Novel lipids and liponanoparticle formulations for delivery of nucleic acids

Abstract

Provided are compounds having the following structure (I) or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof 1 , R 2 , R 3 , L 1 , L 2 , G 1 , G 2 , and G 3 as defined herein. Also provided are uses of the compounds as components of lipid nanoparticle formulations for delivery of therapeutic agents, compositions comprising the compounds, and methods of their use and manufacture.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201680063235.2, filed on October 28, 2016, entitled "Novel Lipids and Lipid Nanoparticle Formulations for Delivery of Nucleic Acids". background Technical Field

[0003] This invention generally relates to novel cationic lipids that can be used to bind with other lipid components (e.g., neutral lipids, cholesterol, and polymer-conjugated lipids) to form lipid nanoparticles with oligonucleotides, thereby facilitating the intracellular delivery of therapeutic nucleic acids (e.g., oligonucleotides, messenger RNA) in vitro and in vivo.

[0004] Related fields description

[0005] Numerous challenges remain regarding the delivery of nucleic acids to influence desired responses in biological systems. Nucleic acid-based therapy holds immense potential, but more efficient delivery of nucleic acids to appropriate sites within cells or organisms is needed to realize this potential. Therapeutic nucleic acids include, for example, messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNases, plasmids, immunostimulatory nucleic acids, antagomir, antimir, mimics, supermir, and aptamers. Some nucleic acids, such as mRNA or plasmids, can be used to achieve the expression of specific cellular products, as they are useful for treating diseases, for example, those related to protein or enzyme deficiencies. The therapeutic applications of translatable nucleotide delivery are extremely broad because constructs can be synthesized to produce any selected protein sequence (whether or not it is inherent to the system). The expression products of these nucleic acids can enhance the presence of proteins, replace missing or non-functional protein forms, or introduce new proteins and associated functions into cells or organisms.

[0006] Some nucleic acids, such as miRNA inhibitors, can be used to achieve the expression of specific cellular products regulated by miRNAs, as they are useful for treating diseases such as those related to protein or enzyme deficiencies. The therapeutic applications of miRNA inhibition are extremely broad because constructs can be synthesized to inhibit one or more miRNAs that thereby regulate the expression of mRNA products. Inhibition of endogenous miRNAs can increase the expression of their downstream target endogenous proteins and restore appropriate function in cells or organisms, serving as a means of treating diseases associated with specific miRNAs or miRNA groups.

[0007] Other nucleic acids can downregulate the intracellular levels of specific mRNAs, and as a result, downregulate the synthesis of the corresponding proteins through processes such as RNA interference (RNAi) or complementary binding of antisense RNA. The therapeutic applications of antisense oligonucleotides and RNAi are also extremely broad, as oligonucleotide constructs can be synthesized using any nucleotide sequence directed against the target mRNA. Targets can include mRNAs from normal cells, mRNAs associated with diseases (e.g., cancer), and mRNAs of infectious agents (e.g., viruses). To date, antisense oligonucleotide constructs have shown the ability to specifically downregulate target proteins through the degradation of homologous mRNAs in in vitro and in vivo models. Furthermore, antisense oligonucleotide constructs are currently being evaluated in clinical investigations.

[0008] However, the use of oligonucleotides in therapeutic settings currently faces two challenges. First, free RNA is readily digested by nucleases in plasma. Second, the ability of free RNA to enter intracellular compartments containing relevant translation mechanisms is limited. Lipid nanoparticles, formed from cationic lipids and other lipid components such as neutral lipids, cholesterol, PEG, PEGylated lipids, and oligonucleotides, have been used to prevent RNA degradation in plasma and promote cellular uptake of oligonucleotides.

[0009] There remains a need for improved cationic lipids and lipid nanoparticles for delivering oligonucleotides. Preferably, these lipid nanoparticles provide an optimized drug-to-lipid ratio, protect nucleic acids from degradation and clearance in serum, are suitable for systemic or local delivery, and provide intracellular delivery of nucleic acids. Furthermore, these lipid-nucleic acid particles should be well-tolerated and provide a sufficient therapeutic index so that patient treatment at an effective dose of nucleic acid is not associated with unacceptable toxicity and / or risk to the patient. The present invention provides these and related advantages.

[0010] Overview

[0011] In summary, the present invention provides lipid compounds, including their stereoisomers, pharmaceutically acceptable salts, or tautomers, which can be used alone or in combination with other lipid components such as neutral lipids, charged lipids, steroids (including, for example, all steroids) and / or their analogues, and / or polymerically conjugated lipids to form lipid nanoparticles for delivering therapeutic agents. In some instances, lipid nanoparticles are used to deliver nucleic acids, such as antisense RNA and / or messenger RNA. Methods for using such lipid nanoparticles to treat various diseases or conditions, such as infectious solids and / or diseases or conditions caused by protein deficiency, are also provided.

[0012] In one embodiment, a compound having the following structure (I) is provided:

[0013]

[0014] Or its drug-acceptable salts, tautomers or stereoisomers, wherein R 1 R 2 R 3 L 1 L 2 G 1 G 2 and G 3 As defined in this article.

[0015] Pharmaceutical compositions comprising one or more of the aforementioned structures (I) and therapeutic agents are also provided. In some embodiments, the pharmaceutical composition further comprises one or more components selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. Such compositions are useful for forming lipid nanoparticles for delivering therapeutic agents.

[0016] In other embodiments, the present invention provides a method for administering a therapeutic agent to a patient in need, the method comprising preparing a composition of lipid nanoparticles comprising a compound containing structure (I) and a therapeutic agent, and delivering the composition to the patient.

[0017] These and other aspects of the invention will become apparent from the following detailed description.

[0018] Brief description of several views in the attached diagram

[0019] In the accompanying drawings, the same reference numerals denote similar elements. The dimensions and relative positions of the elements in the drawings are not necessarily drawn to scale, and some of these elements have been arbitrarily enlarged and placed to improve the readability of the drawings. Moreover, the specific shapes of the drawn elements are not intended to express information about the actual shape of the particular element, and are only selected for ease of identification in the drawings. Figure 1 The time course of luciferase expression in mouse liver is shown.

[0020] Figure 2 The calculation of pKa for MC3, a representative example associated with the disclosed lipids, is illustrated.

[0021] Figure 3 Comparative luciferase activity data for selected lipids are provided. Invention Details

[0022] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the invention. However, those skilled in the art will understand that the invention can be practiced without these details.

[0023] This invention is partly based on the discovery of novel cationic (amino) lipids, which offer advantages when used in lipid nanoparticles for the in vivo delivery of active agents or therapeutic agents (e.g., nucleic acids) to mammalian cells. In particular, embodiments of the invention provide nucleic acid-lipid nanoparticle compositions comprising one or more of the novel cationic lipids described herein, which provide enhanced nucleic acid activity and improved in vivo tolerability of the composition, resulting in a significantly improved therapeutic index compared to previously described nucleic acid-lipid nanoparticle compositions.

[0024] In certain embodiments, the present invention provides novel cationic lipids capable of formulating improved compositions for in vitro and in vivo delivery of mRNA and / or other oligonucleotides. In some embodiments, these improved lipid nanoparticle compositions are used to express proteins encoded by mRNA. In other embodiments, these improved lipid nanoparticle compositions are used to upregulate endogenous protein expression by delivering miRNA inhibitors that target a specific miRNA or a group of miRNAs (which regulate one or more target mRNAs). In other embodiments, these improved lipid nanoparticle compositions are used to downregulate (e.g., silence) protein and / or mRNA levels of target genes. In some other embodiments, the lipid nanoparticles are also used to deliver mRNA and plasmids for transgenic expression. In other embodiments, the lipid nanoparticle compositions are used to induce pharmacological effects resulting from protein expression, such pharmacological effects being, for example, the production of increased red blood cells by delivering suitable erythropoietin mRNA, or the prevention of infection by delivering mRNA encoding suitable antigens or antibodies.

[0025] The lipid nanoparticles and compositions of the present invention can be used for a variety of purposes, including the delivery of encapsulated or associated (e.g., complexed) therapeutic agents such as nucleic acids to cells in vitro and in vivo. Therefore, embodiments of the present invention provide methods for treating or preventing diseases and conditions of a subject by contacting a subject in need with lipid nanoparticles encapsulated with or associated with a suitable therapeutic agent, wherein the lipid nanoparticles comprise one or more novel cationic lipids described herein.

[0026] As described herein, embodiments of the lipid nanoparticles of the present invention are particularly useful for the delivery of nucleic acids, including, for example, mRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomirs / 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 invention 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 or associate nucleic acids that are expressed to produce the desired protein (e.g., messenger RNA or plasmid encoding the desired protein) or expressed to inhibit the process terminating mRNA expression (e.g., miRNA inhibitors). Optionally, the lipid nanoparticles and compositions of the present invention can be used to reduce the expression of target genes and target 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 or associate nucleic acids (e.g., antisense oligonucleotides or small interfering RNA (siRNA)) that reduce the expression of target genes. The lipid nanoparticles and compositions of the present invention can also be used, alone or in combination, to co-deliver different nucleic acids (e.g., mRNA and plasmid DNA), for example, to provide the effect of co-localization of different nucleic acids (e.g., mRNA encoding suitable gene-modifying enzymes and DNA fragments incorporated into the host genome).

[0027] Nucleic acids used in conjunction with this invention can be prepared using any available technology. For mRNA, the primary preparation method is, but is not limited to, enzymatic synthesis (also known as in vitro transcription), which currently represents the most efficient method for producing long-sequence-specific mRNA. In vitro transcription describes a method for template-directed synthesis of RNA molecules using an engineered DNA template containing an upstream phage promoter sequence (e.g., including but not limited to those from T7, T3, and SP6 E. coli phages) linked to a downstream sequence encoding a target gene. 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, 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).

[0028] RNA transcription occurs in vitro using a linearized DNA template, in the presence of appropriate RNA polymerases and rNTPs (adenosine, guanosine, uridine, and cytidine ribonucleoside triphosphates), under conditions that support polymerase activity while minimizing potential degradation of the resulting mRNA transcript. Various commercially available kits and reagents can be used for in vitro transcription, including, but not limited to, the RiboMax large-scale RNA production system (Promega) and the MegaScript transcription kit (Life Technologies), and reagents including RNA polymerases and rNTPs. Methods for in vitro transcription of mRNA are well known in the art. (See, for example, Losick, R., 1972, In vitro transcription, Ann Rev Biochem v. 41409-46; Kamakaka, RT and Kraus, WL 2001. In Vitro Transcription. Current Protocols in Cell Biology. 2:11.6:11.6.1-11.6.17; Beckett, B. and Masquida, B., (2010) Synthesis of RNA by In Vitro Transcription in RNAin Methods in Molecular Biology v. 703 (edited by Neilson, H.), New York, NY Humana Press, 2010; Brunelle, JL and Green, R., 2013, Chapter Five - In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology v. 530, 101-114; all of the above are incorporated herein by reference).

[0029] The desired in vitro transcribed mRNA is then purified from undesirable components of transcription or related reactions, including unincorporated rNTPs, proteases, salts, short RNA oligonucleotides, etc. Techniques for isolating mRNA transcripts are well known in the art. Well-known procedures include phenol / chloroform extraction or precipitation with alcohols (ethanol, isopropanol) or lithium chloride in the presence of monovalent cations. Other non-limiting examples of purification procedures that may be used include size exclusion chromatography (Lukavsky, PJ and Puglisi, JD, 2004, Large-scale preparation and purification of polyacrylamide-free RNAoligonucleotides, RNA v.10, 889-893), silica-based affinity chromatography, and polyacrylamide gel electrophoresis (Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LDI RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and invitro RNA syntheses Methods v.941 Conn GL (ed.), New York, NY Humana Press, 2012). Purification can be performed using a variety of commercially available kits, including, but not limited to, the SV total separation system (Promega) and the in vitro transcription cleaning and concentration kit (Norgen Biotek).

[0030] Furthermore, while reverse transcription can produce large amounts of mRNA, the product can contain numerous aberrant RNA impurities associated with undesirable polymerase activity, which may need to be removed from the full-length mRNA preparation. These impurities include short RNAs resulting from failed transcription initiation, as well as double-stranded RNAs (dsRNAs) generated by RNA-dependent RNA polymerase activity, transcription initiated by RNA from the RNA template, and self-complementary 3' elongation. These contaminants with dsRNA structures have been shown to lead to undesirable immunostimulatory activity by interacting with various innate immune sensors in eukaryotic cells, which function to recognize specific nucleic acid structures and induce potent immune responses. This, in turn, can significantly reduce mRNA translation due to reduced protein synthesis 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 tunable-ratio HPLC purification (see, for example, Kariko, K., Muramatsu, H., Ludwig, J., and Weissman, D., 2011, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA, Nucl Acid Res, v.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, PH editor), 2013). HPLC-purified mRNA has been reported to be translated at much higher levels, particularly in primary cells and in vivo.

[0031] Numerous modifications have been described in this art for altering specific properties of in vitro transcribed mRNA and improving its utility. These modifications include, but are not limited to, modifications to the 5' and 3' ends of mRNA. Endogenous eukaryotic mRNA typically contains a cap structure at the 5' end of mature molecules, which plays a crucial role in mediating the binding of mRNA cap-binding proteins (CBPs), thereby enhancing mRNA stability and translation efficiency in cells. Therefore, capped mRNA transcripts achieve the highest levels of protein expression. The 5' cap contains a 5'-5'-triphosphate linker between a 5'-majority nucleotide and a guanine nucleotide. The conjugated guanine nucleotide is methylated at the N7 position. Additional modifications include methylation of the last and penultimate 5'-majority nucleotides at the 2'-hydroxyl group.

[0032] Several different cap structures can be used to generate the 5'-cap of in vitro transcribed mRNA. The 5'-capping of the synthesized mRNA can be performed co-with transcription using chemical cap analogs (i.e., capping during in vitro transcription). For example, the anti-reverse cap analog (ARCA) contains a 5'-5'-guanine triphosphate-guanine linker, where one guanine contains an N7 methyl group and a 3'-O-methyl group. However, up to 20% of the transcript remains uncapped during this co-transcriptional process, and the synthesized cap analogs do not resemble the 5'-cap structure of real cellular mRNA, potentially reducing translatability and cellular stability. Alternatively, the synthesized mRNA molecule can also be enzymatically capped post-transcriptionally. This can generate a more realistic 5'-cap structure that more structurally or functionally mimics the endogenous 5'-cap, which has enhanced binding to cap-binding proteins, increased half-life, reduced sensitivity to 5' endonucleases, and / or reduced 5' uncapping. Many synthetic 5'-cap analogues have been developed and are known in the field to enhance mRNA stability and translatability (see, for example, Grudzien-Nogalska, E., Kowalska, J., Su, W., Kuhn, AN, Slepenkov, SV, Darynkiewicz, E., Sahin, U., Jemielity, J., and Rhoads, RE, Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PHEd), 2013).

[0033] At the 3' end, a long chain of adenine nucleotides (polyadenylation tail) is typically added to the mRNA molecule during RNA processing. Immediately after transcription, the 3' end of the transcript is cleaved to release the 3' hydroxyl group, and towards the 3' end, polyadenylation polymerase adds the adenine nucleotide chain to the RNA in a process known as polyadenylation. Poly(A) tails have been widely shown to enhance translation efficiency and mRNA stability (see Bernstein, P. and Ross, J., 1989, Poly(A), poly(A)binding protein and the regulation of mRNA stability, Trends Bio Sci v. 14373-377; 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 ineukaryotes, scavenger in bacteria, Cell, v. 111, 611-613).

[0034] Polyadenylation of in vitro transcribed mRNA can be achieved using various methods, including, but not limited to, cloning a poly(T) fragment into a DNA template or adding it post-transcriptionally using a poly(A) polymerase. The first approach allows in vitro transcription of mRNA with a poly(A) tail of a defined length (depending on the size of the poly(T) fragment), but requires additional template manipulation. The latter approach involves using a poly(A) polymerase that catalyzes the incorporation of adenine residues into the 3' end of RNA to enzymatically add the poly(A) tail to in vitro transcribed mRNA without requiring additional DNA template manipulation, resulting in mRNA with poly(A) tails of varying lengths. 5'-capping and 3'-poly(A) tailing can be performed using various commercially available kits and reagents, various ARCA caps, poly(A) polymerases, etc., including, but not limited to, the Poly(A) Polymerase Tailing Kit (EpiCenter), the mMESSAGE mMACHINE T7 Ultra Kit, and the Poly(A) Tailing Kit (LifeTechnologies).

[0035] In addition to 5' caps and 3' polyadenylation, other modifications to 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 eukaryotic cells, triggering a potent innate immune response. Because most naturally derived nucleic acids contain modified nucleosides, the ability to distinguish pathogenic DNA and RNA from self-DNA and RNA has been shown to be at least partially based on structural and nucleoside modifications. Conversely, in vitro synthesized RNA lacks these modifications, thus making it immunostimulatory and consequently inhibiting the efficient mRNA translation outlined above. Introducing modified nucleosides into in vitro transcribed mRNA can be used to block the recognition and activation of RNA sensors, thereby alleviating this undesirable immunostimulatory activity and enhancing translational capacity (see, for example, Kariko, K. and Weissman, D. 2007, Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: implication for therapeutic RNA development, Curr Opin Drug Discov Devel, v.10 523-532; Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology). v.969 (Rabinovich, PH, ed.), 2013; Kariko, K., Muramatsu, H., Welsh, FA, Ludwig, J., Kato, H., Akira, S., Weissman, D., 2008, Incorporation of Pseudouridine Into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability, MolTher v.16, 1833-1840. Modified nucleosides and nucleotides used for the synthesis of modified RNA can be prepared, monitored, and used using methods and procedures generally known in the art.Many types of nucleoside modifications can be used, either alone or in combination with other modified nucleosides, to incorporate to some extent into in vitro transcribed mRNA (see, for example, US2012 / 0251618). It has been reported that the in vitro synthesis of nucleoside-modified mRNA reduces the ability to activate immune sensors, while simultaneously enhancing translational capabilities.

[0036] Other components of mRNA that can be modified to provide benefits in terms of translatability and stability include the 5' and 3' untranslated regions (UTRs). Optimization of either or only the UTR (favorable 5' and 3' UTRs can be obtained from cellular or viral RNA) has been shown to enhance mRNA stability and translation efficiency of in vitro transcribed mRNA (see, e.g., Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH ed.), 2013).

[0037] Besides mRNA, other nucleic acid payloads can also be used in this invention. For oligonucleotides, preparation methods include, but are not limited to, chemical synthesis and enzymatic or chemical cleavage of longer precursors, and in vitro transcription as described above. Methods for synthesizing DNA and RNA nucleotides are widely used and well known in the art (see, for example, Gait, MJ, ed., Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, DC: IRL Press, 1984; and Herdewijn, P., ed., Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, NJ) Totowa, NJ: Humana Press, 2005; both are incorporated herein by reference).

[0038] For plasmid DNA, the preparations used in conjunction with this invention typically involve, but are not limited to, in vitro amplification and isolation of plasmid DNA in a liquid culture medium containing bacteria containing the target plasmid. The presence of genes within the target plasmid encoding resistance to specific antibiotics (penicillin, kanamycin, etc.) allows those bacteria containing the target plasmid to grow selectively in antibiotic-containing media. Methods for isolating plasmid DNA are widely used and well known in the art (see, for example, Heilig, J., Elbing, KL and Brent, R (2001) Large-Scale Preparation of Plasmid DNA. Current Protocols in Molecular Biology. 41:II:1.7:1.7.1-1.7.16; Rozkov, A., Larsson, B., 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 a variety of commercially available kits and reagents, including, but not limited to, Plasmid Plus (Qiagen), GenJET plasmid MaxiPrep (Thermo), and Pure Yield MaxiPrep (Promega) kits.

[0039] Various exemplary embodiments of the present invention, including cationic lipids, lipid nanoparticles, and compositions comprising lipid nanoparticles, as well as their use in delivering active agents such as nucleic acids (e.g., therapeutic agents) to regulate gene and protein expression, are described in further detail below.

[0040] As used herein, unless otherwise specified, the following terms have the meanings assigned to them.

[0041] Unless the context otherwise requires, the word “comprise” and its variations, such as “including” and “containing”, are interpreted in an open and inclusive sense, that is, “including, but not limited to”.

[0042] In this specification, references to "an embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Therefore, the phrases "in one embodiment" or "in an embodiment" appearing throughout this specification do not necessarily refer to the same embodiment. Furthermore, the particular feature, structure, or characteristic can be combined with one or more embodiments in any suitable manner.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. As used in this specification and claims, unless the context clearly indicates otherwise, the singular forms “a”, “an”, and “described” include plural references.

[0044] The phrase "inducing the expression of a desired protein" refers to the ability of nucleic acids to increase the expression of a desired protein. To test the extent of protein expression, a test sample (e.g., a cell sample in a culture medium expressing the desired protein) or a test mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model) is exposed to nucleic acids (e.g., nucleic acids binding to the lipids of the present invention). The expression of the desired protein in the test sample or test animal is compared to the expression of the desired protein in a control sample (e.g., a cell sample in a culture medium 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., a mouse) or a non-human primate (e.g., a monkey) model) that has not been exposed to or treated with nucleic acids. 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 specified as a value of 1.0. In a particular embodiment, the induction of desired protein expression is achieved when the ratio of the desired protein expression level in the test sample or test mammal to that in the control sample or control mammal is greater than 1, for example, about 1.1, 1.5, 2.0, 5.0, or 10.0. The induction of desired protein expression is also achieved when the desired protein is not present in the control sample or control mammal, or when any measurable level of the desired protein is detected in the test sample or test mammal. Those skilled in the art will understand that suitable assays for determining protein expression levels in a sample include, for example, dot blots, northern blots, 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.

[0045] The phrase "inhibits target gene expression" refers to the ability of nucleic acids to silence, reduce, or inhibit the expression of a target gene. To test the degree of gene silencing, a test sample (e.g., a cell sample in a culture medium 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., a mouse) or a non-human primate (e.g., a monkey) model) is exposed to nucleic acids that silence, reduce, or inhibit the expression of the target gene. The expression of the target gene in the test sample or test animal is compared to the expression of the target gene in a control sample (e.g., a cell sample in a culture medium 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., a mouse) or a non-human primate (e.g., a monkey) model) that has not been exposed to or treated with nucleic acids. The expression of the target gene in the control sample or control mammal can be specified as a value of 100%. In a specific embodiment, the expression of a target gene is silenced, suppressed, or reduced when the expression level of the target gene 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 a control sample or control mammal that has not been exposed to or administered nucleic acid. In other words, in the test sample or test mammal, the nucleic acid is able to silence, reduce, or suppress the expression of the target gene 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%. Suitable assays for determining target gene expression levels include, but are not limited to, tests for protein or mRNA levels using techniques known to those skilled in the art, such as dot blot, northern blot, in situ hybridization, ELISA, immunoprecipitation, enzyme function assays, and phenotypic assays.

[0046] An "effective amount" or "therapeutic effective amount" of an active agent or therapeutic agent (e.g., a therapeutic nucleic acid) is an amount sufficient to produce a desired effect, such as an increase or inhibition of target sequence expression compared to the normal expression level of the target sequence 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. An increase in expression is achieved when, prior to contact with nucleic acid, the expression product is present at a certain level, and the fold increase in the value obtained using a nucleic acid such as mRNA relative to the control is approximately 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000, or greater. When the values ​​obtained using nucleic acids such as antisense oligonucleotides are approximately 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% relative to the control, inhibition of target gene or target sequence expression is achieved. Suitable assays for measuring target gene or target sequence expression include, for example, tests of protein or RNA levels using techniques known to those skilled in the art, such as dot blotting, northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of a suitable reporter protein, and phenotypic assays.

[0047] As used herein, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form, and includes DNA, RNA, and their hybrids. DNA can be in the following forms: antisense molecules, plasmid DNA, cDNA, PCR products, or vectors. RNA can be in the following forms: 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 links, said nucleotide analogs or modified backbone residues or links being synthetic, naturally occurring, or non-natural, and having binding properties similar to a reference nucleic acid. Examples of such analogs include, but are not limited to, phosphate thioesters, aminophosphate esters, methylphosphate esters, chiral-methylphosphate esters, 2'-O-methylribonucleotides, and peptide-nucleic acids (PNAs). Unless specifically defined, the term covers nucleic acids containing analogs of known natural nucleotides having binding properties similar to a reference nucleic acid. Unless otherwise stated, a particular nucleic acid sequence also implicitly encompasses variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences, as well as explicitly stated sequences. In particular, degenerate codon substitutions can be achieved by generating sequences in which three of one or more selected (or all) codons are substituted with mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). A “nucleotide” contains a sugar (deoxyribose (DNA) or ribose (RNA)), a base, and a phosphate group. Nucleotides are linked together by phosphate groups. "Base" includes purines and pyrimidines, and further includes natural compounds such as 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 configure new reactive groups, such as, but not limited to, amines, alcohols, thiols, carboxylates, and hydrocarbon halides.

[0048] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that contains a partial or full-length coding sequence necessary to produce a polypeptide or precursor polypeptide.

[0049] As used in this article, “gene product” refers to gene products such as RNA transcripts or polypeptides.

[0050] The term “lipid” refers to a group of organic compounds, including but not limited to esters of fatty acids, and is typically characterized by poor solubility in water but soluble in a wide variety of organic solvents. They are generally classified into at least three categories: (1) “simple lipids”, which include fats and oils as well as waxes; (2) “compound lipids”, which include phospholipids and glycolipids; and (3) “derived lipids”, such as steroids.

[0051] "Steroids" are compounds that contain the following carbon skeleton:

[0052]

[0053] Non-limiting examples of steroids include cholesterol.

[0054] "Cationic lipids" refer to lipids capable of carrying a positive charge. Exemplary cationic lipids include one or more positively charged amine groups. Preferred cationic lipids are ionizable so that they can exist in a positively charged or neutral form depending on the pH value. The ionization of cationic lipids affects the surface charge of lipid nanoparticles under different pH conditions. This charge state can affect plasma protein uptake, blood clearance, and tissue distribution (Semple, SC et al., Adv. DrugDeliv Rev 32:3-17 (1998)) and the ability to form endosomolytic non-bilayer structures (Hafez, IM et al., Gene Ther 8:1188-1196 (2001)), which is crucial for intracellular delivery of nucleic acids.

[0055] The term "polymer-conjugated lipid" refers to a molecule comprising both a lipid moiety and a polymer moiety. An example of a polymer-conjugated lipid is a polyethylene glycol-modified lipid. The term "polyethylene glycol-modified lipid" also refers to a molecule comprising both a lipid moiety and a polyethylene glycol moiety. Polyethylene glycol-modified lipids are known in the art and include, for example, 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG).

[0056] The term "neutral lipid" refers to any of a variety of lipid substances that exist as uncharged or neutral zwitterionic forms at a selected pH. At physiological pH, such lipids include, but are not limited to, phosphatidylcholines such as 1,2-distearyl-sn-glycero-3-phosphate choline (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphate choline (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphate choline (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate choline (POPC), 1,2-dioleoyl-sn-glycero-3-phosphate choline (DOPC), phosphatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphate ethanolamine (DOPE), sphingomyelin (SM), ceramides, steroids such as sterols and their derivatives. Neutral lipids can be synthetic or of natural origin.

[0057] The term "charged lipid" refers to any of a variety of lipid substances that exist in a positively or negatively charged form, independent of pH within a useful physiological range, such as pH ~3 to pH ~9. Charged lipids can be synthetic or of natural origin. Examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyltrimethylammonium propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, and dimethylaminoethanecarbamoylsterol (e.g., DC-Chol).

[0058] The term "lipid nanoparticle" refers to a particle having at least one dimension on the nanometer scale (e.g., 1 nm to 1,000 nm) that comprises one or more compounds of structure (I) or other specific cationic lipids. In some embodiments, the lipid nanoparticles are contained in formulations that can be used to deliver active agents or therapeutic agents, such as nucleic acids (e.g., mRNA), to target sites (e.g., cells, tissues, organs, tumors, etc.). In some embodiments, the lipid nanoparticles of the present invention comprise nucleic acids. Such lipid nanoparticles typically comprise compounds of structure (I) and one or more excipients selected from neutral lipids, charged lipids, cholesterol, and polymer-conjugated lipids. In some embodiments, active agents or therapeutic agents, such as nucleic acids, may be encapsulated in the lipid portion of the lipid nanoparticle, or encapsulated in an aqueous space encapsulated by some or all of the lipid portions of the lipid nanoparticle, thereby protecting them from enzymatic degradation or other undesirable effects induced by mechanisms of the host organism or cells, such as adverse immune responses.

[0059] In various embodiments, the lipid nanoparticles have the following average diameters: about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm 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 some embodiments, when nucleic acids are present in lipid nanoparticles, they resist degradation by nucleases in aqueous solutions. For example, U.S. Patent Publications 2004 / 0142025 and 2007 / 0042031, and PCT Publications WO2013 / 016058 and WO2013 / 086373 disclose lipid nanoparticles containing nucleic acids and methods for their preparation, the entire disclosure of which is incorporated herein by reference for all purposes.

[0060] As used herein, "lipid-encapsulated" refers to lipid nanoparticles that provide complete, partial, or both encapsulation for active agents or therapeutic agents such as nucleic acids (e.g., mRNA). In one embodiment, the nucleic acid (e.g., mRNA) is completely encapsulated within the lipid nanoparticle.

[0061] As used herein, the term "aqueous solution" refers to a composition containing water.

[0062] For nucleic acid-lipid nanoparticles, "serum stable" means that the nucleotides do not significantly degrade after exposure to serum or after exposure to nuclease assays that significantly degrade free DNA or RNA. Suitable assays include, for example, standard serum assays, DNase assays, or RNase assays.

[0063] As used herein, “systemic delivery” refers to the delivery of a therapeutic product that results in widespread exposure of the active agent within a living organism. Some administration techniques may result in systemic delivery of certain agents but not others. Systemic delivery means that a useful, preferably therapeutic, amount of the agent is exposed to most sites of the body. Systemic delivery of lipid nanoparticles can be performed 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 performed via intravenous delivery.

[0064] As used herein, “local delivery” refers to the direct delivery of an active agent to a target site within a living organism. For example, agents can be delivered locally by direct injection into disease sites such as tumors, other target sites such as sites of inflammation, or target organs such as the liver, heart, pancreas, and kidneys. Local delivery can also include local application or injection techniques, such as intramuscular, subcutaneous, or intradermal injection. Local delivery does not preclude systemic pharmacological effects.

[0065] "Hydrocarbon group" refers to a straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, which may be saturated or unsaturated (i.e., containing one or more double bonds (alkenyl) and / or triple bonds (alkynyl)) and has, for example, one to twenty-four carbon atoms (C1-C2). 24 Hydrocarbon group), four to twenty carbon atoms (C4-C) 20 Hydrocarbon group), six to sixteen carbon atoms (C6-C) 16 Hydrocarbon group), six to nine carbon atoms (C6-C9 hydrocarbon group), one to fifteen carbon atoms (C1-C9 hydrocarbon group) 15 Hydrocarbon group), one to twelve carbon atoms (C1-C2) 12 Hydrocarbon groups (one to eight carbon atoms (C1-C8 hydrocarbon groups) or one to six carbon atoms (C1-C6 hydrocarbon groups) are attached to the remainder of the molecule by single bonds, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, vinyl, prop-1-enyl, but-1-enyl, pent-1-enyl, pent-1,4-dienyl, ethynyl, propynyl, butynyl, pentyynyl, hexynyl, etc. Unless otherwise expressly stated in this specification, hydrocarbon groups are optionally substituted.

[0066] "Hydroxy" or "hydronyl chain" refers to a straight or branched divalent hydrocarbon chain in which the remainder of the molecule is attached to a free radical group. It consists only of carbon and hydrogen, and is either saturated or unsaturated (i.e., contains one or more double bonds (alkenyl groups) and / or triple bonds (ynyne groups)), and has, for example, one to twenty-four carbon atoms (C1-C2). 24 (hydrocarbon group), one to fifteen carbon atoms (C1-C) 15 (hydrocarbon group), one to twelve carbon atoms (C1-C) 12The alkylene group (C1-C8 alkylene), consisting of one to eight carbon atoms, one to six carbon atoms (C1-C6 alkylene), two to four carbon atoms (C2-C4 alkylene), or one to two carbon atoms (C1-C2 alkylene), such as methylene, ethylene, propyleneene, n-butylene, vinylene, propenylene, n-butynylene, propynylene, n-butynylene, etc. The alkylene chain is attached to the remainder of the molecule by a single or double bond, and also to a free radical group by a single or double bond. The connection point between the alkylene chain and the remainder of the molecule, and to the free radical group, can be through one carbon atom or any two carbon atoms in the chain. Unless otherwise expressly stated in this specification, the alkylene chain may be optionally substituted.

[0067] "Cycloalkyl" or "carbocyclic" refers to a stable, non-aromatic monocyclic or polycyclic alkyl group consisting only of carbon and hydrogen atoms. It may include fused or bridged ring systems having 3 to 15 carbon atoms, preferably 3 to 10, which may be saturated or unsaturated and are linked to the remainder of the molecule by single bonds. Monocyclic groups include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic groups include, for example, adamantyl, norbornyl, decahydronaphthyl, 7,7-dimethyl-bicyclo[2.2.1]heptyl, etc. Unless otherwise specified in the specification, the cyclic alkyl group may be optionally substituted.

[0068] "Ringed hydrocarbon group" is a divalent cyclic hydrocarbon group. Unless otherwise specified in the specification, the ringed hydrocarbon group may be optionally substituted.

[0069] As used herein, the term "substituted" means any of the aforementioned groups (e.g., hydrocarbon, alkylene, cycloalkyl, or cycloalkylene groups) in which at least one hydrogen atom is substituted by a bond to a non-hydrogen atom, such as, but not limited to: halogen atoms such as F, Cl, Br, or I; oxo groups (=O); hydroxyl groups (-OH); C1-C... 12 Hydrocarbon group; Cyclic hydrocarbon group; -(C=O)OR'; –O(C=O)R'; -C(=O)R'; -OR'; -S(O) x R';-S-SR';-C(=O)SR';-SC(=O)R';-NR'R';-NR'C(=O)R';-C(=O)NR'R';-NR'C(=O)NR'R';-OC(=O)NR'R';-NR'C(=O)OR';-NR'S(O) x NR'R';-NR'S(O) x R'; and -S(O) x NR'R', where R' independently represents H, C1-C each time it appears. 15 The substituent is a hydrocarbon group or a cyclic hydrocarbon group, and x is 0, 1, or 2. In some embodiments, the substituent is C1-C.12 Hydrocarbon group. In other embodiments, the substituent is a cyclic hydrocarbon group. In other embodiments, the substituent is a halogroup, such as a fluorinated group. In other embodiments, the substituent is an oxogroup. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkyloxy group (-OR'). In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group (-NR'R').

[0070] The terms "optional" or "optionally" (e.g., optionally substituted) mean that the event described thereafter may or may not occur, and the description includes instances where the event or condition occurs and instances where the event or condition does not occur. For example, "optionally substituted hydrocarbon group" means that the hydrocarbon group may or may not be substituted, and the description includes substituted hydrocarbon groups and unsubstituted hydrocarbon groups.

[0071] The term "prodrug" is intended to indicate a compound that can be converted into the bioactive compound of the present invention under physiological conditions or by solvation. Therefore, the term "prodrug" refers to a pharmaceutically acceptable metabolic precursor of the compound of the present invention. When administered to a subject in need, the prodrug may be inactive but is converted in vivo into the active compound of the present invention. Prodrugs are typically rapidly converted in vivo to the parent compound of the present invention, for example, by hydrolysis in the blood. The prodrug compound typically offers advantages such as solubility, tissue compatibility, or delayed release in mammalian organisms (see Bundgard, H., Design of Prodrugs (1985), 7-9, 21-24 (Elsevier, Amsterdam). Discussions of prodrugs are provided in Higuchi, T. et al., ACSSymposium Series, Volume 14, and Bioreversible Carriers in Drug Design, eds. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

[0072] The term "prodrug" is also intended to include any covalently bonded carrier that, when administered to a mammalian subject, releases the active compound of the present invention in vivo. Prodrugs of the compounds of the present invention can be prepared by modifying the functional groups present in the compounds of the present invention, in such a way that the modification is performed by conventional procedures or cleaved in vivo to form the parent compound of the present invention. Prodrugs include the following compounds of the present invention: wherein a hydroxyl, amino, or thiol group is bonded to any of the following groups, which, when administered to a mammalian subject, cleave to form a free hydroxyl, free amino, or free thiol group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohols or amides with amine functional groups in the compounds of the present invention.

[0073] The present invention disclosed herein is also intended to cover all pharmaceutically acceptable compounds of structure (I) that are isotopically labeled by substituting one or more atoms into atoms having different atomic weights or mass numbers. Examples of isotopes that may be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, respectively, for example... 2 H, 3 H, 11 C 13 C 14 C 13 N、 15 N、 15 O、 17 O、 18 O、 31 P, 32 P, 35 S, 18 F, 36 Cl、 123 I and 125 I. These radiolabeled compounds can be used to help determine or measure the effects of a compound by characterization, for example, the site or mode of action, or binding affinity to a pharmacologically important site of action. Certain isotope-labeled compounds of structure (I) or (II), such as those incorporating a radioisotope of structure (I) or (II), are useful in studies of drug and / or substrate tissue distribution. Radioisotope tritium (i.e., 3 H) and carbon-14 (i.e., ... 14 C), which are particularly useful for this purpose because they are easy to incorporate and have existing detection methods.

[0074] Using, for example, deuterium (i.e., 2 Heavier isotope substitutions of H can provide certain therapeutic advantages attributable to stronger metabolic stability, such as increased in vivo half-life or reduced dose requirements, and are therefore preferred in some cases.

[0075] Using positron emission of isotopes (e.g.) 11 C 18 F, 15 O and 13 N) substitution can be useful in positron emission tomography (PET) studies used to examine substrate acceptor occupancy. Compounds of structure (I) can typically be prepared using conventional techniques known to those skilled in the art, or by methods similar to those described in the preparations and examples set forth below, using an appropriate isotopically labeled reagent instead of the previously used unlabeled reagent.

[0076] The present invention disclosed herein is also intended to cover in vivo metabolites of the disclosed compounds. Such products can be obtained, for example, from the administered compound, primarily through enzymatic processes such as oxidation, reduction, hydrolysis, amidation, esterification, etc. Therefore, the present invention includes compounds produced by methods comprising administering the compounds of the present invention to mammals for a sufficient time to produce their metabolites. Such products are typically identified by administering a radiolabeled compound of the present invention at a detectable dose to animals such as rats, mice, guinea pigs, monkeys, or to humans for a sufficient duration to allow metabolism to occur, and separating its metabolites from urine, blood, or other biological samples.

[0077] "Stable compound" and "stable structure" are intended to indicate compounds that are stable enough to be isolated from the reaction mixture to useful purity and to be retained in the formulation as an effective therapeutic agent.

[0078] “Mammals” include humans; as well as domesticated animals, such as laboratory animals and domestic pets (e.g., cats, dogs, pigs, cattle, sheep, goats, horses, rabbits), and non-domesticated animals, such as wild animals.

[0079] "Drug-acceptable carriers, diluents, or excipients" include, but are not limited to, any adjuvant, carrier, excipient, gliding agent, sweetener, diluent, preservative, dye / coloring agent, 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 domestic animals.

[0080] "Drug-acceptable salts" include acid addition salts and base addition salts.

[0081] "Pharmaceutical-acceptable acid addition salts" refer to those salts that retain the biological effects and properties of the free base, are not biologically or otherwise undesirable, and form with the following inorganic and organic acids, such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., and such organic acids as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetaminobenzoic acid, camphoric acid, camphor-10-sulfonic acid, decanoic acid, hexanoic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclohexane, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyl... Ethylenesulfonic acid, formic acid, fumaric acid, galactosic acid, gentian acid, glucoheponic acid, gluconic acid, glucuronic acid, glutamic acid, glutamate, 2-oxoglutamate, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, mesylic acid, mucoic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, dihydroxynaphthalic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanate, p-toluenesulfonic acid, trifluoroacetic acid, undecenoic acid, etc.

[0082] "Pharmaceutical-acceptable base addition salts" refer to salts that retain the biological effects and properties of the free acid, and are not biologically or otherwise undesirable. These salts are prepared by the addition of an inorganic or organic base to a free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, the following primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, and salts of basic ion exchange resins: for example, ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, tannin, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benzylamine, benzathine penicillin, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0083] Crystallization typically produces solvates of the compounds of the present invention. As used herein, the term "solvate" refers to an aggregate comprising one or more molecules of the compound of the present invention and one or more solvent molecules. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of the present invention can exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc., and in the corresponding solvated forms. The compounds of the present invention may be true solvates, while in other cases, the compounds of the present invention may retain only adventitious water, or a mixture of water and adventitious solvent.

[0084] "Pharmaceutical composition" refers to a formulation of the compounds of the present invention with a medium generally recognized in the art for delivering bioactive compounds to mammals (e.g., humans). Therefore, such media include all pharmaceutically acceptable carriers, diluents, or excipients.

[0085] "Effective amount" or "therapeutic effective amount" refers to the amount of the compound of the present invention that, when administered to a mammal (preferably a human), is sufficient to achieve a therapeutic effect in the mammal (preferably a human). The amount of the lipid nanoparticles of the present invention constituting a "therapeutic effective amount" will depend on the compound, the condition and its severity, the method of administration, and the age of the mammal to be treated, but can be conventionally determined by those skilled in the art based on their own knowledge and the present disclosure.

[0086] As used herein, “treating” or “treatment” encompasses treatment of the relevant disease or condition in mammals (preferably humans) suffering from the relevant disease or condition, and includes:

[0087] (i) To prevent the occurrence of diseases or conditions in mammals, especially when these mammals are predisposed to the condition but have not yet been diagnosed with it;

[0088] (ii) Suppress the disease or condition, that is, prevent its development;

[0089] (iii) Alleviate the disease or condition, that is, cause the disease or condition to subside; or

[0090] (iv) To alleviate symptoms caused by the disease or condition, i.e., to relieve pain without addressing the underlying disease or condition. As used herein, the terms “disease” and “condition” may be used interchangeably or may be different because a particular disease or condition may not have a known pathogen (and therefore the cause has not yet been identified), and is therefore not yet considered a disease, but only as an undesirable condition or syndrome in which a clinician has identified more or less a particular group of symptoms.

[0091] The compounds of the present invention, or pharmaceutically acceptable salts thereof, may contain one or more asymmetric centers and thus may produce enantiomers, diastereomers, and other stereoisomers, which, for amino acids, may be defined according to absolute stereochemistry as (R)- or (S)-, or (D)- or (L)-. The present invention is intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques such as chromatography and fractional crystallization. Conventional techniques for the preparation / separation of single enantiomers include chiral synthesis from suitable optically pure precursors, or resolution using, for example, chiral high-performance liquid chromatography (HPLC) of racemates (or racemates of salts or derivatives). When the compounds described herein contain alkene double bonds or other geometrically asymmetric centers, unless otherwise specified, it is intended that the compound includes E- and Z-type geometric isomers. Similarly, it is intended to include all tautomerisms.

[0092] "Stereoisomers" refer to compounds composed of identical atoms bonded by the same bonds but with different three-dimensional structures, and they cannot be interconverted. This invention covers various stereoisomers and mixtures thereof, and includes "enantiomers," which are two stereoisomers whose molecules are nonsuperimposeable mirror images of each other.

[0093] "Tautomer" refers to the transfer of a proton from one atom of a molecule to another atom of the same molecule. This invention includes tautomers of any of the compounds described.

[0094] compound

[0095] On the one hand, this invention provides novel lipid compounds that can combine with other lipid components (e.g., neutral lipids, charged lipids, steroids, and / or polymer-conjugated lipids) to form lipid nanoparticles with oligonucleotides. Without being bound by theory, it is envisioned that these lipid nanoparticles protect oligonucleotides from degradation in serum and provide efficient delivery of oligonucleotides to cells both in vitro and in vivo.

[0096] In one embodiment, the compound has the following structure (I):

[0097]

[0098] Or an acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein:

[0099] L 1 or L2 One of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-、-C(=O)NR a -、NR a C(=O)NR a -、-OC(=O)NR a -or-NR a C(=O)O-, and L 1 or L 2 The other two are -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, and -S(O). x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-、-C(=O)NR a -、NR a C(=O)NR a -、-OC(=O)NR a -or

[0100] -NR a C(=O)O- or bond;

[0101] G 1 and G 2 Each is independently unsubstituted C1-C 12 Alkylene or C1-C 12 alkenyl;

[0102] G 3 For C1-C 24 Alkylene, C1-C 24 C3-C8 cycloalkylene, C3-C8 cycloalkylene;

[0103] R a For H or C1-C 12 hydrocarbon group;

[0104] R 1 and R 2 Each independently is C6-C 24 Alkyl or C6-C 24 alkenyl;

[0105] R 3 For H, OR 5 CN, -C(=O)OR 4 -OC(=O)R 4 Or –NR 5 C(=O)R4 ;

[0106] R 4 For C1-C 12 hydrocarbon group;

[0107] R 5 It is an H or C1-C6 hydrocarbon group; and

[0108] x is 0, 1, or 2.

[0109] In some of the foregoing embodiments, the compound has one of the following structures (IA) or (IB):

[0110]

[0111] in:

[0112] A is a 3- to 8-membered ring hydrocarbon group or a subcyclic hydrocarbon group;

[0113] R 6 Each time it appears, it is independently H, OH, or Cl-C. 24 hydrocarbon group;

[0114] n is an integer from 1 to 15.

[0115] In some of the foregoing embodiments, the compound has structure (IA), and in other embodiments, the compound has structure (IB).

[0116] In the other embodiments described above, the compound has one of the following structures (IC) or (ID):

[0117]

[0118] Where y and z are each an independent integer from 1 to 12.

[0119] In any of the foregoing embodiments, L 1 or L 2 One of them is -O (C = O)-. For example, in some implementations, L 1 and L 2 Each of these is -O (C = O)-. In any of the different implementations described above, L 1 and L 2 Each is independently -(C=O)O- or -O(C=O)-. For example, in some implementations, L 1 and L 2 Each of them is -(C=O)O-.

[0120] In some of the different embodiments described above, the compound has one of the following structures (IE) or (IF):

[0121]

[0122] In some of the foregoing embodiments, the compound has one of the following structures: (IG), (IH), (II), or (IJ):

[0123]

[0124] In some of the foregoing embodiments, n is an integer from 2 to 12, such as 2 to 8 or 2 to 4. For example, in some embodiments, n is 3, 4, 5, or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0125] In some other of the aforementioned embodiments, y and z are each independently an integer from 2 to 10. For example, in some embodiments, y and z are each independently an integer from 4 to 9 or from 4 to 6.

[0126] In some of the aforementioned implementation schemes, R 6 For H. In other of the aforementioned embodiments, R 6 For C1-C 24 Hydrocarbon group. In other embodiments, R 6 It is OH.

[0127] In some implementations, G 3 G3 is not replaced. In other embodiments, G3 is replaced. In various different embodiments, G... 3 For straight-chain C1-C 24 Alkylene or straight-chain C1-C 24 Alkenyl group.

[0128] In some other of the aforementioned implementations, R 1 or R 2 Or both are C6-C 24 Alkenyl. For example, in some embodiments, R 1 and R 2 Each of them independently has the following structure:

[0129]

[0130] in:

[0131] R 7a and R 7b Each occurrence is independently H or Cl-C. 12 hydrocarbon group; and

[0132] a is an integer from 2 to 12.

[0133] Where R7a R 7b a and a are each chosen to make R 1 and R 2 Each contains 6 to 20 carbon atoms independently. For example, in some embodiments, a is an integer from 5 to 9 or from 8 to 12.

[0134] In some of the aforementioned implementation schemes, R appears at least once. 7a For example, in some implementations, R is H. 7a H is present each time it occurs. In the other different embodiments described above, R occurs at least once. 7b It is a C1-C8 hydrocarbon group. For example, in some embodiments, the C1-C8 hydrocarbon group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0135] In different implementation schemes, R 1 or R 2 Or both have one of the following structures:

[0136]

[0137] In some of the aforementioned implementation schemes, R 3 For OH, CN, -C(=O)OR 4 -OC(=O)R 4 Or –NHC(=O)R 4 In some implementations, R 4 It can be methyl or ethyl.

[0138] In various embodiments, the compound has one of the structures shown in Table 1 below.

[0139] Table 1

[0140] Representative compounds

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148] It should be understood that any embodiment of the compound of structure (I) as described above, and any specific substituents and / or variables of the compound of 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 invention not expressly set forth above. Furthermore, in cases where a series of substituents and / or variables are listed for any specific R group, L group, G group, A group, or variable a, n, x, y, or z in a particular embodiment and / or claim, it should be understood that each individual substituent and / or variable can be deleted from the specific embodiment and / or claim, and the enumeration of remaining substituents and / or variables will be considered within the scope of the invention.

[0149] It should be understood that, in this specification, combinations of substituents and / or variables of the formulas are permitted only if these contributions result in a stable compound.

[0150] In some embodiments, a composition is provided comprising any one or more compounds of structure (I) and a therapeutic agent. For example, in some embodiments, the composition comprises any one of compounds of structure (I) and a therapeutic agent, as well as one or more excipients selected from neutral lipids, steroids, and polymer-conjugated lipids. Other pharmaceutically acceptable excipients and / or carriers are also included in various embodiments of the composition.

[0151] In some embodiments, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the neutral lipid is DSPC. In various embodiments, the molar ratio of the compound to the neutral lipid is from about 2:1 to about 8:1.

[0152] In various embodiments, the composition further comprises a steroid or a steroid analog. In some embodiments, the steroid or steroid analog is cholesterol. In some of these embodiments, the molar ratio of the compound to cholesterol is from about 5:1 to 1:1.

[0153] In various implementations, the polymer-conjugated lipids are polyethylene glycol-modified lipids. For example, some embodiments include polyethylene glycol-modified diacylglycerols (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), polyethylene glycol-modified phosphatidylethanolamine (PEG-PE), PEG-S-DAG, such as 4-O-(2',3'-di(tetradecyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (salt) (PEG-S-DMG), polyethylene glycol-modified ceramides (PEG-cer), or PEG-dialkoxypropylcarbamates, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecyloxy)propyl)carbamate or 2,3-di(tetradecyloxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of the compound to the polyethylene glycol-modified lipid is from about 100:1 to about 20:1.

[0154] In some embodiments, the composition comprises a polyethylene glycol-modified lipid having the following structure (II):

[0155]

[0156] Or a drug-acceptable salt, tautomer, or stereoisomer thereof, wherein:

[0157] R 8 and R 9 Each is independently a straight-chain or branched, saturated or unsaturated hydrocarbon chain containing 10 to 30 carbon atoms, wherein the hydrocarbon chain is optionally interrupted by one or more ester bonds; and

[0158] w has an average value of 30 to 60.

[0159] In some implementation schemes, R 8 and R 9 Each is independently a straight-chain, saturated hydrocarbon chain containing 12 to 16 carbon atoms. In some embodiments, w has an average value of 43 to 53. In other embodiments, the average value w is about 45. In other different embodiments, the average value w is about 49.

[0160] In some embodiments of the aforementioned compositions, the therapeutic agent comprises a nucleic acid. For example, in some embodiments, the nucleic acid is selected from antisense RNA and messenger RNA.

[0161] In other different embodiments, the present invention relates to a method of administering a therapeutic agent to a patient in need, the method comprising preparing or providing any of the above-described compositions and administering the composition to the patient.

[0162] For application purposes, the compounds of the present invention (typically in the form of lipid nanoparticles combined with a therapeutic agent) can be applied as crude chemicals or formulated as pharmaceutical compositions. The pharmaceutical compositions of the present invention comprise a compound of structure (I) and one or more pharmaceutically acceptable carriers, diluents, or excipients. The compound of structure (I) is present in the composition in an amount that effectively forms lipid nanoparticles and delivers a therapeutic agent, for example, for treating a specific disease or condition. Appropriate concentrations and dosages can be readily determined by those skilled in the art.

[0163] The compositions of the present invention can be administered by any acceptable method of administration of an agent with similar efficacy. The pharmaceutical compositions of the present invention can be formulated into solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalers, gels, microspheres, and aerosols. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, oral, rectal, vaginal, and intranasal routes. The term parenteral, as used herein, includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection, or infusion techniques. The pharmaceutical compositions of the present invention are formulated to allow the active ingredient contained therein to be bioavailable after administration to a patient. The composition to be administered to a subject or patient is in the form of one or more dose units, wherein, for example, tablets may be single dose units, while containers of the compounds of the present invention in aerosol form may contain multiple dose units. Current methods for preparing these dosage forms are known or will be obvious to those skilled in the art; see, for example, Remington: The Science and Practice of Pharmacy, 20th edition (Philadelphia College of Pharmacy and Science, 2000). In any case, the composition to be administered will contain a therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof, in order to treat the relevant disease or condition according to the teachings of the present invention.

[0164] The pharmaceutical compositions of the present invention can be in solid or liquid form. On one hand, the carrier is a microparticle, such that the composition is, for example, a tablet or powder. The carrier can be a liquid, in which case the composition is, for example, an oral syrup, an injectable liquid, or an aerosol suitable for, for example, inhalation administration.

[0165] When intended for oral administration, the pharmaceutical composition is preferably in solid or liquid form, wherein the solid or liquid form is considered herein to include semi-solid, semi-liquid, suspension and gel forms.

[0166] As solid compositions intended for oral administration, pharmaceutical compositions can be formulated into powders, granules, compressed tablets, pills, capsules, chewing gum, sheets, etc. These solid compositions typically contain one or more inert diluents or edible carriers. Additionally, one or more of the following may be present: binders, such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, tragacanth gum, or gelatin; excipients, such as starch, lactose, or dextrin; disintegrants, such as alginate, sodium alginate, Primogel, corn starch, etc.; lubricants, such as magnesium stearate or Sterotex; flow aids, such as colloidal silica; sweeteners, such as sucrose or saccharin; flavoring agents, such as peppermint, methyl salicylate, or orange flavorings; and coloring agents.

[0167] When the pharmaceutical composition is in the form of a capsule (e.g., a gelatin capsule), it may contain a liquid carrier other than the materials described above, such as polyethylene glycol or oil.

[0168] The pharmaceutical composition may be in liquid form, such as an elixir, syrup, solution, emulsion, or suspension. As two examples, the liquid may be for oral administration or for injection delivery. When intended for oral administration, the preferred composition contains one or more of the following, in addition to the compounds of the present invention: a sweetener, a preservative, a coloring / staining agent, and a flavor enhancer. In compositions administered by injection, one or more of the following may be included: a surfactant, a preservative, a wetting agent, a dispersant, a suspending agent, a buffer, a stabilizer, and an isotonic agent.

[0169] The liquid pharmaceutical compositions of the present invention, whether in the form of a 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; non-volatile oils, such as synthetic mono- or diglycerides that can be used as solvents or suspension media, polyethylene glycol, glycerol, propylene glycol, or other solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates; and agents for regulating tension, such as sodium chloride or glucose; and agents used as cryoprotectants, such as sucrose or trehalose. Parenteral preparations may be packaged in ampoules made of glass or plastic, disposable syringes, or multi-dose vials. Physiological saline is a preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

[0170] Liquid pharmaceutical compositions of the present invention intended for parenteral or oral administration should contain an amount of the compound of the present invention to obtain a suitable dose.

[0171] The pharmaceutical compositions of the present invention are intended for topical application, in which case the carrier may suitably comprise a solution matrix, emulsion matrix, ointment matrix, or gel matrix. For example, the matrix may comprise one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickeners may be present in the pharmaceutical compositions for topical application. If intended for transdermal application, the composition may comprise a transdermal patch or an iontophoresis device.

[0172] The pharmaceutical compositions of the present invention are intended for rectal administration, for example, in the form of suppositories, which dissolve and release the drug in the rectum. Compositions for rectal administration may contain an oily matrix as a suitable, non-irritating excipient. Such matrices include, but are not limited to, lanolin, cocoa butter, and polyethylene glycol.

[0173] The pharmaceutical compositions of the present invention may include a variety of materials that modify the physical form of solid or liquid dosage units. For example, the composition may include a material that forms a coating shell around the active ingredient. The material forming the coating shell is generally inert and may be selected from, for example, sugars, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule.

[0174] Pharmaceutical compositions of the present invention in solid or liquid form may include agents that bind to and thereby facilitate the delivery of the compounds of the present invention. Suitable agents that can act with this ability include monoclonal or polyclonal antibodies or proteins.

[0175] The pharmaceutical compositions of the present invention can consist of dosage units that can be administered as aerosols. The term aerosol is used to refer to a variety of systems, ranging from colloidal systems to systems consisting of pressurized packaging. Delivery can be made by liquefied or compressed gas, or by a suitable pump system for dispersing the active ingredient. Aerosols of the compounds of the present invention can be delivered in single-phase, two-phase, or three-phase systems to deliver the active ingredient. Aerosol delivery includes necessary containers, activators, valves, sub-containers, etc., which together can form a kit. Preferred aerosols can be determined by those skilled in the art without extensive experimentation.

[0176] The pharmaceutical compositions of the present invention can be prepared by methods well known in the pharmaceutical industry. For example, pharmaceutical compositions intended for injection can be prepared by conjugating the lipid nanoparticles of the present invention with sterile, distilled water or other carriers to form a solution. Surfactants can be added to promote the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the compounds of the present invention to promote the dissolution or homogeneous suspension of said compounds in an aqueous delivery system.

[0177] The compositions of the present invention or pharmaceutically acceptable salts thereof are administered in therapeutically effective amounts, said amounts which will vary depending on a variety of factors, including the activity of the specific therapeutic agent used; the metabolic stability and duration of action of the therapeutic agent; the patient's age, weight, general health condition, sex, and diet; the manner and timing of administration; the rate of excretion; the combination of drugs; the severity of the specific condition or disease; and the subject of treatment.

[0178] The compositions of the present invention can also be administered concurrently with, before, or after the administration of one or more other therapeutic agents. Such combination therapies include single-dose formulations of the composition of the present invention and one or more additional active agents, as well as formulations of the composition of the present invention and each active agent in its own separate pharmaceutical dosage form. For example, the composition of the present invention and other active agents can be administered to a patient together as a single oral dosage composition (e.g., tablets or capsules), or the individual agents can be administered in different oral dosage forms. When using different dosage forms, the compounds of the present invention and one or more additional active agents can be administered substantially at the same time (i.e., simultaneously) or at staggered times (i.e., sequentially); it should be understood that combination therapies include all of these administration regimens.

[0179] The methods for preparing the above compounds and compositions are described below and / or are known in the art.

[0180] Those skilled in the art will recognize that, in the methods described herein, the functional groups of the intermediate compounds may require protection by suitable protecting groups. Such functional groups include hydroxyl, amino, mercapto, and carboxylic acids. Suitable protecting groups for hydroxyl groups include trialkylsilyl or diarylalkylsilyl (e.g., tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or trimethylsilyl), tetrahydropyranyl, benzyl, etc. Suitable protecting groups for amino, amidine, and guanidine groups include tert-butoxycarbonyl, benzyloxycarbonyl, etc. Suitable protecting groups for mercapto groups include -C(O)-R (where R is a hydrocarbon, aryl, or aromatic group), p-methoxybenzyl, triphenylmethyl, etc. Suitable protecting groups for carboxylic acids include hydrocarbon, aryl, or aromatic esters. Protecting groups may 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, TW and PGMUTZ, Protective Groups in Organic Synthesis (1999), 3rd edition, Wiley. As those skilled in the art will recognize, protecting groups can also be polymeric resins, such as Wang resin, Rink resin or 2-chlorotriphenylmethyl-chloro resin.

[0181] Those skilled in the art will also recognize that while such protected derivatives of the compounds of the present invention may not thereby possess pharmacological activity, they can be administered to mammals and subsequently metabolized in vivo to form the pharmacologically active compounds of the present invention. Such derivatives can therefore be described as "prodrugs." All prodrugs of the compounds of the present invention are included within the scope of this invention.

[0182] Furthermore, all compounds of the present invention existing in the form of free bases or free acids can be converted into pharmaceutically acceptable salts by treatment with suitable inorganic or organic bases or acids according to methods known to those skilled in the art. Salts of the compounds of the present invention can be converted into their free base or acid forms using standard techniques.

[0183] The following general reaction scheme 1 exemplarily illustrates a method for preparing the compounds of the present invention, namely compounds of structure (I).

[0184]

[0185] Or its drug-acceptable salts, tautomers or stereoisomers, wherein R 1 R 2 R 3 L 1 L 2 G 1 G 2 and G 3 As defined herein. It should be understood that those skilled in the art can prepare these compounds by similar methods or by combining other methods known to them. It should also be understood that those skilled in the art can prepare other compounds of structure (I) not explicitly described below in a similar manner as described below, by using appropriate starting components and modifying the synthesis parameters as needed. Typically, starting components may be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, or synthesized from sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Edition (Wiley, December 2000)), or prepared as described herein.

[0186] General Reaction Scheme 1

[0187]

[0188] Universal Reaction Scheme I provides an exemplary method for preparing compounds of structure (I). G in the universal reaction scheme... 1 G 3 R 1 and R 3 As defined herein, and G1' refers to a shorter homologue of one carbon atom of G1. A compound of structure A-1 is purchased, or a compound of structure A-1 is prepared according to methods known in the art. The reaction of A-1 with diol A-2 under suitable condensation conditions (e.g., DCC) yields ester / alcohol A-3, which can then be oxidized (e.g., PCC) to aldehyde A-4. The reaction of A-4 with amine A-4 under reductive amination conditions yields a compound of structure (I).

[0189] It should be noted that various alternative strategies for preparing compounds of structure (I) are available to those skilled in the art. For example, compounds containing L can be prepared using suitable raw materials according to similar methods. 1 and L 2 Unlike esters, other compounds with structure (I) are present. Furthermore, General Reaction Scheme 1 describes the preparation of compounds with structure (I), wherein G... 1 and G 2 The same; however, this is not a necessary aspect of the invention, and modifications to the above reaction scheme can produce a result in G. 1 and G 2 These are different compounds. The use of protecting groups and other modifications to the general reaction scheme described above, as needed, will be readily apparent to those skilled in the art.

[0190] The following embodiments are provided for illustrative purposes and are not intended to be limiting.

[0191] Example 1

[0192] In vivo evaluation of luciferase mRNA using lipid nanoparticle compositions: Cationic lipids, DSPC, cholesterol, and PEG-lipids were dissolved in ethanol at molar ratios of 50:10:38.5:1.5 or 47.5:10:40.8:1.7. Lipid nanoparticles (LNPs) were prepared at a total lipid to mRNA weight ratio of approximately 10:1 to 30:1. Briefly, mRNA was diluted to 0.2 mg / mL in 10 mM to 50 mM citrate buffer (pH 4). Using a syringe pump, the ethanol solution of lipids was mixed with the aqueous mRNA solution at a ratio of approximately 1:5 to 1:3 (volume / volume) at a total flow rate of at least 15 mL / min. The ethanol was then removed, and PBS was used instead of external buffer for dialysis. Finally, the lipid nanoparticles were filtered through a sterile filter with 0.2 μm pores. The particle size of the lipid nanoparticles, determined by quasi-elastic light scattering using Malvern Zetasizer Nano ZS (Malvern, UK), was approximately 55-95 nm in diameter, and in some cases, approximately 70-90 nm.

[0193] The study was conducted in 6-8 week old female C57BL / 6 mice (Charles River) and 8-10 week old CD-1 (Harlan) mice (Charles River), following guidelines established by the Laboratory Animal Control Committee (ACC) and the Canadian Council for Animal Control (CCAC). Different doses of mRNA-lipid nanoparticles were administered systemically via tail vein injection, and animals were euthanized at specific time points post-administration (e.g., 4 hours). Liver and spleen were collected in pre-weighed tubes, their weight determined, and immediately rapidly frozen in liquid nitrogen and stored at -80°C until analysis. For liver, approximately 50 mg was cut for analysis in 2 mL FastPrep tubes (MP Biomedicals, Solon OH). Add 1 / 4” ceramic balls (MP Biomedicals) to each tube and add 500 μL of Glo lysis buffer-GLB (Promega, Madison WI) equilibrated to room temperature to the liver tissue. Homogenize the liver tissue at 2 x 6.0 m / s for 15 seconds using a FastPrep24 instrument (MP Biomedicals). Incubate the homogenate at room temperature for 5 minutes, then dilute 1:4 in GLB and evaluate using the SteadyGlo luciferase assay system (Promega). Specifically, react 50 μL of the diluted tissue homogenate with 50 μL of SteadyGlo substrate, shake for 10 seconds, incubate for 5 minutes, and then quantify using a CentroXS3 LB 960 spectrophotometer (Berthold Technologies, Germany). Quantification was performed using a BCA protein assay kit (Pierce, Rockford). The amount of protein measured was determined using IL (in luciferase). The relative luminescence unit (RLU) was then normalized to the total μg of the measured protein. To convert RLU to ng luciferase, a standard curve was generated using QuantiLum recombinant luciferase (Promega). Figure 1 The data provided selected a four-hour time point for evaluating the efficacy of lipid formulations.

[0194] FLuc mRNA (L-6107) from Trilink Biotechnologies expresses the luciferase protein, originally isolated from the firefly (Photinus pyralis). Fluc is commonly used in mammalian cell cultures to measure gene expression and cell viability. It emits bioluminescence in the presence of its substrate, luciferin. This capped and polyadenylated mRNA is completely replaced by 5-methylcytidine and pseudouridine.

[0195] Example 2

[0196] The pK of the prepared lipids A As described elsewhere in this paper, the pKa of the formulated cationic lipids was correlated with their effectiveness for delivering nucleic acids using LNPs (see Jayaraman et al., Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semil et al., Nature Biotechnology 28, 172-176 (2010)). The preferred pKa range was ~5 to ~7. The pKa of each cationic lipid in the lipid nanoparticles was determined using fluorescence analysis based on 2-(p-toluidine)-6-naphthalenesulfonic acid (TNS). a As described in Example 1, lipid nanoparticles containing cationic lipids / DSPC / cholesterol / PEG-lipids (50 / 10 / 38.5 / 1.5 mol%) at a total lipid concentration of 0.4 mM in PBS were prepared using an ordered method. TNS was prepared into a 100 μM stock solution in distilled water. The vesicles were diluted to a 2 mL buffer solution containing 24 μM lipids, which contained 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl, with a pH of 2.5 to 11. Equal portions of TNS solution were added to produce a final concentration of 1 μM, and after vortex mixing, fluorescence intensity was measured at room temperature using an SLM Aminco Series 2 fluorescence spectrophotometer with excitation and emission wavelengths of 321 nm and 445 nm. S-shaped best-fit analysis was applied to the fluorescence data, and pK... a The pH value at which half of the maximum fluorescence intensity is produced is measured (see [reference]). Figure 2 ).

[0197] Example 3

[0198] Prior to determining the efficacy of lipid nanoparticle formulations containing various cationic lipids using a rodent model of in vivo luciferase mRNA expression, the cationic lipids shown in Table 2 were tested with nucleic acids. For comparative purposes, these lipids were also used to formulate lipid nanoparticles containing FLuc mRNA (L-6107) using an in-line mixing method, as described in Example 1 and PCT / US10 / 22614 (which are incorporated herein by reference in their entirety). The lipid nanoparticles were formulated using the following molar ratio: 50% cationic lipid / 10% distearate phosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid (“PEG-DMG”, i.e., (1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol, with an average PEG molecular weight of 2000). Relative activity was determined by measuring luciferase expression in the liver 4 hours after administration via tail vein injection, as described in Example 1. The activity was compared at doses of 0.3 and 1.0 mg mRNA / kg, expressed as ng luciferase / g liver as measured 4 hours after administration as described in Example 1.

[0199] Table 2

[0200] Lipids compared to mRNA exhibiting activity

[0201]

[0202]

[0203] The representative compounds of the present invention shown in Table 3 were formulated using the following molar ratios: A) 50% cationic lipid / 10% distearate phosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid (“PEG-DMA” 2-[2-(ω-methoxy(polyethylene glycol) 2000 [Ethoxy]-N,N-bistetradecylacetamide) or B) 47.5% cationic lipid / 10% DSPC / 40.8% cholesterol / 1.7% PEG lipid. As described in Example 1, relative activity was determined by measuring luciferase expression in the liver 4 hours after administration via tail vein injection. The activity was compared at doses of 0.3 and 1.0 mg mRNA / kg, and expressed as ng luciferase / g liver as measured 4 hours after administration as described in Example 1. Figure 3 The graph of the selected data is shown (from top to bottom: triangle = compound 3; circle = compound 2; cross = compound 1; square = MC3).

[0204] Table 3

[0205] Novel cationic lipids and related activities

[0206]

[0207]

[0208]

[0209]

[0210] Example 4

[0211] Synthesis of 6-(2'-hexyldecanoyloxy)hex-1-aldehyde

[0212] A solution of hexyl-1,6-diol (27.6 g) in dichloromethane (475 mL) was treated with 2-hexyldecanoic acid (19.8 g), DCC (18.2 g), and DMAP (11.3 g). The solution was stirred for three days. The reaction mixture was filtered, and hexane (500 mL) was added to the filtrate. The mixture was stirred, and the precipitate was precipitated. The supernatant was decanted and washed with dilute hydrochloric acid. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed to yield 30 g of crude product.

[0213] The crude product was dissolved in dichloromethane (200 mL) and treated with pyridinium chlorochromate (15 g) for two hours. Diethyl ether (600 mL) was added, and the supernatant was filtered through a silica gel bed. The solvent was removed from the filtrate, and the resulting oil was dissolved in hexane. The suspension was filtered through a silica gel stopper to remove the solvent. The residue was passed through a silica gel column (80 g) using hexane followed by dichloromethane as the eluent. 24 g of 6-(2'-hexyldecanoyloxy)hex-1-aldehyde was obtained as a colorless oil.

[0214] Example 5

[0215] Synthesis of 4-(2'-hexyldecanoyloxy)but-1-aldehyde

[0216] A solution of butane (12.5 g) in 200 mL of dichloromethane was treated with 2-hexyldecanoic acid (9.2 g), DCC (8.8 g), and DMAP (4.9 g). The solution was stirred overnight. The reaction mixture was filtered to remove the solvent. The residue was dissolved in dichloromethane and washed with dilute hydrochloric acid. The organic phase was dried over anhydrous magnesium sulfate, filtered through a silica gel bed, and the solvent was removed.

[0217] The crude product was dissolved in dichloromethane (150 mL) and treated with pyridinium chlorochromate (6 g) for one hour. Diethyl ether (450 mL) was added, and the supernatant was filtered through a silica gel bed. The solvent was removed from the filtrate, and the resulting oil was dissolved in hexane. The suspension was filtered through a silica gel bed to remove the solvent, yielding 11 g of 4-(2'-hexyldecanoyloxy)but-1-aldehyde as a colorless oil.

[0218] Example 6

[0219] Synthesis of Compound 1

[0220] A solution of 6-(2'-hexyldecanoyloxy)hex-1-aldehyde (3.0 g), acetic acid (0.21 g), and ethanolamine (0.14 g) in dichloromethane (50 mL) was treated overnight with sodium triacetoxyborohydride (1.4 g). The solution was washed with a dilute aqueous sodium hydroxide solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a methanol / dichloromethane (0–8 / 100–92%) gradient to produce compound 1 (0.63 g) as a colorless oil.

[0221] Example 7

[0222] Synthesis of Compound 2

[0223] A solution of 6-(2'-hexyldecanoyloxy)hex-1-aldehyde (3.0 g), acetic acid (0.33 g), and 3-aminoprop-1-ol (0.17 g) in dichloromethane (20 mL) was treated with sodium triacetoxyborohydride (1.3 g) for one hour. The solution was washed with a dilute aqueous sodium hydroxide solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a methanol / dichloromethane (0-8 / 100-92%) gradient to produce compound 2 (1.1 g) as a colorless oil.

[0224] Example 8

[0225] Synthesis of Compound 3

[0226] A solution of 6-(2'-hexyldecanoyloxy)hex-1-aldehyde (2.4 g), acetic acid (0.33 g), and 4-aminobut-1-ol (0.23 g) in dichloromethane (20 mL) was treated with sodium triacetoxyborohydride (1.3 g) for two hours. The solution was washed with an aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a methanol / dichloromethane (0–8 / 100–92%) gradient to produce compound 3 (0.4 g) as a colorless oil.

[0227] Example 9

[0228] Synthesis of Compound 4

[0229] A solution of 2.4 g of 4-(2'-hexyldecanoyloxy)but-1-aldehyde, 0.30 g of acetic acid, and 0.22 g of 4-aminobut-1-ol in dichloromethane (20 mL) was treated with sodium triacetoxyborohydride (1.3 g) for two hours. The solution was washed with dilute aqueous sodium hydroxide solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a methanol / dichloromethane (0-8 / 100-92%) gradient. A portion of the purified fraction was passed through a second column using an acetic acid / methanol / dichloromethane (2-0 / 0-10 / 98-90%) gradient. The purified fraction was washed with aqueous sodium bicarbonate solution to give compound 4 (0.9 g) as a colorless oil.

[0230] Example 10

[0231] Synthesis of Compound 5

[0232] A solution of 4-(2'-hexyldecanoyloxy)but-1-aldehyde (2.4 g), acetic acid (0.31 g), and 3-aminoprop-1-ol (0.17 g) in dichloromethane (20 mL) was treated with sodium triacetoxyborohydride (1.4 g) for one hour. The solution was washed with an aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a methanol / dichloromethane (0-8 / 100-92%) gradient. A portion of the purified fraction was passed through a second column using an acetic acid / methanol / dichloromethane (2-0 / 0-8 / 98-92%) gradient. The purified fraction was washed with an aqueous sodium bicarbonate solution to give compound 5 (0.57 g) as a colorless oil.

[0233] Example 11

[0234] Synthesis of Compound 6

[0235] A solution of 4-(2'-hexyldecanoyloxy)but-1-aldehyde (2.4 g), acetic acid (0.30 g), and ethanolamine (0.14 g) in dichloromethane (20 mL) was treated with sodium triacetoxyborohydride (1.3 g) for two hours. The solution was washed with an aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a methanol / dichloromethane (0-10 / 100-90%) gradient. A portion of the purified fraction was passed through a second column using an acetic acid / methanol / dichloromethane (2-0 / 0-9 / 98-92%) gradient. The purified fraction was washed with an aqueous sodium bicarbonate solution to yield compound 6 (0.2 g) as a colorless oil.

[0236] Example 12

[0237] Synthesis of Compound 7

[0238] A solution of 6-(2'-hexyldecanoyloxy)hex-1-aldehyde (2.4 g), acetic acid (0.14 g), and 5-aminopentan-1-ol (0.24 g) in dichloromethane (20 mL) was treated with sodium triacetoxyborohydride (1.3 g) for two hours. The solution was washed with an aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a methanol / dichloromethane (0–8 / 100–92%) gradient to produce compound 7 (0.5 g) as a colorless oil.

[0239] Example 13

[0240] Synthesis of Compound 8

[0241] A solution of 20 mL of dichloromethane containing 2.4 g of 6-(2'-hexyldecanoyloxy)hex-1-aldehyde, 0.17 g of acetic acid, and 0.26 g of 6-aminohexyl-1-ol was treated with sodium triacetoxyborohydride (1.3 g) for two hours. The solution was washed with an aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a methanol / dichloromethane (0-8 / 100-92%) gradient to produce compound 8 (0.5 g) as a colorless oil.

[0242] Example 14

[0243] Synthesis of Compound 9

[0244] A solution of 2.4 g of 6-(2'-hexyldecanoyloxy)hex-1-aldehyde and 0.35 g of trans-2-aminocyclohexanol hydrochloride in dichloromethane (10 mL) / tetrahydrofuran (10 mL) was treated with sodium triacetoxyborohydride (1.3 g) for 1.5 h. The solution was washed with an aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using a methanol / dichloromethane (0–8 / 100–92%) gradient to produce compound 9 (0.6 g) as a colorless oil.

[0245] Example 15

[0246] Synthesis of Compound 10

[0247] To a solution of 2-aminoethanol (106 mg, 1.75 mmol) in anhydrous THF (15 mL), 2-octyldodecyl 6-bromohexanoate (2 equivalents, 1.66 g, 3.5 mmol), potassium carbonate (2 equivalents, 3.5 mmol, 477 mg), and cesium carbonate (0.3 equivalents, 0.525 mmol, 171 mg) were added, and the mixture was heated at 63°C (oil bath) for 16 h. Trace amounts of tetrabutylammonium iodide were added to the mixture, and the mixture was refluxed for 4 days. The solvent was evaporated under reduced pressure, and the residue was placed in a mixture of hexane and ethyl acetate (approximately 9:1) and washed with water and brine. The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure to obtain an oil (1.6 g). The residue (1.6 g) was purified by column chromatography (MeOH in chloroform, 0 to 4%) on silica gel. This produces compound 10 (700 mg, 0.82 mmol, 47%), which is a colorless oil.

[0248] Example 16

[0249] Synthesis of Compound 11

[0250] Add 2-hexyldecyl 6-bromohexanoate (1.9 equivalence, 1.52 g, 3.62 mmol), potassium carbonate (1.9 equivalence, 3.62 mmol, 500 mg), cesium carbonate (0.3 equivalence, 0.57 mmol, 186 mg), and sodium iodide (10 mg) to 15 mL of anhydrous THF solution of 2-aminoethanol (116 mg, 1.9 mmol, 115 μL), and heat under reflux at Ar for 6 days. Evaporate the solvent under reduced pressure, place the residue in hexane, and wash with water and brine. Separate the organic layer, dry over anhydrous sodium sulfate, filter, and evaporate under reduced pressure to obtain a colorless oil. Purify the crude product by rapid column chromatography on silica gel (MeOH in chloroform, 0 to 4%) to produce compound 11 (936 mg, 1.27 mmol, 70%) as a colorless oil.

[0251] Example 17

[0252] Synthesis of Compound 12

[0253] Compound 12 was prepared in a similar manner to that of compound 11, yielding 538 mg of colorless oil (0.86 mmol, 57%).

[0254] Example 18

[0255] Synthesis of Compound 13

[0256] To a solution of 2-aminoethanol (171 mg, 2.81 mmol, 169 μL) in anhydrous THF (30 mL), 2-octyldodecyl 4-bromobutyrate (1.9 equivalence, 2.386 g, 5.33 mmol), potassium carbonate (1.9 equivalence, 5.33 mmol, 736 mg), cesium carbonate (0.3 equivalence, 0.84 mmol, 275 mg), and sodium iodide (10 mg) were added, and the mixture was heated under reflux at Ar for 16 h. TLC (hexane / ethyl acetate = 9:1, CHCl3 / MeOH = 19:1) showed the production of significant amounts of 2-octyl-1-dodecanol. The mixture was cooled and filtered. The filtrate was concentrated, and the residue was dissolved in 2-octyl-1-dodecanol (2.1 g). Some 4A molecular sieve beads and N,N-diisopropylethylamine (1.9 equivalence, 5.33 mmol, 683 mg, 0.92 mL) were added. The mixture was sealed and heated at 62°C for 4 days. The reaction mixture was cooled. Hexane was added. The hexane solution was decanted and concentrated to dryness. The residue was purified by column chromatography (MeOH in chloroform, 0 to 4%) on silica gel to give compound 13 (282 mg, 0.35 mmol, 13%) as a colorless oil.

[0257] Example 19

[0258] Synthesis of Compound 14

[0259] To a solution of heptadecano-9-yl-6-bromohexanoate (2 equivalents, 1.13 g, 2.61 mmol) in anhydrous THF (15 mL), 2-aminoethanol (1 eq. 1.31 mmol, 79.7 mg), potassium carbonate (2 equivalents, 2.61 mmol, 361 mg), cesium carbonate (0.3 equivalents, 0.39 mmol, 128 mg), and sodium iodide (6 mg) were added. The mixture was heated under reflux at Ar for 7 days. The solvent was evaporated under reduced pressure, and the residue was placed in hexane / ethyl acetate (approximately 10%) and washed with water and brine. The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure to obtain an oil (1 g). The residue (1 g) was purified by gravity column chromatography (MeOH in DCM, 0 to 4%) on silica gel. This yielded compound 14 as a colorless oil (757 mg 0.99 mmol, 76%).

[0260] Example 20

[0261] Synthesis of Compound 15

[0262] Add 4-amino-1-butanol (1 equivalent, 1.5 mmol, 0.134 mg, 139 μL), potassium carbonate (2 equivalents, 3 mmol, 415 mg), cesium carbonate (0.3 equivalents, 0.45 mmol, 146 mg), and sodium iodide (6 mg) to 15 mL of anhydrous THF (opened 2 months ago). Refluxing the mixture under Ar for 6 days. Evaporate the solvent under reduced pressure, place the residue in a mixture of hexane and ethyl acetate (approximately 10%), and wash with water and brine. Separate the organic layer, dry over anhydrous sodium sulfate, filter, and evaporate under reduced pressure to obtain an oil (1.12 g). Purify the residue by column chromatography on silica gel (MeOH in chloroform, 0 to 5%). This yields compound 15 as a colorless oil (487 mg, 0.66 mmol, 44%). 1 HNMR (400MHz, CDCl3) δ: 5.99 (s, 1H), 3.98 (d, 5.8Hz, 4H), 3.56 (t sample, 4.8Hz, 2H), 2.48-2.41 (m, 6H), 2 .33(t,7.4Hz,4H),1.70-1.57(m,10H),1.55-1.47(m,4H),1.35-1.21(48H),0.89(t sample,6.8Hz,12H).

[0263] Example 21

[0264] Synthesis of Compound 16

[0265] To a solution of 3-amino-1-propanol (0.37 mmol, 28 mg) in anhydrous acetonitrile (15 mL), 2-hexyldecyl 6-bromohexanoate (1.9 equivalents, 294 mg, 0.7 mmol), N,N-diisopropylethylamine (2 equivalents, 0.74 mmol, 96 mg), and sodium iodide (5 mg) were added, and the mixture (two layers) was heated in a pressure flask at 59 °C (oil bath) for 3 days. The mixture was concentrated, and the residue was placed in a mixture of hexane and ethyl acetate (approximately 5:1, 100 mL), washed with water and brine, dried over sodium sulfate, filtered, and concentrated. A slightly yellow oil (approximately 300 mg) was obtained. The crude product (300 mg) was purified by rapid column chromatography on silica gel (MeOH in chloroform, 0 to 4.4%). This yielded compound 16 as a colorless oil (95 mg, 0.13 mmol, 36%). 1H NMR (400MHz, CDCl3) δ: 5.61-5.44 (br.s, 1H), 3.97 (d, 5.8Hz, 4H), 3.80 (t sample, 5.1Hz, 2H), 2.63 (t sample, 5.6Hz, 2H), 2.43-2.39 (m, 4H), 2.32 (t, 7.5Hz, 4H), 1.70-1.59 (m, 8H), 1.55-1.45 (m, 4H), 1.36-1.21 (52H), 0.89 (t sample, 6.8Hz, 12H).

[0266] Example 22

[0267] Synthesis of Compound 17

[0268] Add 4-amino-1-butanol (1 equivalent, 1.57 mmol, 140 mg, 145 μL), potassium carbonate (2 equivalents, 3.14 mmol, 434 mg), cesium carbonate (0.3 equivalents, 0.47 mmol, 153 mg), and sodium iodide (6 mg) to 15 mL of anhydrous THF solution of 2-hexyldecyl 6-bromohexanoate (2 equivalents, 1.32 g, 3.14 mmol, 145 μL), to 15 mL of anhydrous THF solution. Heat the mixture in Ar at 75 °C (oil bath) in a pressure-resistant round-bottom flask for 6 days. Cool and concentrate the reaction mixture. Place the residue in a mixture of hexane and ethyl acetate (approximately 9:1), wash with water and brine, dry over sodium sulfate, filter, and concentrate to dryness (1.28 g colorless oil). Purify the crude product by rapid column chromatography on silica gel (MeOH in chloroform, 0 to 5%). This yields compound 17 as a colorless oil (581 mg, 0.76 mmol, 48%). 1 HNMR (400MHz, CDCl3) δ: 6.43-6.17 (br.s, 1H), 3.97 (d, 5.8Hz, 4H), 3.55 (t sample, 4.7Hz, 2H), 2.46-2.40 (m, 6 H),2.31(t,7.5Hz,4H),1.70-1.59(m,10H),1.55-1.45(m,4H),1.36-1.21(52H),0.89(t sample,6.7Hz,12H).

[0269] Example 23

[0270] Synthesis of Compound 20

[0271] Add 4-amino-1-butanol (1 equivalent, 3.45 mmol, 308 mg), potassium carbonate (2 equivalents, 6.9 mmol, 954 mg), cesium carbonate (0.3 equivalents, 1.04 mmol, 337 mg), and sodium iodide (10 mg) to 30 mL of anhydrous THF solution of 2-hexyldecyl 8-bromooctanoate (2 equivalents, 3.09 g, 6.9 mmol, 3.9 mg). Heat the mixture in a pressure-resistant round-bottom flask in Ar at 64–70 °C (oil bath) for 6 days. Cool and concentrate the mixture. Place the residue in a mixture of hexane and ethyl acetate (9:1), wash with water and brine, dry over sodium sulfate, filter, and concentrate to dryness (colorless oil). Purify the crude product by rapid drying column chromatography on silica gel (MeOH in chloroform, 0–4.2%). This yields compound 20 (1.28 g, 1.56 mmol, 45%) as a colorless oil. 1 HNMR(400MHz, CDCl3)δ:6.64-6.45(br.s,1H),3.97(d,5.8Hz,4H),3.62-3.51(br.2H),3.07-2. 34(br.6H),2.30(t,7.5Hz,4H),1.71-1.40(m,14H),1.39-1.19(m,60H),0.89(t sample,6.8Hz,12H).

[0272] Example 24

[0273] Synthesis of 9-(2'-ethylhexanoyloxy)non-1-aldehyde

[0274] A solution of nonan-1,9-diol (10.1 g) in dichloromethane (150 mL) was treated with 2-ethylhexanoic acid (9.0 g), DCC (14.3 g), and DMAP (9.1 g). The solution was stirred overnight. The reaction mixture was filtered to remove the solvent. The residue was suspended in hexane and filtered. The filtrate was washed with dilute hydrochloric acid. The organic phase was dried over anhydrous magnesium sulfate, filtered through a silica gel bed to remove the solvent. The crude product was passed through a silica gel column using a methanol / dichloromethane (0–8%) gradient to produce 9-(2'-ethylhexanoyloxy)nonan-1-ol (7.2 g) as an oil.

[0275] 9-(2'-Ethylhexanoyloxy)non-1-ol was dissolved in dichloromethane (100 mL) and treated with pyridinium chlorochromate (7.5 g) for one hour. Hexane (400 mL) was added, and the supernatant was filtered through a silica gel bed. The solvent was removed from the filtrate, and the resulting oil was dissolved in hexane. The suspension was filtered through a silica gel bed to remove the solvent, yielding 6 g of 9-(2'-Ethylhexanoyloxy)non-1-aldehyde as a colorless oil.

[0276] Example 25

[0277] Synthesis of 9-(2'-Butyloctyloxy)non-1-aldehyde

[0278] A solution of nonan-1,9-diol (12.0 g) in dichloromethane (150 mL) was treated with 2-butyloctanoic acid (5.0 g), DCC (7.7 g), and DMAP (4.5 g). The solution was stirred overnight. The reaction mixture was filtered to remove the solvent. The residue was suspended in hexane and filtered. The filtrate was washed with dilute hydrochloric acid. The organic phase was dried over anhydrous magnesium sulfate, filtered through a silica gel bed, and the solvent was removed. The crude product was passed through a silica gel column using a methanol / dichloromethane (0–4%) gradient to produce 9-(2'-butyloctanoyloxy)nonan-1-ol (6 g) as an oil.

[0279] 9-(2'-Butyloctyloxy)non-1-ol was dissolved in dichloromethane (100 mL) and treated overnight with pyridinium chlorochromate (3.8 g). Hexane (300 mL) was added, and the supernatant was filtered through a silica gel bed. The solvent was removed from the filtrate, and the resulting oil was dissolved in hexane. The suspension was filtered through a silica gel bed to remove the solvent, yielding 9-(2'-Butyloctyloxy)non-1-aldehyde (3.1 g) as a colorless oil.

[0280] Example 26

[0281] Synthesis of 6-(2'-Butyloctanoyloxy)hex-1-aldehyde

[0282] A solution of hexane-1,6-diol (9.4 g) in 150 mL of dichloromethane was treated with 2-butyloctanoic acid (5.0 g), DCC (7.6 g), and DMAP (4.8 g). The solution was stirred overnight. The reaction mixture was filtered to remove the solvent. The residue was suspended in hexane and filtered. The filtrate was washed with dilute hydrochloric acid. The organic phase was dried over anhydrous magnesium sulfate, filtered through a silica gel bed to remove the solvent. The crude product was passed through a silica gel column using a methanol / dichloromethane (0–4%) gradient to produce an oily 6-(2'-butyloctanoyloxy)hexane-1-ol (4.5 g).

[0283] 6-(2'-Butyloctyloxy)hex-1-ol was dissolved in dichloromethane (100 mL) and treated with pyridinium chlorochromate (4.8 g) for two hours. Hexane (300 mL) was added, and the supernatant was filtered through a silica gel bed. The solvent was removed from the filtrate, and the resulting oil was dissolved in hexane. The suspension was filtered through a silica gel bed to remove the solvent, yielding 3.9 g of 6-(2'-Butyloctyloxy)hex-1-aldehyde as a colorless oil.

[0284] Example 27

[0285] Synthesis of 6-(2'-octyldodecanoyloxy)hex-1-aldehyde

[0286] A solution of hexane-1,6-diol (11.5 g) in dichloromethane (150 mL) / THF (20 mL) was treated with 2-octyldodecanoic acid (9.9 g), DCC (7.5 g), and DMAP (4.7 g). The solution was stirred overnight. The reaction mixture was filtered to remove the solvent. The residue was suspended in hexane and filtered. The filtrate was washed with dilute hydrochloric acid. The organic phase was dried over anhydrous magnesium sulfate, filtered through a silica gel bed, and the solvent was removed. The crude product was passed through a silica gel column using a methanol / dichloromethane (0–4%) gradient to produce 6-(2'-octyldodecanoyloxy)hexane-1-ol (7.4 g) in an oily form.

[0287] 6-(2'-Octyldodecanoyloxy)hex-1-ol was dissolved in dichloromethane (100 mL) and treated with pyridinium chlorochromate (4.0 g) for two hours. Diethyl ether (300 mL) was added, and the supernatant was filtered through a silica gel bed. The solvent was removed from the filtrate, and the resulting oil was dissolved in hexane. The suspension was filtered through a silica gel bed to remove the solvent, yielding 5.3 g of 6-(2'-Octyldodecanoyloxy)hex-1-aldehyde as a colorless oil.

[0288] Example 28

[0289] Synthesis of 6-(2'-decyltetradecanoyloxy)hex-1-aldehyde

[0290] A solution of hexane-1,6-diol (9.6 g) in 150 mL of dichloromethane was treated with 2-decyltetradecanoic acid (6.1 g), DCC (4.9 g), and DMAP (3.1 g). The solution was stirred overnight. The reaction mixture was filtered to remove the solvent. The residue was suspended in hexane and filtered. The filtrate was washed with dilute hydrochloric acid. The organic phase was dried over anhydrous magnesium sulfate, filtered through a silica gel bed, and the solvent was removed. The crude product was passed through a silica gel column using a methanol / dichloromethane (0–4%) gradient to produce 6-(2'-decyltetradecanoyloxy)hexane-1-ol (4.6 g).

[0291] 6-(2'-decyltetradecanoyloxy)hex-1-ol was dissolved in dichloromethane (100 mL) and treated with pyridinium chlorochromate (3.2 g) for two hours. Hexane (300 mL) was added, and the supernatant was filtered through a silica gel bed. The solvent was removed from the filtrate, and the resulting product was dissolved in hexane. The suspension was filtered through a silica gel bed to remove the solvent, yielding 6-(2'-decyltetradecanoyloxy)hex-1-aldehyde (4.2 g).

[0292] Example 29

[0293] Synthesis of 12-(2'-hexyldecanoyloxy)dodecyl-1-aldehyde

[0294] A solution of dodecane-1,12-diol (25.0 g) in dichloromethane (300 mL) / THF (100 mL) was treated with 2-hexyldecanoic acid (10.6 g), DCC (10.2 g), and DMAP (7.5 g). The solution was stirred overnight. The reaction mixture was filtered to remove the solvent. The residue was suspended in hexane and filtered. The filtrate was washed with water. The organic phase was dried over anhydrous magnesium sulfate, filtered through a silica gel bed, and the solvent was removed. The crude product was passed through a silica gel column using hexane followed by dichloromethane to produce an oily 12-(2'-hexyldecanoyloxy)dodecane-1-ol (7.9 g).

[0295] 12-(2'-hexyldecanoyloxy)dodecane-1-ol was dissolved in dichloromethane (150 mL) and treated with pyridinium chlorochromate (4.0 g) for three hours. Hexane (300 mL) was added, and the supernatant was filtered through a silica gel bed. The solvent was removed from the filtrate, and the resulting oil was dissolved in hexane. The suspension was filtered through a silica gel bed to remove the solvent, yielding 3.9 g of 12-(2'-hexyldecanoyloxy)dodecane-1-aldehyde as a colorless oil.

[0296] Example 30

[0297] Synthesis of 9-(2'-hexyldecanoyloxy)non-1-aldehyde

[0298] A solution of nonan-1,9-diol (46.8 g) in dichloromethane (600 mL) was treated with 2-hexyldecanoic acid (25.0 g), DCC (22.0 g), and DMAP (15.0 g). The solution was stirred overnight. The reaction mixture was filtered to remove the solvent. The residue was suspended in hexane and filtered. The filtrate was washed with dilute hydrochloric acid. The organic phase was dried over anhydrous magnesium sulfate, filtered through a silica gel bed, and the solvent was removed. The crude product was passed through a silica gel column using hexane followed by a methanol / dichloromethane (0–8%) gradient to produce 9-(2'-hexyldecanoyloxy)nonan-1-ol (22 g) as an oil.

[0299] 5.0 g of 9-(2'-hexyldecanoyloxy)non-1-ol was dissolved in 50 mL of dichloromethane and treated with pyridinium chlorochromate salt (2.7 g) for one hour. 200 mL of hexane was added, and the supernatant was filtered through a silica gel bed. The solvent was removed from the filtrate, and the resulting oil was dissolved in hexane. The suspension was filtered through a silica gel bed to remove the solvent, yielding 3.6 g of 9-(2'-hexyldecanoyloxy)non-1-aldehyde as a colorless oil.

[0300] Example 31

[0301] Synthesis of Compound 22

[0302] A solution of 9-(2'-hexyldecanoyloxy)non-1-aldehyde (2.2 g), acetic acid (0.15 g), and 4-aminobut-1-ol (0.20 g) in dichloromethane (20 mL) was treated overnight with sodium triacetoxyborohydride (1.30 g). The solution was washed with an aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using an acetic acid / methanol / dichloromethane gradient (2-0 / 0-12 / 98-88%). The pure fraction was washed with an aqueous sodium bicarbonate solution to give compound 22 (0.93 g) as a colorless oil.

[0303] Example 32

[0304] Synthesis of Compound 23

[0305] A solution of 12-(2'-hexyldecanoyloxy)dodecyl-1-aldehyde (2.0 g), acetic acid (0.09 g), and 4-aminobut-1-ol (0.14 g) in dichloromethane (20 mL) was treated overnight with sodium triacetoxyborohydride (0.71 g). The solution was washed with an aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using an acetic acid / methanol / dichloromethane (2-0 / 0-12 / 98-88%) gradient. The pure fraction was washed with an aqueous sodium bicarbonate solution to give compound 23 (1.0 g) as a colorless oil.

[0306] Example 33

[0307] Synthesis of Compound 24

[0308] A solution of 9-(2'-ethylhexanoyloxy)non-1-aldehyde (3.0 g), acetic acid (0.11 g), and 4-aminobut-1-ol (0.17 g) in dichloromethane (50 mL) was treated overnight with sodium triacetoxyborohydride (0.89 g). The solution was washed with an aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using an acetic acid / methanol / dichloromethane gradient (2-0 / 0-10 / 98-90%). The pure fraction was washed with an aqueous sodium bicarbonate solution to give compound 24 (0.69 g) as a colorless oil.

[0309] Example 34

[0310] Synthesis of Compound 25

[0311] A solution of 9-(2'-butyloctyloxy)non-1-aldehyde (2.6 g), acetic acid (0.20 g), and 4-aminobut-1-ol (0.26 g) in dichloromethane (50 mL) was treated overnight with sodium triacetoxyborohydride (1.42 g). The solution was washed with an aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using an acetic acid / methanol / dichloromethane gradient (2-0 / 0-12 / 98-88%). The pure fraction was washed with an aqueous sodium bicarbonate solution to give compound 25 (0.82 g) as a colorless oil.

[0312] Example 35

[0313] Synthesis of Compound 26

[0314] A solution of 6-(2'-octyldodecanoyloxy)hex-1-aldehyde (2.7 g), acetic acid (0.20 g), and 4-aminobut-1-ol (0.20 g) in dichloromethane (20 mL) was treated overnight with sodium triacetoxyborohydride (1.30 g). The solution was washed with an aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using an acetic acid / methanol / dichloromethane gradient (2-0 / 0-12 / 98-88%). The pure fraction was washed with an aqueous sodium bicarbonate solution to give compound 26 (0.21 g) as a colorless oil.

[0315] Example 36

[0316] Synthesis of Compound 27

[0317] A solution of 6-(2'-decyltetradecanoyloxy)hex-1-aldehyde (2.1 g), acetic acid (0.11 g), and 4-aminobut-1-ol (0.13 g) in dichloromethane (30 mL) was treated overnight with sodium triacetoxyborohydride (0.70 g). The solution was washed with an aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using an acetic acid / methanol / dichloromethane gradient (2-0 / 0-12 / 98-88%). The pure fraction was washed with an aqueous sodium bicarbonate solution to give compound 27 (0.90 g) as a colorless oil.

[0318] Example 37

[0319] Synthesis of Compound 28

[0320] A solution of 6-(2'-butyloctyloxy)hex-1-aldehyde (2.0 g), acetic acid (0.13 g), and 3-aminoprop-1-ol (0.13 g) in dichloromethane (20 mL) was treated overnight with sodium triacetoxyborohydride (1.0 g). The solution was washed with an aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using an acetic acid / methanol / dichloromethane gradient (2-0 / 0-8 / 98-92%). The pure fraction was washed with an aqueous sodium bicarbonate solution to give compound 28 (0.77 g) as a colorless oil.

[0321] Example 38

[0322] Synthesis of Compound 30

[0323] A solution of 6-(2'-hexyldecanoyloxy)hex-1-aldehyde (2.4 g), acetic acid (0.15 g), and 3-aminoprop-1,2-diol (0.21 g) in dichloromethane (20 mL) was treated overnight with sodium triacetoxyborohydride (1.76 g). The solution was washed with an aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using an acetic acid / methanol / dichloromethane gradient (2-0 / 0-12 / 98-88%). The pure fraction was washed with an aqueous sodium bicarbonate solution to give compound 30 (0.60 g) as a colorless oil.

[0324] Example 39

[0325] Synthesis of Compound 31

[0326] A solution of 6-(2'-hexyldecanoyloxy)hex-1-aldehyde (2.4 g), acetic acid (0.15 g), and 2-aminobut-1-ol (0.20 g) in dichloromethane (20 mL) was treated with sodium triacetoxyborohydride (1.1 g) for two hours. The solution was washed with dilute aqueous sodium hydroxide solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using an acetic acid / methanol / dichloromethane (2-0 / 0-4 / 98-96%) gradient. The pure fraction was washed with aqueous sodium bicarbonate solution to give compound 31 (0.31 g) as a colorless oil.

[0327] Example 40

[0328] Synthesis of Compound 37

[0329] A solution of 6-(2'-octyldodecanoyloxy)hex-1-aldehyde (2.7 g), acetic acid (0.20 g), and 3-aminoprop-1-ol (0.17 g) in dichloromethane (20 mL) was treated overnight with sodium triacetoxyborohydride (1.3 g). The solution was washed with an aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using an acetic acid / methanol / dichloromethane (2-0 / 0-12 / 98-88%) gradient. The pure fraction was washed with an aqueous sodium bicarbonate solution to yield compound 37 (0.22 g) as a colorless oil.

[0330] Example 41

[0331] Synthesis of Compound 38

[0332] A solution of 12-(2'-hexyldecanoyloxy)dodecyl-1-aldehyde (1.8 g), acetic acid (0.08 g), and 3-aminoprop-1-ol (0.11 g) in dichloromethane (10 mL) was treated overnight with sodium triacetoxyborohydride (0.64 g). The solution was washed with an aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using an acetic acid / methanol / dichloromethane gradient (2-0 / 0-10 / 98-90%). The pure fraction was washed with an aqueous sodium bicarbonate solution to give compound 38 (0.83 g) as a colorless oil.

[0333] Example 42

[0334] Synthesis of Compound 39

[0335] A mixture (two layers) of ethyl 4-aminobutyrate hydrochloride (1.28 mmol, 214 mg), 2-hexyldecyl 6-bromohexanoate (1.9 equivalences, 2.43 mmol, 1.02 g), N,N-diisopropylethylamine (3.5 equivalences, 4.48 mmol, 579 mg), and sodium iodide (5 mg) in anhydrous acetonitrile (15 mL) was heated in a pressure flask at 60 °C for 2 days. The mixture was cooled and concentrated. The residue was placed in a mixture of hexane and ethyl acetate (approximately 5:1, 100 mL), washed with water and brine, dried over sodium sulfate, filtered, and concentrated. A brown oil (approximately 1.04 g) was obtained. The crude product was purified by rapid column chromatography on silica gel (MeOH in DCM, 0 to 3.5%). This yielded compound 39 (334 mg, 0.41 mmol, 43%) as a colorless oil. 1H NMR (400MHz, CDCl3) δ: 4.13 (q, 7.1Hz, 2H), 3.97 (d, 5.8Hz, 4H), 2.43–2.34 (m, 6H), 2.33–2.28 (m, 6H), 1.73 (quintet, 7.3Hz, 2H), 1.68–1.58 (m, 6H), 1.47–1.37 (m, 4H), 1.36–1.20 (54H), 0.89 (t sample, 6.8Hz, 12H).

[0336] Example 43

[0337] Synthesis of Compound 40

[0338] A solution of 6-(2'-hexyldecanoyloxy)hex-1-aldehyde (2.4 g), acetic acid (0.15 g), and 1-aminobut-2-ol (0.10 g) in dichloromethane (20 mL) was treated with sodium triacetoxyborohydride (1.8 g) for two hours. The solution was washed with an aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using an acetic acid / methanol / dichloromethane gradient (2-0 / 0-8 / 98-92%). The pure fraction was washed with an aqueous sodium bicarbonate solution to give compound 40 (0.85 g) as a colorless oil.

[0339] Example 44

[0340] Synthesis of Compound 41

[0341] A solution of 6-(2'-hexyldecanoyloxy)hex-1-aldehyde (2.4 g), acetic acid (0.19 g), and 3-methoxypropylamine (0.21 g) in dichloromethane (20 mL) was treated overnight with sodium triacetoxyborohydride (1.8 g). The solution was washed with an aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using an acetic acid / methanol / dichloromethane (2-0 / 0-8 / 98-92%) gradient. The pure fraction was washed with an aqueous sodium bicarbonate solution to give compound 41 (0.77 g) as a colorless oil.

[0342] Example 45

[0343] Synthesis of Compound 42

[0344] A solution of 6-(2'-butyloctyloxy)hex-1-aldehyde (2.0 g), acetic acid (0.13 g), and 4-aminobut-1-ol (0.20 g) in dichloromethane (20 mL) was treated overnight with sodium triacetoxyborohydride (1.03 g). The solution was washed with an aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using an acetic acid / methanol / dichloromethane gradient (2-0 / 0-8 / 98-92%). The pure fraction was washed with an aqueous sodium bicarbonate solution to give compound 42 (0.54 g) as a colorless oil.

[0345] Example 46

[0346] Synthesis of Compound 43

[0347] A solution of 9-(2'-ethylhexanoyloxy)non-1-aldehyde (3.0 g), acetic acid (0.11 g), and 3-aminoprop-1-ol (0.14 g) in dichloromethane (50 mL) was treated overnight with sodium triacetoxyborohydride (0.91 g). The solution was washed with an aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using an acetic acid / methanol / dichloromethane (2-0 / 0-6 / 98-94%) gradient. The pure fraction was washed with an aqueous sodium bicarbonate solution to give compound 43 (1.01 g) as a colorless oil.

[0348] Example 47

[0349] Synthesis of Compound 44

[0350] A solution of 6-(2'-decyltetradecanoyloxy)hex-1-aldehyde (2.1 g), acetic acid (0.11 g), and 3-aminoprop-1-ol (0.11 g) in dichloromethane (30 mL) was treated overnight with sodium triacetoxyborohydride (0.71 g). The solution was washed with an aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using an acetic acid / methanol / dichloromethane (2-0 / 0-8 / 98-96%) gradient. The pure fraction was washed with an aqueous sodium bicarbonate solution to give compound 44 (1.07 g) as a colorless oil.

[0351] Example 48

[0352] Synthesis of Compound 45

[0353] A solution of 9-(2'-butyloctyloxy)non-1-aldehyde (2.6 g), acetic acid (0.17 g), and 3-aminoprop-1-ol (0.21 g) in dichloromethane (50 mL) was treated overnight with sodium triacetoxyborohydride (1.34 g). The solution was washed with an aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using an acetic acid / methanol / dichloromethane (2-0 / 0-8 / 98-96%) gradient. The pure fraction was washed with an aqueous sodium bicarbonate solution to give compound 45 (1.1 g) as a colorless oil.

[0354] Example 49

[0355] Synthesis of Compound 46

[0356] Add 2-hexyldecyl 8-bromooctanoate (1.8 equivalents, 1.27 g, 2.84 mmol), potassium carbonate (1.9 equivalents, 3 mmol, 414 mg), cesium carbonate (0.3 equivalents, 0.47 mmol, 154 mg), and sodium iodide (10 mg) to 15 mL of a 2-propanol solution of 2-aminoethanol (96.5 mg, 1.58 mmol, 95.4 μL, MW 61.08, d 1.012) and heat for 3 days (oil bath, 60 °C). Concentrate the mixture and place the residue in THF (10 mL). Add more aminoethanol (80 mg, 1.3 mmol) to the mixture. Continue heating at 70 °C for another 3 days. After a total of 6 days, cool, filter, and concentrate the reaction mixture. Purify the residue on silica gel by rapid dry column chromatography (methanol in chloroform, 1% to 4.2%). This produces compound 46 (334 mg, 0.42 mmol, 30%), which is a colorless oil. 1 HNMR(400MHz, CDCl3)δ:4.09-4.06(m,2H),3.97(d,5.8Hz,4H),3.39-3.36(m,2H),3.31-3. 23(m,4H),2.31(t,7.5Hz,4H),1.88-1.56(m,12H),1.43-1.19(59H),0.89(t sample,6.8Hz,12H).

[0357] Example 50

[0358] Synthesis of Compound 47

[0359] A solution of 6-(2'-hexyldecanoyloxy)hex-1-aldehyde (3.0 g), acetic acid (0.20 g), and 3-aminopropionitrile (0.21 g) in dichloromethane (30 mL) was treated overnight with sodium triacetoxyborohydride (1.3 g). The solution was washed with an aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using an acetic acid / methanol / dichloromethane (2-0 / 0-6 / 98-94%) gradient. The pure fraction was washed with an aqueous sodium bicarbonate solution to give compound 47 (0.29 g) as a colorless oil.

[0360] Example 51

[0361] Synthesis of Compound 48

[0362] A solution of 6-(2'-hexyldecanoyloxy)hex-1-aldehyde (3.0 g) and ethyl 4-aminobutyrate hydrochloride (0.46 g) in dichloromethane (30 mL) was treated overnight with sodium triacetoxyborohydride (1.4 g). The solution was washed with aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel column using an acetic acid / methanol / dichloromethane (2-0 / 0-8 / 98-92%) gradient. The pure fraction was washed with aqueous sodium bicarbonate solution to give compound 48 (0.80 g) as a colorless oil.

[0363] Example 52

[0364] Synthesis of Compound 49

[0365] Add 4-amino-1-butanol (1 equivalent, 2.4 mmol, 214 mg, 221 μL), potassium carbonate (2 equivalents, 4.8 mmol, 664 mg), cesium carbonate (0.3 equivalents, 0.72 mmol, 234 mg), and sodium iodide (approximately 5 mg) to 20 mL of anhydrous THF solution of 2-butyloctyl 8-bromooctanoate (2 equivalents, 0.72 mmol, 234 mg). Heat the mixture in a pressure-resistant round-bottom flask (oil bath, 80 °C) for 6 days. Cool and concentrate the reaction mixture. Place the residue in a mixture of hexane and ethyl acetate (approximately 5:1), wash with water and brine, dry over sodium sulfate, filter, and concentrate. Purify the residue by rapid column chromatography on silica gel (methanol in chloroform, 1 to 4%). This yields compound 49 (857 mg, 1.21 mmol, 50%) as a colorless oil. 1HNMR (400MHz, CDCl3) δ: 6.55 (br.s, 1H), 3.97 (d, 5.8Hz, 4H), 3.55 (not well resolved) triplet,2H),2.45-2.40(m.6H),2.30(t,7.5Hz,4H),1.71-1.58(m,10H),1.51-1.42(m,4H),1.39-1.19(m,44H),0.93-0.87(m,12H).

[0366] Other embodiments may be combined to provide alternative embodiments. To the extent inconsistent with the specific teachings and definitions herein, U.S. Patent Application No. 62 / 247,616, filed October 28, 2015, and U.S. Patent Application No. 62 / 328,244, filed April 27, 2016; all U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referenced in this specification and / or listed in the filing data sheet are incorporated herein by reference in their entirety. Other embodiments may be provided by modifying aspects of the embodiments using the concepts of various patents, applications, and publications, as necessary. These and other changes to the embodiments may be made in accordance with the detailed description above. Generally, the terms used in the claims should not be construed as limiting the claims to the specific embodiments disclosed in this specification and the claims, but rather should be construed as encompassing all possible embodiments and the full scope of the equivalents to which those claims are entitled. Therefore, the claims are not limited by this disclosure.

Claims

1. Compounds having the following structure (IF): (IF) Or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, wherein: G 1 and G 2 Each is independently unsubstituted C4-C 12 Alkylene; G 3 It is a C3-C6 alkylene group; R 1 and R 2 Each of the C6-C branches is independent. 24 alkyl; R 3 is H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 ; R 4 For C1-C 12 hydrocarbon group; and R 5 It is an H or C1-C6 hydrocarbon group.

2. The compound of claim 1, having the following structure (IH): (IH) in: R 6 H, OH or C1-C 24 hydrocarbon group; y and z are each independent integers from 4 to 12; and n is an integer from 3 to 6.

3. The compound of claim 2, wherein n is 3.

4. The compound of claim 2, wherein n is 4.

5. The compound of claim 2, wherein y and z are each independently an integer from 4 to 9.

6. The compound of claim 1, wherein R 1 and R 2 Each of them independently has the following structure: in: R 7a and R 7b Each occurrence is independently H or Cl-C. 12 hydrocarbon group; and a is an integer from 2 to 12. Where R 7a R 7b a and a are each chosen to make R 1 and R 2 Each is independently a branched alkyl group containing 6 to 20 carbon atoms.

7. The compound of claim 6, wherein a is an integer from 8 to 12.

8. The compound of claim 6, wherein R appears at least once. 7a For H.

9. The compound of claim 6, wherein R 7a It is H each time it appears.

10. The compound of claim 6, wherein R appears at least once. 7b It is a C1-C8 hydrocarbon group.

11. The compound of claim 10, wherein the C1-C8 hydrocarbon group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

12. The compound of claim 1, wherein R 1 or R 2 Or both have one of the following structures: ； ； ； ； ； ； ； ； ； 。 13. The compound of claim 1, wherein R 3 It is OH.

14. The compound of claim 1, wherein R 3 For CN.

15. The compound of claim 1, wherein R 3 -C(=O)OR 4 -OC(=O)R 4 or -NHC(=O)R 4 .

16. The compound of claim 15, wherein R 4 It can be methyl or ethyl.

17. The compound of claim 1, having one of the following structures: ； ； ； ； ； ; or 。 18. A composition comprising the compound of any one of claims 1-17 and a therapeutic agent, wherein the therapeutic agent comprises a nucleic acid.

19. The composition of claim 18, further comprising one or more excipients selected from neutral lipids, steroids, and polymerically conjugated lipids.

20. The composition of claim 19, wherein the composition comprises one or more neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.

21. The composition of claim 20, wherein the neutral lipid is DSPC.

22. The composition of claim 19, wherein the molar ratio of the compound to the neutral lipid is 2:1 to 8:

1.

23. The composition of claim 19, wherein the steroid is cholesterol.

24. The composition of claim 23, wherein the molar ratio of the compound to cholesterol is 5:1 to 1:

1.

25. The composition of claim 19, wherein the polymer-conjugated lipid is a polyethylene glycol-modified lipid.

26. The composition of claim 25, wherein the molar ratio of the compound to the polyethylene glycol-modified lipid is from 100:1 to 20:

1.

27. The composition of claim 25, wherein the polyethylene glycol-modified lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, or PEG dialkoxypropyl carbamate.

28. The composition of claim 25, wherein the polyethylene glycol-modified lipid has the following structure (II): (II) Or a drug-acceptable salt, tautomer, or stereoisomer thereof, wherein: R 8 and R 9 Each is independently a straight-chain or branched, saturated or unsaturated hydrocarbon chain containing 10 to 30 carbon atoms, wherein the hydrocarbon chain is optionally interrupted by one or more ester bonds; and w has an average value of 30 to 60.

29. The composition of claim 28, wherein R 8 and R 9 Each is an independent straight-chain, saturated hydrocarbon chain containing 12 to 16 carbon atoms.

30. The composition of claim 28, wherein the average value w is about 49.

31. The composition of claim 18, wherein the nucleic acid is selected from antisense RNA and messenger RNA.

32. Use of the composition of any one of claims 18-31 in the preparation of a medicament for administering a therapeutic agent to a patient in need.

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