'-O-methylribonucleotides, and peptide-nucleic acids (PNAs). Unless otherwise specified, the term encompasses nucleic acids that contain known analogs of natural nucleotides having similar binding properties to the reference nucleic acid. Unless otherwise specified, a given nucleic acid sequence implicitly includes not only the explicitly indicated sequence but also its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologues, single nucleotide polymorphisms, and complementary sequences. Specifically, degenerate codon substitutions can be performed by generating a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). A "nucleotide" consists of the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked via phosphate groups.Examples of "bases" include purines and pyrimidines, as well as the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and their natural analogues, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications that introduce new reactive groups, such as, but are not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0037] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence containing a partial or full-length coding sequence necessary for the production of polypeptides or polypeptide precursors.

[0038] In this specification, “gene product” means a product of a gene, such as an RNA transcript or polypeptide.

[0039] The term "lipids" refers to a group of organic compounds that include, but are not limited to, fatty acid esters, and are generally poorly soluble in water but soluble in many organic solvents. They are usually classified into at least three classes: (1) "simple lipids" (such as oils and waxes); (2) "complex lipids" (such as phospholipids and glycolipids); and (3) "derived lipids" (such as steroids).

[0040] "Steroids" have the following carbon skeleton: [ka] It is a compound containing [this substance]. Examples of steroids, though not limited to this, include cholesterol.

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

[0042] The term "polymer-bound lipid" refers to a molecule that contains both a lipid portion and a polymer portion. An example of a polymer-bound 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 this art, and examples include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG).

[0043] The term "neutral lipids" refers to any of several lipid species that exist in either an uncharged or neutral zwitterionic form at a selected pH. 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), and 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, and sterols; and their derivatives. Neutral lipids may 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 occurring. Examples of charged lipids include phosphatidylserine, phosphatidylic acid, phosphatidylglycerol, phosphatidylinositol, hemisuccinate sterols, dialkyltrimethylammonium propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, and dimethylaminoethanecarbamoyl sterol (e.g., DC-Chol).

[0045] The term “lipid nanoparticles” means particles having at least one dimension on the order of nanometers (e.g., 1 to 1,000 nm), comprising one or more 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 may be used to deliver activators or therapeutic agents, such as nucleic acids (e.g., mRNA), to a target site of interest (e.g., cells, tissues, organs, tumors, etc.). In some embodiments, the lipid nanoparticles comprise the compound of structure (I) and nucleic acids. Such lipid nanoparticles typically comprise the compound of structure (I) and one or more excipients selected from neutral lipids, charged lipids, steroids, and polymer-binding lipids. In some embodiments, the activator or therapeutic agent, such as nucleic acids, can be encapsulated in the lipid portion of the lipid nanoparticle, or in an 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 induced by host organism or cellular mechanisms, such as adverse immune responses.

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

[0047] In this specification, “lipid encapsulation” means lipid nanoparticles that provide an activator or therapeutic agent, such as a nucleic acid (e.g., mRNA), involving complete encapsulation, partial encapsulation, or both. In one embodiment, the nucleic acid (e.g., mRNA) is completely encapsulated in lipid nanoparticles.

[0048] In this specification, the term "aqueous solution" means a composition containing water.

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

[0050] In this specification, “systemic delivery” means the delivery of a therapeutic product that can expose an active agent to a wide area within a living organism. Some administration techniques can allow certain drugs to be delivered systemically, while others cannot. Systemic delivery means that a useful, preferably therapeutic, amount of the drug is exposed to most parts of the body. Systemic delivery of lipid nanoparticles can be done by any means known in the art, including, for example, intravenous, intra-arterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery.

[0051] In this specification, “local delivery” means the direct delivery of an active agent to a target site within a living organism. For example, a drug can be delivered locally by direct injection to a disease site such as a tumor, another target site such as an inflammatory site, or a 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 preclude systemic pharmacological effects.

[0052] "Alkyl" refers to a group of a saturated, linear or branched hydrocarbon chain consisting only of carbon and hydrogen atoms, for example, 1 to 24 carbon atoms (C1-C1). 24 Alkyl), 4-20 carbon atoms (C4-C 20 Alkyl), 6-16 carbon atoms (C6-C 16 Alkyl), 6-9 carbon atoms (C6-C9 alkyl), 1-15 carbon atoms (C1-C 15 Alkyl), 1 to 12 carbon atoms (C1-C 12Alkyl groups have 1 to 8 carbon atoms (C1-C8 alkyl) or 1 to 6 carbon atoms (C1-C6 alkyl), which are bonded to the rest of the molecule by single bonds, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, etc. Unless otherwise specified herein, alkyl groups are substituted or unsubstituted.

[0053] "Alkylene" refers to a saturated, linear or branched divalent hydrocarbon chain consisting only of carbon and hydrogen atoms, with the rest of the molecule bonded to other groups, for example, 1 to 24 carbon atoms (C1-C1). 24 Alkylene), 1 to 15 carbon atoms (C1-C 15 Alkylene), 1 to 12 carbon atoms (C1-C 12 Alkylenes have 1 to 8 carbon atoms (C1-C8 alkylenes), 1 to 6 carbon atoms (C1-C6 alkylenes), 2 to 4 carbon atoms (C2-C4 alkylenes), and 1 to 2 carbon atoms (C1-C2 alkylenes), such as methylene, ethylene, propylene, and n-butylene. Alkylene chains are bonded to the rest of the molecule via single bonds and to other groups via single bonds. The bonding points of the alkylene chain to the rest of the molecule and other groups can be via one carbon or any two carbons in the chain. Unless otherwise specified herein, alkylene chains are substituted or unsubstituted.

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

[0055] In this specification, the term “substituted” means, but is not limited to, any of the above groups (e.g., alkyl or alkylene) in which at least one hydrogen atom is replaced by a bond to a non-hydrogen atom, but substitution can be a halogen atom, e.g., F, Cl, Br, or I; an oxo group (=O); a hydroxyl group (-OH); a carboxyl group (-CO2H); or C1-C 12 alkyl;-(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' is independently H, C1-C each time it appears. 15 It is alkyl, and x is 0, 1, or 2. In some embodiments, the substituent is C1-C 12 It is an alkyl group. In other embodiments, the substituent is a halo group such as fluoro. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group (-OR'). In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group (-NR'R').

[0056] "Optional" or "optionally" (e.g., optionally substituted) means that the event of the situation described thereafter may or may not occur, and that description includes both cases in which such event or situation occurs and cases in which it does not occur. For example, "optionally substituted alkyl" means that the alkyl group may or may not be substituted, and that the description includes both substituted alkyl groups and alkyl groups without substituents.

[0057] The disclosures herein also mean to encompass all pharmaceutically acceptable compounds of the compound of structure (I) that are isotope-labeled by replacing one or more atoms with atoms of different atomic masses or mass numbers. Examples of isotopes that can be incorporated into the disclosed compounds include, for example, isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, respectively. 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 36 Cl, 123 I, and 125 These radiolabeled compounds may be useful in determining or measuring the efficacy of a compound by characterizing, for example, the site of action or mechanism of action, or the binding affinity to a pharmacologically important site of action. Specific isotope-labeled compounds of structure (I), (IA), or (IB), such as compounds incorporating radioisotopes, are useful in studying drug and / or substrate tissue distribution. The radioisotope tritium, i.e. 3 H, and carbon-14, i.e. 14 C is particularly useful for this purpose in terms of ease of integration and detection preparation.

[0058] Deuterium, that is 2 Substitution with heavier isotopes, such as 1H, may result in greater metabolic stability, leading to certain therapeutic benefits such as increased in vivo half-life or reduced required doses, and may therefore be preferable in certain situations.

[0059] 11 C, 18 F, 15 O, 13Substitution with positron-emitting isotopes such as 16N may be useful in positron emission tomography (PET) studies to investigate substrate-receptor occupancy. The isotope-labeled compound of structure (I) can generally be prepared by using a suitable isotope-labeled reagent in place of the previously used unlabeled reagent, either by conventional techniques known to those skilled in the art or by processes similar to those described in the following manufacturing methods and examples.

[0060] "Stable compound" and "stable structure" refer to a compound that is robust enough to withstand isolation from a reaction mixture to a useful purity and formulation into an effective therapeutic agent.

[0061] "Mammals" include both humans and domesticated animals such as laboratory animals and household pets (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits), as well as non-domesticated animals such as wild animals.

[0062] "Pharmacologically acceptable carriers, diluents or excipients" include, but are not limited to, adjuvants, carriers, excipients, flow enhancers, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, humectants, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers that are approved by the U.S. Food and Drug Administration as acceptable for use in human or livestock animals.

[0063] "Pharmacologically acceptable salts" include both addition salts of acids and bases.

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

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

[0066] Crystallization often produces solvates of the compounds of the Disclosure (i.e., compounds of structure (I)). In this specification, the term “solvate” means an aggregate comprising one or more molecules of the compounds of the 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 Disclosure may exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, and the corresponding solvates. While the solvates of the compounds of the Disclosure may be true solvates, in other cases, the compounds of the Disclosure may simply retain incidental water or be a mixture of water and several incidental solvents.

[0067] "Pharmaceutical composition" means a formulation of the compounds of this disclosure and a medium generally accepted in the art for delivering the bioactive compounds to a mammal (e.g., human). Such a medium includes all pharmaceutically acceptable carriers, diluents, or excipients for that purpose.

[0068] “Effective dose” or “therapeutic dose” means the amount of the compound of the Disclosure that, when administered to a mammal, preferably a human, is sufficient to treat a mammal, preferably a human. The amount of lipid nanoparticles of the Disclosure that constitutes a “therapeutic dose” varies depending on the compound, the disease and its severity, the method of administration, and the age of the mammal being treated, but a person skilled in the art can determine it in the course of business, taking into account their knowledge and the Disclosure.

[0069] In this specification, “to treat” or “to treat” encompasses the treatment of the disease or illness in a mammal (preferably human) having the disease or illness of the interest, and includes: (i) To prevent the occurrence of disease or illness in a mammal, especially if that mammal is susceptible to the disease but has not yet been diagnosed as having it; (ii) To suppress a disease or illness (i.e., to stop its onset); (iii) to alleviate a disease or illness (i.e., to cause a regression of a disease or illness); or, (iv) Relieving symptoms caused by a disease or illness (i.e., relieving pain without addressing the underlying disease or illness). In this specification, the terms “disease” and “illness” may be used interchangeably, or they may differ in that a particular illness or condition may not have a known causative agent (and therefore its etiology is not yet understood), and thus it may be recognized only as an undesirable illness or syndrome in which some specific symptoms have been identified by a clinician, but which is not yet recognized as a disease.

[0070] The compounds of this disclosure, or any pharmaceutically acceptable salts thereof, may contain one or more stereocenters and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined with respect to absolute stereochemistry as (R)- or (S)-, or in the case of amino acids as (D)- or (L)-. This 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 suitable optically pure precursors, or separation of racemics (or racemics of salts or derivatives) using, for example, chiral high-performance liquid chromatography (HPLC). Where a compound described herein contains an olefin double bond or other geometrically asymmetric center, unless otherwise specified, the compound is intended to include both E and Z geometric isomers. Similarly, it is also intended to include all tautomer forms.

[0071] A "stereoisomer" refers to a compound that consists of the same atoms bonded together by the same bonds, but has different, incompatible three-dimensional structures. This disclosure intends to cover various stereoisomers and mixtures thereof, and includes "enantiomers," which means two stereoisomers that are mirror images of each other and cannot be superimposed.

[0072] "Tautomerism" refers to a proton shift from one atom in a molecule to another atom in the same molecule. This disclosure includes tautomers of any of the aforementioned compounds.

[0073] compound In one embodiment, the 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 containing therapeutic agents, such as oligonucleotides. While not constrained by theory, these lipid nanoparticles are thought to protect therapeutic agents from degradation in serum and provide effective delivery of oligonucleotides to cells 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 These are, independently, C1-C6 alkylenes; L 1 and L 2 These are, independently, -O(C=O)- or -(C=O)O-; R 1a and R 1b Each instance of this occurs independently of (a)H, or C1-C. 12 (b) R 1a is H, or C1-C 12 It is alkyl, R 1b It, together with the carbon atom to which it is bonded, and adjacent R 1band together with the carbon atom to which it is attached forms R 1b is taken to form a carbon-carbon double bond; R 2a and R 2b each independently, upon each occurrence, is (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, together with adjacent R 2b and together with the carbon atom to which it is attached forms R 2b is taken to form a carbon-carbon double bond; R 3a and R 3b each independently, upon each occurrence, is (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, together with adjacent R 3b and together with the carbon atom to which it is attached forms R 3b is taken to form a carbon-carbon double bond; R 4a and R 4b each independently, upon each occurrence, is (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, together with adjacent R 4b and together with the carbon atom to which it is attached forms 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 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 It is alkyl; R 10 C1-C 15 It is alkyl; R 11 is H, or a C1-C6 alkyl group; R 12 It is a C1-C6 alkyl group; X is -(C=O)-, or a direct bond; and, a, b, c, and d are each independent integers between 1 and 24; Here, each alkyl and alkylene may be independently and optionally substituted. The compound represented by the structure shown, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

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

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

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

[0078] In various different embodiments, X is -(C=O)-, but 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 embodiments of these embodiments, R 10 is a linear C1-C 15 It is alkyl, for example, a linear C6-C 10 It is alkyl. In other such embodiments, R 10 is methyl, or R 10 This is branch C2-C 15 It is alkyl, for example, branched C 10 -C 15 It is alkyl.

[0080] In yet another embodiment of the above embodiment, R 7 -NR 9 (C=O) or -(C=O)NR 9 R 10 In some of these embodiments, R 9 H is H. In other embodiments of these, R 9 and R 10 These 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 It is alkyl, R 1b It, together with the carbon atom to which it is bonded, and adjacent R 1b and together with the carbon atom to which it is bonded, R 1b A carbon atom is removed, forming 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 It is alkyl, R 4b It, together with the carbon atom to which it is bonded, and adjacent R 4b and together with the carbon atom to which it is bonded, R 4b A carbon atom is removed, forming a carbon-carbon double bond.

[0083] In yet another embodiment, R 2a and R 2b For at least one occurrence of R 2a is H, or C1-C 12 It is alkyl, R 2b It, together with the carbon atom to which it is bonded, and adjacent R 2b and together with the carbon atom to which it is bonded, R 2b A carbon atom is removed, forming 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 It is alkyl, R 3b It, together with the carbon atom to which it is bonded, and adjacent R 3b and together with the carbon atom to which it is bonded, R 3b A carbon atom is removed, forming a carbon-carbon double bond.

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

[0086] In other, more specific embodiments, see below: [ka] or both, independently, as follows: [ka] It is one of the structures shown.

[0087] In a further embodiment, a, b, c, and d are each independently integers between 2 and 12. In a further embodiment, a, b, c, and d are each independently integers between 4 and 10, 5 and 10, 6 and 10, 4 and 9, 5 and 9, or 6 and 9. In yet another embodiment, b and c are each independently 5, 6, 7, 8, 9, or 10.

[0088] In some embodiments, R 5 or R6 One of them is methyl. In other embodiments, R 5 and R 6 Each of them is methyl.

[0089] In some embodiments, R 8 It 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 a more different embodiment, R 8 -NR 11 R 12 In some of these embodiments described above, R 11 and R 12 Each is independently H or C1-C8 alkyl. In these other embodiments, R 11 and R 12 Each is independently H or C1-C3 alkyl. For example, in some embodiments, the C1-C8 alkyl or C1-C3 alkyl is either unsubstituted or substituted with a hydroxyl group. In other different such embodiments, R 11 and R 12 These are methyl compounds.

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

[0092] In any particular embodiment of the above compound, R 8 The following: -OH, or [ka] It is one of the structures shown.

[0093] In various different embodiments, the present disclosure provides compounds represented by one of the structures listed in Table 1 below, or pharmaceutically acceptable salts thereof, or tautomers 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 compound of structure (I) above, and any specific substituents and / or variables in the compound of structure (I) above, can independently be combined with other embodiments and / or substituents and / or variables of the compound of structure (I) to form embodiments of the present disclosure not specifically described above. Furthermore, if a list of substituents and / or variables is given for a particular R group, G group, L group, or variable a, b, c, d, or n in a particular embodiment and / or claim, it is understood that each individual substituent and / or variable may be removed from the particular embodiment and / or claim, and the remaining list of substituents and / or variables is considered to be within the scope of the present disclosure.

[0096] In this specification, it is understood that combinations of substituents and / or variables in the formulas described are permissible only if such contributions result in a stable compound.

[0097] In some embodiments, lipid nanoparticles comprising a compound of structure (I) are provided. The lipid nanoparticles may optionally contain excipients selected from neutral lipids, steroids, and polymer-bound lipids.

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

[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 analogue. In certain embodiments, the steroid or steroid analogue is cholesterol. In some of these embodiments, the molar ratio of the compound to cholesterol is in the range of about 5:1 to 1:1.

[0101] In various embodiments, the polymer-bound lipid is a PEGylated lipid. For example, in some embodiments, PEGylated diacylglycerol (PEG-DAG), e.g., 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG-succinate diacylglycerol (PEG-S-DAG), e.g., 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanediol The compounds include PEG-S-DMG, PEG-ceramide, 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 is in the range of 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 Each is independently a linear or branched saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain may optionally have one or more ester bonds interspersed; and The average value of w is in the range of 30 to 60. This includes pegylated lipids represented by , or their pharmaceutically acceptable salts, tautomers, or stereoisomers.

[0103] In some embodiments, R 8 and R 9 Each of these is 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, for example, about 49.

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

[0105] In some related embodiments, lipid nanoparticles are provided that comprise any one of the compounds of the embodiments described above (e.g., the compound of structure (I)). In some embodiments, the lipid nanoparticles further comprise a therapeutic agent (e.g., nucleic acids such as antisense RNA and messenger RNA).

[0106] In some embodiments, the lipid nanoparticles further comprise 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 a more specific embodiment, the neutral lipid is DSPC.

[0107] In some more specific embodiments, the molar ratio of the compound to the neutral lipid is in the range of about 2:1 to about 8:1. In some embodiments, the steroid is cholesterol. In some embodiments, the molar ratio of the compound to cholesterol is in the range of 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 is in the range of 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 Each is independently a linear or branched saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain may optionally have one or more ester bonds interspersed; and The average value of w is in the range of 30 to 60. These are pegylated lipids represented by , or their pharmaceutically acceptable salts, tautomers, or stereoisomers.

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

[0111] In other different embodiments, the Disclosure relates to a method for administering a therapeutic agent to a patient in need thereof, comprising preparing or providing one of the aforementioned compositions, and administering the composition to the patient.

[0112] For administration purposes, embodiments of the compounds of this disclosure (typically in the form of lipid nanoparticles combined with a therapeutic agent) may be administered as raw chemicals or formulated as pharmaceutical compositions. Pharmaceutical compositions of embodiments of this disclosure comprise the 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 illness of interest. Appropriate concentrations and doses can be readily determined by those skilled in the art.

[0113] The administration of the compositions of the embodiments of this disclosure can be carried out through any acceptable method of administration of a drug to provide similar utility. The pharmaceutical compositions of the embodiments of this disclosure can be formulated into solid, semi-solid, liquid, or gaseous formulations, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes for administering such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, oral cavity, rectal, vaginal, and nasal. In this specification, the term parenteral includes subcutaneous, intravenous, intramuscular, intradermal, intrasternal, and intrasternal injection or infusion techniques. The pharmaceutical compositions of the embodiments of this disclosure are formulated so that the active ingredients contained herein are bioavailable when the composition is administered to a patient. In some embodiments, the composition administered to a subject or patient may take 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 embodiments of this disclosure in aerosol form may hold multiple unit dosage forms. Methods for actually producing such dosage forms are known or obvious to those skilled in the art. See, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). In some embodiments, the administered composition in each case contains a therapeutically effective amount of the compound of the Disclosure or a pharmaceutically acceptable salt thereof for the treatment of the disease or illness of interest in accordance with the teachings of the Disclosure.

[0114] The pharmaceutical compositions of the embodiments of this disclosure may be in solid or liquid form. In one embodiment, the carrier is particulate, and the composition is, for example, in the form of a tablet or powder. Alternatively, the carrier may be liquid, and the composition may be, for example, an oral syrup, an injection solution, or an aerosol useful for, for example, inhalation administration.

[0115] When intended for oral administration, the pharmaceutical composition of a particular embodiment is preferably in either solid or liquid form, and this includes semi-solid, semi-liquid, suspension, and gel forms, which are considered as either solid or liquid in this specification.

[0116] As solid compositions for oral administration, pharmaceutical compositions of several embodiments can be formulated in the form of powders, granules, compressed tablets, pills, capsules, chewing gum, wafers, and the like. Such solid compositions typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders, e.g., carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth gum, or gelatin; excipients, e.g., starch, lactose, or dextrin; disintegrants, e.g., alginic acid, sodium alginate, Primogel, corn starch, etc.; lubricants, e.g., magnesium stearate, or Sterotex; flow enhancers, e.g., colloidal silicon dioxide; sweeteners, e.g., sucrose, or saccharin; flavorings, e.g., peppermint, methyl salicylate, or orange flavor; and colorants.

[0117] In some embodiments, when the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may include a liquid carrier such as polyethylene glycol or oil, in addition to the raw materials of the types described above.

[0118] The pharmaceutical compositions of some embodiments may be in liquid form, such as elixirs, syrups, solutions, emulsions, or suspensions. The liquids may be, as two examples, for oral administration or for delivery by injection. When intended for oral administration, preferred compositions include, in addition to the compound of structure (I), one or more of the following: sweeteners, preservatives, dyes / colorants, and flavoring agents. Compositions intended for administration by injection may include one or more of the following: surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents.

[0119] Liquid pharmaceutical compositions of embodiments of this disclosure, whether they are in the form of solutions, suspensions or other methods, may contain one or more of the following adjuvants: sterile diluents, e.g., water for injection, saline, preferably physiological saline, Ringer's solution, isotonic sodium chloride; non-volatile oils, e.g., synthetic monoglycerides or diglycerides, polyethylene glycol, glycerin, propylene glycol, or other solvents that can function as a solvent or suspension medium; antimicrobial agents, e.g., benzyl alcohol or methylparaben; antioxidants, e.g., ascorbic acid or sodium bisulfite; chelating agents, e.g., ethylenediaminetetraacetic acid; buffers, e.g., acetates, citrates, or phosphates, and tonic modifiers such as sodium chloride or dextrose; agents acting as antifreeze, e.g., sucrose or trehalose. Parenteral formulations may be placed in glass or plastic ampoules, disposable syringes, or multi-dose vials. Physiological saline is a preferred adjuvant. Pharmaceutical compositions for injection are preferably sterile.

[0120] The pharmaceutical compositions of embodiments of this disclosure may be intended for topical administration, in which case the carrier may optionally include a solution, emulsion, ointment, or gel base. The base may include, for example, diluents such as petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, water, and alcohol, as well as one or more emulsifiers and stabilizers. Thickeners may be present in the pharmaceutical composition for topical administration. When intended for transdermal administration, the composition may include a transdermal patch or an iontophoresis device.

[0121] The pharmaceutical compositions of embodiments of this disclosure may include a variety of materials that alter the physical form of a solid or liquid dosing unit. For example, the composition may include a material that forms a coating shell around the active ingredient. The material forming the coating shell is typically inert and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule.

[0122] Pharmaceutical compositions of embodiments of the present disclosure, in solid or liquid form, may include agents that bind to the compounds of the present disclosure, thereby assisting in the delivery of LNPs. Suitable agents capable of acting in this capacity include monoclonal or polyclonal antibodies, or proteins.

[0123] The pharmaceutical compositions of embodiments of this disclosure may consist of dosing units that can be administered as aerosols. The term aerosol is used to describe a variety of systems, ranging from colloidal to systems consisting of pressurized packaging. Delivery may be by liquefaction or compressed gas that disperses the active ingredient, or by a suitable pumping system. The aerosols of LNPs of embodiments of this disclosure may be delivered in single-phase, two-phase, or three-phase systems for delivering the active ingredient. Delivery of the aerosol may include necessary containers, activators, valves, sub-containers, etc., which together may form a kit. Those skilled in the art can obtain a preferred aerosol without excessive experimentation.

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

[0125] The compositions of embodiments of this disclosure, or pharmaceutically acceptable salts thereof, are administered in therapeutically effective doses, which vary depending on a variety of factors including the activity of the particular therapeutic agent used: factors such as the metabolic stability and duration of action of the therapeutic agent; the patient's age, weight, usual health condition, sex, and diet; the manner and timing of administration; the rate of excretion; the combination of drugs; the severity of a particular disorder or disease; and the subject being treated.

[0126] The compositions of the embodiments of this disclosure may also be administered concurrently with, before, or after the administration of one or more other therapeutic agents. Such combination therapies include the administration of a single pharmaceutical formulation of the compositions of the embodiments of this disclosure and one or more additional activators, as well as the administration of separate pharmaceutical formulations of the compositions of the embodiments of this disclosure and each activator. For example, the compositions of the embodiments of this disclosure and other activators may be administered together to a patient in a single oral formulation such as a tablet or capsule, or each agent may be administered in a separate oral formulation. When separate formulations are used, the compounds of the embodiments of this disclosure and one or more additional activators may be administered essentially simultaneously, i.e., together, or separately at staggered times, i.e., consecutively, and combination therapy is understood to include all such regimens.

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

[0128] It will be understood by those skilled in the art that in the methods described herein, the functional groups of intermediate compounds may need to be protected by appropriate protecting groups. Such functional groups include hydroxy, amino, mercapto, and carboxylic acids. Suitable protecting groups for hydroxyl groups include trialkylsilyl or diarylalkylsilyl (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl, or trimethylsilyl), tetrahydropyranyl, and benzyl. Suitable protecting groups for amino, amidino, and guanidino groups include t-butoxycarbonyl and benzyloxycarbonyl. Suitable protecting groups for mercapto groups include -C(0)-R'' (where R'' is alkyl, aryl, or arylalkyl), p-methoxybenzyl, and trityl. Suitable protecting groups for carboxylic acids include alkyl, aryl, or arylalkyl esters. Protecting groups can be added or removed according to the standard techniques known to those skilled in the art and described herein. For the use of protecting groups, see Green, TW and PGM Wutz, Protective Groups in Organic Synthesis (1999), 3 rd As described in detail in Ed., Wiley, as those skilled in the art will understand, the protecting group may also be a polymer resin such as Wang resin, Rink resin, or 2-chlorotrityl chloride resin.

[0129] Such protected derivatives of the compounds of the Disclosure may not possess pharmacological activity themselves, but they may be administered to mammals and subsequently metabolized in the body to form pharmacologically active compounds of the Disclosure. Therefore, such derivatives can be described as “prodrugs.” All prodrugs of the compounds of the Disclosure are included within the scope of the Disclosure.

[0130] Furthermore, compounds of the embodiments of this disclosure, existing in the form of a free base or acid, can be converted to their pharmaceutically acceptable salts by treatment with a suitable inorganic or organic base or acid using methods known in the art. Salts of compounds of the embodiments of this disclosure can be converted to their free base or acid form by standard techniques.

[0131] The following general reaction scheme 1 applies to the compound of this 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 are as defined herein. This document describes exemplary methods for preparing the compounds shown, or their pharmaceutically acceptable salts, tautomers, or stereoisomers. Those skilled in the art will understand that these compounds can be prepared by similar methods or in combination with other methods known to those skilled in the art. They will also understand that other compounds of structure (I) not specifically shown below can be prepared by using appropriate starting components and, as necessary, modifying the parameters of the synthesis, in a manner similar to that described below. Generally, starting components can be obtained from suppliers such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, or synthesized according to sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)), or prepared as described in this disclosure.

[0132] General reaction scheme 1 [ka]

[0133] General reaction scheme 1 provides an exemplary method for producing the compound of structure (I) (i.e., A5). a, b, c, d, G in general reaction scheme 1 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 10Z is as defined herein, and Z is sufficient G for bonding with the NH group of A3. 1 This represents an activated analog of (e.g., alkylene or alkene ending in aldehyde, acid halide, acrylate, etc.). The intermediates and reagents required for the preparation of the compound by the general reaction scheme 1 (e.g., A1 and A2) can be purchased or prepared according to the following examples or methods known to those skilled in the art.

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

[0135] The following examples are provided for illustrative purposes only and are not limited thereto.

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

[0137] The 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), following guidelines established by the Institutional Animal Care Board (ACC) and the Canadian Animal Care Council (CCAC). Various doses of mRNA-lipid nanoparticles were administered systemically by tail vein injection, and the animals were euthanized at a specific time point after administration (e.g., 4 hours). The liver and spleen were collected in pre-weighed tubes, weighed, immediately rapid-frozen in liquid nitrogen, and stored at -80°C until analytical processing.

[0138] For liver samples, approximately 50 mg was processed for analysis in 2 mL FastPrep tubes (MP Biomedicals, Solon OH). A 1 / 4-inch ceramic sphere (MP Biomedicals) was added to each tube, and 500 μL of Glo Lysis Buffer-GLB (Promega, Madison, WI), equilibrated to room temperature, was added to the liver tissue. The liver tissue was homogenized twice in a FastPrep24 instrument (MP Biomedicals) at 6.0 m / s for 15 seconds each time. After incubating the homogenate at room temperature for 5 minutes, it was 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 measured using the CentroXS assay system. 3 Quantitative analysis was performed using an LB960 luminometer (Berthold Technologies, Germany). The amount of protein being assayed was measured using the BCA protein assay kit (Pierce, Rockford IL). Next, relative luminescence units (RLU) were normalized to the total protein ug being assayed. A standard curve was created using QuantiLum Recombinant Luciferase (Promega) to convert RLU to luciferase ng.

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

[0140] Example 2 PK of formulated lipids A Measurement As explained elsewhere, the pK of formulated cationic lipids a pK correlates with the effectiveness of LNPs in 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 is ~5 to ~7. The pK of each cationic lipid a The fluorescence intensity was measured using lipid nanoparticles with a fluorescence-based assay for 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). Lipid nanoparticles containing cationic lipids / DSPC / cholesterol / PEG lipids (50 / 10 / 38.5 / 1.5 mol%) at a total lipid concentration of 0.4 mM were prepared in PBS using 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 with 2 mL of buffer containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl, with a pH in the range of 2.5 to 11. Aliquots of the TNS solution were added to a final concentration of 1 μM, and then vortex mixing was performed. The fluorescence intensity was measured at room temperature using an SLM Aminco Series 2 Luminescence Spectrophotometer with excitation and emission wavelengths of 321 nm and 445 nm. Sigmoid best-fit analysis was applied to the fluorescence data, and the pKa was measured as the pH that produced half of the maximum fluorescence intensity.

[0141] Example 3 Measurement of the efficacy of lipid nanoparticle formulations containing various cationic lipids using an in vivo rodent model expressing luciferase mRNA. The representative compounds of this disclosure shown in Table 2 are in the following molar ratios: 50% cationic lipid / 10% distearoyl phosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid 2-[2-(ω-methoxy(polyethylene glycol)] 2000The formulations were prepared using either ethoxy-N,N-ditetradecylacetamide or 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 by 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 the measured value of ng luciferase / g liver 4 hours after administration, as described in Example 1. The compound numbers in Table 2 refer to the compound numbers in Table 1.

[0142] Table 2. Novel cationic lipids and their activity [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 under N2 while stirring, and the reaction mixture was heated under reflux for 4 hours. After the reaction was complete, the crude product was concentrated to obtain 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 dichloromethane solution (0°C) containing dioctylamine with triethylamine (1 equivalent) as a base. The reaction mixture was stirred at 0°C for 1 hour, and then stirred at room temperature for another 1 hour. The reaction mixture was filtered, and the resulting solution was washed with hydrochloric acid (1N HCl), and then washed with saturated NaHCO3 solution and brine. The solvent was evaporated under reduced pressure to obtain the crude product (colorless liquid), which was used in the next step without further purification.

[0146] Synthesis of bis(2-butyloctyl)7-oxotridecanediote To an anhydrous DCM 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), DCC (4.27 g, 20.69 mmol) was added. The resulting mixture was stirred overnight at room temperature. The solid (DCU) was then filtered and washed with DCM. The filtrate was concentrated. The residue (oil / solid) was purified by silica gel column chromatography (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 DCE solution of 3-(dimethylamino)-1-propylamine (0.09 g, 0.88 mmol) and bis(2-butyloctyl)7-oxotridecanedioate (0.37 g, 0.63 mmol) was treated overnight with sodium triacetoxyborohydride (0.20 g, 0.94 mmol) and AcOH (55 μL, 0.98 mmol). The solution was washed with a dilute aqueous sodium hydroxide solution (1 N NaOH). The organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered, and the solvent was removed. The residue was passed through a small silica gel pad and washed with a mixture of DCM / MeOH / Et3N (85:15:1). The filtrate was concentrated to obtain the desired product as a slightly yellowish oil (240 mg, 0.35 mmol, 56%).

[0148] I-7 synthesis A solution of bis(2-butyloctyl)7-((3-(dimethylamino)propyl)amino)tridecanedioate (210 mg, 0.30 mmol) and N,N-dioctylacrylamide (1.5 equivalents, 136 mg, 0.46 mmol) in EtOH (10 mL) was stirred overnight at room temperature. The reaction mixture was heated under reflux for 7 days. After the reaction was complete, the solvent was removed. The residue was dissolved in a mixture of hexane and siRNA (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 silica gel pad. The pad was washed with a mixture of hexane / ethyl acetate / triethylamine (80:20:1). The washing solution was concentrated to obtain the desired crude product.

[0149] The crude product was further purified by flash-dry column chromatography on silica gel (0-5% MeOH in chloroform). This yielded the target 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 silica gel column chromatography (0% to 5% MeOH in DCM) to obtain 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 to remove the solvent. The crude product (colorless liquid) was used in the next step without further purification.

[0153] I-19 synthesis 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 according to the procedure of Example 4) in 1,2-dichloroethane (10 mL) was stirred for 15 minutes, then sodium triacetoxyborohydride (0.41 g, 1.97 mmol) was added all at once, and the mixture was stirred for a further 16 hours at room temperature. The mixture was concentrated. The residue was placed 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 obtain a colorless oil. The crude product was purified by flash column chromatography on silica gel (0-5% MeOH in chloroform) to obtain the target 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-oxohexanamide) nonadecanedioate (Compound I-14)

Chemical formula

[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 minutes at room temperature. The mixture was stirred for 2 hours, then methanol (0.5 mL) was added to remove excess acyl chloride. The resulting mixture was stirred for an additional 1 hour, then filtered through a pad of silica gel and washed with a mixture of hexane / EtOAc / Et3N (70:30:1) and concentrated. The residue was passed through a silica gel column (0 - 4% MeOH gradient in DCM) to afford Compound I-14 as a colorless oil (0.28 g, 0.30 mmol, 89%). 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-oxohexaamide)nonadecanedioate (compound I-15) Compound I-15 was prepared according to the general procedure of Example 8 and obtained as 0.18 g of 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-oxohexaamide)tridecanediote (compound I-16) Compound I-16 was prepared according to the general procedure of Example 8 and obtained as 0.27 g of 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-oxooctaneamide)tridecanediote (compound I-17) Compound I-17 was prepared according to the general procedure of Example 8 and obtained as 0.08 g of colorless oil, 0.08 mmol, 75%. 1 1H NMR (400 MHz, CDCl3) 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-oxooctaneamide)tridecanediote (compound I-18) Compound I-18 was prepared according to the general procedure of Example 8 and obtained as 0.15 g of 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-oxohexanamide)nonadecanedioate (compound I-1) [ka]

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

[0164] Synthesis of compound I-1 A solution of bis(2-butyloctyl)10-((4-(dimethylamino)butyl)amino)nonadecanedioate (1 equivalent, 220 mg, 0.28 mmol, prepared according to the general procedure described above) and intermediate C (2.75 equivalents, 0.77 mmol, 140 mg) in EtOH (10 mL) was placed in a sealed pressure flask and stirred at room temperature under Ar for 4 days. The reaction mixture was concentrated. The residue was purified twice by flash-dry column chromatography on silica gel (hexane-siRNA-Et3N, 95:5:0~80:20:1, and 0~5% 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 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)

Chemical Structure

[0169] Synthesis of compound I-23 A solution of octanal (3.5 equivalents, 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 described above) in 1,2-dichloroethane (5 mL) was stirred for 15 minutes, and then sodium triacetoxyborohydride (3.5 equivalents, 0.9 mmol, 190 mg) was added all at once. Stirring was continued at room temperature for 16 hours. The reaction mixture was concentrated. The residue was purified twice by flash-dry column chromatography on silica gel (hexane-Â-Et3N, 95:5:0~80:20:1, and 0~5% 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 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 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] Further embodiments can be provided by combining the various embodiments described above. 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. The aspects of the embodiments may be modified to provide yet another embodiment using concepts from various patents, applications, and documents as needed. These and other modifications may be made to the embodiments in light of the detailed description above. In general, the terms used in the following claims should not be construed as limiting the claims to any particular embodiment disclosed in the specification and claims, but rather as including all possible embodiments, along with the entire scope of equivalents to which such claims are granted. Thus, the claims are not limited by this disclosure.

Claims

1. Equation (I): 【Chemistry 1】 [In the formula, G １ and G ２ Each of them is independent of C １ -C ６ It is alkylene; L １ and L ２ These are independently -O(C=O)- or -(C=O)O-; R １ａ and R １ｂ each independently, every time it appears, is (a) H, or C １ -C １２ alkyl; or (b) R １ａ is H, or C １ -C １２ alkyl, and R １ｂ together with the carbon atom to which it is attached and the adjacent R １ｂ and together with the carbon atom to which it is attached and R １ｂ are taken to form a carbon-carbon double bond; R ２ａ and R ２ｂ Each instance of this is independently (a) H or C １ -C １２ (b) R ２ａ is H, or C １ -C １２ It is alkyl, R ２ｂ It, together with the carbon atom to which it is bonded, and adjacent R ２ｂ and together with the carbon atom to which it is bonded, R ２ｂ It is removed, forming a carbon-carbon double bond; R ３ａ and R ３ｂ Each instance of this is independently (a) H or C １ -C １２ (b) R ３ａ is H, or C １ -C １２ It is alkyl, R ３ｂ It, together with the carbon atom to which it is bonded, and adjacent R ３ｂ and together with the carbon atom to which it is bonded, R ３ｂ It is removed, forming a carbon-carbon double bond; R ４ａ and R ４ｂ Each instance of this is independently (a) H or C １ -C １２ (b) R ４ａ is H, or C １ -C １２ It is alkyl, R ４ｂ It, together with the carbon atom to which it is bonded, and adjacent R ４ｂ and together with the carbon atom to which it is bonded, R ４ｂ It is removed, forming a carbon-carbon double bond; R ５ and R ６ These are, 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 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 C １ -C １５ It is alkyl; R １０ C １ -C １５ It is alkyl; R １１ is H, or C １ -C ６ It is alkyl; R １２ C １ -C ６ It is alkyl; X is a -(C=O)- or direct bond; and, a, b, c, and d are each independent integers between 1 and 24; Here, each alkyl and alkylene may be independently and optionally substituted. A compound represented by the structure shown, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

2. Structure (IA) or (IB) below: 【Chemistry 2】 The compound according to claim 1, represented by one of the above, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

3. G １ However, C ２ -C ３ The compound according to claim 1 or 2, wherein the compound is alkylene.

4. G １ However, C ４ -C ６ The compound according to claim 1 or 2, wherein the compound is alkylene.

5. G ２ However, C ２ -C ４ The compound according to any one of claims 1 to 4, which is an alkylene.

6. G ２ is C ２ -C ３ alkylene, or C ３ -C ４ alkylene, the compound according to claim 5

7. The compound according to any one of claims 1 to 6, wherein X is -(C=O)-.

8. The compound according to any one of claims 1 to 6, wherein X is a direct bond.

9. R ７ However, -O(C=O)R １０ , or -(C=O) OR １０ The compound according to any one of claims 1 to 8.

10. R １０ However, linear C １ -C １５ The compound according to claim 9, wherein it is alkyl.

11. R １０ is a linear C ６ -C １０ alkyl, the compound according to claim 10.

12. R １０ The compound according to claim 10, wherein the compound is methyl.

13. R １０ However, branch C ２ -C １５ The compound according to claim 10, wherein it is alkyl.

14. R １０ However, branch C １０ -C １５ The compound according to claim 13, wherein it is alkyl.

15. R ７ However, -NR ９ (C=O), or -(C=O)NR ９ R １０ The compound according to any one of claims 1 to 8.

16. R ９ The compound according to claim 15, wherein H is present.

17. R ９ and R １０ However, each is independent of C ６ -C １０ The compound according to claim 15, wherein it is alkyl.

18. R １ａ and R １ｂ For at least one occurrence of R １ａ However, H, or C １ -C １２ It is alkyl, R １ｂ However, together with the carbon atom to which it is bonded, the adjacent R １ｂ and together with the carbon atom to which it is bonded, R １ｂ The compound according to any one of claims 1 to 17, wherein a carbon-carbon double bond is formed by the removal of a carbon-carbon double bond.

19. R ４ａ and R ４ｂ For at least one occurrence of R ４ａ However, H, or C １ -C １２ It is alkyl, R ４ｂ However, together with the carbon atom to which it is bonded, the adjacent R ４ｂ and together with the carbon atom to which it is bonded, R ４ｂ The compound according to any one of claims 1 to 18, wherein a carbon-carbon double bond is formed by the removal of a carbon atom.

20. R ２ａ and R ２ｂ For at least one occurrence of R ２ａ However, H, or C １ -C １２ It is alkyl, R ２ｂ However, together with the carbon atom to which it is bonded, the adjacent R ２ｂ and together with the carbon atom to which it is bonded, R ２ｂ The compound according to any one of claims 1 to 19, wherein a carbon-carbon double bond is formed by the removal of a carbon-carbon double bond.

21. R ３ａ and R ３ｂ For at least one occurrence of R ３ａ However, H, or C １ -C １２ It is alkyl, R ３ｂ However, together with the carbon atom to which it is bonded, the adjacent R ３ｂ and together with the carbon atom to which it is bonded, R ３ｂ The compound according to any one of claims 1 to 20, wherein a carbon-carbon double bond is formed by the removal of a carbon-carbon double bond.

22. R １ａ , R １ｂ , R ２ａ , R ２ｂ , R ３ａ , R ３ｂ , R ４ａ , and R ４ｂ However, each time it appears, it is independently H or C １ -C １２ A compound according to any one of claims 1 to 17, wherein it is alkyl.

23. R ２ａ , R ２ｂ , R ３ａ , and R ３ｂ The compound according to claim 22, wherein each instance is H.

24. R １ａ and R ４ａ The compound according to claim 22 or 23, wherein each instance is H.

25. R １ｂ and R ４ｂ At least one of them is C １ -C ８ The compound according to any one of claims 22 to 24, wherein it is alkyl.

26. C １ -C ８ The compound according to claim 25, wherein the alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

27. the below described: 【Transformation 3】 However, or both, independently, as follows: 【Chemistry 4】 A compound according to any one of claims 22 to 26, which is one of the structures shown.

28. The compound according to any one of claims 1 to 27, wherein a, b, c, and d are each independently integers from 2 to 12.

29. The compound according to any one of claims 1 to 27, wherein a, b, c, and d are each independently integers from 4 to 10, 5 to 10, 6 to 10, 4 to 9, 5 to 9, or 6 to 9.

30. R ５ or R ６ The compound according to any one of claims 1 to 29, wherein one of the compounds is methyl.

31. R ５ and R ６ The compound according to any one of claims 1 to 30, wherein each of them is methyl.

32. R ８ The compound according to any one of claims 1 to 31, wherein the compound is an OH group.

33. R ８ However, -N(R １１ )(C=O)R １２ The compound according to any one of claims 1 to 31.

34. R ８ However, -(C=O)NR １１ R １２ The compound according to any one of claims 1 to 31.

35. R ８ However, -NR １１ R １２ The compound according to any one of claims 1 to 31.

36. R １１ and R １２ However, each is independent of H or C １ -C ８ The compound according to any one of claims 33 to 35, wherein it is alkyl.

37. R １１ and R １２ However, each is independent of H or C １ -C ３ The compound according to any one of claims 33 to 36, wherein it is alkyl.

38. C １ -C ８ Alkyl or C １ -C ３ The compound according to claim 36 or 37, wherein the alkyl group is either unsubstituted or substituted with a hydroxyl group.

39. R １１ and R １２ The compound according to any one of claims 33 to 36, wherein each of the compounds is methyl.

40. R ８ is -(C=O)OR １２ The compound according to any one of claims 1 to 31.

41. R ８ However, -O(C=O)R １２ The compound according to claim 40.

42. R ８ However, see below: -OH, or 【Transformation 5】 A compound according to any one of claims 1 to 31, which is one of the structures shown.

43. A compound selected from the compounds listed in Table 1.

44. Lipid nanoparticles comprising a compound according to any one of claims 1 to 43, and a therapeutic agent.

45. Lipid nanoparticles according to claim 44, further comprising one or more excipients selected from neutral lipids, steroids, and polymer-bound lipids.

46. Lipid nanoparticles according to claim 45, wherein the composition comprises one or more neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPPC, DOPE, and SM.

47. Lipid nanoparticles according to claim 46, wherein the neutral lipid is DSPC.

48. Lipid nanoparticles according to any one of claims 44 to 47, wherein the molar ratio of the compound to the neutral lipid is in the range of about 2:1 to about 8:

1.

49. Lipid nanoparticles according to any one of claims 45 to 48, wherein the steroid is cholesterol.

50. The lipid nanoparticles according to claim 49, wherein the molar ratio of the compound to cholesterol is in the range of 5:1 to 1:

1.

51. Lipid nanoparticles according to any one of claims 45 to 50, wherein the polymer-bound lipid is a pegylated lipid.

52. The lipid nanoparticle according to claim 51, wherein the molar ratio of the compound to the PEGylated lipid is in the range of about 100:1 to about 20:

1.

53. Lipid nanoparticles according to any one of claims 51 or 52, wherein the pegylated lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-Cer, or PEG-dialkyloxypropylcarbamate.

54. The pegylated lipid has the following structure (II): 【Transformation 6】 [In the formula, R ８ and R ９ Each is independently an alkyl, alkenyl, or alkynyl, which is a linear or branched chain of 10 to 30 carbon atoms, where the alkyl, alkenyl, or alkynyl may have one or more ester bonds interposed between them; and The average value of w is between 30 and 60. Lipid nanoparticles according to any one of claims 51 or 52, which are pegylated lipids represented by , or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof.

55. R ８ and R ９ The lipid nanoparticles according to claim 54, wherein each is independently a linear alkyl chain containing 12 to 16 carbon atoms.

56. Lipid nanoparticles according to any one of claims 54 or 55, wherein the average w is approximately 49.

57. Lipid nanoparticles according to any one of claims 44 to 56, wherein the therapeutic agent comprises nucleic acid.

58. Lipid nanoparticles according to claim 57, wherein the nucleic acid is selected from antisense RNA and messenger RNA.

59. A pharmaceutical composition comprising lipid nanoparticles according to any one of claims 44 to 58, and a pharmaceutically acceptable diluent or excipient.

60. A method for treating or preventing a disease in a patient requiring such treatment, comprising administering to the patient a lipid nanoparticle according to any one of claims 44 to 58, or a pharmaceutical composition according to claim 59.

61. A method for vaccinating a patient in need of vaccination against a viral pathogen, comprising administering to the patient a lipid nanoparticle according to any one of claims 44 to 58, or a pharmaceutical composition according to claim 59, wherein the therapeutic agent is a viral antigen or a nucleic acid capable of transcribing a viral antigen.

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