Lipids for lipid nanoparticle formulations

Abstract

The present invention provides compounds having the following structure (I): # imgabs0 # or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein R1a, R1b, R2, R3, L1a, L1b, n1 and X are as defined herein. Also provided are uses of the compounds as a component of lipid nanoparticle formulations for the delivery of therapeutic agents, compositions comprising the compounds, and uses and methods of preparation thereof.

Description

background Technical Field

[0002] The present disclosure generally relates to novel cationic lipids that can be used in combination with other lipid components (such as neutral lipids, cholesterol, and polymer-conjugated lipids) to form lipid nanoparticles with oligonucleotides to facilitate the intracellular delivery of therapeutic nucleic acids (e.g., oligonucleotides, messenger RNA) in vitro and in vivo.

[0003] Related technical description

[0004] In biological systems, there are many challenges related to nucleic acid delivery to affect the desired response. Nucleic acid-based therapy has great potential, but there is still a need to more effectively deliver nucleic acids to appropriate sites in cells or organisms to realize this potential. Therapeutic nucleic acids include, for example, messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNA enzymes, plasmids, immunostimulatory nucleic acids, antagomirs, antimirs, mimics, supermirs and aptamers. Some nucleic acids, such as mRNA or plasmids, can be used to affect the expression of specific cell products, which will help treat diseases such as those associated with protein or enzyme deficiency. The therapeutic application of translatable nucleotide delivery is very extensive because constructs can be synthesized to produce any selected protein sequence, whether or not it is inherent to the system. The expression product of the nucleic acid can increase the existing level of the protein, replace a missing or non-functional version of the protein, or introduce new proteins and related functions in a cell or organism.

[0005] Some nucleic acids, such as miRNA inhibitors, can be used to affect the expression of specific cellular products regulated by miRNAs, which will help treat diseases associated with, for example, protein or enzyme deficiencies. The therapeutic applications of miRNA inhibition are very broad, as constructs can be synthesized to inhibit one or more miRNAs, thereby regulating the expression of mRNA products. Inhibition of endogenous miRNAs can enhance the expression of their downstream target endogenous proteins and restore normal function in cells or organisms as a means of treating diseases associated with specific miRNAs or groups of miRNAs.

[0006] Other nucleic acids can down-regulate the intracellular level of specific mRNA through the process of complementary binding such as RNA interference (RNAi) or antisense RNA, and the result is to down-regulate the synthesis of the corresponding protein. The therapeutic application of antisense oligonucleotides and RNAi is also very extensive, because the oligonucleotide construct can be synthesized with any nucleotide sequence for the target mRNA. The target may include mRNA from normal cells, mRNA related to disease states (such as cancer) and mRNA of infectious factors (such as viruses). So far, antisense oligonucleotide constructs have been shown in both in vitro and in vivo models to specifically down-regulate the ability of target proteins by degrading homologous mRNA. In addition, antisense oligonucleotide constructs are currently being evaluated in clinical studies.

[0007] However, the use of oligonucleotides in therapeutic settings currently faces two problems. First, free RNA is susceptible to nuclease digestion in plasma. Second, free RNA has limited ability to enter intracellular compartments where the relevant translation machinery exists. Lipid nanoparticles formed by cationic lipids and other lipid components (such as neutral lipids, cholesterol, PEG, PEGylated lipids) with oligonucleotides have been used to block RNA degradation in plasma and promote cellular uptake of oligonucleotides.

[0008] Still need improved cationic lipids and lipid nanoparticles for delivering oligonucleotides.Preferably, these lipid nanoparticles will provide the best drug to lipid ratio, protect nucleic acid from degradation and removal in serum, be suitable for systemic delivery, and provide intracellular delivery of nucleic acid.In addition, these lipid nucleic acid particles should have good tolerance and provide enough therapeutic index, so that the patient can be treated with effective doses of nucleic acid without unacceptable toxicity and / or risk to the patient.The present disclosure provides these advantages and related advantages. Summary of the invention

[0009] In short, the present disclosure provides lipid compounds, including stereoisomers, pharmaceutically acceptable salts or tautomers thereof, which can be used alone or in combination with other lipid components (such as neutral lipids, charged lipids, steroids (including, for example, all sterols) and / or their analogs, and / or polymer-coupled lipids) to form lipid nanoparticles for the delivery of therapeutic agents. In some cases, lipid nanoparticles are used to deliver nucleic acids, such as antisense RNA and / or messenger RNA. Methods for treating various diseases or conditions using such lipid nanoparticles are also provided, such as those caused by infectious entities and / or protein deficiencies.

[0010] In one embodiment, a compound having the following structure (I) is provided:

[0011]

[0012] or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein R 1a , R 1b , R 2 , R 3 , L 1a , L 1b , n1 and X are as defined herein.

[0013] Also provided are pharmaceutical compositions and therapeutic agents comprising one or more of the compounds of the aforementioned structure (I). In some embodiments, the pharmaceutical composition further comprises one or more components selected from neutral lipids, charged lipids, steroids, and polymer-coupled lipids. Such compositions can be used to form lipid nanoparticles for delivering therapeutic agents.

[0014] In other embodiments, the present disclosure provides a method of administering a therapeutic agent to a patient in need thereof, the method comprising preparing a composition comprising a lipid nanoparticle of a compound of structure (I) and a therapeutic agent, and delivering the composition to the patient. Such methods can be used to induce expression of a protein in a subject, such as expression of an antigen for vaccination or a gene editing protein.

[0015] These and other aspects of the disclosure will become apparent upon reference to the following detailed description. DETAILED DESCRIPTION OF THE INVENTION

[0017] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the present disclosure. However, it will be understood by those skilled in the art that the embodiments of the present disclosure may be implemented without these details.

[0018] The present disclosure is based in part on the discovery of novel cationic (amino) lipids, which provide advantages when used in lipid nanoparticles to deliver active agents or therapeutic agents (such as nucleic acids) to mammalian cells in vivo. In particular, embodiments of the present disclosure provide nucleic acid lipid nanoparticle compositions comprising one or more novel cationic lipids described herein, which provide increased nucleic acid activity and improved tolerability of the composition in vivo compared to the nucleic acid lipid nanoparticle compositions previously described, resulting in a significant increase in the therapeutic index.

[0019] In a specific embodiment, the disclosure provides novel cationic lipids that can be formulated for improved compositions for in vitro and in vivo delivery of mRNA and / or other oligonucleotides. In some embodiments, these improved lipid nanoparticle compositions can be used for the expression of proteins encoded by mRNA. In other embodiments, these improved fat nanoparticle compositions can be used for upregulating endogenous protein expression by delivering a targeted regulation of a target mRNA or several mRNAs targeting a specific miRNA or a group of miRNAs. In other embodiments, these improved lipid nanoparticles can be used for downregulating (e.g., silencing) protein levels and / or mRNA levels of target genes. In some other embodiments, lipid nanoparticles can also be used for delivering mRNA and plasmids to express transgenics. In other embodiments, lipid nanoparticle compositions can be used for inducing pharmacological effects caused by protein expression, such as increasing the yield of erythrocytes by delivering a suitable erythropoietin mRNA, or preventing infection by delivering mRNA encoding a suitable antibody.

[0020] The lipid nanoparticles and compositions of the present disclosure can be used for various purposes, including delivering encapsulated or associated (e.g., complexed) therapeutic agents (e.g., nucleic acids) to cells in vitro and in vivo. Therefore, embodiments of the present disclosure provide methods for treating or preventing a disease or condition in a subject in need thereof by contacting the subject with a lipid nanoparticle encapsulating or associated with a suitable therapeutic agent, wherein the lipid nanoparticle comprises one or more of the novel cationic lipids described herein.

[0021] As described herein, embodiments of the lipid nanoparticles of the present disclosure are particularly suitable for the delivery of nucleic acids, including, for example, mRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antigomirs / antimirs), messenger RNA interference complementary RNA (micRNA), DNA, multivalent RNA, dicer substrate RNA, complementary DNA (cDNA), etc. Therefore, the lipid nanoparticles and compositions of the present disclosure can be used to induce the expression of desired proteins in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more novel cationic lipids described herein, wherein the lipid nanoparticles encapsulate nucleic acids or are associated with nucleic acids, and the nucleic acids (e.g., messenger RNA or plasmids encoding the desired proteins) are expressed to produce the desired proteins. Alternatively, the lipid nanoparticles and compositions of the present disclosure can be used to reduce the expression of target genes and proteins in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more novel cationic lipids described herein, wherein the lipid nanoparticles encapsulate nucleic acids or are associated with nucleic acids, and the nucleic acids (e.g., antisense oligonucleotides or small interfering RNA (siRNA)) reduce target gene expression. The lipid nanoparticles and compositions of the present disclosure can also be used for the co-delivery of different nucleic acids (e.g., mRNA and plasmid DNA), either alone or in combination, which can be used to provide effects that require co-localization of different nucleic acids (e.g., mRNA encoding a suitable gene modification enzyme and a DNA fragment for incorporation into the host genome).

[0022] Nucleic acids for use in the present disclosure can be prepared according to any available technology. For mRNA, the main method of preparation is, but is not limited to, enzymatic synthesis (also known as in vitro transcription), which is currently the most efficient method for producing long sequence-specific mRNA. In vitro transcription describes the process of directing template synthesis of RNA molecules from an engineered DNA template that includes an upstream phage promoter sequence (e.g., including but not limited to promoter sequences from T7, T3, and SP6 E. coli phages) linked to a downstream sequence encoding a gene of interest. Template DNA for in vitro transcription can be prepared from a variety of 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 invitro 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).

[0023] RNA transcription is performed in vitro using a linear DNA template in the presence of a corresponding RNA polymerase and adenosine, guanosine, uridine and cytidine ribonucleoside triphosphates (rNTPs) under conditions that support polymerase activity while minimizing potential degradation of the resulting mRNA transcript. In vitro transcription can be performed using a variety of commercially available kits, including but not limited to the RiboMax large-scale RNA production system (Promega), the MegaScript transcription kit (Life Technologies), and commercially available reagents including RNA polymerase and rNTPs. Methods for in vitro transcription of mRNA are well known in the art (see, e.g., Losick, R., 1972, In vitro transcription, Ann Rev Biochem v. 41 409-46; Kamakaka, RT and Kraus, WL 2001. In Vitro Transcription. Current Protocols in Cell Biology. 2: 11.6: 11.6.1–11.6.17; Beckert, B. And Masquida, B., (2010) Synthesis of RNA by In Vitro Transcription in RNA in Methods in Molecular Biology v. 703 (Neilson, H. Ed), New York, NY Humana Press, 2010; Brunelle, J. Land and Green, R., 2013, Chapter Five—In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology v.530, 101-114; all of which are incorporated herein by reference).

[0024] The desired in vitro transcribed mRNA is then purified from undesired components of the transcription or related reactions (including unincorporated rNTPs, proteases, salts, short RNA oligonucleotides, etc.). Techniques for the isolation of mRNA transcripts are well known in the art. Well-known methods include phenol / chloroform extraction or precipitation with any alcohol (ethanol, isopropanol) in the presence of monovalent cations or precipitation with lithium chloride. In addition, non-limiting examples of purification methods that can be used include size exclusion chromatography (Lukavsky, PJ and Puglisi, JD, 2004, Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides, RNA v.10, 889-893), silica-based affinity chromatography and polyacrylamide gel electrophoresis (Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LDIN 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 performed using various commercially available kits, including but not limited to SV Total Isolation System (Promega) and In Vitro Transcription Removal and Concentration Kit (Norgen Biotek).

[0025] In addition, although reverse transcription can produce a large amount of mRNA, the product may contain many abnormal RNA impurities associated with undesirable polymerase activity, which may need to be removed from the full-length mRNA preparation. These include short RNAs produced by transcription initiation failure, as well as double-stranded RNA (dsRNA) produced by RNA-dependent RNA polymerase activity, transcription initiated by RNA from RNA templates, and self-complementary 3' extension. It has been shown that these pollutants with dsRNA structure can cause undesirable immunostimulatory activity by interacting with various innate immune sensors in eukaryotic cells, the function of which is to recognize specific nucleic acid structures and induce effective immune responses. This in turn can greatly reduce mRNA translation because protein synthesis can be reduced during innate cellular immune responses. Therefore, additional techniques for removing these dsRNA contaminants have been developed and are known in the art, including but not limited to scalable HPLC purification (see, e.g., Kariko, K., Muramatsu, H., Ludwig, J. And Weissman, D., 2011, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA, Nucl Acid Res, v.39e142; 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, PHEd), 2013). It is reported that HPLC purified mRNA is translated at a higher level, particularly in primary cells and in vivo.

[0026] A large number of various modifications for changing the specific properties of in vitro transcribed mRNA and improving its effectiveness have been described in the art. These include, but are not limited to, modifications to the 5' and 3' ends of the mRNA. Endogenous eukaryotic mRNAs typically contain a cap structure at the 5' end of the mature molecule, which plays an important role in mediating the binding of mRNA cap binding protein (CBP), which in turn is responsible for enhancing the stability of mRNA in cells and the efficiency of mRNA translation. Therefore, the highest level of protein expression is achieved by capping mRNA transcripts. The 5' cap contains a 5'-5'-triphosphate bond between the nucleotides and the guanine nucleotides at the 5' end. The conjugated guanine nucleotides are methylated at the N7 position. Additional modifications include methylation of the most terminal and penultimate 5' nucleotides on the 2' hydroxyl group.

[0027] A variety of different cap structures can be used to generate 5' caps for in vitro transcribed synthetic mRNAs. 5' capping of synthetic mRNAs can be performed co-transcriptionally (i.e., capping during in vitro transcription) with chemical cap analogs. For example, the anti-reverse cap analog (ARCA) cap contains a 5'-5'-triphosphate guanine-guanine bond, in which one guanine contains an N7 methyl group and a 3'-O-methyl group. However, during this co-transcription process, up to 20% of the transcripts remain uncapped, and the synthetic cap analogs are different from the 5' cap structure of the real cell mRNA, which may reduce translatability and cellular stability. Alternatively, synthetic mRNA molecules can also be enzymatically capped after transcription. These may produce more realistic 5' cap structures that more closely mimic endogenous 5' caps in structure or function, which enhances the binding of cap-binding proteins, increases half-life, reduces sensitivity to 5' endonucleases and / or reduces 5' decapping. A variety of synthetic 5' cap analogs have been developed and are known in the art to enhance the stability and translatability of mRNA (see, e.g., Grudzien-Nogalska, E., Kowalska, J., Su, W., Kuhn, AN, Slepenkov, SV, Darynkiewicz, E., Sahin, U., Jemielity, J., and Rhoads, RE, Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v. 969 (Rabinovich, PHEd), 2013).

[0028] During RNA processing, a long chain of adenine nucleotides (poly-A tail) is usually added to the mRNA molecule at the 3' end. Immediately after transcription, the 3' end of the transcript is cleaved, releasing the 3' hydroxyl group, and poly-A polymerase adds the adenine nucleotide chain to the RNA in a process called polyadenylation. The poly-A tail has been widely shown to improve 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).

[0029] Poly(A) tailing of in vitro transcribed mRNA can be achieved using a variety of methods, including but not limited to cloning a poly(T) stretch into a DNA template, or by post-transcriptional addition using a poly(A) polymerase. The first scenario allows for in vitro transcription of mRNA with a poly(A) tail of defined length, depending on the size of the poly(T) stretch, but requires additional manipulation of the template. The latter scenario involves enzymatic addition of a poly(A) tail to in vitro transcribed mRNA using a poly(A) polymerase, which catalyzes the incorporation of an adenine residue at the 3' end of the RNA, does not require additional manipulation of the DNA template, but results in mRNA with poly(A) tails of uneven length. 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), the mMESSAGEmMACHINE T7 Ultra High Efficiency Kit, and the poly(A) tailing kit (Life Technologies), as well as with commercially available reagents, various ARCA caps, poly(A) polymerases, and the like.

[0030] In addition to 5' capping and 3' polyadenylation, other modifications of in vitro transcripts have been reported to provide benefits related to translation efficiency and stability. It is well known in the art that pathogenic DNA and RNA can be recognized by various sensors within eukaryotes and trigger strong innate immune responses. The ability to distinguish between pathogenic and self DNA and RNA has been shown to be based at least in part on structure and nucleoside modifications, as most nucleic acids from natural sources contain modified nucleosides. In contrast, RNA synthesized in vitro lacks these modifications, thus making it immunostimulatory, which in turn can inhibit efficient mRNA translation as described above. The introduction of modified nucleosides into in vitro transcribed mRNA can be used to prevent recognition and activation of RNA sensors, thereby alleviating this undesirable immunostimulatory activity and enhancing translation capacity (see, 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, PHEd), 2013); Kariko, K., Muramatsu, H., Welsh, FA, Ludwig, J., Kato, H., Akira, S., Weissman, D., 2008, Incorporation of Pseudouridine Into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability, Mol Ther v.16, 1833-1840). The modified nucleosides and nucleotides used to synthesize modified RNA can be prepared, monitored and utilized using general methods and procedures known in the art.A variety of nucleoside modifications can be used that can be incorporated into in vitro transcribed mRNA to some extent, either alone or in combination with other modified nucleosides (see, e.g., U.S. Publication No. 2012 / 0251618). In vitro synthesis of nucleoside-modified mRNA has been reported to have reduced ability to activate immune sensors, accompanied by enhanced translational capacity.

[0031] Other components of mRNA that can be modified to provide benefits in terms of translatability and stability include 5' and 3' untranslated regions (UTRs). Whether it is the optimization of both or separate UTRs (favorable 5' and 3' UTRs can be obtained from cells or viral RNAs), it has been shown that mRNA stability and translation efficiency of in vitro transcribed mRNA can be improved (see, e.g., Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of longRNA containing modified nucleosides in Synthetic Messenger RNA and CellMetabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PHEd), 2013).

[0032] In addition to mRNA, other nucleic acid payloads may also be used in the present disclosure. For oligonucleotides, preparation methods include, but are not limited to, chemical synthesis and enzymatic, chemical cleavage of longer precursors, in vitro transcription as described above, and the like. Methods for synthesizing DNA and RNA nucleotides are widely used and well known in the art (see, 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 are incorporated herein by reference).

[0033] For plasmid DNA, the preparations used in the present disclosure generally utilize, but are not limited to, in vitro amplification and isolation of plasmid DNA in liquid cultures of bacteria containing the target plasmid. The presence of genes encoding resistance to specific antibiotics (penicillin, kanamycin, etc.) in the target plasmid allows those bacteria containing the target plasmid to selectively grow in cultures containing antibiotics. Methods for isolating plasmid DNA are widely used and well known in the art (see, e.g., Heilig, J., Elbing, KLand 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., S., 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 US6197553B1). Plasmid isolation can be performed using various commercially available kits, including but not limited to Plasmid Plus (Qiagen), GenJET Plasmid MaxiPrep (Thermo) and PureYield MaxiPress (Promega) kits, as well as commercially available reagents.

[0034] Various exemplary embodiments of the cationic lipids, lipid nanoparticles, and compositions comprising the cationic lipids and lipid nanoparticles disclosed herein, as well as their use for delivering active agents or therapeutic agents (such as nucleic acids) to regulate gene and protein expression, are described in further detail below.

[0035] Unless otherwise stated, the following terms used herein have the meanings ascribed to them.

[0036] Unless the context requires otherwise, in this specification and the claims, the word "comprise" and variations such as "include" and "comprising" should be construed in the open and inclusive sense, ie, "including, but not limited to."

[0037] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Therefore, the phrases "in one embodiment" or "in an embodiment" appearing in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, in one or more embodiments, the particular features, structures, or characteristics may be combined in any suitable manner.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present disclosure belongs. In the specification and claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

[0039] The phrase "inducing the expression of a desired protein" refers to the ability of a nucleic acid to increase the expression of a desired protein. To detect the extent of protein expression, a test sample (e.g., a cell sample expressing the desired protein in culture) or a test mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g., mouse) or a non-human primate (e.g., monkey) model) is contacted with a nucleic acid (e.g., a nucleic acid bound to a lipid of the present disclosure). The expression of the desired protein in the test sample or test animal is compared with the expression of the desired protein in a control sample (e.g., a cell sample in culture expressing the desired protein) or a control mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g., mouse) or a non-human primate (e.g., monkey) model) that is not contacted with the nucleic acid or is not administered with the nucleic acid. When the desired protein is present in the control sample or control mammal, the expression of the desired protein in the control sample or control mammal can be assigned a value of 1.0. In certain embodiments, when the ratio of the desired protein expression in the test sample or test mammal to the desired protein expression level in the control sample or control mammal is greater than 1 (e.g., about 1.1, 1.5, 2.0, 5.0, or 10.0), induction of the expression of the desired protein is achieved. When the desired protein is not present in the control sample or control mammal, any measurable level of the desired protein detected in the test sample or test mammal achieves induction of the expression of the desired protein. One of ordinary skill in the art will appreciate appropriate assays for determining protein expression levels in a sample, such as dot blots, 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.

[0040] 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. In order to examine the extent of gene silencing, a test sample (e.g., a cell sample in a culture expressing the target gene) or a test mammal (e.g., a mammal, such as a human or an animal model, such as a rodent (e.g., mouse) or a non-human primate (e.g., monkey) model) is contacted with a 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 with the expression of the target gene in a control sample (e.g., a cell sample in a culture expressing the target gene) or a control mammal (e.g., a mammal, such as a human or an animal model, such as a rodent (e.g., mouse) or a non-human primate (e.g., monkey) model) that is not exposed to the nucleic acid or to which the nucleic acid is not administered. The expression of the target gene in the control sample or control mammal can be assigned a value of 100%. In certain embodiments, the target gene expression is silenced, inhibited or reduced when the target gene expression level in the test sample or test mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 0% relative to the target gene expression level in the control sample or control mammal. In other words, the nucleic acid is capable of silencing, reducing or inhibiting the expression of the target gene in the test sample or test mammal by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% relative to the target gene expression level in the control sample or control mammal that is not exposed to the nucleic acid or is not administered with the nucleic acid. Suitable assays for determining target gene expression levels include, but are not limited to, detecting protein or mRNA levels using techniques known to those skilled in the art, such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those skilled in the art.

[0041] An "effective amount" or "therapeutically effective amount" of an active agent or therapeutic agent (such as a therapeutic nucleic acid) is an amount sufficient to produce a desired effect, for example, an increase or inhibition of target sequence expression compared to normal expression levels detected in the absence of nucleic acid. An increase in target sequence expression is achieved when any measurable level is detected in the absence of expression product in the absence of nucleic acid. In the case where the expression product is present at a certain level before contact with the nucleic acid, expression is increased when the value obtained with the nucleic acid (such as mRNA) is increased by about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000 or more relative to the control fold. When the value obtained with nucleic acid (such as antisense oligonucleotide) relative to the control is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 0%, the inhibition of target gene or target sequence expression is achieved. Suitable assays for measuring target gene or target sequence expression include, for example, using those techniques known to those skilled in the art to detect protein or RNA levels, such as dot blots, northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of suitable reporter proteins, and phenotypic assays.

[0042] The term "nucleic acid" as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form, and includes DNA, RNA and hybrids thereof. DNA can be in the form of an antisense molecule, plasmid DNA, cDNA, PCR product or vector. RNA can be in the form of a 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 nucleic acids containing known nucleotide analogs or modified backbone residues or bonds, which are synthetic, naturally occurring and non-naturally occurring, and have binding properties similar to reference nucleic acids. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2'-O-methyl ribonucleotides and peptide nucleic acids (PNAs). Unless otherwise specified, the term includes nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids. Unless otherwise indicated, a specific nucleic acid sequence also implicitly includes its appropriately modified variants (e.g., degenerate codon replacements), alleles, homologous sequences, single nucleotide polymorphisms, and complementary sequences, as well as sequences clearly indicated. Specifically, degenerate codon replacements can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem., 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8: 91-98 (1994)). "Nucleotide" comprises sugar deoxyribose (DNA) or ribose (RNA), bases, and phosphate groups. Nucleotides are linked together by phosphate groups. "Bases" include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine and natural analogs, as well as synthetic derivatives of purines and pyrimidines, including but not limited to modifications that place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0043] The term "gene" refers to a nucleic acid (eg, DNA or RNA) sequence that contains a partial or full-length coding sequence necessary to produce a polypeptide or precursor polypeptide.

[0044] "Gene product," as used herein, refers to the product of a gene, such as an RNA transcript or a polypeptide.

[0045] The term "lipid" refers to a group of organic compounds, including but not limited to fatty acid esters, which are generally characterized by being poorly soluble in water but soluble in many organic solvents. They are generally divided into at least three categories: (1) "simple lipids," which include fats, oils, and waxes; (2) "complex lipids," which include phospholipids and glycolipids; and (3) "derivative lipids," such as steroids.

[0046] "Steroids" are chemical compounds containing the following carbon skeleton:

[0047]

[0048] Non-limiting examples of steroids include cholesterol and the like.

[0049] "Cationic lipid" refers to a lipid that can be positively charged. Exemplary cationic lipids include 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 according to the pH value. The ionization of cationic lipids affects the surface charge of lipid nanoparticles under different pH conditions. This charge state can affect plasma protein absorption, blood clearance and tissue distribution (Semple, SC, et al., Adv. Drug Deliv Rev 32: 3-17 (1998)) that are crucial for the intracellular delivery of nucleic acids and the ability to form an intraosmotic non-bilayer structure (Hafez, IM, et al., Gene Ther. 8: 1188-1196 (2001)).

[0050] The term "lipid nanoparticle" refers to a particle having at least one nanometer size (e.g., 1nm to 1000nm) comprising one or more or other specified cationic lipids in a compound of formula (I). In some embodiments, the lipid nanoparticle is contained in a formulation that can be used to deliver an active agent or therapeutic agent (e.g., nucleic acid (e.g., mRNA)) to a target site of interest (e.g., cell, tissue, organ, tumor, etc.). In some embodiments, the lipid nanoparticle of the present disclosure comprises nucleic acid. Such lipid nanoparticles typically comprise a compound of structure (I) and one or more excipients selected from neutral lipids, charged lipids, steroids, and polymer-coupled lipids. In some embodiments, an active agent or therapeutic agent (e.g., nucleic acid) can be encapsulated in the lipid portion of the lipid nanoparticle or in an aqueous space encapsulated by some or all of the lipid portions of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects caused by a host organism or cellular mechanism, such as adverse immune responses.

[0051] In various embodiments, the lipid nanoparticles have an average diameter of 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. In certain embodiments, when nucleic acid is present in lipid nanoparticles, it resists degradation by nucleases in aqueous solution. Lipid nanoparticles comprising nucleic acid and methods for preparing the same are disclosed in, for example, U.S. Patent Publication Nos. 2004 / 0142025, 2007 / 0042031 and PCT Publication Nos. WO 2013 / 016058 and WO 2013 / 086373, the entire disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0052] As used herein, "lipid encapsulation" refers to lipid nanoparticles that provide active or therapeutic agents, such as nucleic acids (e.g., mRNA), with complete encapsulation, partial encapsulation, or both. In one embodiment, nucleic acids (e.g., mRNA) are completely encapsulated in lipid nanoparticles.

[0053] The term "polymer-coupled lipid" refers to a molecule comprising a lipid portion and a polymer portion. An example of a polymer-coupled lipid is a pegylated lipid. The term "pegylated lipid" refers to a molecule comprising a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG) and the like.

[0054] The term "neutral lipid" refers to any of a variety of lipid species that exist in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, but are not limited to, phosphatidylcholines such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), fatty acylethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM), ceramides, steroids such as sterols and their derivatives. Neutral lipids can be synthetic or naturally derived.

[0055] The term "charged lipid" refers to any of a variety of lipid species that exist in a positively or negatively charged form independent of pH within a useful physiological range (e.g., pH about 3 to pH about 9). Charged lipids can be synthetic or naturally derived. Examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyltrimethylammonium propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, dimethylaminoethanecarbamoylsterol (e.g., DC-Chol).

[0056] As used herein, the term "aqueous solution" refers to a composition comprising water.

[0057] "Serum stable" in relation to nucleic acid lipid nanoparticles means that the nucleotides are not significantly degraded after exposure to serum or nuclease assays that would significantly degrade free DNA or RNA. Suitable assays include, for example, standard serum assays, DNase assays, or RNase assays.

[0058] "Systemic delivery" as used herein refers to the delivery of therapeutic products that may result in extensive exposure of active agents in an organism. Some administration techniques can result in systemic delivery of certain drugs, but other drugs cannot. Systemic delivery means exposing a useful, preferably therapeutic, dose to most parts of the body. Systemic delivery of lipid nanoparticles can be carried out 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.

[0059] As used herein, "local delivery" refers to the direct delivery of an active agent to a target site in an organism. For example, an agent can be delivered locally by direct injection into a disease site (such as a tumor), other target sites (such as an inflammatory site), or a target organ (such as the liver, heart, pancreas, kidney, etc.). Local delivery can also include local application or local injection techniques, such as intramuscular, subcutaneous, or intradermal injections. Local delivery does not exclude systemic pharmacological effects.

[0060] "Alkyl" refers to a saturated (i.e., containing no double bonds and / or triple bonds) straight or branched chain hydrocarbon radical consisting solely of carbon and hydrogen atoms and having from 1 to 24 carbon atoms (C1-C 24 alkyl), 1 to 12 carbon atoms (C1-C 12 The alkyl group may be an alkyl radical having 1 to 8 carbon atoms (C1-C8 alkyl), 1 to 8 carbon atoms (C1-C8 alkyl), or 1 to 6 carbon atoms (C1-C6 alkyl) and connected to the rest of the molecule by a single bond, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, etc. Unless otherwise specifically stated in the specification, the alkyl group may be optionally substituted.

[0061] "Cycloalkyl" or "carbocycle" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting only of carbon and hydrogen atoms, which may include fused or bridged ring systems, having 3 to 15 carbon atoms, preferably 3 to 10 carbon atoms and which is saturated or unsaturated, and connected to the rest of the molecule by a single bond. Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. Polycyclic radicals include, for example, adamantyl, norbornyl, decahydronaphthyl, 7,7-dimethylbicyclo[2.2.1]heptyl, etc. Unless otherwise specifically stated in the specification, cycloalkyl groups may be optionally substituted.

[0062] "Alkylene" or "alkylene chain" refers to a straight or branched divalent saturated hydrocarbon chain that connects the rest of the molecule to a radical group consisting of only carbon and hydrogen. In some embodiments, the alkylene chain has 1 to 24 carbon atoms (C1-C 24 alkylene), 1 to 15 carbon atoms (C1-C 15 alkylene), 1 to 12 carbon atoms (C1-C 12The alkylene chain may be a C1-C8 alkylene chain or a C1-C6 alkylene chain or a C2-C4 alkylene chain or a C2-C4 alkylene chain or a C1-C2 alkylene chain or a C2-C2 alkylene chain or a C1-C8 ...

[0063] "Alkylene oxide" refers to an alkylene group as defined herein, wherein at least one carbon-carbon bond is replaced by a carbon-oxygen-carbon bond. Examples of alkylene oxides include ethylene oxide, methylene oxide, propylene oxide, and the like. Multiple repeats of alkylene oxide groups are included in the definition of alkylene oxides. For example, polyethylene oxides and ethylene oxides with fewer repeating units, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating ethylene oxide units are included in the alkylene oxides. Unless otherwise specifically stated in the specification, alkylene oxides may be optionally substituted.

[0064] "Aryl" refers to a carbocyclic ring system radical containing hydrogen, 6 to 18 carbon atoms and at least one aromatic ring. For the purposes of this disclosure, an aryl radical is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems. Aryl radicals include, but are not limited to, those derived from aceanthrene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, Aromatic radicals of fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene and triphenylene.

[0065] "Arylalkyl" refers to a group of the formula -R b -R c The free radical, R b is an alkylene or alkenylene group as defined above, and R c is one or more aryl radicals as defined above, for example benzyl, diphenylmethyl, etc. Unless otherwise specifically stated in the specification, an aralkyl group is optionally substituted.

[0066] As used herein, the term "substituted" means any of the above groups (e.g., alkyl, cycloalkyl, or heterocyclyl) wherein at least one hydrogen atom is replaced by a bond to a non-hydrogen atom, such as, but not limited to, a halogen atom, such as F, Cl, Br, and I; an oxygen group (=O); a hydroxyl group (-OH); an alkoxy group (-OR a, where R a It is C1-C 12 alkyl or cycloalkyl); carboxyl group (-OC(=O)R a OR -C(=O)OR a , where R a Yes H, C1-C 12 alkyl or cycloalkyl); amine group (-NR a R b , where R a and R b Each independently is H, C1-C 12 alkyl or cycloalkyl); C1-C 12 In some embodiments, the substituent is C1-C 12 In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halogen group, such as fluorine. In other embodiments, the substituent is an oxygen group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group. In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amino group.

[0067] "Optional" or "optionally" (e.g., optionally substituted) means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur. For example, "optionally substituted alkyl" means that the alkyl radical may or may not be substituted, and that the description includes substituted alkyl radicals and unsubstituted alkyl radicals. In some embodiments, "optionally substituted" means that the specified radical is substituted with one or more substituents selected from the group consisting of halogen (e.g., F, Cl, Br, and I), oxygen (=O), hydroxyl (-OH), alkoxy (-OR a , where R a It is C1-C 12 Alkyl), cycloalkoxy (-OR a , where R a is C3-C8 cycloalkyl), carboxyl (-OC(=O)R a OR – C(=O)OR a , where R a Yes H, C1-C 12 Alkyl, or C3-C8 cycloalkyl), amino (-NR a R b , where R a and R b Each independently is H, C1-C 12 alkyl or C3-C8 cycloalkyl), C1-C 12 Alkyl and C3-C8 cycloalkyl.

[0068] In some embodiments, "optionally substituted" means substituted with one or more halogen substituents. In some embodiments, "optionally substituted" means substituted with one or more oxygen substituents. In some embodiments, "optionally substituted" means substituted with one or more hydroxy substituents. In certain embodiments, "optionally substituted" means substituted with one or more alkoxy substituents. In some embodiments, "optionally substituted" means substituted with one or more cycloalkoxy substituents. In certain embodiments, "optionally substituted" means substituted with one or more carboxyl substituents. In some embodiments, "optionally substituted" means substituted with one or more amine substituents. In certain embodiments, "optionally substituted" means substituted with one or more C1-C 12 Alkyl substituents are substituted. In some embodiments, "optionally substituted" means substituted with one or more C3-C8 cycloalkyl substituents.

[0069] When a functional group is described as "optionally substituted," and conversely, a substituent on the functional group is also "optionally substituted," and so on, for purposes of this disclosure, such repetitions are limited to five, preferably, such repetitions are limited to two. In some embodiments, such repetitions are limited to one. In some embodiments, such repetitions are limited to zero.

[0070] The present disclosure is also meant to include all pharmaceutically acceptable compounds of structure (I) compounds that are isotopically labeled by replacing one or more atoms with atoms having a different atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as 2 H. 3 H. 11 C. 13 C. 14 C. 13 N. 15 N. 15 O. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F. 36 Cl, 123 I, and 125 I. These radiolabeled compounds can be used to help determine or measure the effectiveness of a compound, for example by characterizing the site or mode of action, or the binding affinity to a site of pharmacological importance. Certain isotopically labeled compounds of structure (I), for example those incorporating a radioactive isotope, are useful in drug and / or substrate tissue distribution studies. The radioactive isotope tritium (i.e.3 H) and carbon-14 (i.e. 14 C) are particularly suitable for this purpose in view of their ease of incorporation and ready means of detection.

[0071] Using heavier isotopes, such as deuterium (i.e. 2 H) substitutions may offer certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and therefore may be preferred in some circumstances.

[0072] Using positron emitting isotopes (such as 11 C. 18 F. 15 O and 13 N) substitution can be used in positron emission tomography (PET) studies to examine substrate receptor occupancy. Isotopically labeled compounds of structure (I) can generally be prepared by conventional techniques known to those skilled in the art or by methods analogous to those described in the Preparations and Examples, as set forth hereinafter, using an appropriate isotopically labeled reagent in place of the unlabeled reagent previously used.

[0073] The disclosure is also intended to include in vivo metabolites of the disclosed compounds. Such products may be oxidation, reduction, hydrolysis, amidation, esterification of the administered compound, for example, caused by a predominantly enzymatic process. Therefore, the disclosure includes compounds produced by the following method, which includes administering the disclosed compound to a mammal for a period of time sufficient to produce its metabolites. Such products are typically identified by the following method: administering a radiolabeled compound of the present invention to an animal (such as a rat, mouse, guinea pig, monkey or human) at a detectable dose, allowing enough time for metabolism to occur, and separating its conversion products from urine, blood or other biological samples.

[0074] "Stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.

[0075] "Mammal" includes humans and domestic animals, such as laboratory animals and household pets (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits), as well as non-domestic animals, such as wild animals, etc.

[0076] "Pharmaceutically acceptable carriers, diluents or excipients" include, but are not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the U.S. Food and Drug Administration for use in humans or livestock.

[0077] "Pharmaceutically acceptable salts" include acid addition salts and base addition salts.

[0078] "Pharmaceutically acceptable acid addition salts" refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed from inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid , cyclamic acid, dodecyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, etc.

[0079] "Pharmaceutically acceptable base addition salts" refer to those salts which retain the biological effectiveness and properties of the free acids and which are not biologically or otherwise undesirable. These salts are prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, phenethylbenzylamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0080] Crystallization will typically produce a solvate of the disclosed compound. As used herein, the term "solvate" refers to an aggregate comprising one or more molecules of the disclosed compound and one or more molecules of a solvent. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the disclosed compound may exist as a hydrate, including a monohydrate, a dihydrate, a hemihydrate, a sesquihydrate, a trihydrate, a tetrahydrate, etc., and corresponding solvated forms. The disclosed compound may be a true solvate, while in other cases, the disclosed compound may retain only exogenous water, or a mixture of water plus some exogenous solvent.

[0081] "Pharmaceutical composition" refers to a preparation of the disclosed compounds and a medium generally accepted in the art for delivering the biologically active compound to a mammal (eg, a human). Such a medium includes all pharmaceutically acceptable carriers, diluents or excipients.

[0082] "Effective amount" or "therapeutically effective amount" means that, when administered to a mammal (preferably a human), the amount of the compound of the present disclosure is sufficient to achieve treatment in the mammal (preferably a human). The amount of lipid nanoparticles of the present disclosure that constitutes a "therapeutically effective amount" will vary depending on the compound, the condition and its severity, the mode of administration, and the age of the mammal to be treated, but a person of ordinary skill in the art can routinely determine the amount of lipid nanoparticles based on his or her knowledge and the present disclosure.

[0083] As used herein, "treatment" or "treating" encompasses the treatment of a disease or condition in a mammal, preferably a human, suffering from the disease or condition of interest, and includes:

[0084] (i) preventing a disease or condition from occurring in a mammal, particularly where such mammal is susceptible to the condition but has not yet been diagnosed as having the condition;

[0085] (ii) inhibiting a disease or condition, i.e. preventing its development;

[0086] (iii) alleviate the disease or condition, i.e. cause regression of the disease or condition; or

[0087] (iv) Relief of symptoms caused by a disease or condition, i.e., relief of pain without addressing the underlying disease or condition. As used herein, the terms "disease" and "condition" may be used interchangeably, or may be different, in that a particular disease or condition may have no known causative agent (and thus a cause has not been determined), and therefore it is not yet considered a disease, but rather an adverse condition or syndrome in which clinicians have identified a more or less specific set of symptoms.

[0088] The compounds of the present disclosure, or their pharmaceutically acceptable salts, may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms, which may be defined as (R)- or (S)-, or as (D)- or (L)- for amino acids, in terms of absolute stereochemistry. The present disclosure is intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques (e.g., chromatography and fractional crystallization). Conventional techniques for preparing / isolating individual enantiomers include chiral synthesis or resolution of racemates (or racemates of salts or derivatives) from suitable optically pure precursors using, for example, chiral high pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, unless otherwise indicated, the compounds shall include both E and Z geometric isomers. Likewise, all tautomeric forms are intended to be included.

[0089] "Stereoisomers" refer to compounds composed of the same atoms joined by the same bonds, but having different three-dimensional structures that are not interchangeable. The present disclosure contemplates various stereoisomers and mixtures thereof, and includes "enantiomers," which refer to two stereoisomers whose molecules are non-superimposable mirror images of each other.

[0090] "Tautomer" refers to a proton shift from one atom of a molecule to another atom of the same molecule. The present disclosure includes tautomers of any of the described compounds.

[0091] Compound

[0092] In one aspect, the disclosure provides novel lipid compounds that can be combined with other lipid components (such as neutral lipids, charged lipids, steroids and / or polymer-coupled lipids) to form lipid nanoparticles with oligonucleotides. Without wishing to be bound by theory, it is believed that these lipid nanoparticles can protect oligonucleotides from degradation in serum and effectively deliver oligonucleotides to cells in vitro and in vivo.

[0093] One embodiment provides a compound having the following structure (I):

[0094]

[0095] or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

[0096] L 1a and L 1b Each is independently an optionally substituted C3-C 12 alkyl;

[0097] R 1a Yes -C(=O)OR 4a or –O(C=O)R 4a ;

[0098] R 1b Yes -C(=O)OR 4b or –O(C=O)R 4b ;

[0099] R 2 Yes - NR 6 (C=O)R 5 、-(C=O)N(R 6 )R 5 OR -(C=O)OR 7 ;

[0100] R 3 and R 6 Each is independently hydrogen or an optionally substituted C1-C6 alkyl group;

[0101] R 4a , R 4b and R 5 are each independently optionally substituted alkyl;

[0102] R 7 is an optionally substituted aralkyl group;

[0103] n1 is 2, 3, 4, 5, or 6; and

[0104] X is C2-C6 alkylene or C4-C 20 Alkylene oxide.

[0105] One embodiment provides a compound having the following structure (I):

[0106]

[0107] or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

[0108] L 1a and L 1b Each is independently an optionally substituted C3-C 12 alkyl;

[0109] R 1a Yes -C(=O)OR 4a or -O(C=O)R 4a ;

[0110] R 1b Yes -C(=O)OR 4b or -O(C=O)R 4b ;

[0111] R 2 Yes - NR 6 (C=O)R 5 、-(C=O)N(R 6 )R 5 OR -(C=O)OR 7 ;

[0112] R 3 and R 6 are each independently hydrogen or an optionally substituted C1-C 12 alkyl;

[0113] R 4a , R 4b and R 5 are each independently optionally substituted alkyl;

[0114] R 7 is an optionally substituted C1-C6 alkyl group or an optionally substituted aralkyl group;

[0115] n1 is 2, 3, 4, 5, or 6; and

[0116] X is C2-C6 alkylene or C4-C 20 Alkylene oxide.

[0117] In some embodiments, wherein X is:

[0118] or

[0119] in:

[0120] n2 is 2, 3, 4, 5, or 6;

[0121] n3 is 0, 1, 2, 3, or 4;

[0122] n4 is 2, 3, or 4; and

[0123] n5 is 2, 3, 4 or 5.

[0124] In some embodiments, L 1a is C5-C9 alkyl. 1b is a C5-C9 alkyl group. 1a is C5, C6, C7 or C9 alkyl. 1b is C5, C6, C7 or C9 alkyl. 1a is a C5 alkyl group. In certain embodiments, L 1a is a C6 alkyl group. 1ais a C7 alkyl group. In certain embodiments, L 1a is a C9 alkyl group. 1b is a C5 alkyl group. In certain embodiments, L 1b is a C6 alkyl group. 1b is a C7 alkyl group. In certain embodiments, L 1b is a C9 alkyl group. 1a is unsubstituted. In certain embodiments, L 1b is unsubstituted. In some embodiments, L 1a In certain embodiments, L 1b It is unbranched.

[0125] In some embodiments, R 1a One of them is -O(C=O)R 4a In certain embodiments, R 1a One of them is -(C=O)OR 4a In some embodiments, R 1b Yes-O(C=O)R 4b In certain embodiments, R 1b One of them is -(C=O)OR 4b .

[0126] In some embodiments, R 4a It is C8-C 24 In certain embodiments, R 4a It is C 10 -C 18 In certain embodiments, R 4a It is C 11 -C 16 In some embodiments, R 4a It is C 11 In certain embodiments, R 4a It is C 15 In some embodiments, R 4a It is C 16 In certain embodiments, R 4b It is C8-C 24 In some embodiments, R 4b It is C 10 -C 18 In certain embodiments, R 4b It is C 11 -C 16 In some embodiments, R 4b It is C 11 In certain embodiments, R4b It is C 15 In some embodiments, R 4b It is C 16 alkyl.

[0127] In certain embodiments, R 4a In some embodiments, R 4b In certain embodiments, R 4a is unsubstituted. In some embodiments, R 4b is unsubstituted. In certain embodiments, R 4a Has one of the following structures:

[0128]

[0129] In some embodiments, R 4b Has one of the following structures:

[0130]

[0131] In some embodiments, R 2 Yes - NR 6 (C=O)R 5 In certain embodiments, R 2 is -(C=O)N(R 6 )R 5 In some embodiments, R 5 It is C2-C 16 In certain embodiments, R 5 It is C4-C 13 In some embodiments, R 5 It is C4, C7, C8, C 10 or C 13 In certain embodiments, R 5 is unsubstituted. In some embodiments, R 5 In some embodiments, R 5 In certain embodiments, R 5 In some embodiments, R 5 Has one of the following structures:

[0132]

[0133] or

[0134] In certain embodiments, R 5 In some embodiments, R 5Has one of the following structures:

[0135]

[0136] or

[0137] In some embodiments, R 6 is C1-C6 alkyl. 6 It is C1-C 10 In certain embodiments, R 6 is C1-C4 alkyl. 6 Is C1, C2, C3, C6, C8 or C 10 In some embodiments, R 6 In certain embodiments, R 6 is methyl or n-butyl. 6 is unsubstituted. In some embodiments, R 6 In some embodiments, R 6 is a C1-C6 alkyl group substituted with one or more hydroxyl groups. 6 is a C2, C3, C4 or C6 alkyl group substituted with one or more hydroxyl groups. 6 is hydrogen. In some embodiments, R 2 is -(C=O)OR 7 .

[0138] In some embodiments, R 7 It is C1-C3 alkyl or C7-C 16 In certain embodiments, R 7 It is C7-C 16 In some embodiments, R 7 is C1-C3 alkyl. 7 is unsubstituted.

[0139] In certain embodiments, R 7 is -CH3 or has the following structure:

[0140]

[0141] In certain embodiments, R 7 Has the following structure:

[0142]

[0143] In some embodiments, R 3 is an optionally substituted C1-C6 alkyl. 3 is optionally substituted methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl. 3 is an optionally substituted methyl group. 3 is a C1-C6 alkyl group substituted with one or more hydroxyl groups. 3 is a C2 or C4 alkyl group substituted with one or more hydroxyl groups. 3 is unsubstituted. In some embodiments, R 3 It's hydrogen.

[0144] In some embodiments, X is For example, in some aspects, the compound has the following structure (II):

[0145]

[0146] or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof. For example, in some of these embodiments, n2 is 3, 4 or 5.

[0147] In other embodiments, X is In some such embodiments, the compound has the following structure (III):

[0148]

[0149] or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof. In different of these embodiments, n3 is 0 or 1. In other embodiments, n4 is 2 or 3. In some other different embodiments, n5 is 3.

[0150] In some embodiments, n1 is 3, 4, or 5. In certain embodiments, n1 is 2.

[0151] In some embodiments, the compound has one of the structures listed in Table 1 below, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166] It should be understood that any embodiment of the compound of structure (I) as described above, and any specific substituents and / or variables in the compound structure (I) as described above, can be independently combined with other embodiments and / or substituents and / or variables of the compound of structure (I) to form embodiments of the present disclosure not specifically described above. In addition, in a specific embodiment and / or claim, if a list of substituents and / or variables for any specific R group, L group or variable n1-n2 is listed, it should be understood that each individual substituent and / or variable can be deleted from the specific embodiment and / or claim, and the remaining list of substituents and / or variables will be deemed to be within the scope of the embodiments of the present disclosure.

[0167] It is understood that throughout this specification, combinations of substituents and / or variables of the described formulas are permissible only if such combinations result in stable compounds.

[0168] For the purpose of administration, the compounds of the present disclosure (usually in the form of lipid nanoparticles combined with therapeutic agents) can be used as raw chemical or can be formulated into pharmaceutical compositions. The pharmaceutical compositions of the present disclosure comprise compounds of structure (I) and one or more pharmaceutically acceptable carriers, diluents or excipients. The compounds of structure (I) are present in the composition in an amount effective to form lipid nanoparticles and deliver the therapeutic agent, for example, for the treatment of a particular disease or condition of interest. Suitable concentrations and dosages can be easily determined by those skilled in the art.

[0169] Embodiments provide compositions comprising a compound of structure (I) and a therapeutic agent. In some embodiments, the composition further comprises one or more excipients selected from neutral lipids, steroids, and polymer-conjugated lipids.

[0170] In some embodiments, the therapeutic agent comprises a nucleic acid. In certain embodiments, the nucleic acid is selected from antisense RNA and messenger RNA.

[0171] In certain embodiments, the composition comprises one or more neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In certain embodiments, the neutral lipid is DSPC. In some embodiments, the molar ratio of the compound to the neutral lipid ranges from about 2:1 to about 8:1. In certain embodiments, the steroid is cholesterol. In some embodiments, the molar ratio of the compound to cholesterol ranges from about 2:1 to 1:1. In certain embodiments, the polymer-coupled lipid is a pegylated lipid. In various embodiments, the polymer-coupled lipid is a pegylated lipid. For example, some embodiments include PEGylated diacylglycerols (PEG-DAG) such as 1-(monomethoxypolyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinyl diacylglycerols (PEG-S-DAG) such as 4-O-[2',3'-di(tetradecanoyloxy)propyl]-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG)), PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamate such as ω-methoxy(polyethoxy)ethyl-N-[2,3-di(tetradecanoyloxy)propyl]carbamate or 2,3-di(tetradecanoyloxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. In some embodiments, the molar ratio of the compound to the PEGylated lipid ranges from about 100: 1 to about 10: 1 or from about 100: 1 to about 25: 1. In some embodiments, the PEGylated lipid is PEG-DMG. In certain embodiments, the PEGylated lipid has the following structure (II):

[0172]

[0173] or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

[0174] R 8 and R 9 are each independently an unbranched or branched alkyl, alkenyl or alkynyl group containing from 10 to 30 carbon atoms, wherein the alkyl, alkenyl or alkynyl group is optionally interrupted by one or more ester bonds; and

[0175] The average value of z ranges from 30 to 60.

[0176] In some embodiments, R 8 and R 9 Each is independently an unbranched alkyl chain containing 12 to 16 carbon atoms. In some embodiments, the average z is about 45 (e.g., 43, 44, 45, 46, or 47). In some embodiments, the average z is about 43 to 47. In some embodiments, the average z is about 40 to 50.

[0177] The synthesis of PEGylated lipids can be found in US Pat. No. 9,738,593, the disclosure of which is incorporated herein by reference.

[0178] The application of the composition of the present disclosure can be carried out by any acceptable mode of application for similar purposes. The pharmaceutical composition of the present disclosure can be formulated into a preparation in solid, semisolid, liquid or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols. Typical routes of applying such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhaled, parenteral, sublingual, oral, rectal, vaginal and intranasal. The term parenteral as used herein includes subcutaneous injection, intravenous injection, intramuscular injection, intradermal injection, intrasternal injection or infusion technology. The pharmaceutical composition of the present disclosure is formulated to allow the active ingredients contained therein to be bioavailable when the composition is applied to the patient. The composition to be applied to a subject or a patient adopts the form of one or more dosage units, wherein, for example, a tablet can be a single dosage unit, and a container in the form of aerosol of the disclosed compound can accommodate multiple dosage units. Actual methods of preparing such dosage forms are known or will be apparent to those skilled in the art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). In any case, the composition to be administered comprises a therapeutically effective amount of a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, for treating a disease or condition of interest in accordance with the teachings of the present disclosure.

[0179] Pharmaceutical compositions of the present disclosure can be in the form of solid or liquid. In one aspect, the carrier is a microparticle, so the composition is, for example, in tablet or powder form. The carrier can be a liquid, wherein the composition is, for example, an oral syrup, an injectable liquid, or, for example, an aerosol for inhalation administration.

[0180] When used for oral administration, the pharmaceutical composition is preferably in solid or liquid form, with semi-solid, semi-liquid, suspension and gel forms included within the solid or liquid forms contemplated herein.

[0181] As a solid composition for oral administration, the pharmaceutical composition can be formulated into the form of powder, granules, compressed tablets, pills, capsules, chewing gum, wafers, etc. Such solid compositions generally contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders, such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, gum or gelatin; excipients, such as starch, lactose or dextrin; disintegrants, such as alginic acid, sodium alginate, Primogel, corn starch, etc.; lubricants, such as magnesium stearate or Sterotex; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; flavoring agents, such as mint, methyl salicylate or orange flavor; and coloring agents.

[0182] When the pharmaceutical composition is in the form of a capsule (eg, a gelatin capsule), it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.

[0183] The pharmaceutical composition can be in liquid form, for example, elixir, syrup, solution, emulsion or suspension. As two examples, the liquid can be used for oral administration or delivered by injection. When used for oral administration, in addition to the compounds of the present disclosure, the preferred composition also includes one or more of a sweetener, a preservative, a dye / colorant and a flavor enhancer. In the composition for administration by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersant, a suspending agent, a buffer, a stabilizer and an isotonic agent can be included.

[0184] Liquid pharmaceutical compositions of the present disclosure, whether in solution, suspension or other similar form, may include one or more of the following adjuvants: sterile diluents, such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride; fixed oils, such as synthetic monoglycerides or diglycerides, polyethylene glycol, glycerol, propylene glycol or other solvents that can be used as solvents or suspension media; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates and agents for adjusting tension, such as sodium chloride or glucose; agents used as cryoprotectants, such as sucrose or trehalose. Parenteral injection preparations can be packaged in ampoules, disposable syringes or multidose vials made of glass or plastic. Physiological saline is a preferred adjuvant. The injectable pharmaceutical composition is preferably sterile.

[0185] Liquid pharmaceutical compositions of the present disclosure for parenteral injection or oral administration should contain an amount of the disclosed compound to obtain a suitable dosage.

[0186] Pharmaceutical compositions of the present disclosure can be used for topical administration, in which case the carrier can suitably include a solution, an emulsion, an ointment or a gel matrix. For example, the matrix can include one or more of: vaseline, lanolin, polyethylene glycol, beeswax, mineral oil, a diluent (such as water and alcohol), and an emulsifier and a stabilizer. A thickener can be present in a pharmaceutical composition for topical administration. If used for transdermal administration, the composition can include a transdermal patch or an iontophoresis device.

[0187] Pharmaceutical compositions of the present disclosure can be used for rectal administration, for example, in the form of suppositories that will melt in the rectum and release the drug. Compositions for rectal administration can include an oily base as a suitable non-irritating excipient. Such bases include, but are not limited to, lanolin, cocoa butter, and polyethylene glycol.

[0188] Pharmaceutical compositions of the present disclosure can include various materials in modified physical forms of solid or liquid dosage units. For example, the composition can include materials forming a coating shell around the active ingredient. The materials forming the coating shell are usually inert and can be selected from, for example, sugar, shellac and other enteric coating agents. Alternatively, the active ingredient can be encapsulated in a gelatin capsule.

[0189] Pharmaceutical compositions of the present disclosure in solid or liquid form may include agents that bind to the compounds of the present disclosure to aid in the delivery of the compounds. Suitable agents that may perform this function include monoclonal or polyclonal antibodies or proteins.

[0190] Pharmaceutical compositions of the present disclosure can be composed of dosage units that can be administered as aerosols. The term aerosol is used to represent various systems, ranging from systems of colloidal nature to systems consisting of pressurized packaging. Delivery can be carried out by liquefied or compressed gas or by a suitable pump system that distributes active ingredients. Aerosols of compounds of the present disclosure can be delivered in a single-phase, two-phase or three-phase system to deliver active ingredients. The delivery of aerosols includes necessary containers, activators, valves, sub-containers, etc., which can form a kit together. Those skilled in the art can determine preferred aerosols without excessive experimentation.

[0191] The pharmaceutical composition of the present disclosure can be prepared by methods well known in the pharmaceutical field. For example, the pharmaceutical composition for administration by injection can be prepared by mixing the lipid nanoparticles of the present disclosure with sterile distilled water or other carriers to form a solution. Surfactants can be added to promote the formation of uniform solutions or suspensions. Surfactants are compounds that non-covalently interact with the disclosed compounds to promote dissolution or uniform suspension of the compounds in aqueous delivery systems.

[0192] The compositions of the present disclosure, or pharmaceutically acceptable salts thereof, are administered in a therapeutically effective amount, which will vary depending on a variety of factors, including the activity of the specified therapeutic agent employed; the metabolic stability and duration of action of the therapeutic agent; the age, weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disease or condition; and the treatment the subject is receiving.

[0193] The compositions of the present disclosure may also be administered at the same time as one or more other therapeutic agents are administered, before or after one or more other therapeutic agents are administered. Such combination therapy includes administering a single pharmaceutical dosage formulation of the compositions of the present disclosure and one or more additional active agents, as well as administering the compositions of the present disclosure and each active agent in its own separate pharmaceutical dosage formulation. For example, the compositions of the present disclosure and other active agents may be administered to the patient together in the form of a single oral dosage composition (such as a tablet or capsule), or each agent may be administered in a separate oral dosage formulation. In the case of using a separate dosage formulation, the compounds of the present disclosure and one or more additional active agents may be administered substantially simultaneously, i.e., simultaneously or at separate staggered times, i.e., sequentially; it should be understood that combination therapy includes all of these schemes.

[0194] Methods for preparing the above-mentioned compounds and compositions are described herein below and / or methods known in the art.

[0195] Those skilled in the art will appreciate that in the methods described herein, the functional groups of the intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxyl, amino, sulfhydryl and carboxylic acid. Suitable protecting groups for hydroxyl include trialkylsilyl or diarylalkylsilyl (e.g., tert-butyldimethylsilyl, tert-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, etc. Suitable protecting groups for amino, amidino and guanidino include tert-butyloxycarbonyl, benzyloxycarbonyl, etc. Suitable protecting groups for sulfhydryl include -C (O) R "(wherein R " is alkyl, aryl or aralkyl), p-methoxybenzyl, trityl, etc. Suitable protecting groups for carboxylic acid include alkyl, aryl or aralkyl esters. Protecting groups can be added or removed according to standard techniques known to those skilled in the art and described herein. The use of protecting groups is described in detail in Green, TWand PGM Wutz, Protective Groups in Organic Synthesis (1999), 3 rd Ed., Wiley. It will be appreciated by those skilled in the art that the protecting group may also be a polymer resin, such as Wang resin, Rink resin or 2-chlorotrityl chloride resin.

[0196] It will also be understood by those skilled in the art that, although such protected derivatives of the compounds of the present disclosure may therefore not have pharmacological activity, they can be administered to mammals and then metabolized in vivo to form pharmacologically active compounds of the present disclosure. Therefore, such derivatives can be described as "prodrugs". All prodrugs of the compounds of the present disclosure are included within the scope of the present disclosure.

[0197] In addition, by treatment with an appropriate inorganic base or acid or an organic base or acid by methods known to those skilled in the art. All salts of the disclosed compounds can be converted to their free base or acid form by standard techniques.

[0198] The following reaction schemes illustrate methods for preparing compounds of the present disclosure, namely compounds of structure (I):

[0199]

[0200] Where R 1a , R 1b , R2, R 3 , L 1a , L 1b , n1 and n2 are as defined herein. It should be understood that those skilled in the art may be able to prepare these compounds by similar methods or by combining other methods known to those skilled in the art. It should also be understood that by using appropriate starting components and modifying synthesis parameters as required, those skilled in the art will be able to prepare other compounds of structure (I) not specifically shown below in a manner similar to that described below. In general, starting components can be obtained from the following sources, such as Sigma-Aldrich, Lancaster Synthesis, Inc., Maybridge, MatrixScientific, TCI and U.S. Fluorochem, 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 the present disclosure.

[0201] General reaction scheme 1

[0202]

[0203] Embodiments of compounds of structure (I) can be prepared according to General Reaction Scheme 1 ("Method A"). R in General Reaction Scheme 1 1a , R 1b , R2, R 3 , L 1a , L1b and X are as defined herein.

[0204] Referring to general reaction scheme 1, compound starting material A1 can be purchased from commercial sources or prepared according to methods familiar to those skilled in the art. A1 is reacted with compound A2 under appropriate alkaline conditions (e.g., potassium carbonate and cesium carbonate) and heated at 70°C to obtain compound A3. A3 is then treated dropwise with thionyl chloride in an ice bath to convert the hydroxyl group to chloride A4, which is then reacted with amine A5 using appropriate conditions (e.g., heating) to obtain a compound of structure (I) as shown in the figure.

[0205] It should be noted that a variety of alternative strategies for preparing compounds of structure (I) may be employed by one of ordinary skill in the art. For example, using appropriate starting materials, other compounds of structure (I) may be prepared according to similar methods, such as shown in the examples below. The use of protecting groups and other modifications to the general reaction scheme described above as needed will be apparent to one of ordinary skill in the art. The following examples are provided for illustrative purposes and not for limiting purposes.

[0206] Example 1

[0207] Evaluation of luciferase mRNA in vivo using lipid nanoparticle compositions

[0208] Lipids, DSPC, cholesterol and PEG lipids of structure (I) are dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 or 47.5:10:40.7:1.8. Lipid nanoparticles (LNP) are prepared at a weight ratio of about 10:1 to 40:1 of total lipid to mRNA. In brief, mRNA is diluted to 0.2 mg / mL in a 10 mM to 50 mM citrate buffer at pH 4 or a 10 mM to 25 mM acetate buffer at pH 4. An ethanol lipid solution is mixed with an mRNA aqueous solution at a ratio of about 1:5 to 1:3 (vol / vol) with a syringe pump, and a total flow rate is higher than 15 mL / min. Ethanol is then removed, and the external buffer is replaced with PBS by dialysis. Finally, the lipid nanoparticles are filtered through a 0.2 μm pore sterile filter.

[0209] Studies were performed in female C57BL / 6 mice (Charles River) aged 6 to 8 weeks or CD-1 (Harlan) mice (Charles Rivers) aged 8 to 10 weeks in accordance with the guidelines set by the Institutional Animal Care Committee (ACC) and the Canadian Council on Animal Care (CCAC). Different doses of mRNA lipid nanoparticles were systemically administered by tail vein injection, and animals were euthanized at designated time points (e.g., 4 hours) after administration. The liver and spleen were collected in pre-weighed tubes, the weight was determined, and they were immediately snap-frozen in liquid nitrogen and stored at -80°C until processed for analysis.

[0210] For liver, approximately 50 mg was cut into 2 mL FastPrep tubes (MP Biomedicals, Solon OH) for analysis. A 1 / 4" ceramic ball (MP Biomedicals) was added to each tube, and 500 μL of Glo Lysis Buffer-GLB (Promega, Madison WI) equilibrated to room temperature was added to the liver tissue. The liver tissue was homogenized at 2×6.0 m / s for 15 seconds using a FastPrep24 instrument (MP Biomedicals). The homogenate was incubated at room temperature for 5 minutes, then diluted 1:4 in GLB and evaluated using the SteadyGlo luciferase assay system (Promega). Specifically, 50 μL of the diluted tissue homogenate was reacted with 50 μL SteadyGlo substrate, shaken for 10 seconds, then incubated for 5 minutes, and then measured using a CentroXS3 LB 960 luminometer (Berthold, Germany). Technologies) for quantification. The amount of protein was determined by using a BCA protein assay kit (Pierce, Rockford, IL). The relative luminescence unit (RLU) was then standardized to the total μg protein measured. In order to convert RLU to ng luciferase, a standard curve was generated using QuantiLum recombinant luciferase (Promega).

[0211] FLuc mRNA (L-6107) from Trilink Biotechnologies expresses the luciferase protein originally isolated from the firefly Photinus pyralis. FLuc is commonly used in mammalian cell culture to measure gene expression and cell viability. It emits bioluminescence in the presence of the substrate luciferin. This capped and polyadenylated mRNA is completely substituted with 5-methylcytidine and pseudouridine.

[0212] Example 2

[0213] Evaluation of Immunoglobulin G (IgG) mRNA in vivo using lipid nanoparticle compositions

[0214] Lipids, DSPC, cholesterol and PEG lipids of structure (I) are dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 or 47.5:10:40.7:1.8. Lipid nanoparticles (LNP) are prepared at a weight ratio of about 10:1 to 40:1 of total lipid to mRNA. In brief, mRNA is diluted to 0.2 mg / mL in a 10 mM to 50 mM citrate buffer at pH 4 or a 10 mM to 25 mM acetate buffer at pH 4. An ethanol lipid solution is mixed with an mRNA aqueous solution at a ratio of about 1:5 to 1:3 (vol / vol) with a syringe pump, and a total flow rate is higher than 15 mL / min. Ethanol is then removed, and the external buffer is replaced with PBS by dialysis. Finally, the lipid nanoparticles are filtered through a 0.2 μm pore sterile filter.

[0215] Studies were conducted in 6- to 8-week-old CD-1 / ICR mice (Envigo) according to the guidelines set by the Institutional Animal Care Committee (ACC) and the Canadian Council on Animal Care (CCAC). Different doses of mRNA lipid nanoparticles were systemically administered by tail vein injection, and the animals were euthanized at a specified time point (e.g., 24 hours) after administration. Whole blood was collected, and serum was subsequently separated by centrifugation of tubes of whole blood at 2000 x g for 10 minutes at 4°C and stored at -80°C until used for analysis.

[0216] For immunoglobulin G (IgG) ELISA (Life Diagnostics Human IgG ELISA Kit), serum samples were diluted 100 to 15,000 times with 1× diluent. 100 μL of diluted serum was dispensed in duplicate into a 96-well plate coated with anti-human IgG along with human IgG standards and incubated at 25°C for 45 minutes at 150 rpm on a plate shaker. The wells were washed 5 times with 1× wash buffer using a plate washer (400 μL / well). 100 μL of HRP conjugate was added to each well and incubated on a plate shaker under the same conditions as above. The wells were washed again 5 times with 1× wash buffer using a plate washer (400 μL / well). 100 μL of TMB reagent was added to each well and incubated on a plate shaker under the same conditions as above. The reaction was stopped by adding 100 μL of stop solution to each well. The absorbance was read at 450 nm (A450) using a microplate reader. The amount of human IgG in mouse serum was determined by plotting the A450 values ​​of the assay standards against the human IgG concentration.

[0217] Example 3

[0218] Determination of pKa of formulated lipids

[0219] As described elsewhere, the pKa of the formulated lipids is correlated with the effectiveness of LNPs for delivering nucleic acids (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semplet al, Nature Biotechnology 28, 172–176 (2010)). In some embodiments, the preferred range of pKa is from about 5 to about 7. The pKa of each lipid was determined in the lipid nanoparticles using an assay based on 2-(p-toluidinyl)-6-naphthalenesulfonic acid (TNS) fluorescence. Lipid nanoparticles were prepared using the series of processes described in Example 1, which contained structure (I) compound / DSPC / cholesterol / PEG-lipid (50 / 10 / 38.5 / 1.5 or 47.5:10:40.7:1.8 mol %) at a total lipid concentration of 0.4 mM in PBS. TNS was prepared as a 100 μM stock solution in distilled water. The vesicles were diluted to 24 μM lipid in 2 mL of buffer solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl in a pH range of 2.5 to 11. An aliquot of TNS solution was added to a final concentration of 1 μM, and the fluorescence intensity after vortex mixing was measured at room temperature in a SLM Aminco Series 2 luminescence spectrophotometer using excitation and emission wavelengths of 321 nm and 445 nm. The fluorescence data were subjected to sigmoidal best fit analysis, and the pKa was measured as the pH value that produced half-maximal fluorescence intensity.

[0220] Example 4

[0221] Determining the Efficacy of Lipid Nanoparticle Formulations Containing Different Cationic Lipids Using an In Vivo Luciferase / IgG mRNA Expression Rodent Model

[0222] Representative compounds of the present disclosure shown in Table 2 were formulated using the following molar ratios: 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid 2-[2-(ω-methoxy(polyethylene glycol) 2000)ethoxy)]-N,N-di-tetradecyl acetamide or 47.5% cationic lipid / 10% DSPC / 40.7% cholesterol / 1.8% PEG lipid. Activity was determined by measuring luciferase expression in the liver 4 hours after administration by tail vein injection as described in Example 1 or by measuring the amount of human IgG in mouse serum as described in Example 2. Activity was compared at a dose of 1.0 or 0.5 or 0.3 mg mRNA / kg and expressed as ng luciferase / g liver measured 4 hours after administration as described in Example 1 or expressed as μg IgG / mL serum measured 24 hours after administration as described in Example 2. The compound numbers in Table 2 refer to the compound numbers in Table 1.

[0223] Table 2. Novel cationic lipids and their related activities

[0224]

[0225]

[0226] Example 5

[0227] Synthesis of bis(2-hexyldecyl) 6,6'-((2-((4-octanamidobutyl)amino)ethyl)azadiyl) dihexanoate (Compound I-2)

[0228]

[0229] Synthesis of compound 4-1

[0230] Acetyl chloride (10 mL) was slowly added (about 15 minutes) to a solution of octanoic acid (50 mmol, 7.21 g, 7.92 mL) in 200 mL of methanol cooled with ice salt under Ar. Stir for 20 minutes, then remove the cooling bath. Allow the solution to stand overnight at room temperature (20 hours). Methanol was removed under reduced pressure. Saturated sodium bicarbonate (100 mL) and hexane (200 mL) were added to the residue. The hexane extract was washed with brine (70 mL), dried with sodium sulfate, filtered and concentrated to give methyl octanoate as a colorless oil. Methyl octanoate was dried overnight on a high vacuum line (oil pump) (6.439 g, 40.7 mmol, 93%) and used without further purification.

[0231] Under reflux, methyl octanoate (20 mmol, 3.16 g) in methanol (20 ml) was slowly added (15 min) to a solution of 1,4-butanediamine (5 eq. 100 mmol, 8.8 g) in 50 ml of methanol. Reflux was continued for 48 hours. The solvent was then evaporated under reduced pressure and the residue was dissolved in a mixture of water (70 mL) and ethyl acetate (100 mL). A small amount of citric acid was added. The two phases were separated and the aqueous phase was extracted twice with ethyl acetate. The combined ethyl acetate extracts were washed with water and brine, dried over sodium sulfate and filtered. Concentration gave a mixture of oil and white solid. The residue was dissolved in DCM and MeOH (19:1). The solution was passed through a silica gel pad (1.5 cm high × 6.5 cm wide) and washed with a mixture of MeOH (5%) and DCM until TLC showed that all the diacylated products were eluted. Then C H The pad was washed with a mixture of Cl3 / EtOH / water / NH3 (30 / 25 / 3 / 2, 225 mL) to elute the desired product 4-1. The fractions containing the product were combined and concentrated to dryness (white solid, 2.52 g, 11.8 mmol, 59%).

[0232] Synthesis of compound 4-2

[0233] 2-Hexyldecyl-6-bromohexanoate (1.9eq, 4.37g, 10.4mmol), potassium carbonate (1.9eq, 1.44g, 10.4mmol, MW 138.21), cesium carbonate (0.3eq, 534mg, 1.64mmol, MW325.82) and sodium iodide (30mg) were added to a solution of 2-aminoethanol (333mg, 5.46mmol) in 35ml anhydrous THF, and the mixture was heated at 70°C for 6 days in a sealed pressure bottle. The solvent was evaporated under reduced pressure, and the residue was dissolved in a mixture of hexane and ethyl acetate (about 6%), washed with water and brine. The organic layer was separated and dried over anhydrous sodium sulfate. The dried extract was filtered through a silica gel pad under reduced pressure. The pad was then washed with hexane-EtOAc-Et3N (95:5:0 to 80:20:1). The portions containing the desired product were combined and concentrated to dryness. A colorless oil (1.766 g, 2.39 mmol, 46%) was obtained.

[0234] Synthesis of compound 4-3

[0235] Under argon atmosphere, to ice-cooled 4-2 (2.16g, 2.93mmol) solution in 8mL CHCl3, thionyl chloride (3eq, 8.79mmol, 1.05g) in 35mL chloroform is added dropwise (about 1 to 2min). After addition is completed, the ice bath is removed, and the reaction mixture is stirred at room temperature (20°C) for 16 hours. The concentrated mixture obtains a viscous dark oil. The residue is dissolved in chloroform and Et3N (0.6mL) mixture, and purified by silica gel flash column chromatography (0 to 1% MeOH in chloroform). This obtains the desired product, which is a brown oil (1.786g, 2.36mmol, 80%).

[0236] Synthesis of Compound I-2

[0237] A solution of 4-3 (1 eq, 522 mg, 0.67 mmol), 4-1 (3 eq, 2.07 mmol, 444 mg) and N,N-diisopropylethylamine (3 equiv. 2.07 mmol, 267 mg, 0.36 mL; MW 129.25, d 0.742) in acetonitrile (15 mL) was sealed and heated at 65 ° C overnight. The reaction mixture was cooled and concentrated. The residue was placed in a mixture of ethyl acetate and hexane (80:20) and filtered through a short silica gel column (2.5 cm high × 3 cm wide). The column was then eluted with the same solvent mixture. The fractions containing the less polar product were combined and concentrated (433 mg, light yellow oil, dialkylated product 4-4). The column was then eluted with a mixture of DCM-MeOH-NH3 (95:5:0.5). All parts containing the desired product are combined and concentrated; the residue (oil / solid) is dissolved in a mixture of hexane and ethyl acetate (about 98:2) and filtered. The filtrate is concentrated to give a viscous brown oil (Compound I-2). The yellow oil (444 mg) is further purified by silica gel flash drying column chromatography (1% to 5% MeOH in chloroform). The parts containing the desired product are combined and concentrated (288 mg, 0.23 mmol, yellow oil). 1 HNMR (400MHz,CDCl3)δ:6.24-6.13(m,1H,NHCO),3.96(d,5.8Hz,4H),3.32-3.19(m,2H),2.64-2.59(m,4H),2.54-2.50(m,2H),2.44-2.34(m,4H),2.30(t,7.5Hz,4H),2.18-2.09(m,2H),1.67-1.50(m,13H),1.47-1.38(m,4H),1.38-1.08(m,60H),0.90-0.86(m,15H). ESI-MS: molecular weight C58 H 115 N3O5[M+H] + : Calculated value 934.9; measured value 935.1.

[0238] Example 6

[0239] Synthesis of bis(2-hexyldecyl) 6,6'-((2-(methyl(4-octanamidobutyl)amino)ethyl)azadiyl) dihexanoate (Compound I-1)

[0240]

[0241] Synthesis of Compound I-1

[0242] To a solution of compound I-2 (223 mg, 0.23 mmol) in THF (5 mL) was added formaldehyde solution (about 6 mmol, 500 mg of a 37 wt.% solution in water) at room temperature. The resulting mixture was stirred for 30 minutes, and then sodium triacetoxyborohydride (5 eq., 1.2 mmol, 243 mg) was introduced. After stirring overnight, the mixture was dissolved in a mixture of hexane and ethyl acetate (100 mL) and washed with a saturated sodium bicarbonate solution. After drying with sodium sulfate, the solution was filtered through a silica gel short column (230 to 400 mesh grade silica gel, 2.5 cm high × 3 cm wide) under reduced pressure. The column was then eluted with a mixture of hexane, ethyl acetate and Et3N (80:20:1, 200 mL). The column was subsequently eluted with a mixture of DCM and MeOH (97:3, 100 mL). All parts were combined and concentrated (220 mg, yellow oil). The crude product was further purified by flash column chromatography on silica gel (0 to 5% MeOH in chloroform). This gave the desired product as a colorless oil (165 mg, 016 mmol, 42%). 1 H NMR (400MHz,CDCl3)δ:6.18-6.10(m,1H,NHCO),3.96(d,5.8Hz,4H),3.29-3.18(m,2H),2.55-2.50(m,2H),2.44-2.38(m,6H),2.37-2.33(m,2H),2.30(t,7.5Hz,4H),2.21(s,3H),2.16-2.10(m,2H),1.67-1.58(m,8H),1.55-1.48(m,4H),1.48-1.39(m,4H),1.34-1.08(m,60H),0.90-0.85(m,15H). ESI-MS: molecular weight C 59 H 117 N3O5[M+H] +Calculated value: 948.9; measured value: 949.1.

[0243] Example 7

[0244] Bis(2-hexyldecyl)6,6'-((2-((6-(benzyloxy)-6-oxohexyl)(methyl)amino)ethyl)azadiyl)dihexanoate (Compound I-9)

[0245]

[0246] Synthesis of compound 6-1

[0247] By methylamine (5eq, 75mmol, 37.5mL, 2M solution in THF), benzyl-6-bromohexanoate (4.2g, 14.7mmol), NaI (40mg) and N, N-diisopropylethylamine (2.6mL) mixture sealed in pressure bottle, and stirred at room temperature overnight. After the reaction mixture was concentrated, the residue was purified by silica gel flash drying column chromatography (DCM / MeOH / Et3N, 99:1:0 to 80:20:1). This obtained the desired product, which was a light yellow oil (1.95g, 8.3mmol, 56%).

[0248] Synthesis of Compound I-9

[0249] A mixture of 4-3 (1.141 g, 1.51 mmol), 6-1 (3 eq. 4.53 mmol, 1.066 g), N,N-diisopropylethylamine (3 equiv., 4.53 mmol, 584 mg, 0.788 mL) and sodium iodide (60 mg) in acetonitrile (20 mL) was sealed and heated at 76 ° C (oil bath) for 24 hours. The reaction mixture was cooled and concentrated the next day. The crude product was purified by silica gel flash drying column chromatography (230 to 400 mesh grade silica gel, hexane / EtOAc / Et3N, 90:10:0 to 80:20:1). This gave the desired product as a light yellow oil (1.20 g, 1.25 mmol, 83%). 1 H NMR(400MHz, CDCl3@7.26ppm)δ:7.39-7.29(m,5H),5.11(s,2H),3.96(d,5.8Hz,4H),2.54-2.49(m,2H),2.4 3-2.27(m,14H),2.20(s,3H),1.70-1.58(m,8H),1.51-1.38(m,6H),1.36-1.18(m,54H),0.90-0.85(m,12H).

[0250] Example 8

[0251] Synthesis of bis(2-hexyldecyl)6,6'-((2-(methyl(4-tetradecylaminobutyl)amino)ethyl)azadiyl]dihexanoate

[0252]

[0253] Synthesis of Compound I-3

[0254] A mixture of 4-3 (372 mg, 0.49 mmol), N-[4-(methylamino)butyl]tetradecanoamide (343 mg, 1.1 mmol), N,N-diisopropylethylamine (equiv., 2.1 mmol, 271 mg, 0.37 mL) and sodium iodide (10 mg) in acetonitrile (10 mL) was sealed and heated at 76 ° C (oil bath) for 24 hours. The mixture was cooled and concentrated the next day. The residue was mixed in a mixture of hexane / EtOAc / Et3N / MeOH (70:30:1:1) and filtered through a short silica gel column (230 to 400 mesh grade silica gel). The column was then eluted with the same solvent mixture. All fractions containing the product were combined and concentrated. The product (418 mg) was further purified by silica gel flash column chromatography (MeOH in chloroform with trace Et3N, 0 to 5%). This gave the desired product as a colorless oil (294 mg, 0.28 mmol, 58%). 1 H NMR (400MHz, CDCl3@7.26ppm)δ:6.16-6.08(m,1H,NHCO),3.96(d,5.8Hz,4H),3.28-3.19(m,2H),2.55-2.50(m,2H),2.44-2.32(m,8H),2.30(t,7.5Hz,4H),2.21(s,3H),2.17-2.09(m,2H),1.67-1.58(m,8H),1.55-1.37(m,8H),1.36-1.08(m,72H),0.90-0.86(m,15H). ESI-MS: molecular weight C 65 H 129 N3O5[M+H] + Calculated value: 1033.0; measured value: 1033.2.

[0255] Example 9

[0256] Synthesis of bis(2-hexyldecyl) 8,8'-((2-(methyl(3-nonanamidopropyl)amino)ethyl)azadiyl) dioctanoate (Compound I-4)

[0257]

[0258] Synthesis of compound 8-1

[0259] This intermediate was prepared in a similar manner to the preparation of compound 4-3 in Example 5.

[0260] Synthesis of Compound I-4

[0261] The mixture of 8-1 (344 mg, 0.42 mmol), N-[3-(methylamino)propyl]nonylamide (3 eq. 1.27 mmol, 290 mg), N,N-diisopropylethylamine (0.37 mL) and sodium iodide (10 mg) in acetonitrile (10 mL) was sealed and heated at 80 ° C for 24 hours. The mixture was cooled and concentrated the next day. The residue was dissolved in a mixture of hexane / EtOAc / Et3N (70: 30: 1) and filtered through a short silica gel column (230 to 400 mesh silica gel). The column was then eluted with the same solvent mixture. All parts containing the product were combined and concentrated into 332 mg of yellow oil. The product was further purified by silica gel rapid drying column chromatography (methanol solution in chloroform, containing trace Et3N, 0 to 5%). This obtained the desired product, which was a slightly yellow oil (96 mg, 0.095 mmol, 23%). 1 H NMR (400MHz, CDCl3@7.26ppm)δ:7.11-7.05(m,1H,NHCO),3.96(d,5.8Hz,4H),3.37-3.30(m,2H),2.55-2.49(m,2H),2.48-2.36(m,8H),2.29(t,7.5Hz,4H),2.22(s,3H),2.16-2.09(m,2H),1.67-1.58(m,10H),1.46-1.37(m,4H),1.34-1.08(m,70H),0.90-0.85(m,15H). ESI-MS: molecular weight C 63 H 125 N3O5[M+H] + Calculated value: 1005.0; measured value: 1005.2.

[0262] Example 10

[0263] Synthesis of bis(2-hexyldecyl) 8,8'-((2-(methyl(4-octanamidobutyl)amino)ethyl)azadiyl) dioctanoate (Compound I-5)

[0264]

[0265] Synthesis of compound 9-1

[0266] This intermediate was prepared in a similar manner to the preparation of compound 1-2 in Example 5.

[0267] Synthesis of Compound I-5

[0268] To a solution of 9-1 (122 mg, 0.12 mmol) in THF (6 mL) was added formaldehyde-HCHO solution (460 mg of a 37 wt.% solution in water) at room temperature. The resulting mixture was stirred for 30 minutes, and then sodium triacetoxyborohydride (5 eq., 0.6 mmol, 122 mg) was introduced. The resulting mixture was stirred overnight at room temperature. The mixture was placed in hexane and washed with a saturated sodium bicarbonate solution. The organic phase was dried over Na2SO4 and concentrated to give 122 mg of a yellow oil. The oil was dissolved in a mixture of hexane / EtOAc / Et3N (70:30:1) and filtered through a short silica gel column (230 to 400 mesh grade silica gel). The column was then eluted with the same solvent mixture. All product-containing portions were combined and concentrated to give 106 mg of a yellow oil. The product was further purified by silica gel rapid drying column chromatography (methanol solution in chloroform, containing trace amounts of Et3N, 0 to 5%). This gave the desired product as a colorless oil (43 mg). 1 HNMR (400MHz, CDCl3@7.26ppm)δ:6.21-6.14(m,1H,NHCO),3.96(d,5.8Hz,4H),3.27-3.20(m,2H),2.55-2.50(m,2H),2.45-2.33(m,8H),2.29(t,7.5Hz,4H),2.22(s,3H),2.16-2.10(m,2H),1.67-1.58(m,8H),1.55-1.48(m,4H),1.46-1.37(m,4H),1.35-1.18(m,68H),0.90-0.85(m,15H). ESI-MS: molecular weight C 63 H 125 N3O5[M+H] + Calculated value: 1005.0; measured value: 1005.2.

[0269] Embodiment 11

[0270] Synthesis of bis(2-hexyldecyl)6,6'-((2-(([6-[(decylamino)-6-oxohexyl)(methyl)amino)ethyl)azadiyl]dihexanoate (Compound I-7)

[0271]

[0272] Synthesis of compound 10-1

[0273] To a solution of compound I-9 (prepared according to the steps of Example 10, 1.019 g, 1.07 mmol) in EtOH / EtOAc (1:10 mL) was added 10% Pd / C (25 mg) and the mixture was stirred under hydrogen. The reaction was monitored by TLC. Two additional portions of Pd / C (30 mg each) were added during the reaction. After 9 days, TLC showed that the reaction was about 90% complete. The mixture was passed through a celite pad (e.g. ) and washed the pad with DCM. The filtrate was concentrated to give a yellow oil. The crude product was purified by silica gel flash column chromatography (0 to 10% methanol in DCM). This gave the desired product as a yellow oil (751 mg, 0.87 mmol, 81%).

[0274] Synthesis of Compound I-7

[0275] At room temperature, oxalyl chloride (5eq, 1.45mmol, 184mg, 0.13mL) was added to a solution of 10-1 (250mg, 0.29mmol) in DCM (6mL) and DMF (about 0.01mL). After stirring at room temperature for 16 hours, the mixture was concentrated under reduced pressure. The remaining oil was dissolved in 5mL DCM and added to a solution of 1-decylamine (1.5eq, 0.44mmol, 68mg) and triethylamine (0.12mL) in DCM (5mL). The resulting mixture was stirred at room temperature for 30 minutes, then concentrated. The residue was dissolved in a mixture of hexane / EtOAc / Et3N (80:20:1) and filtered through a short silica gel column (230 to 400 mesh grade silica gel). The same solvent mixture elution column was then used. All parts containing the product were merged and concentrated to obtain 254mg brown oil. The product was further purified by flash column chromatography on silica gel (methanol in chloroform, trace Et3N, 0 to 5%). This gave the desired product as a colorless oil (218 mg, 0.22 mmol, 75%). 1 HNMR (400MHz, CDCl3@7.26ppm)δ:5.54-5.46(m,1H,NHCO),3.96(d,5.8Hz,4H),3.27-3.19(m,2H),2.55-2.50(m,2H),2.44-2.37(m,6H),2.35-2.27(m,6H),2.21(s,3H),2.19-2.13(m,7.6Hz,2H),1.69-1.57(m,8H),1.52-1.39(m,8H),1.34-1.08(m,68H),0.90-0.85(m,15H). ESI-MS: molecular weight C 63 H 125N3O5[M+H] + Calculated value: 1005.0; measured value: 1005.2.

[0276] Example 12

[0277] Synthesis of bis(2-hexyldecyl)6,6'-((2-(methyl(6-[methyl(octyl)amino)-6-oxohexyl)amino)ethyl)azadiyl]dihexanoate (Compound I-10)

[0278]

[0279] Synthesis of Compound I-10

[0280] At room temperature, oxalyl chloride (5eq, 1.45mmol, 184mg, 0.13mL) was added to a solution of 10-1 (prepared according to the steps of Example 11, 250mg, 0.29mmol) in DCM (6mL) and DMF (0.01mL). After stirring at room temperature for 16 hours, the mixture was concentrated under reduced pressure. The remaining oil was dissolved in 5mL DCM and added to a solution of N-methyloctylamine (1.5eq, 0.44mmol, 63mg) and triethylamine (0.12mL) in DCM (5mL). The resulting mixture was stirred at room temperature for 30 minutes and then concentrated. The residue was dissolved in a mixture of hexane / EtOAc / Et3N (80:20:1) and filtered through a silica gel short column (230 to 400 mesh silica gel). The same solvent mixture was then used to elute the column. All parts containing the product were combined and concentrated to obtain a brown oil. The product (brown oil) was further purified by silica gel column chromatography (0 to 5% MeOH in DCM). This gave the desired product as a yellow oil (146 mg, 0.15 mmol, 51%). 1 HNMR (400MHz, CDCl3@7.26ppm)δ:3.96(d,5.8Hz,4H),3.34,3.32(2 sets of triplet peaks,ratio=1:1,7.5Hz,2H),2.95,2.90(2 sets of singlet peaks,ratio=1:1,3H),2.55-2.50(m,2H),2.43-2.36(m,8H),2.35-2.27(m,6H),2.21(s,3H),1.70-1.54(m,8H),1.54-1.38(m,8H),1.37-1.08(m,64H),0.91-0.85(m,15H). ESI-MS: molecular weight C 62 H 123 N3O5[M+H] + Calculated value: 991.0; measured value: 991.1.

[0281] Embodiment 13

[0282] Synthesis of bis(2-hexyldecyl)6,6'-((2-((6-((2-ethylhexyl)amino)-6-oxohexyl)(methyl)amino)ethyl)azadiyl)dihexanoate (Compound I-11)

[0283]

[0284] Synthesis of Compound I-11

[0285] At room temperature, oxalyl chloride (5eq, 1.45mmol, 184mg, 0.13mL) was added to a solution of 10-1 (prepared according to the steps of Example 11, 250mg, 0.29mmol) in DCM (6mL) and DMF (about 0.01mL). After stirring at room temperature for 16 hours, the mixture was concentrated under reduced pressure. The remaining oil was dissolved in 5mL DCM and added to a solution of 2-ethyl-1-hexylamine (1.5eq, 0.44mmol, 57mg) and triethylamine (0.12mL) in DCM (5mL). The resulting mixture was stirred at room temperature for 2 hours and then concentrated. The residue was dissolved in a mixture of hexane / EtOAc / Et3N (80:20:1) and filtered through a short silica gel column (230 to 400 mesh silica gel). The same solvent mixture was then used for elution. All parts containing the product were combined and concentrated to obtain 258mg brown oil. The product was further purified by flash column chromatography on silica gel (MeOH in chloroform, trace Et3N, 0 to 5%). This gave the desired product as a slightly yellow oil (184 mg, 0.19 mmol, 65%). 1 H NMR (400MHz, CDCl3@7.26ppm)δ:5.43(s,1H),3.96(d,5.8Hz,4H),3.25-3.13(m,2H),2.55-2.49(m,2H),2.43-2.37(m,6H),2.35-2.27(m,6H),2.21(s,3H),2.17(t,7.6Hz,2H),1.69-1.58(m,8H),1.52-1.38(m,7H),1.37-1.18(m,62H),0.91-0.85(m,18H). ESI-MS: molecular weight C 61 H 121 N3O5[M+H] + Calculated value: 976.9; measured value: 977.1.

[0286] Embodiment 14

[0287] Synthesis of ((2-((4-(dibutylamino)-4-oxobutyl)(methyl)amino)ethyl)azepinediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (Compound I-8)

[0288]

[0289] Synthesis of compound 13-2

[0290] A mixture of 13-1 (620 mg, 1.33 mmol), 2-aminoethanol (0.68 mmol, 42 mg), N,N-diisopropylethylamine (0.18 mL) and anhydrous acetonitrile (10 mL) was heated in a sealed pressure bottle (oil bath 80 ° C) for 1 day. The reactant was cooled and concentrated under reduced pressure. The residue was dissolved in a mixture of hexane / EtOAc / Et3N (80:20:1) and filtered through a short silica gel column (230 to 400 mesh grade silica gel). The column was then eluted with the same solvent mixture. All parts containing the product were combined and concentrated to give a colorless oil (0.395 g), which was used for the next step without further purification.

[0291] Synthesis of compound 13-3

[0292] Thionyl chloride (3eq, 1.88mmol, 223mg, 0.137mL) in 10mL chloroform was added dropwise to an ice-cold solution of 13-2 (462mg, 0.62mmol) in 2mL CHCl in a few minutes. After the addition was completed, the ice bath was removed and the reaction mixture was stirred at room temperature (20°C) for 16 hours. The reactant was concentrated under reduced pressure. The residue was dissolved in a mixture of hexane / EtOAc / Et3N (80:20:1) and filtered through a silica gel pad. The column was then washed with the same solvent mixture. The filtrate was concentrated to obtain a yellow oil (445mg), which can be used in the next step without further purification.

[0293] Synthesis of compound 13-4

[0294] A mixture of 13-3 (222 mg, 0.29 mmol), 4-amino-N, N-dibutylbutyramide (3 eq, 0.88 mmol, 188 mg), N, N-diisopropylethylamine (0.25 mL) and sodium iodide (10 mg) in acetonitrile (10 mL) was sealed and heated at 76 ° C for 24 hours. The resulting mixture was concentrated. The residue was dissolved in a mixture of hexane and ethyl acetate (85:15) and filtered through a silica gel short column (230 to 400 mesh silica gel). The column was then eluted with a mixture of hexane / EtOAc / Et3N (85:15:0 ​​to 70:30:1). All parts containing the desired product were combined and concentrated to give a yellow oil (109 mg, 0.12 mmol, 40%).

[0295] Synthesis of Compound I-8

[0296] Formaldehyde-HCHO solution (250 mg of 37 wt.% solution in water) was added to a solution of 13-4 (109 mg, 0.12 mmol) in THF (5 mL) at room temperature. The resulting mixture was stirred for 30 minutes, and then sodium triacetoxyborohydride (5 eq, 0.6 mmol, 122 mg) was introduced. After stirring overnight, the crude mixture was put into a mixture of hexane and ethyl acetate (about 90:10, 100 mL) and washed with saturated sodium bicarbonate solution. The extract was concentrated to give a yellow oil / solid. The residue was dissolved in a mixture of hexane / EtOAc / Et3N (80:20:1) and filtered through a short silica gel column (230 to 400 mesh silica gel). The column was then eluted with the same solvent mixture. All parts containing the product were combined and concentrated to give a brown oil (100 mg). The product was further purified by silica gel column chromatography (0 to 6% MeOH in DCM; 0 to 5% methanol in chloroform). This gave the desired product as a yellowish oil (30 mg, 0.03 mmol, 26%). 1 H NMR (400MHz, CDCl3@7.26ppm)δ:4.06(t,6.7Hz,4H),3.33-3.26(m,2H),3.25-3.18(m,2H),2.55-2.50(m,2H),2.47-2.35(m,8H),2.35-2.25(m,4H),2.23(s,3H),1.81(quintet,7.4Hz,2H),1.67-1.37(m,20H),1.37-1.08(m,52H),0.97-0.85(m,18H). ESI-MS: molecular weight C 59 H 117 N3O5[M+H] + Calculated value: 948.9; measured value: 949.1.

[0297] Embodiment 15

[0298] Synthesis of ((2-(methyl(4-octanamidobutyl)amino)ethyl)azadiyl)bis(nonane-9,1-diyl)bis(2-butyloctanoate) (Compound I-6)

[0299]

[0300] Synthesis of compounds 14-1, 14-2 and 14-3

[0301] These intermediates were prepared in a similar manner to the preparation of compounds 13-2, 13-3 and 13-4 in Example 14.

[0302] Synthesis of Compound I-6

[0303] To a solution of 14-3 (152 mg, 0.17 mmol) in THF (6 mL) was added formaldehyde-HCHO solution (420 mg of a 37 wt.% solution in water) at room temperature. The resulting mixture was stirred for 30 minutes, and then sodium triacetoxyborohydride (5 eq, 0.6 mmol, 122 mg, MW 211.94) was introduced. After stirring overnight, the crude mixture was placed in a mixture of hexane (60 mL) and washed with a saturated sodium bicarbonate solution. The aqueous phase was extracted with hexane. The combined extracts were filtered through a silica gel short column (230 to 400 mesh silica gel). The column was then eluted with a mixture of hexane / EtOAc / Et3N (80:20:1). All product-containing portions were combined and concentrated to give a brown oil (129 mg). The product was further purified by silica gel column chromatography (0 to 5% methanol solution in chloroform). This gave the desired product as a yellowish oil (76 mg, 0.08 mmol, 49%). 1 H NMR (400MHz, CDCl3@7.26ppm)δ:6.20(br.1H,NHCO),4.06(t,6.7Hz,4H),3.28-3.20(m,2H),2.56-2.51(m,2H),2.48-2.26(m,10H),2.22(s,3H),2.16-2.10(t,2H),1.67-1.55(m,10H),1.55-1.50(m,4H),1.48-1.37(m,8H),1.37-1.12(m,52H),0.90-0.85(m,15H). ESI-MS: molecular weight C 57 H 113 N3O5[M+H] + Calculated value: 920.9; measured value: 921.1.

[0304] Example 16

[0305] 6-((2-((3-(N,N-dihexylcarbamoyl)propyl)-N-methylamino)ethyl)(6-(1-hexylnonylcarbonyloxy)hexyl])amino)hexyl-2-hexyldecanoate (Compound I-53)

[0306]

[0307] Synthesis of compound 16-1

[0308] 6-Chloro-1-hexanol (5.0 g, 4.87 mL, 36.6 mmol, 1 eq), 2-hexyldecanoic acid (14.1 g, 16.1 mL, 54.9 mmol, 1.5 eq) and N,N-dimethyl-4-pyridinamine (DMAP; 2.2 g, 18.3 mmol, 0.5 eq) were dissolved in DCM (40 mL). 1-[(cyclohexylimino)methenamino]cyclohexane (DCC; 8.3 g, 40.3 mmol, 1.1 eq) was added in one portion and the reaction mixture was stirred at room temperature overnight. Afterwards, the reaction mixture was diluted with hexane (200 mL) and the mixture was filtered by passing through a small pad of diatomaceous earth (such as ) to remove the solid. After removing the solvent under vacuum, the residue was purified by automated flash chromatography (220 g SiO2 column; 0 to 15% EtOAc in hexane, eluting the target with 4% to 6% EtOAc in hexane) to give 6-chlorohexyl-2-hexyldecanoate (Compound 16-1; 10.0 g, 73%). ESI-MS: Molecular weight C 22 H 43 ClO2[M+H] + Calculated value: 375.33; measured value: 375.43.

[0309] Synthesis of compound 16-2

[0310] 6-Chlorohexyl-2-hexyldecanoate (compound 16-1; 2.0 g, 5.33 mmol, 2 eq), 2-aminoethanol (195 mg, 3.2 mmol, 1.2 eq), N-ethylbis(isopropyl)amine (DIPEA; 1.4 g, 1.91 mL, 10.9 mmol, 4.1 eq) and potassium iodide (930 mg, 5.6 mmol, 2.1 eq) in acetonitrile (6.4 mL) were placed in a microwave reactor and heated to 140 ° C for 30 minutes. The reaction was carried out three times (3×2.0 g of 6-chlorohexyl-2-hexyldecanoate). After cooling the reaction mixture, it was combined and the acetonitrile was removed under vacuum. The residue was partitioned between EtOAc and water, the organic layer was dried with anhydrous Na2SO4, and the solvent was removed under vacuum. The residue was purified by automated flash chromatography (80 g SiO2 column; 0 to 30% EtOAc in hexanes containing 1% Et3N) to give 6-((6-(1-hexylnonylcarbonyloxy)hexyl)(2-hydroxyethyl)amino)hexyl-2-hexyldecanoate (compound 16-2; 2.5 g, 42%). ESI-MS: molecular weight C 46 H 91 NO5[M+H] + Calculated value: 738.70; measured value: 738.83.

[0311] Synthesis of compound 16-3

[0312] SOCl2 (1.49 g, 908 μL, 12.5 mmol, 3 eq) was added dropwise to an ice-cold solution of 16-2 (3.08 g, 4.17 mmol, 1 eq) in DCM (50 mL). Afterwards, the ice bath was removed and the reaction mixture was stirred at room temperature overnight. DCM and SOCl2 were removed under vacuum and the crude dark red oil was purified by automated flash chromatography (80 g SiO2 column; 0 to 30% EtOAc in hexane, containing 1% Et3N; 8% to 12% EtOAc to elute the target) to give 6-((2-chloroethyl)(6-(1-hexylnonylcarbonyloxy)hexyl)amino)hexyl)-2-hexyldecanoate (compound 16-3; 2.8 g, 89%). ESI-MS: molecular weight C 46 H 90 NClO4[M+H] + Calculated value: 756.66; measured value: 756.80.

[0313]

[0314] Synthesis of compound 16-4

[0315] 4-(tert-Butyloxycarbonylamino)butyric acid (1.0 g, 4.92 mmol, 1 eq), dihexylamine (1.19 g, 1.49 mL, 6.40 mmol, 1.3 eq) and DIPEA (1.27 g, 1.71 mL, 9.84 mmol, 2 eq) were mixed in DCM (15 mL) and HATU (2.25 g, 5.90 mmol, 1.2 eq) was subsequently added. The resulting mixture was stirred at room temperature for 1.5 hours, then diluted with EtOAc (15 mL) and washed with water (10 mL). The aqueous layer was washed with 2×15 mL EtOAc. The combined organic phases were washed with anhydrous N a2 SO4 was dried and the solvent was removed. The resulting light yellow oil was purified by automated flash chromatography (80 g SiO2 column; 0 to 100% EtOAc in hexane, eluting the target with 29% to 39% EtOAc) to give 3-(N,N-dihexylcarbamoyl)propylamino tert-butylcarboxylate (Compound 16-4, 1.49 g, 82%) as a clear light yellow oil. ESI-MS: Molecular weight C 21 H 42 N2O3[M+H] + Calculated value: 371.32; measured value: 371.53.

[0316] Synthesis of compound 16-5

[0317] Compound 16-4 (about 1.49 g, 4.02 mmol, 1 eq) was dissolved in DCM (35 mL), and TFA (12.1 g, 8.16 mL, 107 mmol, 27 eq) was then added. The resulting mixture was stirred at room temperature overnight, and then 10 mL of water was added, followed by solid NaHCO3, until no more gas evolution was observed. Finally, the pH was adjusted to 11 by adding solid K2CO3. The organic layer and the aqueous layer were separated, and the aqueous layer was washed with 3×15 mL EtOAc. The combined organic phases were dried over anhydrous sodium sulfate, and the solvent was removed to give N,N-dihexyl 4-aminobutanamide (compound 16-5: 987 mg, 91%) as a clear yellow oil. ESI-MS: molecular weight C 16 H 34 N2O[M+H] + Calculated value: 271.27; measured value: 271.37.

[0318] Synthesis of compound 16-6

[0319] A mixture of compound 16-3 (400 mg, 529 μmol, 1 eq), compound 16-5 (357 mg, 1.32 mmol, 2.5 eq), potassium iodide (87.8 mg, 529 μmol, 1 eq) and DIPEA (137 mg, 185 μL, 1.06 mmol, 2 eq) in acetonitrile (5 mL) was heated to 140 ° C for 30 minutes in a microwave reactor. The resulting mixture was concentrated and distributed between EtOAc and water, and the aqueous layer was washed with 3×15 mL EtOAc. The combined organic phase was dried over anhydrous Na2SO4 and the solvent was removed. The resulting yellow oil was purified by automated flash chromatography (40 g SiO2 column; 0 to 100% EtOAc in hexanes containing 1% Et3N; eluting the target with 56% to 86% EtOAc and 1% Et3N) to give 6-((2-(3-(N,N-dihexylcarbamoyl)propylamino)ethyl)(6-(1-hexylnonylcarbonyloxy)hexyl)amino)hexyl-2-hexyldecanoate (Compound 16-6; 141 mg, 27%) as a yellow oil. ESI-MS: molecular weight C 62 H 123 N3O5[M+H] + Calculated value: 990.95; measured value: 991.29.

[0320] Synthesis of Compound I-53

[0321] To a solution of compound 16-6 (141 mg, 142 μmol, 1 eq) in methanol (1.0 mL) was added a formaldehyde solution in water (13.3 mol / L, 390 μL, 37 eq). The mixture was stirred at room temperature for 35 minutes, followed by the introduction of sodium triacetoxyborohydride (127 mg, 598 μmol, 4.2 eq), and then further stirred at room temperature for 4 hours. The resulting mixture was diluted with EtOAc and washed with a saturated NaHCO3 solution (10 mL) in water, and the aqueous layer was washed with 3×15 mL EtOAc. The combined organic phases were dried over anhydrous Na2SO4, and the solvent was removed. The resulting oil was purified by automated flash chromatography (25 g SiO2 column; 0 to 100% EtOAc and 1% Et3N in hexanes, containing 1% Et3N; eluting the target with 23% to 44% EtOAc and 1% Et3N) to give 6-((2-((3-(N,N-dihexylcarbamoyl)propyl)-N-methylamino)ethyl)(6-(1-hexylnonylcarbonyloxy)hexyl)amino)hexyl-2-hexyldecanoate (Compound I-53; 93.2 mg, 65%) as a clear, colorless oil. 1H NMR (400 MHz, CDCl3) δ 4.09 (t, J = 6.7 Hz, 4H), 3.36-3.28 (m, 2H), 3.28-3.19 (m, 2H), 2.63-2.29 (m, 13H), 2.26 (s, 3H), 1.85 (q, J = 7.3 Hz, 2H), 1.73-1.19 (m, 87H), 0.92 (dt, J = 10.2, 6.7 Hz, 18H). ESI-MS: molecular weight C 63 H 125 N3O5[M+H] + Calculated value: 1004.97; measured value: 1005.25.

[0322] Embodiment 17

[0323] 6-((2-((3-(N,N-diethylcarbamoyl)propyl)-N-methylamino)ethyl)(6-(1-hexylnonylcarbonyloxy)hexyl)amino)hexyl-2-hexyldecanoate (Compound I-52)

[0324]

[0325] Synthesis of compound 17-6

[0326] 4-(tert-Butyloxycarbonylamino)butyric acid (1.02 g, 5.02 mmol, 1 eq), diethylamine (477 mg, 675 μL, 6.52 mmol, 1.3 eq) and DIPEA (1.3 g, 1.75 mL, 10.0 mmol, 2 eq) were mixed in DCM (15.3 mL) and HATU (2.29 g, 6.02 mmol, 1.02 eq) was then added. The resulting mixture was stirred at room temperature for 1.5 hours, then diluted with EtOAc (15 mL) and washed with water (10 mL). The aqueous layer was washed with 3×15 mL EtOAc. The combined organic phases were washed with anhydrous N a2 SO4 was dried and the solvent was removed. The resulting light yellow oil was purified by automated flash chromatography (80 g SiO2 column; 0 to 100% EtOAc in hexane, eluting the target with 78% to 84% EtOAc) to give 3-(N,N-diethylcarbamoyl)propylamino tert-butyl carboxylate (Compound 17-6; 1.11 g, 85%) as a light yellow oil. ESI-MS: Molecular weight C 13 H 26 N2O3[M+H] + Calculated value: 259.20; measured value: 259.31.

[0327] Synthesis of compound 17-7

[0328] Compound 17-6 (1.11 g, 4.3 mmol, 1 eq) was dissolved in DCM (25.0 mL), and then TFA (9.8 g, 6.58 mL, 85.9 mmol, 20 eq) was added. The resulting mixture was stirred at room temperature overnight, and then 10 mL of water was added, followed by solid NaHCO3, until no more gas evolution was observed. Finally, the pH was adjusted to 11 by the addition of solid K2CO3. The organic and aqueous layers were separated, and the aqueous layer was washed with 3×15 mL EtOAc. The combined organic phases were dried over anhydrous Na2SO4, and the solvent was removed to obtain N,N-diethyl 4-aminobutanamide (compound 17-7; 370 mg, 54%) as a clear light yellow oil. ESI-MS: molecular weight C8H 18 N2O[M+H] + Calculated value: 159.15; measured value: 159.14.

[0329] Synthesis of compound 17-8

[0330] A mixture of compound 16-3 (413 mg, 546 μmol, 1 eq), compound 17-7 (345 mg, 2.18 mmol, 4 eq), potassium iodide (94.5 mg, 569 μmol, 1.04 eq) and DIPEA (139 mg, 188 μL, 1.08 mmol, 1.97 eq) in acetonitrile (3.2 mL) was heated to 140 ° C for 30 minutes in a microwave reactor. The resulting mixture was concentrated and partitioned between EtOAc and water, and the aqueous layer was washed with 3×15 mL EtOAc. The combined organic phases were dried over anhydrous Na2SO4 and the solvent was removed. The resulting yellow oil was purified by automated flash chromatography (40 g SiO2 column; 0 to 100% EtOAc and 1% Et3N in hexanes, containing 1% Et3N, eluting the target with 100% EtOAc and 1% Et3N) to give 6-((2-(3-(N,N-diethylcarbamoyl)propylamino)ethyl)(6-(1-hexylnonylcarbonyloxy)hexyl)amino)hexyl-2-hexyldecanoate (Compound 17-8; 241 mg, 50%) as a brown-orange oil. ESI-MS: molecular weight C 54 H 107 N3O5[M+H] + Calculated value 878.82; measured value 879.11.

[0331] Synthesis of Compound I-52

[0332] To a solution of compound 17-8 (240 mg, 273 μmol, 1 eq) in methanol (4.5 mL) was added a formaldehyde solution in water (13.3 mol / L, 614 μL, 30 eq). The mixture was stirred at room temperature for 30 minutes, followed by the introduction of sodium triacetoxyborohydride (251 mg, 1.18 30 mmol, 4.3 eq) and then stirred at room temperature overnight. The resulting mixture was concentrated, diluted with EtOAc, and washed with a saturated NaHCO3 solution (10 mL) in water, and the aqueous layer was washed with 4×15 mL EtOAc. The combined organic phases were dried over anhydrous Na2SO4, and the solvent was removed. The resulting oil was purified by automated flash chromatography (40 g column; 0 to 100% EtOAc and 1% Et3N in hexanes, containing 1% Et3N; eluting the target with 40% to 44% EtOAc and 1% Et3N) to give 6-((2-((3-(N,N-diethylcarbamoyl)propyl)-N-methylamino)ethyl)(6-(1-hexylnonylcarbonyloxy)hexyl)amino)hexyl-2-hexyldecanoate (Compound I-52; 182 mg, 75%) as a clear light yellow oil. 1 HNMR (400MHz,CDCl3)δ4.05(s,4H),3.48-3.20(m,4H),2.58-2.27(m,18H),2.23(s,3H),1.87-1.21(m,70H),1.16(t,J=7.1Hz,3H),1.10(t,J=7.1Hz,3H),0.92-0.82(m,12H).ESI-MS: molecular weight C 55 H 109 N3O5[M+H] + Calculated value 892.84; measured value 893.10.

[0333] Embodiment 18

[0334] ((4-(((5-(dihexylamino)-5-oxopentyl)(methyl)amino)butyl)azepinediyl)bis((hexane-6,1-diyl)bis(2-hexyldecanoate) (Compound I-47)

[0335]

[0336] Synthesis of compound 18-2

[0337] 6-Chlorohexyl-2-hexyldecanoate (Compound 16-1; 1.2 g, 3.25 mmol, 2 eq), tert-butyl (4-aminobutyl) (methyl) carbamate amine (395 mg, 1.995 mmol, 1.2 eq), N-ethyldi(isopropyl)amine (DIPEA; 862 mg, 1.16 mL, 6.67 mmol, 4.1 eq) and KI (567 mg, 3.42 mmol, 2.1 eq) were mixed in acetonitrile (3.9 mL) and reacted in a microwave reactor (140 ° C, 30 minutes). After cooling, the solvent was removed under vacuum and the residue was distributed between EtOAc and water. The organic layer was dried over anhydrous Na2SO4. After removing the solvent under vacuum, the residue was purified by automated flash chromatography (40 g SiO2 column; 0 to 50% EtOAc in hexane, containing 1% Et3N; eluting the target with 15% to 20% EtOAc) to give (4-((tert-butyloxycarbonyl)(methyl)amino)butyl)azepinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (Compound 18-2; 460 mg, 32%). ESI-MS: molecular weight C 54 H 106 N2O6[M+H] + Calculated value: 879.81; found: 880.08. TLC (MeOH:DCM=1:9) Rf=0.5.

[0338] Synthesis of compound 18-3

[0339] ((4-((tert-butyloxycarbonyl)(methyl)amino)butyl)azepinediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)) (Compound 18-2; 460 mg, 523 μmol, 1 eq) was dissolved in DCM (7.3 mL); TFA (895 mg, 0.6 mL, 7.85 mmol, 15 eq) was added, and the mixture was stirred at room temperature overnight. Afterwards, 10 mL of water was added, and then solid N a HCO3. When no bubbles were observed after adding a fresh amount of NaHCO3, the pH was adjusted to 11 by adding 1 M NaOH. The layers were separated; the DCM layer was dried over anhydrous Na2SO4. The solvent was removed under vacuum to give crude 6-((6-(1-hexylnonylcarbonyloxy)hexyl))(4-(methylamino)butyl)amino)hexyl-2-hexyldecanoate (compound 18-3; 0.4 g, 98%), which was further used as is. ESI-MS: molecular weight C 49 H 98 N2O4[M+H] + Calculated value: 779.76; measured value: 779.98.

[0340]

[0341] Synthesis of compound 18-4

[0342] 5-Bromopentanoic acid (219 mg, 1.21 mmol, 1 eq), dihexylamine (269 mg, 338 μL, 1.45 mmol, 1.2 eq) and DIPEA (313 mg, 423 μL, 2.42 mmol, 2 eq) were dissolved in DCM (11 mL). 1,1,3,3-tetramethyl-2-(3H-1,2,3,4-tetraazaindene-3-yl)-3-isourea hexafluorophosphate (1-) (HATU; 598 mg, 1.57 mmol, 1.3 eq) was added to the solution in one portion. The reaction mixture was stirred at room temperature for 15 minutes. Thereafter, the reaction mixture was washed with saturated aqueous NaHCO3 solution (5 mL). The aqueous layer was extracted with DCM (2×5 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum. The residue was purified by automated flash chromatography (12 g SiO2 column; 0 to 50% EtOAc in hexane, eluting the target with 25% to 30% EtOAc) to give 5-bromo-N,N-dihexylpentanamide (compound 18-4; 290 mg, 68%). ESI-MS: molecular weight C 17 H 34 BrNO[M+H] + Calculated value: 348.19; measured value: 348.30.

[0343] Synthesis of Compound I-47

[0344] Compound 18-3 (290 mg, 373 μmol, 1.3 eq), N, N-dihexyl-5-bromopentanamide (compound 18-4; 99.8 mg, 287 μmol, 1 eq) and DIPEA (74.1 mg, 0.1 mL, 576 μmol, 2 eq) were mixed in acetonitrile (745 μL) and reacted in a microwave reactor (140 ° C, 30 minutes). After cooling, the solvent was removed under vacuum and the residue was distributed between EtOAc and water. The organic layer was dried over anhydrous Na2SO4. After removing the solvent under vacuum, the residue was purified by automated flash chromatography (12 g SiO2 column; 0 to 50% EtOAc in hexanes containing 1% Et3N; eluting the target with 18% to 31% EtOAc) to give ((4-((5-(dihexylamino)-5-oxopentyl)(methyl)amino)butyl)azepinediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (Compound I-47; 99 mg, 33%). 1H NMR (400 MHz, CDCl3) δ 4.06 (t, J = 6.7 Hz, 4H), 3.33-3.24 (m, 2H), 3.24-3.14 (m, 2H), 2.45-2.26 (m, 14H), 2.19 (s, 3H), 1.72-1.47 (m, 19H), 1.47-1.16 (m, 73H), 0.93-0.82 (m, 18H). ESI-MS: molecular weight C 66 H 131 N3O5[M+H] + Calculated value: 1047.01; measured value: 1047.27.

[0345] Embodiment 19

[0346] ((4-( 5-(Didecylamino)-5-oxopentyl ) (methyl) (amino)butyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (Compound I-48)

[0347]

[0348] Synthesis of compound 19-1

[0349] 5-Bromopentanoic acid (200 mg, 1.10 mmol, 1 eq), didecylamine (395 mg, 1.33 mmol, 1.2 eq) and DIPEA (286 mg, 386 μL, 2.21 mmol, 2 eq) were dissolved in DCM (10 mL). HATU (546 mg, 1.44 mmol, 1.3 eq) was added to the solution in one portion. The reaction mixture was stirred at room temperature for 15 minutes. Thereafter, the reaction mixture was washed with saturated aqueous NaHCO3 solution (5 mL). The aqueous layer was extracted with DCM (2×5 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum. The residue was purified by automated flash chromatography (12 g SiO2 column; 0 to 30% EtOAc solution in hexane, eluting the target with 11% EtOAc) to give 5-bromo-N,N-didecylpentanamide (compound 19-1; 380 mg, 75%). ESI-MS: molecular weight C 25 H 50 BrNO[M+H] + Calculated value: 460.31; measured value: 460.42.

[0350] Synthesis of Compound I-48

[0351] Compound 18-3 (290 mg, 373 μmol, 1.3 eq), 5-bromo-N, N-didecylpentanamide (compound 19-1; 132 mg, 287 μmol, 1 eq) and DIPEA (74.1 mg, 0.1 mL, 576 μmol, 2 eq) were mixed in acetonitrile (745 μL) and reacted in a microwave reactor (140 ° C, 30 minutes). After cooling, the solvent was removed under vacuum and the residue was distributed between EtOAc and water. The organic layer was dried over anhydrous Na2SO4. After removing the solvent under vacuum, the residue was purified by automated flash chromatography (12 g SiO2 column; 0 to 50% EtOAc in hexanes containing 1% Et3N; eluting the target with 25% to 31% EtOAc) to give ((4-((5-(didecylamino)-5-oxopentyl)(methyl)amino)butyl)-azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (Compound I-47; 99 mg, 33%). 1 H NMR (400 MHz, CDCl3) δ 4.06 (t, J = 6.7 Hz, 4H), 3.34-3.24 (m, 2H), 3.24-3.15 (m, 2H), 2.41-2.25 (m, 14H), 2.20 (s, 3H), 1.77-1.19 (m, 105H), 0.88 (tt, J = 7.1, 2.3 Hz, 18H). ESI-MS: molecular weight C 74 H 147 N3O5[M+H] + Calculated: 1159.14; measured: 1159.29.

[0352] Embodiment 20

[0353] ((4-((5-(dioctylamino)-5-oxopentyl)(methyl)amino)butyl)azepinediyl)bis(hexane-6,1-diyl)bis

[0354] (2-Hexyldecanoate) (Compound I-51)

[0355]

[0356] Synthesis of Compound 20-1

[0357] 5-Bromopentanoic acid (200 mg, 1.10 mmol, 1 eq), dihexylamine (293 mg, 367 μL, 1.22 mmol, 1.1 eq) and DIPEA (286 mg, 386 μL, 2.21 mmol, 2 eq) were dissolved in DCM (10 mL). HATU (546 mg, 1.44 mmol, 1.3 eq) was added to the solution in one portion. The reaction mixture was stirred at room temperature for 15 minutes. Thereafter, the reaction mixture was washed with saturated aqueous NaHCO3 solution (5 mL). The aqueous layer was extracted with DCM (2×5 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum. The residue was purified by automated flash chromatography (12 g SiO2 column; 0 to 50% EtOAc in hexane, eluting the target with 25% to 32% EtOAc) to give 5-bromo-N,N-dioctylpentanamide (compound 20-1; 325 mg, 72%). ESI-MS: Molecular Weight C 21 H 42 BrNO[M+H] + Calculated value: 404.25; measured value: 404.43.

[0358] Synthesis of Compound I-51

[0359] Compound 18-3 (217 mg, 278 μmol, 1.5 eq), N, N-dioctyl-5-bromopentanamide (compound 20-1; 75.0 mg, 185 μmol, 1 eq) and DIPEA (47.9 mg, 64.8 μL, 371 μmol, 2 eq) were mixed in acetonitrile (556 μL) and reacted in a microwave reactor (140 ° C, 30 minutes). After cooling, the solvent was removed under vacuum and the residue was distributed between EtOAc and water. The organic layer was dried over anhydrous Na2SO4. After removing the solvent under vacuum, the residue was purified by automated flash chromatography (12 g SiO2 column; 0 to 50% EtOAc in hexanes containing 1% Et3N; eluting the target with 34% to 38% EtOAc) to give ((4-(5-(dioctylamino)-5-oxopentyl)(methyl)amino)butyl)azepinediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)] (Compound I-51; 110 mg, 54%). 1 H NMR (400 MHz, CDCl3) δ 4.06 (t, J = 6.7 Hz, 4H), 3.40-3.24 (m, 2H), 3.19 (dd, J = 9.2, 6.4 Hz, 2H), 2.55-2.25 (m, 14H), 2.19 (s, 3H), 1.77-1.11 (m, 98H), 0.95-0.77 (m, 18H). ESI-MS: molecular weight C 70 H139 N3O5[M+H] + Calculated value: 1103.08; measured value: 1103.32.

[0360] Embodiment 21

[0361] Bis(2-hexyldecyl)6,6'-((3-((5-methoxy-5-oxopentyl)(methyl)amino)propyl)azepinediyl)dihexanoate (Compound I-50)

[0362]

[0363] Synthesis of compound 21-1

[0364] At 0 to 5 ° C, a solution of di-tert-butyl dicarbonate (15mmol, 3.27g) in dichloromethane (25mL) was added dropwise to a solution of N-methyl-1,3-diaminopropane (15mmol, 1.32g, 1.57mL) in dichloromethane (25mL). After addition, the temperature was allowed to rise to room temperature. A white precipitate gradually appeared. Stirring was continued at room temperature for 16 hours. The white solid was filtered out and washed with DCM. The filtrate was concentrated to give the desired product as a colorless oil (2.31g; 82%), which was used in the next step without further purification.

[0365] Synthesis of compound 21-2

[0366] A mixture of 2-hexyldecyl-6-bromohexanoate (2eq, 3.56g, 8.5mmol), anhydrous acetonitrile (35mL), N,N-diisopropylethylamine (1.85mL) and compound 21-1 (800mg, 4.25mmol) and sodium iodide (0.1eq, 64mg). In a sealed pressure bottle, the mixture was heated at 75°C for 18 hours. TLC (CHCl3 / MeOH=19:1; CHCl3 / EtOH / H2O / NH4OH=30:25:3:2; hexane / ethyl acetate=9:1) showed that there was still a large amount of starting bromide and no starting amine. Another 430mg of compound 21-1 was added and heating was continued for one day. The reaction mixture was concentrated. The residue was dissolved in hexane (100mL) and filtered through a silica gel pad. The pad was then eluted with a gradient mixture of hexanes, EtOAc and Et3N (95:5:0 to 80:20:1). The desired product was obtained as a brown oil (2.381 g, 65%) and used in the next step without further purification.

[0367] Synthesis of compound 21-3

[0368] To a solution of compound 21-2 (2.381 g, about 2.75 mmol) in CH2Cl2 (15 mL) was added TFA (55 mmol, 4.2 mL). After stirring at room temperature for 16 hours, the reaction mixture was diluted with DCM and washed with a saturated bicarbonate solution. The aqueous phase was extracted with DCM. The combined organic phase was dried over sodium sulfate and concentrated. The residue was dissolved in hexane (100 mL) and filtered through a silica gel pad. Then a gradient mixture of hexane and EtOAc (95:5:0 to 80:20) was used followed by a gradient mixture of DCM, MeOH and Et3N (85:15:1) elution pad. The desired product was obtained as a brown oil (1.14 g, 60%). MS (ESI): m / z 765.93 (M+1).

[0369] Synthesis of Compound I-50

[0370] A mixture of 5-bromovalerate (1eq, 102mg, 0.52mmol), compound 21-3 (1eq., 400mg, 0.52mmol), N,N-diisopropylethylamine (1.5eq, 0.14mL), sodium iodide (0.5eq, 39mg) and anhydrous acetonitrile (10mL). In a sealed pressure bottle, the mixture was heated at 80°C for 18 hours and then concentrated. The residue was dissolved in a mixture of hexane, EtOAc and Et3N (80:20:1) and filtered through a silica gel pad. The pad was then washed with the same solvent mixture. The filtrate was concentrated to give a crude product, which was a brown oil (200mg). The product was further purified by silica gel column chromatography (0 to 5% methanol in chloroform). This gave the desired product, which was a colorless oil (113mg, 0.13mmol, 25%). 1 H NMR(400MHz, CDCl3@7.26ppm)δ:3.96(d,5.8Hz,4H),3.66(s,3H),2.42-2.27(m,16H),2.18(s,3H),1.68 -1.57(m,10H),1.53-1.38(m,6H),1.36-1.18(m,52H),0.92-0.85(m,12H).MS(ESI):m / z(M+1),880.10.

[0371] Embodiment 22

[0372] Bis(2-hexyldecyl)6,6'-((3-((5-(dihexylamino]-5-oxopentyl)(methyl)amino)propyl)azepinediyl)dihexanoate (Compound I-49)

[0373]

[0374] Synthesis of Compound I-49

[0375] 5-bromo-N, N-dihexylpentanamide (1eq, 182mg, 0.52mmol), compound 21-3 (prepared according to the steps of Example 21, 1eq, 400mg, 0.52mmol), N, N-diisopropylethylamine (1.5eq, 0.14mL) and sodium iodide (0.5eq, 39mg) and a mixture of anhydrous acetonitrile (10mL). The mixture was heated at 80°C (oil bath 80°C) for 18 hours in a sealed pressure bottle and then concentrated. The residue was dissolved in a mixture of hexane, EtOAc and Et3N (80:20:1) and filtered through a silica gel pad. The pad was then washed with the same solvent mixture. The filtrate was concentrated to obtain a crude product, which was a brown oil (320mg). The product was further purified by silica gel column chromatography (0 to 5% methanol in chloroform). This gave the desired product as a colorless oil (167.6 mg, 0.16 mmol, 27%). 1 H NMR(400MHz, CDCl3@7.26ppm)δ:3.96(d,5.8Hz,4H),3.31-3.25(m,2H),3.22-3.17(m,2H),2.42-2.27( m,16H),2.19(s,3H),1.68-1.57(m,10H),1.55-1.38(m,10H),1.36-1.20(m,64H),0.92-0.85(m,18H). MS(ESI):m / z 1033.3(M+1).

[0376] The various embodiments described above can be combined to provide further embodiments.All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification and / or listed in the Application Data Sheet are incorporated herein by reference in their entirety.

[0377] If necessary, aspects of the embodiments may be modified to adopt concepts from different patents, applications, and publications to provide further embodiments. These and other changes may be made to the embodiments in light of the detailed description above. In general, in the appended claims, the terms used should not be interpreted as limiting the claims to the specific embodiments disclosed in the specification and claims, but should be understood to include all possible embodiments and the full range of equivalents to which such claims are entitled. Therefore, the claims are not limited by the present disclosure.

[0378] This application claims priority to U.S. Provisional Patent Application No. 63 / 404,463, filed on September 7, 2022, the entire contents of which are hereby incorporated by reference.

Claims

1. A compound having the following structure (I): or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: L 1a and L 1b are each independently an optionally substituted C3-C 12 alkyl; R 1a is -C(=O)OR 4a or -O(C=O)R 4a ; R 1b is -C(=O)OR 4b or -O(C=O)R 4b ; R 2 is - NR 6 (C=O)R 5 、-(C=O)N(R 6 )R 5 or -(C=O)OR 7 ; R 3 and R 6 are each independently hydrogen or an optionally substituted C1-C 12 alkyl; R 4a , R 4b and R 5 are each independently an optionally substituted alkyl group; R 7 is an optionally substituted C1-C6 alkyl group or an optionally substituted aralkyl group; n1 is 2, 3, 4, 5 or 6; and X is C2-C6 alkylene or C4-C 20 Alkylene oxide.

2. The compound of claim 1, wherein X is: or in: n2 is 2, 3, 4, 5 or 6; n3 is 0, 1, 2, 3 or 4; n4 is 2, 3 or 4; and n5 is 2, 3, 4 or 5.

3. The compound according to claim 1 or 2, wherein L 1a It is a C5-C9 alkyl group.

4. The compound according to any one of claims 1 to 3, wherein L 1b It is a C5-C9 alkyl group.

5. The compound according to any one of claims 1 to 4, wherein L 1a It is C5-, C6-, C7- or C9-alkyl.

6. The compound according to any one of claims 1 to 5, wherein L 1b It is C5-, C6-, C7- or C9-alkyl.

7. A compound as described in any one of claims 1 to 6, wherein L 1a is unsubstituted.

8. A compound as described in any one of claims 1 to 7, wherein L 1b is unsubstituted.

9. The compound of any one of claims 1 to 8, wherein L 1a It is unbranched.

10. The compound of any one of claims 1 to 9, wherein L 1b It is unbranched.

11. The compound of any one of claims 1 to 10, wherein R 1a Yes-O(C=O)R 4a .

12. The compound of any one of claims 1 to 10, wherein R 1a is -(C=O)OR 4a .

13. A compound as claimed in any one of claims 1 to 12, wherein R 1b Yes-O(C=O)R 4b .

14. A compound as claimed in any one of claims 1 to 12, wherein R 1b One of them is -(C=O)OR 4b .

15. A compound as claimed in any one of claims 1 to 14, wherein R 4a It is C8-C 24 -alkyl.

16. A compound as claimed in any one of claims 1 to 15, wherein R 4a It is C 11 -C 16 -alkyl.

17. A compound as claimed in any one of claims 1 to 16, wherein R 4a It is C 11 -alkyl.

18. A compound as described in any one of claims 1 to 16, wherein R 4a It is C 15 -alkyl.

19. A compound as described in any one of claims 1 to 16, wherein R 4a It is C 16 -alkyl.

20. A compound as claimed in any one of claims 1 to 19, wherein R 4b It is C8-C 24 -alkyl.

21. A compound as described in any one of claims 1 to 20, wherein R 4b It is C 10 -C 18 -alkyl.

22. A compound as described in any one of claims 1 to 21, wherein R 4b It is C 11 -C 16 -alkyl.

23. A compound as described in any one of claims 1 to 22, wherein R 4b It is C 11 -alkyl.

24. A compound as described in any one of claims 1 to 22, wherein R 4b It is C 15 -alkyl.

25. A compound as described in any one of claims 1 to 22, wherein R 4b It is C 16 -alkyl.

26. A compound as described in any one of claims 1 to 25, wherein R 4a It is a branched chain.

27. A compound as described in any one of claims 1 to 26, wherein R 4b It is a branched chain.

28. A compound as described in any one of claims 1 to 27, wherein R 4a is unsubstituted.

29. A compound as described in any one of claims 1 to 28, wherein R 4b is unsubstituted.

30. A compound as described in any one of claims 1 to 29, wherein R 4a Has one of the following structures: or 31. A compound as described in any one of claims 1 to 30, wherein R 4b Has one of the following structures: or 32. A compound as described in any one of claims 1 to 31, wherein R 2 Yes - NR 6 (C=O)R 5 .

33. A compound as described in any one of claims 1 to 31, wherein R 2 is -(C=O)N(R 6 )R 5 .

34. A compound as described in any one of claims 1 to 33, wherein R 5 It is C2-C 16 -alkyl.

35. A compound as described in any one of claims 1 to 34, wherein R 5 It is C4-C 13 -alkyl.

36. A compound as described in any one of claims 1 to 35, wherein R 5 It is C4-, C7-, C8-, C 10 - or C 13 -alkyl.

37. A compound as described in any one of claims 1 to 36, wherein R 5 is unsubstituted.

38. A compound as described in any one of claims 1 to 36, wherein R 5 is replaced.

39. The compound of claim 38, wherein R 5 Replaced by hydroxyl.

40. A compound as described in any one of claims 1 to 39, wherein R 5 It is a branched chain.

41. A compound as described in any one of claims 1 to 39, wherein R 5 It is unbranched.

42. A compound as described in any one of claims 1 to 41, wherein R 5 Has one of the following structures: or 43. A compound as described in any one of claims 1 to 42, wherein R 6 It is C1-C 10 alkyl.

44. A compound as described in any one of claims 1 to 43, wherein R 6 Is C1-, C2-, C3-, C6-, C8- or C 10 -alkyl.

45. A compound as described in any one of claims 1 to 44, wherein R 6 It is unbranched.

46. ​​A compound as described in any one of claims 1 to 45, wherein R 6 It is methyl, ethyl, n-butyl, n-hexyl, n-octyl or n-decyl.

47. A compound as described in any one of claims 1 to 46, wherein R 6 is unsubstituted.

48. A compound as described in any one of claims 1 to 46, wherein R 6 is replaced.

49. A compound as described in any one of claims 1 to 42, wherein R 6 It is a C1-C6 alkyl group substituted by one or more hydroxy groups.

50. A compound as described in any one of claims 1 to 42, wherein R 6 is C2-, C3-, C4- or C6-alkyl substituted by one or more hydroxy groups.

51. A compound as described in any one of claims 1 to 42, wherein R 6 It's hydrogen.

52. A compound as described in any one of claims 1 to 51, wherein R 2 is -(C=O)OR 7 .

53. The compound of claim 52, wherein R 7 It is C1-C3 alkyl or C7-C 16 Aralkyl.

54. A compound as described in any one of claims 52 or 53, wherein R 7 is unsubstituted.

55. A compound as described in any one of claims 52 to 54, wherein R 7 is -CH3 or has the following structure:

56. A compound as described in any one of claims 1 to 55, wherein R 3 is an optionally substituted C1-C6 alkyl group.

57. A compound as described in any one of claims 1 to 56, wherein R 3 is optionally substituted methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl.

58. A compound as described in any one of claims 1 to 57, wherein R 3 is an optionally substituted methyl group.

59. A compound as described in any one of claims 1 to 56, wherein R 3 It is a C1-C6 alkyl group substituted by one or more hydroxy groups.

60. A compound as described in any one of claims 1 to 56, wherein R 3 is a C2- or C4-alkyl radical substituted by one or more hydroxyl groups.

61. A compound as described in any one of claims 1 to 56, wherein R 3 is unsubstituted.

62. A compound as described in any one of claims 1 to 55, wherein R 3 It's hydrogen.

63. A compound as described in any one of claims 2 to 62, wherein X is 64. A compound as described in any one of claims 2 to 63, wherein the compound has the following structure (II): or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

65. A compound as described in any one of claims 2 to 64, wherein n2 is 3, 4 or 5.

66. A compound as described in any one of claims 2 to 62, wherein X is 67. The compound of claim 66, wherein the compound has the following structure (III): or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

68. A compound as described in any one of claims 66 or 67, wherein n3 is 0 or 1.

69. A compound as described in any one of claims 66 to 68, wherein n4 is 2 or 3.

70. A compound as described in any one of claims 66 to 69, wherein n5 is 3.

71. A compound as described in any one of claims 1 to 70, wherein n1 is 2.

72. The compound of claim 1, wherein the compound has one of the following structures: or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

73. A composition comprising a compound as described in any one of claims 1 to 72 and a therapeutic agent.

74. A lipid nanoparticle comprising a compound as described in any one of claims 1 to 72 and a therapeutic agent.

75. The composition or lipid nanoparticle of claim 73 or 74, further comprising one or more excipients selected from neutral lipids, steroids, and polymer-coupled lipids.

76. A composition or lipid nanoparticle as described in claim 73 or 74, wherein the composition comprises one or more neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.

77. The composition or lipid nanoparticle of claim 76, wherein the neutral lipid is DSPC.

78. The composition or lipid nanoparticle of any one of claims 75 to 77, wherein the molar ratio of the compound to the neutral lipid ranges from about 2:1 to about 8:

1.

79. The composition or lipid nanoparticle of any one of claims 75 to 78, wherein the steroid is cholesterol.

80. The composition or lipid nanoparticle of claim 79, wherein the molar ratio of the compound to cholesterol ranges from about 2:1 to 1:

1.

81. compositions or lipid nanoparticles as described in any one in claims 75 to 80, wherein the polymer-coupled lipid is a PEGylated lipid.

82. The composition or lipid nanoparticle of claim 81, wherein the molar ratio of the compound to the PEGylated lipid ranges from about 100:1 to about 10:1 or from about 100:1 to about 25:

1.

83. The composition or lipid nanoparticle of any one of claims 75 to 82, wherein the PEGylated lipid is PEG-DMG, PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer or PEG dialkoxypropyl carbamate.

84. The lipid nanoparticle of claim 83, wherein the pegylated lipid is PEG-DMG.

85. The composition or lipid nanoparticle of any one of claims 75 to 82, wherein the PEGylated lipid has the following structure (II): or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: R 8 and R 9 each independently is an unbranched or branched alkyl, alkenyl or alkynyl group containing from 10 to 30 carbon atoms, wherein the alkyl, alkenyl or alkynyl group is optionally interrupted by one or more ester bonds; and The mean Z values ​​range from 30 to 60.

86. The composition or lipid nanoparticle of claim 85, wherein R 8 and R 9 Each is independently an unbranched alkyl chain containing 12 to 16 carbon atoms.

87. The composition or lipid nanoparticle of any one of claims 85 or 86, wherein the z-average value is about 45.

88. The composition or lipid nanoparticle of any one of claims 73 to 87, wherein the therapeutic agent comprises a nucleic acid.

89. The composition or lipid nanoparticle of claim 88, wherein the nucleic acid is selected from antisense RNA and messenger RNA.

90. A pharmaceutical composition comprising the lipid nanoparticles of any one of claims 74 to 89 and a pharmaceutically acceptable excipient.

91. A method of administering a therapeutic agent to a patient in need thereof, the method comprising preparing or providing the pharmaceutical composition of claim 90 and administering the composition to the patient.

92. A method of inducing protein expression in a patient in need thereof, comprising administering to the patient the pharmaceutical composition of claim 90, wherein the lipid nanoparticles comprise mRNA encoding the protein.

93. The method of claim 92, wherein the protein is an antigen and the method is used to induce an immune response in the patient.

94. The method of claim 92, wherein the protein is an antigen and the method is used to vaccinate the patient against a pathogen.

95. The method of claim 92, wherein the protein is used for gene editing.

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