Novel lipid and lipid nanoparticle formulations for nucleic acid delivery

Abstract

This invention provides the use of compounds as components of lipid nanoparticle formulations for the delivery of therapeutic drugs, compositions containing the compounds, and methods for their use and preparation. [Solution] A compound having the following structural formula (I), or a pharmaceutically acceptable salt thereof, tautomer, or stereoisomer is provided. TIFF2026062680000045.tif2046

Description

[Technical Field]

[0001] The present invention generally relates to novel cationic lipids for forming oligonucleotides and lipid nanoparticles in combination with other lipid components such as neutral lipids, cholesterol, and polymer complex lipids, both in vitro and in vivo, thereby facilitating the intracellular delivery of therapeutic nucleic acids (e.g., oligonucleotides, messenger RNA, etc.). [Background technology]

[0002] Description of related technologies The delivery of nucleic acids to influence desired responses in biological systems presents numerous challenges. While nucleic acid-based therapeutics hold immense potential, realizing this potential still requires more effective delivery of nucleic acids to the appropriate sites within cells or organisms. Examples of therapeutic nucleic acids include messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, immunostimulatory nucleic acids, antagomir, antimir, mimes, supermir, and aptamers. Some nucleic acids, such as mRNA or plasmids, can be used to achieve the expression of specific cell products, for example, to treat diseases associated with protein or enzyme deficiencies. The therapeutic applications of translatable nucleotide delivery are extremely broad, as constructs can be synthesized to produce any chosen protein sequence, whether system-specific or not. Nucleic acid expression products can increase existing protein levels, replace deficient or non-functional versions of proteins, or introduce novel proteins and associated functionalities into cells or organisms.

[0003] Some nucleic acids, such as miRNA inhibitors, can be used to achieve the expression of specific cell products regulated by miRNAs, for example, which may be useful in treating diseases associated with protein or enzyme deficiencies. The therapeutic applications of miRNA inhibition are extremely broad, as constructs can be synthesized to inhibit one or more miRNAs that sequentially regulate the expression of mRNA products. Inhibition of endogenous miRNAs can increase the expression of their downstream target endogenous proteins, restoring proper function in cells or organisms as a means of treating diseases associated with specific miRNAs or groups of miRNAs.

[0004] Other nucleic acids can downregulate the intracellular levels of specific mRNAs, and as a result, can downregulate the synthesis of the corresponding protein through processes such as RNA interference (RNAi) or complementary binding of antisense RNA. The therapeutic applications of antisense oligonucleotides and RNAi are also very broad, as oligonucleotide constructs can be synthesized with any nucleotide sequence directed toward the target mRNA. Targets can include mRNA from normal cells, mRNA associated with disease conditions such as cancer, and mRNA from infectious agents such as viruses. To date, antisense oligonucleotide constructs have demonstrated the ability to specifically downregulate target proteins through the degradation of homologous mRNA in both in vitro and in vivo models. Furthermore, antisense oligonucleotide constructs are currently being evaluated in clinical studies.

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

[0006] Improved cationic lipids and lipid nanoparticles are needed for oligonucleotide delivery. Preferably, these lipid nanoparticles provide an optimal drug-to-lipid ratio, protect nucleic acids from degradation and clearance in serum, are suitable for systemic or local delivery, and provide intracellular delivery of nucleic acids. Furthermore, these lipid-nucleic acid particles should be well tolerable and provide an appropriate therapeutic index so that treatment of a patient with an effective dose of nucleic acid is not associated with unacceptable toxicity and / or risk to the patient. The present invention provides these and related advantages. [Means for solving the problem]

[0007] overview In 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 (e.g., all sterols) and / or their analogues, and / or polymer complex lipids, to form lipid nanoparticles for the delivery of therapeutic agents. In some examples, lipid nanoparticles are used to deliver nucleic acids such as antisense and / or messenger RNA. Methods of using such lipid nanoparticles for the treatment of various diseases or conditions, such as those caused by deficiencies of infectious entities and / or proteins, are also provided.

[0008] In one embodiment, the following structural formula (I): [ka] (I) [In the formula, R 1 , R 2 , R 3 , L 1 , L2 , G 1 , G 2 and G 3 [This is as defined herein] A compound having, or a pharmaceutically acceptable salt thereof, a tautomer, or a stereoisomer thereof, is provided.

[0009] Pharmaceutical compositions and therapeutic agents comprising one or more of the compounds of structural formula (I) are also provided. In some embodiments, the pharmaceutical composition further comprises one or more components selected from neutral lipids, charged lipids, steroids, and polymer complex lipids. Such compositions are useful for forming lipid nanoparticles for therapeutic agent delivery.

[0010] In other embodiments, the present invention provides a method for administering a therapeutic agent to a patient in need, comprising preparing a composition of lipid nanoparticles containing a compound of structural formula (I) and a therapeutic agent, and delivering the composition to the patient.

[0011] These and other aspects of the present invention will become apparent by referring to the following detailed description.

[0012] A brief explanation of some views of the drawing. In the drawings, the same reference number indicates similar elements. The size and relative position of elements in the drawings are not necessarily drawn to scale, and some of these elements are arbitrarily enlarged and positioned to improve the readability of the drawing. Furthermore, the specific shapes of the elements depicted are not intended to convey information about the actual shape of the particular element, but are selected solely to facilitate the understanding of the drawing. [Brief explanation of the drawing]

[0013] [Figure 1] This shows the time course of luciferase expression in mouse liver. [Figure 2] The pKa calculation for MC3, a representative example related to the disclosed lipids, is shown. [Figure 3]This provides comparative luciferase activity data for selected lipids. [Modes for carrying out the invention]

[0014] Detailed description In the following description, certain details are provided to give a complete understanding of the various embodiments of the invention. However, those skilled in the art will understand that the invention can be carried out without these details.

[0015] The present invention is partly based on the discovery of novel cationic (amino) lipids that offer advantages when used in lipid nanoparticles for in vivo delivery of activators or therapeutic agents, such as nucleic acids, to mammalian cells. In particular, embodiments of the present invention provide nucleic acid-lipid nanoparticle compositions comprising one or more novel cationic lipids described herein that provide increased nucleic acid activity and improved compositional tolerance in vivo, resulting in a significant increase in therapeutic index compared to previously described nucleic acid-lipid nanoparticle compositions.

[0016] In certain embodiments, the present invention provides novel cationic lipids that enable the formulation of improved compositions for the in vitro and in vivo delivery of mRNA and / or other oligonucleotides. In some embodiments, these improved lipid nanoparticle compositions are useful for the expression of mRNA-encoded proteins. In other embodiments, these improved lipid nanoparticle compositions are useful for the upregulation of endogenous protein expression by delivering miRNA inhibitors that target one specific miRNA or one target mRNA or a group of miRNAs that control several mRNAs. In other embodiments, these improved lipid nanoparticle compositions are useful for downregulating (e.g., silencing) the protein and / or mRNA levels of a target gene. In some other embodiments, lipid nanoparticles are also useful for the delivery of mRNA and plasmids for the expression of transgenes. In yet another embodiment, lipid nanoparticle compositions are useful for inducing pharmacological effects resulting from protein expression, such as increased red blood cell production by delivery of appropriate erythropoietin mRNA, or protection against infection by delivery of mRNA encoding an appropriate antigen or antibody.

[0017] The lipid nanoparticles and compositions of the present invention can be used for a variety of purposes, both in vitro and in vivo, including the delivery of encapsulated or bound (associated) (e.g., complex) therapeutic agents such as nucleic acids to cells. Accordingly, embodiments of the present invention are methods for treating or preventing a disease or disorder in a subject requiring such treatment, comprising contacting the subject with lipid nanoparticles encapsulating or bound to a suitable therapeutic agent, wherein the lipid nanoparticles comprise one or more novel cationic lipids described herein.

[0018] As described herein, embodiments of the lipid nanoparticles of the present invention are particularly useful for the delivery of nucleic acids such as mRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomyl / antimyl), messenger RNA interfering complementary RNA (micRNA), DNA, multivalent RNA, dicer substrate RNA, and complementary DNA (cDNA). Accordingly, the lipid nanoparticles and compositions of the present invention can be used to induce the expression of a desired protein both in vitro and in vivo by contacting cells with lipid nanoparticles containing one or more novel cationic lipids described herein, wherein the lipid nanoparticles encapsulate or bind nucleic acids expressed to produce the desired protein (e.g., messenger RNA or plasmid encoding the desired protein) or nucleic acids expressed to inhibit the process that terminates mRNA expression (e.g., a miRNA inhibitor). Alternatively, the lipid nanoparticles and compositions of the present invention can be used to reduce the expression of target genes and proteins both in vitro and in vivo by contacting cells with lipid nanoparticles containing one or more novel cationic lipids described herein, wherein the lipid nanoparticles encapsulate or bind nucleic acids (e.g., antisense oligonucleotides or small interfering RNA (siRNA)) that reduce target gene expression. The lipid nanoparticles and compositions of the present invention can be used separately or in combination for co-delivery of different nucleic acids (e.g., mRNA and plasmid DNA) so as to be useful in providing effects that require the co-localization of different nucleic acids (e.g., mRNA encoding a suitable gene-modifying enzyme and a DNA segment for integration into the host genome).

[0019] Nucleic acids for use in this invention can be prepared according to any available technique. For mRNA, the primary methodology for preparation is, but is not limited to, enzymatic synthesis (also known as in vitro transcription), which currently represents the most efficient method for generating long sequence-specific mRNA. In vitro transcription describes the process of template-directed synthesis of RNA molecules from an engineered DNA template (including, but not limited to, those derived from T7, T3, and SP6 colipages) containing an upstream bacteriophage promoter sequence ligated to a downstream sequence encoding the gene of interest. Template DNA can be prepared for in vitro transcription from many sources using appropriate techniques well known in the art, such as plasmid DNA and polymerase chain reaction amplification (see Linpinsel, JL and Conn, GL, General protocols for preparation of plasmid DNA template and Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LD in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941 Conn GL (ed), New York, NY Humana Press, 2012).

[0020] RNA transcription is performed in vitro using a linearized DNA template in the presence of the corresponding RNA polymerase and adenosine, guanosine, uridine, and cytidine ribonucleoside triphosphates (rNTPs), under conditions that support polymerase activity, while minimizing the potential degradation of the obtained mRNA transcript. In vitro transcription can be performed using a variety of commercially available kits, including but not limited to the RiboMax Large Scale RNA Production System (Promega) and the MegaScript Transcription Kit (Life Technologies), as well as commercially available reagents such as RNA polymerase and rNTPs. Methodologies for in vitro transcription of mRNA are well known in the art. (e.g. Losick, R., 1972, In vitro transcription, Ann Rev Biochem v.41 409-46; Kamakaka, RT and Kraus, WL 2001. In Vitro Transcription. Current Protocols in Cell Biology. 2:11.6:11.6.1-11.6.17; Beckert, B. And Masquida, B., (2010) Synthesis of RNA by In Vitro Transcription in RNA in Methods in Molecular Biology v. 703 (Neilson, H. Ed), New York, NY Humana Press, 2010; Brunelle, JL and Green, R., 2013, Chapter Five-In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology v. 530, 101-114; all of which are incorporated herein by reference).

[0021] Next, the mRNA transcribed in vitro is purified from undesirable components of the transcription or related reaction (such as unintegrated rNTPs, protein enzymes, salts, and short RNA oligos). Techniques for isolating mRNA transcripts are well known in the art. Well-known procedures include phenol / chloroform extraction or precipitation with either a monovalent cation or an alcohol (ethanol, isopropanol) in the presence of lithium chloride. Further non-limiting examples of purification procedures that can be used include size exclusion chromatography (Lukavsky, PJ and Puglisi, JD, 2004, Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides, RNA v.10, 889-893), silica affinity chromatography, and polyacrylamide gel electrophoresis (Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LD in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941 Conn GL (ed), New York, NY Humana Press, 2012). Purification can be performed using a variety of commercially available kits, including but not limited to the SV Total Isolation System (Promega) and In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).

[0022] Furthermore, while reverse transcription can produce large amounts of mRNA, the product may contain numerous abnormal RNA impurities associated with undesirable polymerase activity that may need to be removed from the full-length mRNA preparation. These include short RNAs resulting from incomplete transcription initiation, as well as double-stranded RNA (dsRNA) produced by RNA-dependent RNA polymerase activity, RNA-primed transcription from RNA templates, and self-complementary 3' extensions. It has been demonstrated that these contaminants with dsRNA structures can lead to undesirable immunostimulatory activity through interaction with various innate immune sensors within eukaryotic cells that recognize specific nucleic acid structures and function to induce a potent immune response. This, in turn, can dramatically reduce mRNA translation, as protein synthesis is reduced during the innate cellular immune response. Therefore, further techniques for removing these dsRNA contaminants have been developed, and are known in the art, including, but are not limited to, scalable 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. 39 e142; Weissman, D., Pardi, N., Muramatsu, H., and Kariko, K., HPLC Purification of in vitro transcribed long RNA in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH Ed), 2013). HPLC-purified mRNA has been reported to be translated at much higher levels, particularly in primary cells and in vivo.

[0023] A variety of significant modifications used to alter the specific properties of in vitro transcribed mRNA and improve its utility have been described in the Art. These include, but are not limited to, modifications of the 5' and 3' ends of mRNA. Endogenous eukaryotic mRNA typically contains a cap structure at the 5' end of the mature molecule, which plays a crucial role in mediating the binding of mRNA cap-binding proteins (CBPs), and this, in turn, contributes to increasing mRNA stability and the efficiency of mRNA translation in cells. Thus, the highest levels of protein expression are achieved with capped mRNA transcripts. The 5' cap contains a 5'-5'-triphosphate bond between the 5'-most nucleotide and the guanine nucleotide. The complexed guanine nucleotide is methylated at the N7 position. Further modifications include methylation of the last and second-to-last 5'-most nucleotides on the 2'-hydroxyl group.

[0024] Several different cap structures can be used to generate the 5' cap of synthetic mRNA transcribed in vitro. 5' capping of synthetic mRNA can be co-transcribed with a chemical cap analog (i.e., capping during in vitro transcription). For example, an anti-reverse cap analog (ARCA) cap contains a 5'-5'-triphosphate guanine-guanine bond in which one guanine contains both an N7 methyl group and a 3'-O-methyl group. However, during this co-transcription process, up to 20% of the transcript remains uncapped, and the synthetic cap analog is not identical to the 5' cap structure of true cellular mRNA, potentially reducing translational capacity and cellular stability. Alternatively, the synthetic mRNA molecule may be enzymatically capped after transcription. These can produce more closely similar, more authentic 5' cap structures, either structurally or functionally. Endogenous 5'-caps with enhanced binding of cap-binding proteins increase half-life, decrease sensitivity to 5' endonucleases, and / or reduce 5' decapping. Numerous synthetic 5' cap analogs have been developed and are known in the art to improve mRNA stability and translational capacity (see, for example, Grudzien-Nogalska, E., Kowalska, J., Su, W., Kuhn, AN, Slepenkov, SV, Darynkiewicz, E., Sahin, U., Jemielity, J., and Rhoads, RE, Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH Ed), 2013).

[0025] At the 3' end, a long adenine nucleotide (poly-A tail) is typically added to the mRNA molecule during RNA processing. Immediately after transcription, the 3' end of the transcript is cleaved, and in a process called polyadenylation, poly-A polymerase releases the 3' hydroxyl group that attaches the adenine nucleotide chain to the RNA. Poly(A) tails have been widely shown to enhance both the translation efficiency and stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly(A), poly(A) binding protein and the regulation of mRNA stability, Trends Bio Sci v. 14 373-377; Guhaniyogi, J. and Brewer, G., 2001, Regulation of mRNA stability in mammalian cells, Gene, v. 265, 11-23; Dreyfus, M. and Regnier, P., 2002, The poly(A) tail of mRNAs: Bodyguard in eukaryotes, scavenger in bacteria, Cell, v. 111, 611-613).

[0026] Poly(A) tailing of in vitro transcribed mRNA can be achieved using a variety of approaches, including, but not limited to, cloning of a poly(T) tract into a DNA template or post-transcriptional addition using poly(A) polymerase. The first case allows in vitro transcription of mRNA with a poly(A) tail of a defined length, depending on the size of the poly(T) tract, but requires further manipulation of the template. The latter case does not require further manipulation of the DNA template, but involves enzymatically adding a poly(A) tail to in vitro transcribed mRNA using a poly(A) polymerase that catalyzes the incorporation of an adenine residue into the 3' end of the RNA, resulting in mRNA with poly(A) tails of different lengths. 5'-capping and 3'-poly(A) tailing can be performed using a variety of commercially available kits, including but not limited to the poly(A) polymerase tailing kit (EpiCenter), mMESSAGE mMACHINE T7 Ultra kit, and poly(A) tailing kit (Life Technologies), as well as commercially available reagents, various ARCA caps, poly(A) polymerases, etc.

[0027] In addition to 5' capping and 3' polyadenylation, other modifications of in vitro transcripts have been reported to provide benefits related to translational and stability efficiency. It is well known in the art that pathogenic DNA and RNA are recognized by various sensors within eukaryotes, triggering a potent innate immune response. Since most naturally occurring nucleic acids contain modified nucleosides, it has been shown that the ability to distinguish pathogenic from self DNA and RNA is at least partially based on structural and nucleoside modifications. In contrast, RNA synthesized in vitro lacks these modifications and is therefore immunostimulant, which can inhibit effective mRNA translation as outlined above.The introduction of modified nucleosides into in vitro transcribed mRNA can prevent the recognition and activation of RNA sensors and thus can be used to mitigate this undesirable immunostimulatory activity and enhance translational capacity (e.g., Kariko, K. and Weissman, D. 2007, Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: implication for therapeutic RNA development, Curr Opin Drug Discov Devel, v.10 523-532; Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH (See 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, Mol Ther v.16, 1833-1840). Modified nucleosides and nucleotides used in the synthesis of modified RNA can be monitored and utilized using common methods and procedures known in the art. A wide variety of nucleoside modifications are available, which may be incorporated to some extent into in vitro transcribed mRNA, either alone or in combination with other modified nucleosides (see, for example, US2012 / 0251618).In vitro synthesis of nucleoside-modified mRNA has been reported to have reduced ability to activate immune sensors, but with associated enhanced translational capacity.

[0028] Other components of mRNA that can be modified to offer benefits in terms of translatability and stability include the 5' and 3' untranslated regions (UTRs). Optimization of the UTR (advantageous 5' and 3' UTRs can be obtained from cellular or viral RNA), either in both or independently, has been shown to increase mRNA stability and translation efficiency of in vitro transcribed mRNA (see, for example, Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH Ed), 2013).

[0029] In addition to mRNA, other nucleic acid payloads may also be used in the present invention. Regarding oligonucleotides, preparation methods include, but are not limited to, chemical synthesis and enzymatic or chemical cleavage of longer precursors, as well as the in vitro transcription 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 of which are incorporated herein by reference).

[0030] With respect to plasmid DNA, the preparation for use in this invention generally utilizes, but is not limited to, the in vitro extension and isolation of plasmid DNA in a liquid culture of bacteria containing the plasmid of interest. The presence of genes in the plasmid of interest that encode resistance to a specific antibiotic (such as penicillin or kanamycin) allows bacteria containing the plasmid of interest to selectively grow in an antibiotic-containing culture. 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., Gillstroem, S., Bjoernestedt, R. and Schmidt, SR (2008) Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture. Biotechnol. Bioeng., 99:557-566; and 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 PureYield MaxiPrep (Promega) kits.

[0031] Various exemplary embodiments of the cationic lipids, lipid nanoparticles and compositions comprising the present invention, and their use for delivering active substances (e.g., therapeutic agents) such as nucleic acids for regulating gene and protein expression, are described in further detail below.

[0032] As used herein, the following terms have the meanings assigned to them unless otherwise specified.

[0033] Unless otherwise specified in the context, the word "comprise," including "comprises" and "comprising," and its variations thereto, should be interpreted throughout this specification and the claims as having a comprehensive meaning of "inclusive but not limited to."

[0034] Throughout this specification, the expression “one embodiment” or “embodiment” means that certain features, structures, or characteristics described in relation to an embodiment are included in at least one embodiment of the present invention. Therefore, the appearance of the expression “in one embodiment” or “in an embodiment” in various parts of this specification does not necessarily refer to the same embodiment. Furthermore, certain features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention pertains. As used herein and in the claims, the singular forms "a," "an," and "the" include plural references unless explicitly indicated by the context.

[0036] The phrase "induces the expression of a desired protein" refers to the ability of nucleic acids to increase the expression of a desired protein. To examine the degree of protein expression, a test sample (e.g., a sample of cultured cells expressing the desired protein) or a test mammal (e.g., a human, or a mammal such as an animal model, such as a rodent (e.g., mouse) or non-human primate (e.g., monkey) model) is brought into contact with nucleic acids (e.g., nucleic acids combined with the lipids of the present invention). The expression of the desired protein in the test sample or test animal is compared to the expression of the desired protein in a control sample (e.g., a sample of cultured cells expressing the desired protein) or a control mammal (e.g., a human, or a mammal such as an animal model, such as a rodent (e.g., mouse) or non-human primate (e.g., monkey) model) that has not been brought into contact with nucleic acids or has not been administered nucleic acids. If the desired protein is present in the control sample or control mammal, a value of 1.0 can be assigned to the expression of the desired protein in the control sample or control mammal. In certain embodiments, induction of the expression of a desired protein is achieved when the ratio of the expression of the desired protein in the test sample or test mammal to the level of the desired protein expression in the control sample or control mammal is greater than 1, for example, about 1.1, 1.5, 2.0, 5.0, or 10.0. If the desired protein is not present in the control sample or control mammal, induction of the expression of the desired protein is achieved when a measurable level of the desired protein is detected in the test sample or test mammal. Those skilled in the art will understand appropriate assays for determining the level of protein expression in a sample, such as dot blotting, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays, or assays based on reporter proteins that can produce fluorescence or luminescence under appropriate conditions.

[0037] The phrase "inhibits the expression of a target gene" refers to the ability of a nucleic acid to suppress, reduce, or inhibit the expression of a target gene. To investigate the degree of gene silencing, a test sample (e.g., a sample of cultured cells expressing the target gene) or a test mammal (e.g., a human, or a mammal such as an animal model, such as a rodent (e.g., mouse) or non-human primate (e.g., monkey) model) is brought into contact with the nucleic acid that suppresses, reduces, or inhibits the expression of the target gene. The expression of the target gene in the test sample or test animal is compared to the expression of the target gene in a control sample (e.g., a sample of cultured cells expressing the target gene) or a control mammal (e.g., a human, or a mammal such as an animal model, such as a rodent (e.g., mouse) or non-human primate (e.g., monkey) model) that has not been brought into contact with the nucleic acid or has not been administered the nucleic acid. A value of 100% can be assigned to the expression of the target gene in the control sample or control mammal. In certain embodiments, suppression, reduction, or inhibition of target gene expression is achieved when the level of target gene expression in the test sample or test mammal is approximately 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% relative to the level of target gene expression in the control sample or control mammal. In other words, nucleic acids can suppress, reduce, or inhibit the expression of a target gene in a test sample or test mammal by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the level of target gene expression in a control sample or control mammal that has not been in contact with or administered nucleic acids. Appropriate assays for determining the level of target gene expression include, but are not limited to, testing of protein or mRNA levels using techniques known to those skilled in the art, such as dot blotting, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those skilled in the art.

[0038] The “effective dose” or “therapeutic effective dose” of an active agent or therapeutic agent, such as a therapeutic nucleic acid, is an amount sufficient to produce a desired effect, such as an increase or inhibition of the expression of a target sequence compared to the normal expression level detected in the absence of the nucleic acid. For expression products that do not exist in the absence of nucleic acid, an increase in the expression of the target sequence is achieved when a measurable level is detected. If the expression product is present at a certain level before contact with the nucleic acid, Increased expression is achieved when the multiplier of the value obtained using nucleic acids such as mRNA compared to the control is approximately 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000 or higher. Inhibition of target gene or target sequence expression is achieved when the value obtained using nucleic acids such as antisense oligonucleotides compared to the control is approximately 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring the expression of a target gene or target sequence include, but are not limited to, dot blotting, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of a suitable reporter protein, and phenotypic assays known to those skilled in the art, as well as protein or mRNA level testing using techniques known to those skilled in the art.

[0039] As used herein, the term “nucleic acid” means a polymer comprising at least two deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form, and includes DNA, RNA, and hybrids thereof. DNA may be in the form of an antisense molecule, plasmid DNA, cDNA, PCR product, or vector. RNA may be in the form of small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA, dicer substrate RNA, or viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or bindings, which are synthetic, natural, and unnatural, and have similar binding properties to the reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2'-O-methylribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides having similar binding properties to the reference nucleic acid. Unless otherwise specified, a given nucleic acid sequence also implicitly includes its conserved modified variants (e.g., degenerate codon substitutions), alleles, orthologues, single nucleotide polymorphisms, and complementary sequences, as well as explicitly indicated sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). A “nucleotide” consists of the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together via phosphate groups."Bases" include, but are not limited to, natural analogs and synthetic derivatives of the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, purines, and pyrimidines, as well as purines and pyrimidines, and modifications involving novel reactive groups such as amines, alcohols, thiols, carboxylates, and alkyl halides.

[0040] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence containing a partial-length or full-length coding sequence necessary for the production of a polypeptide or precursor polypeptide.

[0041] As used herein, "gene product" refers to the product of a gene, such as an RNA transcript or polypeptide.

[0042] The term "lipids," though not limited, refers to a group of organic compounds that include fatty acid esters and are generally poorly soluble in water but soluble in many organic solvents. They are usually divided into at least three classes: (1) "simple lipids," which include oils and waxes; (2) "complex lipids," which include phospholipids and glycolipids; and (3) "derived lipids," such as steroids.

[0043] "Steroids" have the following carbon skeleton: [ka] It is a compound containing [a certain substance]. Non-specific examples of steroids include cholesterol, etc.

[0044] "Cationic lipids" refer to lipids that can be positively charged. Exemplary cationic lipids contain one or more positively charged amine groups. Preferred cationic lipids are ionizable so that they can exist in a positively charged or neutral form depending on the pH. Ionization of cationic lipids affects the surface charge of lipid nanoparticles under different pH conditions. This charge state can affect the absorption, blood clearance, and tissue distribution of plasma proteins (Semple, SC, et al., Adv. Drug Deliv Rev 32:3-17 (1998)) and the ability to form endosomal soluble non-bilayer structures important for intracellular nucleic acid delivery (Hafez, IM, et al., Gene Ther 8:1188-1196 (2001)).

[0045] The term "polymer complex lipid" refers to a molecule that contains both a lipid portion and a polymer portion. An example of a polymer complex lipid is a PEGylated lipid. The term "PEGylated lipid" refers to a molecule that contains both a lipid portion and a polyethylene glycol portion. PEGylated lipids are well known in the art, and examples include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG).

[0046] The term "neutral lipid" refers to any of many lipid species that exist in either an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, but are not limited to, phosphatidylcholines such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); phosphatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); steroids such as sphingomyelin (SM), ceramides, sterols and their derivatives. Neutral lipids may be synthetic or naturally occurring.

[0047] The term "charged lipid" refers to any of the many lipid species that exist in either a positively charged or negatively charged form, regardless of pH, for example, within a useful physiological range such as pH ~3 to pH ~9. Charged lipids may be synthetic or naturally occurring. Examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyltrimethylammonium propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, and dimethylaminoethanecarbamoylsterol (e.g., DC-Chol).

[0048] The term “lipid nanoparticles” refers to particles having at least one dimension on the order of nanometers (e.g., 1 to 1000 nm) that contain one or more compounds of structural formula (I) or other specific cationic lipids. In some embodiments, lipid nanoparticles are incorporated into formulations that can be used to deliver activators or therapeutic agents, such as nucleic acids (e.g., mRNA), to a target site of interest (e.g., cells, tissues, organs, tumors, etc.). In some embodiments, the lipid nanoparticles of the present invention contain nucleic acids. Such lipid nanoparticles typically contain the compound of structural formula (I) and one or more excipients selected from neutral lipids, charged lipids, steroids, and polymer complex lipids. In some embodiments, the activator or therapeutic agent, such as nucleic acids, may be encapsulated in the lipid portion of the lipid nanoparticle, or in an aqueous space enclosed by some or all of the lipid portion of the lipid nanoparticle, thereby protecting the activator or therapeutic agent from enzymatic degradation or other undesirable effects induced by host organism or cellular mechanisms, such as adverse immune responses.

[0049] In various embodiments, lipid nanoparticles are approximately 30nm to 150nm, 40nm to 150nm, 50nm to 150nm, 60nm to 130nm, 70nm to 110nm, 70nm to 100nm, 80nm to 100nm, 90nm to 100nm, 70nm to 90nm, 80nm to 90nm, 70nm to 80nm, and They have an average diameter of approximately 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially nontoxic. In certain embodiments, when nucleic acids are present in lipid nanoparticles, they are resistant to degradation by nucleases in aqueous solutions. Lipid nanoparticles containing nucleic acids and methods for preparing the same are disclosed, for example, in U.S. Patent Publications 2004 / 0142025, 2007 / 0042031 and International Publications WO 2013 / 016058 and WO 2013 / 086373, the entirety of which is incorporated into this application by whole reference for all purposes.

[0050] As used herein, “lipid encapsulation” refers to lipid nanoparticles that provide an activator or therapeutic agent, such as nucleic acid (e.g., mRNA), by complete encapsulation, partial encapsulation, or both. In one embodiment, nucleic acid (e.g., mRNA) is completely encapsulated in lipid nanoparticles.

[0051] As used herein, the term "aqueous solution" means a composition containing water.

[0052] "Serum stability" associated with nucleic acid-lipid nanoparticles means that nucleotides are not significantly degraded after exposure to serum or nuclease assays that significantly degrade free DNA or RNA. Suitable assays include, for example, standard serum assays, DNAse assays, or RNAse assays.

[0053] As used herein, "systemic delivery" means the delivery of a therapeutic product that can provide broad exposure to an active agent within an organism. Some techniques of administration may result in systemic delivery of a particular agent, while others may not. Systemic delivery means that a useful, preferably therapeutically useful, amount of the agent is exposed to most parts of the body. Systemic delivery of lipid nanoparticles can be effected by any means known in the art, such as intravenous, intra-arterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is effected by intravenous delivery.

[0054] As used herein, "local delivery" means the direct delivery of an active agent to a target site within an organism. For example, the agent can be locally delivered by direct injection to a diseased site such as a tumor, other target sites such as an inflammatory site, or a target organ such as the liver, heart, pancreas, or kidney. Local delivery can also include local application or local injection techniques such as intramuscular injection, subcutaneous injection, or intradermal injection. Local delivery does not exclude systemic pharmacological effects.

[0055] "Alkyl" is, for example, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, pent-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc., for example, 1 to 24 carbon atoms (C1-C 24 alkyl), 4 to 20 carbon atoms (C4-C 20 alkyl), 6 to 16 carbon atoms (C6-C 16 alkyl), 6 to 9 carbon atoms (C6-C9 alkyl), 1 to 15 carbon atoms (C1-C 15 alkyl), 1 to 12 carbon atoms (C1-C 12Alkyl) means a linear or branched hydrocarbon chain radical consisting only of carbon and hydrogen atoms, having 1 to 8 carbon atoms (C1-C8 alkyl) or 1 to 6 carbon atoms (C1-C6 alkyl), and bonded to the rest of the molecule by a single bond, and being saturated or unsaturated (i.e., containing one or more double (alkenyl) and / or triple (alkynyl) bonds). Unless otherwise specified herein, alkyl groups may be optionally substituted.

[0056] "Alkylene" or "alkylene chain" refers to a group of atoms, such as methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, etc., each containing 1 to 24 carbon atoms (C1-C1). 24 Alkylene), 1 to 15 carbon atoms (C1-C 15 Alkylene), 1 to 12 carbon atoms (C1-C 12 Alkylene refers to a linear or branched divalent hydrocarbon chain consisting only of carbon and hydrogen atoms, saturated or unsaturated (i.e., containing one or more double (alkenyl) and / or triple (alkynyl) bonds), having 1 to 8 carbon atoms (C1-C8 alkylene), 1 to 6 carbon atoms (C1-C6 alkylene), 2 to 4 carbon atoms (C2-C4 alkylene), and 1 to 2 carbon atoms (C1-C2 alkylene), with the rest of the molecule bonded to a radical group. The alkylene chain is bonded to the rest of the molecule via single or double bonds and to the radical group via single or double bonds. The bonding points of the alkylene chain to the rest of the molecule and the radical group can be via one carbon or any two carbons in the chain. Unless otherwise specified herein, alkylene chains may be optionally substituted.

[0057] "Cycloalkyl" or "carbocyclic ring" means a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting only of carbon and hydrogen atoms, which may include condensed or bridged ring systems having 3 to 15 carbon atoms, preferably 3 to 10 carbon atoms, and which are saturated or unsaturated and bonded to the rest of the molecule by single bonds. Examples of monocyclic groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of polycyclic groups include adamantyl, norbornyl, dekalinyl, and 7,7-dimethyl-bicyclo[2.2.1]heptanyl. Unless otherwise specified herein, cycloalkyl groups may be optionally substituted.

[0058] "Cycloalkylene" refers to a divalent cycloalkyl group. Unless otherwise specified herein, the cycloalkylene group may be substituted.

[0059] As used herein, the term “substituted” means any of the above groups (e.g., alkyl, alkylene, cycloalkyl, or cycloalkylene) in which at least one hydrogen atom is substituted by bonding to a non-hydrogen atom, but not limited to: halogen atoms such as F, Cl, Br, or I; oxygen group (=O); hydroxyl group (-OH); C1-C 12 Alkyl alkyl group; cycloalkyl 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 ' And so on, here:R ' Each instance of appearance is independent of H, C1-C. 15 It is alkyl or cycloalkyl, where x is 0, 1, or 2. In some embodiments, the substituent is C1-C 12 It is an alkyl group. In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group such as a fluoro group. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group (-OR ' In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group (-NR ' R ' )

[0060] "Optional" or "optionally" (for example, optionally substituted) means that the events of the situation described below may or may not occur, and the description includes both cases in which such events or situations occur and cases in which they do not. For example, "optionally substituted alkyl" means that the alkyl radical may or may not be substituted, and the description includes both substituted and unsubstituted alkyl radicals.

[0061] The term "prodrug" refers to a compound that can be converted to the biologically active compound of the present invention under physiological conditions or by solvolysis. Therefore, the term "prodrug" means a pharmaceutically acceptable metabolic precursor of the compound of the present invention. A prodrug may be inactive when administered to a subject requiring it, but is converted to the active compound of the present invention in vivo. Typically, a prodrug is rapidly converted in vivo, for example, by hydrolysis in the blood, to produce the parent compound of the present invention. Prodrug compounds often offer advantages in mammalian organisms, such as solubility, histocompatibility, or delayed release (see Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam)). A discussion of prodrugs is provided in Higuchi, T., et al., ACS Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

[0062] The term “prodrug” also means that such a prodrug comprises a 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 so that the modifications are cleaved either by conventional means or in vivo to form the parent compounds of the present invention. Examples of prodrugs include compounds of the present invention in which, when the prodrug of the compound of the present invention is administered to a mammalian subject, the hydroxy, amino, or mercapto group is bonded to one of the groups that cleave to form free hydroxy, free amino, or free mercapto groups, respectively. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohol or amide derivatives of the amine functional group in the compounds of the present invention.

[0063] The inventions disclosed herein also encompass all pharmaceutically acceptable compounds of the compound of structural formula (I) that are isotope-labeled by having one or more atoms substituted with atoms having different atomic masses or mass numbers. Examples of isotopes that may be incorporated into the disclosed compounds are, respectively, 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 36 Cl, 123 I, or 125 Examples include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as I. These radiolabeled compounds can be useful in determining or measuring the efficacy of compounds, for example, by characterizing the site or mode of action, or the binding affinity to pharmacologically important sites of action. Certain isotope-labeled compounds of structural formula (I) or (II), such as compounds incorporating radioisotopes, are useful in drug and / or substrate tissue distribution studies. Radioisotope tritium, i.e., 3 H, and carbon-14, that is, 14 C is particularly useful for this purpose in terms of ease of implementation and ease of detection.

[0064] Deuterium, that is, 2 Substitution with heavier isotopes, such as 1H, may offer certain therapeutic benefits resulting from greater metabolic stability, such as an increased in vivo half-life or reduced dose requirements, and may therefore be preferable in some cases.

[0065] 11 C, 18 F, 15 O and 13Substitution with positron-emitting isotopes such as 1N can be useful in positron emission tomography (PET) studies to investigate substrate receptor occupancy. The isotope-labeled compound of structural formula (I) can generally be prepared by conventional techniques known to those skilled in the art, or by methods similar to those described in the following preparations and examples, using a suitable isotope-labeled reagent instead of a previously used unlabeled reagent.

[0066] The present invention as disclosed herein also means encompassing in vivo metabolites of the disclosed compounds. Such products are obtained primarily from enzymatic processes, for example, from oxidation, reduction, hydrolysis, amidation, esterification, etc., of the administered compound. Accordingly, the present invention includes compounds produced by methods comprising administering the compounds of the present invention to a mammal for a period of time sufficient to produce its metabolites. Such products are typically identified by administering the radiolabeled compounds of the present invention to an animal such as a rat, mouse, guinea pig, monkey, or human in a detectable dose, giving sufficient time for metabolism to occur, and isolating the converted products from urine, blood, or other biological samples.

[0067] "Stable compound" and "stable structure" mean that the compound is robust enough to withstand isolation from the reaction mixture to a useful purity and formulation into an effective therapeutic agent.

[0068] "Mammals" include both humans and domesticated animals such as laboratory animals and household pets (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits) and non-domesticated animals such as wild animals.

[0069] "Pharmacologically acceptable carriers, diluents, or excipients" include, but are not limited to, adjuvants, carriers, excipients, flow enhancers, sweeteners, diluents, preservatives, colorants, flavor enhancers, surfactants, humectants, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers that are approved by the U.S. Food and Drug Administration as acceptable for use in humans or livestock.

[0070] "Pharmacologically acceptable salts" include both acid addition salts and base addition salts.

[0071] "Pharmacologically acceptable acid addition salts" are defined as those that retain the biological efficacy and properties of free bases that are biologically ineffective or otherwise undesirable, and include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid (but not limited to these), and acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonate, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, and glucosulfonic acid. This refers to salts formed with organic acids such as (but not limited to) ronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucinic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid.

[0072] A "pharmaceutically acceptable base addition salt" means a salt that preserves the biological efficacy and properties of a free acid that is biologically or otherwise undesirable. These salts are prepared by adding an inorganic or organic base to the free acid. Examples of salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, and aluminum salts. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts. Examples of salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines such as naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydravamin, choline, betaine, benetamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resins. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0073] Often, crystallization 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 compounds 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 may exist as hydrates including monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate, and the corresponding solvated forms. The compounds of the present invention may be true solvates, or in other cases, the compounds of the present invention may simply retain adventitious water, or may be a mixture of water and adventitious solvent.

[0074] "Pharmaceutical composition" means a formulation comprising the compound of the present invention and a medium commonly accepted in the art for the delivery of the biologically active compound to a mammal, such as a human. Such a medium may include any pharmaceutically acceptable carrier, diluent, or excipient.

[0075] "Effective dose" or "therapeutic effective dose" means the amount of the compound of the present invention that, when administered to a mammal, preferably a human, is sufficient to achieve therapy in a mammal, preferably a human. The amount of lipid nanoparticles of the present invention constituting the "therapeutic effective dose" varies depending on the compound, its state and its severity, the mode of administration, and the age of the mammal being treated, but can be routinely determined by those skilled in the art, taking into account their own knowledge and this disclosure.

[0076] As used herein, “to treat” or “to treat” means the treatment of a disease or condition in a mammal, preferably a human, that has the disease or condition in question, and includes the following: (i) In particular, to prevent the occurrence of such disease or condition in mammals when such mammals are susceptible to the condition but have not yet been diagnosed with it; (ii) To inhibit a disease or condition, that is, to prevent its onset; (iii) to alleviate the disease or condition, i.e., to cause regression of the disease or condition; or (iv) Relieving symptoms resulting from a disease or condition, i.e., relieving pain without addressing the underlying disease or condition. Where used herein, the terms “disease” and “condition” may be interchangeable, or they may differ in that a particular disease or condition has no known causative agent (the etiology is not yet understood) and is therefore not recognized as a disease, but only as an undesirable condition or syndrome for which a more or less specific set of symptoms has been identified by a clinician.

[0077] The compounds of the present invention or their pharmaceutically acceptable salts may contain one or more chiral centers, and thus may give rise to enantiomers, diastereomers and other stereoisomers that can be defined from the viewpoint of absolute stereochemistry as (R)- or (S)-, or (D)- or (L)- for amino acids. 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 / isolation of individual enantiomers include chiral synthesis from suitable optically pure precursors, or resolution of racemates (or racemates of salts or derivatives) using chiral high-pressure liquid chromatography (HPLC), for example. Where a compound described herein contains an olefinic double bond or other geometrically asymmetric center, unless otherwise specified, the compound is intended to include both E and Z geometric isomers. Similarly, all tautomers are also intended to be included.

[0078] "Stereoisomers" mean compounds composed of the same atoms bonded by the same bonds, but having different three-dimensional structures and being incompatible with each other. The present invention contemplates various stereoisomers and mixtures thereof, including "enantiomers", which are two stereoisomers whose molecules are mirror images that do not overlap with each other.

[0079] "Tautomers" mean a proton shift from one atom of a molecule to another atom of the same molecule. The present invention includes tautomers of any of the above compounds.

[0080] compound In one aspect, the present invention provides novel lipid compounds that can form oligonucleotide-lipid nanoparticles in combination with other lipid components such as neutral lipids, charged lipids, steroids and / or polymer complex lipids. Without wishing to be bound by theory, these lipid nanoparticles are thought to shield oligonucleotides from degradation in serum and provide effective delivery of oligonucleotides to cells in vitro and in vivo.

[0081] In one embodiment, the compound has the following structural formula (I): [Chemical formula] I [Wherein, L 1 or L 2 One of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -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 the other of L 1 or L 2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x-, -S-S-, -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- or a direct bond; G 1 and G 2 are each independently unsubstituted C1-C 12 alkylene or C1-C 12 alkenylene; G 3 is C1-C 24 alkylene, C1-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; R a is H or C1-C 12 alkyl; R 1 and R 2 are each independently C6-C 24 alkyl or C6-C 24 alkenyl; R 3 is H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 ; R 4 is C1-C 12 alkyl; R 5 is H or C1-C6 alkyl; and x is 0, 1 or 2] A compound having the same, or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

[0082] In some of the foregoing embodiments, the compound has the following structural formula (IA) or (IB):

Chemical formula

[0083] In some of the embodiments described above, the compound has structural formula (IA), and in other embodiments, the compound has structural formula (IB).

[0084] In some of the embodiments described above, the compound has the following structural formula (IC) or (ID): [ka] (I C) or [ka] (ID) [In the formula, y and z are each independent integers between 1 and 12. It has one of the following.

[0085] In any of the embodiments described above, L 1 or L 2 One of them is -O(C=O)-. For example, in some embodiments, L 1 and L 2 Each of them is -O(C=O)-. In any of the several different embodiments described above, L 1 and L 2 Each of these is independently -(C=O)O- or -O(C=O)-. For example, in some embodiments, L 1 and L2 The equation is -(C=O)O-.

[0086] In the various embodiments described above, the compound has the following structural formula (IE) or (IF): [ka] (IE) or [ka] (IF) It has one of the following.

[0087] In some of the embodiments described above, the compound has the following structural formulas: (IG), (IH), (II), or (IJ): [ka] (IG); [ka] (IH); [ka] (II) or [ka] (IJ) It has one of the following.

[0088] In some of the embodiments described above, n is an integer between 2 and 12, for example, between 2 and 8 or between 2 and 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.

[0089] In some other embodiments described above, y and z are independently integers between 2 and 10. For example, in some embodiments, y and z are independently integers between 4 and 9 or between 4 and 6.

[0090] In some of the embodiments described above, R 6 H is H. In other embodiments described above, R is H. 6 C1-C 24 It is alkyl. In other embodiments, R 6 It is OH.

[0091] In some embodiments, G 3 It is not replaced. In other embodiments, G 3 is replaced. In various different embodiments, G 3 is a linear C1-C 24 Alkylene or linear C1-C 24 It is alkenylene.

[0092] In some other embodiments of the access, R 1 or R 2 , or both, C6-C 24 It is an alkenyl. For example, in some embodiments, R 1 and R 2 Each of these has the following structural formula: [ka] [In the formula, R 7a and R 7b Each instance is independently H or C1-C 12 It is alkyl; and a is an integer between 2 and 12; Here, R 7a , R 7b and a are R 1 and R 2 Each of these is independently selected to contain 6 to 20 carbon atoms. It has, for example, in some embodiments, a is an integer between 5 and 9 or between 8 and 12.

[0093] In some of the embodiments described above, R 7a The existence of at least one of is H. For example, in some embodiments, R 7a Each time it appears, it is H. In the other different embodiments described above, R 7b The presence of at least one of the elements is a C1-C8 alkyl group. For example, in some embodiments, the C1-C8 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0094] In different embodiments, R 1 or R 2 , or both, have the following structural formula: [ka] It has one of the following.

[0095] In some of the embodiments described above, R 3 OH, CN, -C(=O)OR 4 -OC(=O)R 4 Or -NHC(=O)R 4 In some embodiments, R 4 It is either methyl or ethyl.

[0096] In various different embodiments, the compound has one of the structures shown in Table 1 below.

[0097] Table 1 Representative compounds [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7]

[0098] It is understood that any embodiment of the compound of structural formula (I) as described above, and any particular substituent and / or variable in the compound of structural formula (I), can independently be combined with substituents and / or variables in other embodiments and / or compounds of structural formula (I) to form embodiments of the invention not specifically described above. Furthermore, if a list of substituents and / or variables for any particular R group, L group, G group, A group, or variable a, n, x, y, or z is enumerated in a particular embodiment and / or claim, each individual substituent and / or variable may be removed from the particular embodiment and / or claim, and the remaining list of substituents and / or variables is considered to be within the scope of the invention.

[0099] In this description, it is understood that combinations of substituents and / or variables in the given formulas are acceptable only if such contributions result in a stable compound.

[0100] In some embodiments, compositions are provided comprising one or more compounds of structural formula (I) and a therapeutic agent. For example, in some embodiments, the composition comprises one of the compounds of structural formula (I) and a therapeutic agent and one or more excipients selected from neutral lipids, steroids, and polymer complex lipids. Other pharmaceutically acceptable excipients and / or carriers are also included in various embodiments of the composition.

[0101] 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 ranges from about 2:1 to about 8:1.

[0102] In various embodiments, the composition further comprises a steroid or steroid analog. In certain embodiments, the steroid or steroid analog is cholesterol. In some of these embodiments, the molar ratio of the compound to cholesterol is in the range of about 5:1 to 1:1.

[0103] In various embodiments, the polymer complex lipid is a PEGylated lipid. For example, some embodiments include PEGylated diacylglycerols (PEG-DAG) such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEGylated diacylglycerol PEG succinate (PEG-S-DAG) such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanediate (PEG-S-DMG), PEGylated ceramide ((PEG-cer), or PEG dialkoxypropyl carbamates such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of the compound to the PEGylated lipid ranges from about 100:1 to about 20:1.

[0104] In some embodiments, the composition is as follows: [ka] (II) [In the formula, R 8 and R 9 Each of these is independently a linear or branched saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, where the alkyl chain is optionally interrupted by one or more ester bonds; and w has an average value in the range of 30-60. This includes PEGylated lipids having the same properties, or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof.

[0105] In some embodiments, R 8 and R 9Each of these is independently a linear, saturated alkyl chain containing 12 to 16 carbon atoms. In some embodiments, w has an average value in the range of 43 to 53. In other embodiments, the average w is about 45. In yet another different embodiment, the average w is about 49.

[0106] In some embodiments of the above composition, the therapeutic agent comprises a nucleic acid. For example, in some embodiments, the nucleic acid is selected from antisense RNA and messenger RNA.

[0107] In other different embodiments, the present invention relates to a method for administering a therapeutic agent to a patient in need thereof, comprising preparing or preparing one of the compositions, and administering the composition to the patient.

[0108] For administration purposes, the compounds of the present invention (typically in the form of lipid nanoparticles combined with a therapeutic agent) may be administered as raw chemicals or formulated as pharmaceutical compositions. The pharmaceutical compositions of the present invention comprise the compound of structural formula (I) and one or more pharmaceutically acceptable carriers, diluents, or excipients. The compound of structural formula (I) forms lipid nanoparticles and is present in the composition in an amount effective to deliver a therapeutic agent for treating a specific disease, for example, the condition of interest. Appropriate concentrations and doses can be readily determined by those skilled in the art.

[0109] The compositions of the present invention may be administered via any of the accepted modes of administration for drugs to perform similar utility. The pharmaceutical compositions of the present invention may be formulated into solid, semi-solid, liquid, or gaseous formulations such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, myrospheres, and aerosols. Typical routes for administering such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and nasal administration. As used herein, the term parenteral includes subcutaneous, intravenous, intramuscular, intradermal, intrasternal, intrasternal injection, or infusion techniques. The pharmaceutical compositions of the present invention are formulated so that when the composition is administered to a patient, the active ingredients contained therein become bioavailable. The composition administered to a subject may take the form of one or more dosing units; for example, a tablet may be a single dosing unit, or a container of the compound of the present invention in aerosol form may hold multiple dosing units. Practical methods for preparing such drug formulations are known or will be apparent 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 administered composition will contain a therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof for the treatment of the disease or condition in question, in accordance with the teachings of the present invention.

[0110] The pharmaceutical composition of the present invention may be solid or liquid. In one embodiment, the carrier is particulate, such that the composition is, for example, in powder form. The carrier may be liquid, and the composition may be, for example, an oral syrup, an injection, or an aerosol, which is useful for, for example, inhalation administration.

[0111] When intended for oral administration, the pharmaceutical composition is preferably either a solid or a liquid, and semi-solid, semi-liquid, suspension, and gel forms are included within the forms that are considered herein to be either solid or liquid.

[0112] As a solid composition for oral administration, the pharmaceutical composition may be formulated in the form of powders, granules, compressed tablets, pills, capsules, chewing gum, wafers, etc. Such a solid composition typically contains one or more inert diluents or food carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethercellulose, microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch, lactose, or dextrin; disintegrants such as alginic acid, sodium alginate, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotex; flow enhancers such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavorings such as peppermint, methyl salicylate, or orange flavor; and colorants.

[0113] If the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may include a liquid carrier such as polyethylene glycol or oil, in addition to the above-mentioned type of material.

[0114] The pharmaceutical composition may be in the form of a liquid, such as an elixir, syrup, solution, emulsifier, or suspension. The liquid may be, as two examples, for oral administration or for delivery by injection. When intended for oral administration, a preferred composition includes, in addition to the compounds of the present invention, one or more sweeteners, preservatives, colorants, and flavor enhancers. In compositions intended for administration by injection, one or more surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents may be included.

[0115] The liquid pharmaceutical composition of the present invention, whether in solution, suspension or other similar form, may contain one or more of the following adjuvants: water for injection, physiological saline, preferably physiological saline, Ringer's solution, isotonic sodium chloride, non-volatile oils such as synthetic mono or diglycerides that can act as a solvent or suspension medium, polyethylene glycol, glycerin, propylene glycol or other solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffering agents such as acetates, citrates or phosphates, and tonicity modifiers such as sodium chloride or dextrose; and agents acting as cryoprotective agents such as sucrose or trehalose. Parenteral formulations may be sealed in glass or plastic ampoules, disposable syringes or multi-dose vials. Physiological saline is a preferred adjuvant. The injectable pharmaceutical composition is preferably sterile.

[0116] A liquid pharmaceutical composition of the present invention intended for either parenteral or oral administration should contain an amount of the compound of the present invention that provides an appropriate dosage.

[0117] The pharmaceutical compositions of the present invention may be intended for topical administration, in which case the carrier may appropriately comprise a solution, emulsion, ointment, or gel base. For example, the base may include one or more of the following: diluents such as petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, water, and alcohol, as well as emulsifiers and stabilizers. Thickeners may be present in the pharmaceutical composition for topical administration. When transdermal administration is intended, the composition may comprise a transdermal patch or an iontophoresis device.

[0118] The pharmaceutical compositions of the present invention may be intended for rectal administration, for example, in the form of suppositories, which dissolve in the rectum and release the drug. Compositions for rectal administration may contain an oily base as a suitable non-irritating excipient. Examples of such bases include, but are not limited to, lanolin, cocoa butter, and polyethylene glycol.

[0119] The pharmaceutical compositions of the present invention may contain various substances that modify the physical form of a solid or liquid dosage unit. For example, the composition may include a material that forms a coating shell around the active ingredient. The material forming the coating shell is typically inert and may be selected from, for example, sugars, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule.

[0120] The pharmaceutical compositions of the present invention, in solid or liquid form, may include agents that bind to the compounds of the present invention and thereby assist in the delivery of the compounds. Suitable agents that can act in this capacity include monoclonal or polyclonal antibodies or proteins.

[0121] The pharmaceutical compositions of the present invention may 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 to systems consisting of pressurized packages. Delivery may be by liquefied or compressed gas, or by a suitable pump system for dispensing the active ingredient. Aerosols of the compounds of the present invention may be delivered in single-phase, two-phase, or three-phase systems to deliver the active ingredient. Aerosol delivery may include necessary containers, activators, valves, subcontainers, etc., which together may form a kit. Those skilled in the art can determine a preferred aerosol without excessive experimentation.

[0122] The pharmaceutical compositions of the present invention can be prepared by methodologies well known in the pharmaceutical field. For example, a pharmaceutical composition intended for administration by injection can be prepared by combining the lipid nanoparticles of the present invention with sterile distilled water or other carriers to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. The surfactant is a compound that interacts non-covalently with the compounds of the present invention to promote the dissolution or homogeneous suspension of the compounds in an aqueous delivery system.

[0123] The compositions of the present invention or their pharmaceutically acceptable salts are administered in therapeutically effective doses that will vary depending on various factors including the activity of the particular therapeutic agent used: metabolic stability and duration of action of the therapeutic agent; the patient's age, weight, overall health, sex, and diet; mode and timing of administration; elimination rate; drug combinations; severity of a particular disorder or condition; and the subject being treated.

[0124] The composition of the present invention may be administered simultaneously with, before, or after the administration of one or more other therapeutic agents. Such combination therapies include the administration of a single pharmaceutical formulation of the composition of the present invention and one or more additional active agents, as well as the administration of each active agent in the composition of the present invention and in a separate pharmaceutical formulation of its own. For example, the composition of the present invention and other active agents may be administered together to a patient in a single oral formulation such as a tablet or capsule, or as each agent in separate oral formulations. When separate formulations are used, the compound of the present invention and one or more additional active agents may be administered essentially at the same time, i.e., simultaneously, or separately alternately, i.e., sequentially; combination therapy is understood to include all of these administration plans.

[0125] Methods for preparing the above compounds and compositions are described later in this specification and / or are known in the art.

[0126] Those skilled in the art will understand that in the methods described herein, it may be necessary to protect the functional groups of intermediate compounds with appropriate protecting groups. Such functional groups include hydroxy, amino, mercapto, and carboxylic acids. Suitable protecting groups for hydroxyl groups include trialkylsilyl or diarylalkylsilyl (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl, or trimethylsilyl), tetrahydropyranyl, and benzyl. Suitable protecting groups for amino, amidino, and guanidino groups include t-butoxycarbonyl and benzyloxycarbonyl. Suitable protecting groups for mercapto groups include -C(O)-R" (wherein R" is alkyl, aryl, or arylalkyl), p-methoxybenzyl, and trityl. Suitable protecting groups for carboxylic acids include alkyl, aryl, or arylalkyl esters. These protecting groups are known to those skilled in the art and can be added or removed according to the standard techniques described herein. The use of protecting groups is described in Green, TW and PGM Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As will be understood by those skilled in the art, the protecting group may be a polymer resin such as Wang resin, Rink resin, or 2-chlorotrityl chloride resin.

[0127] Furthermore, as will be understood by those skilled in the art, such protected derivatives of the compounds of the present invention do not necessarily have pharmacological activity themselves, but they can be administered to mammals and subsequently metabolized in the body to form pharmacologically active compounds of the present invention. Therefore, such derivatives can be described as "prodrugs." All prodrugs of the compounds of the present invention are included within the scope of the present invention.

[0128] Furthermore, all compounds of the present invention, existing in the form of free bases or acids, can be converted to their pharmaceutically acceptable salts by treatment with a suitable inorganic or organic base or acid using methods known to those skilled in the art. Salts of the compounds of the present invention can be converted to their free base or acid form using standard techniques.

[0129] The following general reaction scheme 1 is for the compound of the present invention, i.e., structural formula (I): [ka] (I) [In the formula, R 1 , R 2 , R 3 , L 1 , L 2 , G 1 , G 2 and G 3 [This is as defined herein] Methods for producing the compounds, or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof, are described. Those skilled in the art will understand that these compounds can be produced by similar methods or in combination with other methods known to those skilled in the art. It will also be understood that those skilled in the art can produce other compounds of structural formula (I) not specifically shown below by using appropriate starting components and, as necessary, modifying the parameters of the synthesis, in a manner similar to that described below. Generally, starting components can be obtained from suppliers such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, or synthesized according to sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)), or prepared as described in this invention.

[0130] General reaction scheme 1 [ka]

[0131] General reaction scheme 1 provides an exemplary method for preparing the compound of structural formula (I). G in general reaction scheme 1 1 , G 3 , R 1 and R 3 This is defined herein, and G 1 ' is G 1 This means a homolog of the original with one carbon less. The compound of structural formula A-1 is purchased or prepared according to methods known in the art. The reaction of A-1 with diol A-2 under suitable condensation conditions (e.g., DCC) produces ester / alcohol A-3, which can then be oxidized to aldehyde A-4 (e.g., PCC). The reaction of A-4 with amine A-4 under reductive amination conditions yields the compound of structural formula (I).

[0132] It should be noted that various alternative methods for preparing the compound of structural formula (I) are available to those skilled in the art. For example, L 1 and L 2 Other compounds with structural formula (I) other than esters can be prepared using appropriate starting materials according to similar methods. Furthermore, general reaction scheme 1 is G 1 and G 2 This shows the preparation of a compound having the same structural formula (I); however, this is not a necessary aspect of the present invention, G 1 and G 2 The above reaction scheme can be modified to obtain different compounds. It will be readily apparent to those skilled in the art that protecting groups may be used as needed, and that other modifications may be made to the above general reaction scheme.

[0133] The following examples are provided for illustrative purposes only and are not intended to be limiting. [Examples]

[0134] In vivo evaluation of luciferase mRNA using lipid nanoparticle compositions Cationic lipids, DSPC, cholesterol, and PEG-lipids were solubilized 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 10–50 mM citrate buffer, pH 4. Using a syringe pump, the ethanol-soluble lipid solution was mixed with the mRNA aqueous solution at a total flow rate of over 15 ml / min at a ratio of approximately 1:5 to 1:3 (vol / vol). The ethanol was then removed, and the external buffer was replaced with PBS by dialysis. Finally, the lipid nanoparticles were filtered through a 0.2 μm pore sterile filter. The particle size of the lipid nanoparticles, when measured 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.

[0135] 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) in accordance with guidelines established by the Board of Animal Care (ACC) and the Canadian Council for Animal Health (CCAC). mRNA-lipid nanoparticles were administered systemically by tail vein injection at varying doses, and the animals were euthanized at a specific time point after administration (e.g., 4 hours). The liver and spleen were collected in pre-weighed tubes, weighed, and immediately rapid-frozen in liquid nitrogen, and stored at -80°C until processing for analysis.

[0136] For liver tissue, approximately 50 mg was cut into 2 mL FastPrep tubes (MP Biomedicals, Solon OH) for analysis. A 1 / 4-inch ceramic sphere (MP Biomedicals) was added to each tube, and 500 μL of Glo Lysis Buffer-GLB (Promega, Madison WI), equilibrated to room temperature, was added to the liver tissue. The liver tissue was homogenized at 2 × 6.0 m / s for 15 seconds using a FastPrep24 instrument (MP Biomedicals). After incubating the homogenate at room temperature for 5 minutes, it was diluted 1:4 in GLB and evaluated using the SteadyGlo luciferase assay system (Promega). Specifically, 50 μL of diluted tissue homogenate was reacted with 50 μL of SteadyGlo substrate, shaken for 10 seconds, incubated for 5 minutes, and then quantified using a CentroXS3 LB 960 illuminometer (Berthold Technologies, Germany). The amount of protein assayed was determined using the BCA protein assay kit (Pierce, Rockford IL). Then, relative luminescence units (RLUs) were normalized relative to the total protein assayed in μg. A standard curve was created using QuantiLum Recombinant Luciferase (Promega) to convert RLUs to ng luciferase. Based on the data shown in Figure 1, the 4-hour time point was selected for evaluating the efficacy of the lipid formulation.

[0137] FLuc mRNA (L-6107) from Trilink Biotechnologies expresses the luciferase protein originally isolated from the firefly Photinus pyralis. FLuc is commonly used in mammalian cell culture to measure both gene expression and cell viability. It emits bioluminescence in the presence of the substrate luciferin. This capped and polyadenylated mRNA is completely replaced with 5-methylcytidine and pseudouridine. [Examples]

[0138] PK of formulated lipids a decision As described elsewhere, the pKa of formulated cationic lipids correlates with the efficacy of LNPs for nucleic acid delivery (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176 (2010)). a The preferred range is ~5 to ~7. The pK of each cationic lipid a The fluorescence intensity was measured in lipid nanoparticles using a fluorescence-based assay for 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). Lipid nanoparticles containing cationic lipids / DSPC / cholesterol / PEG-lipids (50 / 10 / 38.5 / 1.5 mol%) in PBS at a total lipid concentration of 0.4 mM were prepared using the in-line process described in Example 1. TNS was prepared as a 100 μM stock solution in distilled water. The vesicles were diluted to 24 μM lipid in 2 mL of buffer solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl (where the pH was in the range of 2.5 to 11). Aliquots of the TNS solution were added to a final concentration of 1 μM, and after vortex mixing, the fluorescence intensity was measured at room temperature using an SLM Aminco Series 2 emission spectrophotometer with excitation and emission wavelengths of 321 nm and 445 nm. S-shaped best fit analysis is applied to fluorescence data, pK a The pH was measured as the value that produced half of the maximum fluorescence intensity (see Figure 2). [Examples]

[0139] Determination of the efficacy of lipid nanoparticle formulations containing various cationic lipids using an involuciferase mRNA expression rodent model. The cationic lipids shown in Table 2 have been previously tested with nucleic acids. For comparative purposes, lipid nanoparticles containing FLuc mRNA (L-6107) were formulated using these lipids by an in-line mixing method, as described in Example 1 and PCT / US10 / 22614 (which is incorporated herein by reference in its entirety). Lipid nanoparticles were formulated using a molar ratio of 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid ("PEG-DMG", i.e., (1-(monomethoxy-polyethylene glycol)-2,3-dimiristoylglycerol) with an average PEG molecular weight of 2000). Relative activity was determined by measuring luciferase expression in the liver 4 hours after administration by tail vein injection, as described in Example 1. Activity was compared at doses of 0.3 and 1.0 mg mRNA / kg and expressed as luciferase ng / liver g measured 4 hours after administration, as described in Example 1.

[0140] Table 2 Comparative lipids showing activity using mRNA [Table 8]

[0141] The representative compounds of the present invention shown in Table 3 were formulated using the following molar ratios: A) 50% cationic lipid / 10% distearoyl phosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid ("PEG-DMA", 2-[2-(ω-methoxy(polyethylene glycol) 2000([Ethoxy]-N,N-ditetradecylacetamide), or B) 47.5% cationic lipid / 10% DSPC / 40.8% cholesterol / 1.7% PEG lipid. Relative activity was determined by measuring luciferase expression in the liver 4 hours after administration by tail vein injection as described in Example 1. Activity was compared at doses of 0.3 and 1.0 mg mRNA / kg and expressed as luciferase ng / g liver measured 4 hours after administration as described in Example 1. A plot of the selected data is shown in Figure 3 (top to bottom: triangle = compound 3; circle = compound 2; cross = compound 1; square = MC3)

[0142] Table 3 Novel Cationic Lipids and Related Activities [Table 9] [Table 10] [Table 11] [Table 12] [Examples]

[0143] Synthesis of 6-(2'-hexyldecanoyloxy)hexane-1-R A solution of hexane-1,6-diol (27.6 g) in methylene chloride (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 3 days. The reaction mixture was filtered and hexane (500 mL) was added to the filtrate. The mixture was stirred and the precipitate was allowed to settle. 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 give 30 g of a crude product.

[0144] The crude product was dissolved in methylene chloride (200 mL) and treated with pyridinium chlorochromate (15 g) for 2 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 plug and the solvent was removed. The residue was passed through a silica gel column (80 g) using hexane followed by methylene chloride as eluents. 6-(2'-Hexyldecanoyloxy)hexan-1-al (24 g) was obtained as a colorless oil.

Example

[0145] Synthesis of 4-(2'-hexyldecanoyloxy)butane-1-R A solution of butane-1,4-diol (12.5 g) in methylene chloride (200 mL) 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 and the solvent was removed. The residue was dissolved in methylene chloride 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.

[0146] The crude product was dissolved in methylene chloride (150 mL) and treated with pyridinium chlorochromate (6 g) for 1 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 and the solvent was removed to give 4-(2'-hexyldecanoyloxy)butan-1-al (11 g) as a colorless oil.

Example

[0147] Synthesis of Compound 1 A solution of 6-(2'-hexyldecanoyloxy)hexane-1-R (3.0 g), acetic acid (0.21 g), and ethanolamine (0.14 g) in methylene chloride (50 mL) was treated overnight with sodium triacetoxyborohydride (1.4 g). 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 / methylene chloride (0-8 / 100-92%) gradient to obtain compound 1 as a colorless oil (0.63 g). [Examples]

[0148] Synthesis of Compound 2 A solution of 6-(2'-hexyldecanoyloxy)hexane-1-R (3.0 g), acetic acid (0.33 g), and 3-aminopropan-1-ol (0.17 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.3 g) for 1 hour. 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 / methylene chloride (0-8 / 100-92%) gradient to obtain compound 2 as a colorless oil (1.1 g). [Examples]

[0149] Synthesis of Compound 3 A solution of 6-(2'-hexyldecanoyloxy)hexane-1-R (2.4 g), acetic acid (0.33 g), and 4-aminobutan-1-ol (0.17 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.3 g) for 2 hours. 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 a methanol / methylene chloride (0-8 / 100-92%) gradient to obtain compound 3 as a colorless oil (0.4 g). [Examples]

[0150] Synthesis of Compound 4 A solution of 4-(2'-hexyldecanoyloxy)butane-1-R (2.4 g), acetic acid (0.30 g), and 4-aminobutan-1-ol (0.22 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.3 g) for 2 hours. The solution was washed with dilute sodium hydroxide aqueous 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 / methylene chloride (0-8 / 100-92%) gradient. The partially purified fraction was passed through a second column using an acetic acid / methylene chloride (2-0 / 0-10 / 98-90%) gradient. The pure fraction was washed with sodium bicarbonate aqueous solution to obtain compound 4 as a colorless oil (0.9 g). [Examples]

[0151] Synthesis of Compound 5 A solution of 4-(2'-hexyldecanoyloxy)butane-1-R (2.4 g), acetic acid (0.31 g), and 3-aminopropan-1-ol (0.17 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.4 g) for 1 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 / methylene chloride (0-8 / 100-92%) gradient. The partially purified fraction was passed through a second column using an acetic acid / methylene chloride (2-0 / 0-8 / 98-92%) gradient. The pure fraction was washed with an aqueous sodium bicarbonate solution to obtain compound 5 as a colorless oil (0.57 g). [Examples]

[0152] Synthesis of Compound 6 A solution of 4-(2'-hexyldecanoyloxy)butan-1-al (2.4 g), acetic acid (0.30 g) and ethanolamine (0.14 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.3 g) for 2 hours. The solution was washed with an aqueous sodium hydrogen carbonate 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 / methylene chloride (0-10 / 100-90%) gradient. The partially purified fraction was passed through a second column using an acetic acid / methanol / methylene chloride (2-0 / 0-9 / 98-92%) gradient. The pure fraction was washed with an aqueous sodium hydrogen carbonate solution, and Compound 6 was obtained as a colorless oil (0.2 g).

Example

[0153] Synthesis of Compound 7 A solution of 6-(2'-hexyldecanoyloxy)hexan-1-al (2.4 g), acetic acid (0.14 g) and 5-aminopentan-1-ol (0.24 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.3 g) for 2 hours. The solution was washed with an aqueous sodium hydrogen carbonate 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 / methylene chloride (0-8 / 100-92%) gradient, and Compound 7 was obtained as a colorless oil (0.5 g).

Example

[0154] Synthesis of compound 8 A solution of 6-(2'-hexyldecanoyloxy)hexan-1-al (2.4 g), acetic acid (0.17 g) and 6-aminohexan-1-ol (0.26 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.3 g) for 2 hours. The solution was washed with an aqueous sodium hydrogen carbonate 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 / methylene chloride (0-8 / 100-92%) gradient, and Compound 8 was obtained as a colorless oil (0.5 g). [Examples]

[0155] Synthesis of compound 9 A solution of 6-(2'-hexyldecanoyloxy)hexane-1-R (2.4 g) and trans-2-aminocyclohexanol hydrochloride (0.35 g) in methylene chloride (10 mL) / tetrahydrofuran (10 mL) was treated with sodium triacetoxyborohydride (1.3 g) for 1.5 hours. 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 a methanol / methylene chloride (0-8 / 100-92%) gradient to obtain compound 9 as a colorless oil (0.6 g). [Examples]

[0156] Synthesis of compound 10 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 heated at 63°C (oil bath) for 16 hours. A trace amount of tetrabutylammonium iodide was added to the mixture, and the mixture was heated under reflux for a further 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 oily substance (1.6 g). The residue (1.6 g) was purified by silica gel column chromatography (MeOH in chloroform, 0-4%). This yielded compound 10 as a colorless oily substance (700 mg, 0.82 mmol, 47%). [Examples]

[0157] Synthesis of compound 11 To a solution of 2-aminoethanol (116 mg, 1.9 mmol, 115 μL) in anhydrous THF (15 mL), 2-hexyldecyl 6-bromohexanoate (1.9 equivalents, 1.52 g, 3.62 mmol), potassium carbonate (1.9 equivalents, 3.62 mmol, 500 mg), cesium carbonate (0.3 equivalents, 0.57 mmol, 186 mg), and sodium iodide (10 mg) were added and heated under argon for 6 days under reflux. The solvent was evaporated under reduced pressure, the residue was transferred to hexane, and washed with water and brine. The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure to obtain a colorless oil. The crude product was purified by silica gel flash column chromatography (MeOH in chloroform, 0-4%) to obtain compound 11 as a colorless oil (936 mg, 1.27 mmol, 70%). [Examples]

[0158] Synthesis of compound 12 Compound 12 was prepared using a method similar to that of Compound 11 to obtain 538 mg of a colorless oily substance, 0.86 mmol, and 57% concentration. [Examples]

[0159] Synthesis of compound 13 To a solution of 2-aminoethanol (171 mg, 2.81 mmol, 169 μL) in anhydrous THF (30 mL), 2-octyldodecyl 4-bromobutyrate (1.9 equivalents, 2.386 g, 5.33 mmol), potassium carbonate (1.9 equivalents, 5.33 mmol, 736 mg), cesium carbonate (0.3 equivalents, 0.84 mmol, 275 mg), and sodium iodide (10 mg) were added and heated under argon for 16 hours under reflux. TLC (hexane / ethyl acetate = 9:1, CHCl3 / MeOH = 19:1) showed that a significant amount of 2-octyl-1-dodecanol was produced. The mixture was cooled and filtered. The filtrate was concentrated and the residue was dissolved in 2-octyl-1-dodecanol (2.1 g). Several beads of 4 Å molecular sieves and N,N-diisopropylethylamine (1.9 equivalents, 5.33 mmol, 683 mg, 0.92 mL) were added. The mixture was sealed and heated at 62°C for a further 4 days. The reaction mixture was cooled. Hexane was added. The hexane solution was decanted and concentrated to dryness. The residue was purified by silica gel column chromatography (MeOH in chloroform, 0-4%) to obtain compound 13 as a colorless oil (282 mg, 0.35 mmol, 13%). [Examples]

[0160] Synthesis of compound 14 To a solution of heptadecan-9-yl-6-bromohexanoate (2 equivalents, 1.13 g, 2.61 mmol) in anhydrous THF (15 mL), 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 argon for 7 days under reflux. The solvent was evaporated under reduced pressure, and the residue was taken to 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 a colorless oil (1 g). The residue (1 g) was purified by gravity column chromatography on silica gel (MeOH in DCM, 0-4%). ​​Compound 14 was obtained as a colorless oil (757 mg, 0.99 mmol, 76%). [Examples]

[0161] Synthesis of compound 15 To a solution of 2-hexyldecyl 5-bromopentanoate (2 equivalents, 1.22 g, 3 mmol) in anhydrous THF (15 mL) (opened 2 months ago), potassium carbonate (2 equivalents, 3 mmol, 415 mg), cesium carbonate (0.3 equivalents, 0.45 mmol, 146 mg), sodium iodide (6 mg), and 4-amino-1-butanol (1 equivalent, 1.5 mmol, 0.134 mg, 139 μL) were added. The mixture was heated under argon for 6 days under reflux. The solvent was evaporated under reduced pressure, and the residue was taken to a mixture of hexane and 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 a colorless oily substance (1.12 g). The residue (1 g) was purified by silica gel column chromatography (MeOH in chloroform, 0-5%). This yielded compound 15 as a colorless oily substance (487 mg, 0.66 mmol, 44%). 1 HNMR (400 MHz, CDCl3) δ:5.99 (s, 1H), 3.98 (d, 5.8 Hz, 4H), 3.56 (t-like, 4.8 Hz, 2H), 2.48-2.41 (m, 6H), 2.33 (t, 7.4 Hz, 4H), 1.70-1.57 (m, 10H), 1.55-1.47 (m, 4H), 1.35-1.21 (48H), 0.89 (t-like, 6.8 Hz, 12H). [Examples]

[0162] Synthesis of compound 16 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. 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 transferred to 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 yellowish oily substance (approximately 300 mg) was obtained. The crude product (300 mg) was purified by silica gel flash chromatography (MeOH in chloroform, 0-4.4%). Compound 16 was obtained as a colorless oily substance (95 mg, 0.13 mmol, 36%). 1 HNMR (400 MHz, CDCl3) δ:5.61-5.44 (br. s, 1H), 3.97 (d, 5.8 Hz, 4H), 3.80 (t-like, 5.1 Hz, 2H), 2.63 (t-like, 5.6 Hz, 2H), 2.43-2.39 (m, 4H), 2.32 (t, 7.5 Hz, 4H), 1.70-1.59 (m, 8H), 1.55-1.45 (m, 4H), 1.36-1.21 (52H), 0.89 (t-like, 6.8 Hz, 12H). [Examples]

[0163] Synthesis of Compound 17 To a solution of 2-hexyldecyl 6-bromohexanoate (2 equivalents, 1.32 g, 3.14 mmol) in 15 ml of anhydrous THF, 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) were added. This mixture was heated in a pressure round-bottom flask at 75°C (oil bath) under argon for 6 days. The reaction mixture was cooled and concentrated. The residue was dissolved in a mixture of hexane and ethyl acetate (approximately 9:1), washed with water and brine, dried over sodium sulfate, filtered, and concentrated to dryness (1.28 g of colorless oil). The crude product was purified by silica gel flash column chromatography (MeOH in chloroform, 0-5%). This yielded compound 17 as a colorless oily substance (581 mg, 0.76 mmol, 48%). 1 HNMR (400 MHz, CDCl3) δ:6.43-6.17 (br. s, 1H), 3.97 (d, 5.8 Hz, 4H), 3.55 (t-like, 4.7 Hz, 2H), 2.46-2.40 (m, 6H), 2.31 (t, 7.5 Hz, 4H), 1.70-1.59 (m, 10H), 1.55-1.45 (m, 4H), 1.36-1.21 (52H), 0.89 (t-like, 6.7 Hz, 12H). [Examples]

[0164] Synthesis of compound 20 To a solution of 2-hexyldecyl 8-bromooctanoate (2 equivalents, 3.09 g, 6.9 mmol) in 30 ml of anhydrous THF, 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) were added. The mixture in a pressure round-bottom flask was heated under argon at 64-70°C (oil bath) for 6 days. The mixture was cooled and concentrated. The residue was taken in a mixture of hexane and ethyl acetate (9:1), washed with water and brine, dried over sodium sulfate, filtered, and concentrated to dryness (colorless oil). The crude product was purified by silica gel flash-dry column chromatography (MeOH in chloroform, 0-4.2%). This yielded compound 20 as a colorless oil (1.28 g, 1.56 mmol, 45%). 1 HNMR (400 MHz, CDCl3) δ:6.64-6.45 (br. s, 1H), 3.97 (d, 5.8 Hz, 4H), 3.62-3.51 (br. 2H), 3.07-2.34 (br. 6H), 2.30 (t, 7.5 Hz, 4H), 1.71-1.40 (m, 14H), 1.39-1.19 (m, 60H), 0.89 (t-like, 6.8 Hz, 12H). [Examples]

[0165] Synthesis of 9-(2'-ethylhexanoyloxy)nonane-1-R A solution of nonane-1,9-diol (10.1 g) in methylene chloride (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 and filtered through a silica gel bed to remove the solvent. The crude product was passed through a silica gel column using a methanol / methylene chloride (0-8%) gradient to obtain 9-(2'-ethylhexanoyloxy)nonane-1-ol as an oil (7.2 g).

[0166] 9-(2'-ethylhexanoyloxy)nonan-1-ol was dissolved in methylene chloride (100 mL) and treated with pyridinium chlorochromate (7.5 g) for 1 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 oily substance was dissolved in hexane. The suspension was filtered through a silica gel bed, and the solvent was removed to obtain 9-(2'-ethylhexanoyloxy)nonan-1-ol as a colorless oily substance (6 g). [Examples]

[0167] Synthesis of 9-(2'-butyloctanoyloxy)nonane-1-R A solution of nonane-1,9-diol (12.0 g) in methylene chloride (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 and filtered through a silica gel bed to remove the solvent. The crude product was passed through a silica gel column using a methanol / methylene chloride (0-4%) gradient to obtain 9-(2'-butyloctanoyloxy)nonane-1-ol as an oil (6 g).

[0168] 9-(2'-butyloctanoyloxy)nonan-1-ol was dissolved in methylene chloride (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 oily substance was dissolved in hexane. The suspension was filtered through a silica gel bed, and the solvent was removed to obtain 9-(2'-butyloctanoyloxy)nonan-1-ol as a colorless oil (3.1 g). [Examples]

[0169] Synthesis of 6-(2'-butyloctanoyloxy)hexane-1-R A solution of hexane-1,6-diol (9.4 g) in methylene chloride (150 mL) 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 and filtered through a silica gel bed to remove the solvent. The crude product was passed through a silica gel column using a methanol / methylene chloride (0-4%) gradient to obtain 6-(2'-butyloctanoyloxy)hexane-1-ol as an oil (4.5 g).

[0170] 6-(2'-butyloctanoyloxy)hexane-1-ol was dissolved in methylene chloride (100 mL) and treated overnight with pyridinium chlorochromate (4.8 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 oily substance was dissolved in hexane. The suspension was filtered through a silica gel bed, and the solvent was removed to obtain 6-(2'-butyloctanoyloxy)hexane-1-ol as a colorless oil (3.9 g). [Examples]

[0171] Synthesis of 6-(2'-octyldodecanoyloxy)hexane-1-R A solution of hexane-1,6-diol (11.5 g) in methylene chloride (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 and filtered through a silica gel bed to remove the solvent. The crude product was passed through a silica gel column using a methanol / methylene chloride (0-4%) gradient to obtain 6-(2'-octyldodecanoyloxy)hexane-1-ol as an oil (7.4 g).

[0172] 6-(2'-octyldodecanoyloxy)hexane-1-ol was dissolved in methylene chloride (100 mL) and treated with pyridinium chlorochromate (4.0 g) for 2 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 oily substance was dissolved in hexane. The suspension was filtered through a silica gel bed, and the solvent was removed to obtain 6-(2'-octyldodecanoyloxy)hexane-1-ol as a colorless oil (5.3 g). [Examples]

[0173] Synthesis of 6-(2'-decyltetradecanoyloxy)hexane-1-R A solution of hexane-1,6-diol (9.6 g) in methylene chloride (150 mL) 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 and filtered through a silica gel bed to remove the solvent. The crude product was passed through a silica gel column using a methanol / methylene chloride (0-4%) gradient to obtain 6-(2'-decyltetradecanoyloxy)hexane-1-ol as an oil (4.6 g).

[0174] 6-(2'-decyltetradecanoyloxy)hexane-1-ol was dissolved in methylene chloride (100 mL) and treated with pyridinium chlorochromate (3.2 g) for 2 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, and the solvent was removed to obtain 6-(2'-decyltetradecanoyloxy)hexane-1-ol as a colorless oil (4.2 g). [Examples]

[0175] Synthesis of 12-(2'-hexyldecanoyloxy)dodecane-1-R A solution of dodecane-1,6-diol (25.0 g) in methylene chloride (300 mL) / THF (100 mL) was treated with 2-hexyldodecanoic 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 and filtered through a silica gel bed to remove the solvent. The crude product was passed through a silica gel column using hexane and then methylene chloride to produce 12-(2'-hexyldecanoyloxy)dodecane-1-ol as an oil (7.9 g).

[0176] 12-(2'-hexyldecanoyloxy)dodecane-1-ol was dissolved in methylene chloride (150 mL) and treated with pyridinium chlorochromate (4.0 g) for 3 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 oily substance was dissolved in hexane. The suspension was filtered through a silica gel bed, and the solvent was removed to obtain 12-(2'-hexyldecanoyloxy)dodecane-1-ol as a colorless oil (3.9 g). [Examples]

[0177] Synthesis of 9-(2'-hexyldecanoyloxy)nonane-1-R A solution of nonane-1,9-diol (46.8 g) in methylene chloride (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 and filtered through a silica gel bed to remove the solvent. The crude product was passed through a silica gel column using a hexane, then methanol / methylene chloride (0-8%) gradient to obtain 9-(2'-hexyldecanoyloxy)nonane-1-ol as an oil (22 g).

[0178] 9-(2'-hexyldecanoyloxy)nonan-1-ol (5.0 g) was dissolved in methylene chloride (50 mL) and treated with pyridinium chlorochromate (2.7 g) for 1 hour. Hexane (200 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, and 9-(2'-hexyldecanoyloxy)nonan-1-ol was obtained as a colorless oil (3.6 g). [Examples]

[0179] Synthesis of compound 22 A solution of 9-(2'-hexyldecanoyloxy)nonane-1-R (2.2 g), acetic acid (0.15 g), and 4-aminobutan-1-ol (0.20 g) in methylene chloride (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 / methylene chloride (2-0 / 0-12 / 98-88%) gradient. The pure fraction was washed with an aqueous sodium bicarbonate solution to obtain compound 22 as a colorless oil (0.93 g). [Examples]

[0180] Synthesis of compound 23 A solution of 12-(2'-hexyldecanoyloxy)dodecane-1-ar (2.0 g), acetic acid (0.09 g), and 4-aminobutan-1-ol (0.14 g) in methylene chloride (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 / methylene chloride (2-0 / 0-12 / 98-88%) gradient. The pure fraction was washed with an aqueous sodium bicarbonate solution to obtain compound 23 as a colorless oil (1.0 g). [Examples]

[0181] Synthesis of Compound 24 A solution of 9-(2'-ethylhexanoyloxy)nonane-1-R (3.0 g), acetic acid (0.11 g), and 4-aminobutan-1-ol (0.17 g) in methylene chloride (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 / methylene chloride (2-0 / 0-10 / 98-88%) gradient. The pure fraction was washed with an aqueous sodium bicarbonate solution to obtain compound 24 as a colorless oil (0.69 g). [Examples]

[0182] Synthesis of Compound 25 A solution of 9-(2'-butyloctanoyloxy)nonane-1-R (2.6 g), acetic acid (0.20 g), and 4-aminobutan-1-ol (0.26 g) in methylene chloride (50 mL) was treated overnight with sodium triacetoxyborohydride (1.42 g). The solution was washed with aqueous sodium bicarbonate. 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 / methylene chloride (2-0 / 0-12 / 98-88%) gradient. The pure fraction was washed with aqueous sodium bicarbonate to obtain compound 25 as a colorless oil (0.82 g). [Examples]

[0183] Synthesis of Compound 26 A solution of 6-(2'-octyldodecanoyloxy)hexane-1-R (2.7 g), acetic acid (0.20 g), and 4-aminobutan-1-ol (0.20 g) in methylene chloride (20 mL) was treated overnight with sodium triacetoxyborohydride (1.30 g). The solution was washed with aqueous sodium bicarbonate. 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 / methylene chloride (2-0 / 0-12 / 98-88%) gradient. The pure fraction was washed with aqueous sodium bicarbonate to obtain compound 26 as a colorless oil (0.21 g). [Examples]

[0184] Synthesis of Compound 27 A solution of 6-(2'-decyltetradecanoyloxy)hexane-1-R (2.1 g), acetic acid (0.11 g), and 4-aminobutan-1-ol (0.13 g) in methylene chloride (30 mL) was treated overnight with sodium triacetoxyborohydride (0.70 g). The solution was washed with aqueous sodium bicarbonate. 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 / methylene chloride (2-0 / 0-12 / 98-88%) gradient. The pure fraction was washed with aqueous sodium bicarbonate to obtain compound 27 as a colorless oil (0.90 g). [Examples]

[0185] Synthesis of compound 28 A solution of 6-(2'-butyloctanoyloxy)hexane-1-R (2.0 g), acetic acid (0.13 g), and 4-aminobutan-1-ol (0.13 g) in methylene chloride (20 mL) was treated overnight with sodium triacetoxyborohydride (1.0 g). The solution was washed with aqueous sodium bicarbonate. 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 / methylene chloride (2-0 / 0-8 / 98-92%) gradient. The pure fraction was washed with aqueous sodium bicarbonate to obtain compound 28 as a colorless oil (0.77 g). [Examples]

[0186] Synthesis of compound 30 A solution of 6-(2'-hexyldecanoyloxy)hexane-1-R (2.4 g), acetic acid (0.15 g), and 3-aminopropane-1,2-diol (0.21 g) in methylene chloride (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 / methylene chloride (2-0 / 0-12 / 98-88%) gradient. The pure fraction was washed with an aqueous sodium bicarbonate solution to obtain compound 30 as a colorless oil (0.60 g). [Examples]

[0187] Synthesis of compound 31 A solution of 6-(2'-hexyldecanoyloxy)hexane-1-R (2.4 g), acetic acid (0.15 g), and 2-aminobutan-1-ol (0.20 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.1 g) for 2 hours. The solution was washed with aqueous sodium bicarbonate. 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 / methylene chloride (2-0 / 0-4 / 98-96%) gradient. The pure fraction was washed with aqueous sodium bicarbonate to obtain compound 31 as a colorless oil (0.31 g). [Examples]

[0188] Synthesis of Compound 37 A solution of 6-(2'-octyldodecanoyloxy)hexane-1-R (2.7 g), acetic acid (0.20 g), and 3-aminopropan-1-ol (0.17 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.3 g) for 2 hours. The solution was washed with aqueous sodium bicarbonate. 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 / methylene chloride (2-0 / 0-12 / 98-88%) gradient. The pure fraction was washed with aqueous sodium bicarbonate to obtain compound 37 as a colorless oil (0.22 g). [Examples]

[0189] Synthesis of compound 38 A solution of 12-(2'-hexyldecanoyloxy)dodecane-1-ar (1.8 g), acetic acid (0.08 g), and 3-aminopropan-1-ol (0.11 g) in methylene chloride (10 mL) was treated overnight with sodium triacetoxyborohydride (0.64 g). The solution was washed with aqueous sodium bicarbonate. 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 / methylene chloride (2-0 / 0-10 / 98-90%) gradient. The pure fraction was washed with aqueous sodium bicarbonate to obtain compound 38 as a colorless oil (0.83 g). [Examples]

[0190] Synthesis of compound 39 A mixture of ethyl 4-aminobutyrate hydrochloride (1.28 mmol, 214 mg), 2-hexyldecyl 6-bromohexanoate (1.9 equivalents, 2.43 mmol, 1.02 g), N,N-diisopropylethylamine (3.5 equivalents, 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 dissolved 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 oily substance (approximately 1.04 g) was obtained. The crude product was purified by silica gel flash column chromatography (MeOH in DCM, 0-3.5%). Compound 39 was obtained as a colorless oily substance (334 mg, 0.41 mmol, 43%). 1 HNMR (400 MHz, CDCl3) δ:4.13 (q, 7.1 Hz, 2H), 3.97 (d, 5.8 Hz, 4H), 2.43-2.34 (m, 6H), 2.33-2.28 (m, 6H), 1.73 (quintet, 7.3 Hz, 2H), 1.68-1.58 (m, 6H), 1.47-1.37 (m, 4H), 1.36-1.20 (54H), 0.89 (t-like, 6.8 Hz, 12H). [Examples]

[0191] Synthesis of compound 40 A solution of 6-(2'-hexyldecanoyloxy)hexane-1-R (2.4 g), acetic acid (0.15 g), and 1-aminobutan-2-ol (0.10 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.8 g) for 2 hours. The solution was washed with aqueous sodium bicarbonate. 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 / methylene chloride (2-0 / 0-8 / 98-92%) gradient. The pure fraction was washed with aqueous sodium bicarbonate to obtain compound 40 as a colorless oil (0.85 g). [Examples]

[0192] Synthesis of compound 41 A solution of 6-(2'-hexyldecanoyloxy)hexane-1-R (2.4 g), acetic acid (0.19 g), and 1-aminobutan-2-ol (0.21 g) in methylene chloride (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 / methylene chloride (2-0 / 0-8 / 98-92%) gradient. The pure fraction was washed with an aqueous sodium bicarbonate solution to obtain compound 41 as a colorless oil (0.77 g). [Examples]

[0193] Synthesis of compound 42 A solution of 6-(2'-butyloctanoyloxy)hexane-1-R (2.0 g), acetic acid (0.13 g), and 4-aminobutan-2-ol (0.20 g) in methylene chloride (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 / methylene chloride (2-0 / 0-8 / 98-92%) gradient. The pure fraction was washed with an aqueous sodium bicarbonate solution to obtain compound 42 as a colorless oil (0.54 g). [Examples]

[0194] Synthesis of compound 43 A solution of 9-(2'-ethylhexanoyloxy)nonane-1-R (3.0 g), acetic acid (0.11 g), and 3-aminopropan-1-ol (0.14 g) in methylene chloride (50 mL) was treated overnight with sodium triacetoxyborohydride (0.91 g). The solution was washed with aqueous sodium bicarbonate. 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 / methylene chloride (2-0 / 0-6 / 98-94%) gradient. The pure fraction was washed with aqueous sodium bicarbonate to obtain compound 43 as a colorless oil (1.01 g). [Examples]

[0195] Synthesis of compound 44 A solution of 6-(2'-decyltetradecanoyloxy)hexane-1-R (2.1 g), acetic acid (0.11 g), and 3-aminopropan-1-ol (0.11 g) in methylene chloride (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 / methylene chloride (2-0 / 0-8 / 98-96%) gradient. The pure fraction was washed with an aqueous sodium bicarbonate solution to obtain compound 44 as a colorless oil (1.07 g). [Examples]

[0196] Synthesis of compound 45 A solution of 9-(2'-butyloctanoyloxy)nonane-1-R (2.6 g), acetic acid (0.17 g), and 3-aminopropan-1-ol (0.21 g) in methylene chloride (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 / methylene chloride (2-0 / 0-8 / 98-96%) gradient. The pure fraction was washed with an aqueous sodium bicarbonate solution to obtain compound 45 as a colorless oil (1.1 g). [Examples]

[0197] Synthesis of compound 46 To a solution of 2-aminoethanol (96.5 mg, 1.58 mmol, 95.4 μL, MW 61.08, d 1.012) in 15 ml of 2-propanol, 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) were added and heated for 3 days (oil bath 60°C). The mixture was concentrated, and the residue was taken in THF (10 mL). To this mixture, aminoethanol (80 mg, 1.3 mmol) was added. Heating was continued at 70°C for a further 3 days. After a total of 6 days, the reaction mixture was cooled, filtered, and concentrated. The residue was purified by silica gel flash-dry column chromatography (methanol in chloroform, 1-4.2%). This yielded compound 46 as a colorless oil (334 mg, 0.42 mmol, 30%). 1 HNMR (400 MHz, CDCl3) δ:4.09-4.06 (m, 2H), 3.97 (d, 5.8 Hz, 4H), 3.39-3.36 (m, 2H), 3.31-3.23 (m, 4H), 2.31 (t, 7.5 Hz, 4H), 1.88-1.56 (m, 12H), 1.43-1.19 (59H), 0.89 (t-like, 6.8 Hz, 12H). [Examples]

[0198] Synthesis of compound 47 A solution of 6-(2'-hexyldecanoyloxy)hexane-1-R (3.0 g), acetic acid (0.20 g), and 3-aminopropionitrile (0.21 g) in methylene chloride (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 / methylene chloride (2-0 / 0-6 / 98-94%) gradient. The pure fraction was washed with an aqueous sodium bicarbonate solution to obtain compound 47 as a colorless oil (0.29 g). [Examples]

[0199] Synthesis of compound 48 A solution of 6-(2'-hexyldecanoyloxy)hexane-1-R (3.0 g) and 4-aminobutyrate ethyl hydrochloride (0.46 g) in methylene chloride (30 mL) was treated overnight with sodium triacetoxyborohydride (1.4 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 / methylene chloride (2-0 / 0-8 / 98-92%) gradient. The pure fraction was washed with an aqueous sodium bicarbonate solution to obtain compound 48 as a colorless oil (0.80 g). [Examples]

[0200] Synthesis of compound 49 To a solution of 2-butyloctyl 8-bromooctanoate (2 equivalents, 1.877 g, 4.8 mmol) in 20 ml of anhydrous THF, 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) were added. The mixture in a pressure round-bottom flask was heated for 6 days (oil bath, 80°C). The reaction mixture was cooled and concentrated. The residue was dissolved in a mixture of hexane and ethyl acetate (approximately 5:1), washed with water and brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by silica gel flash column chromatography (methanol in chloroform, 1-4%). ​​Compound 49 was obtained as a colorless oil (857 mg, 1.21 mmol, 50%). 1 HNMR (400 MHz, CDCl3) δ:6.55 (br. s, 1H), 3.97 (d, 5.8 Hz, 4H), 3.55 (not well resolved triplet, 2H), 2.45-2.40 (m. 6H), 2.30 (t, 7.5 Hz, 4H), 1.71-1.58 (m, 10 H), 1.51-1.42 (m, 4H), 1.39-1.19 (m, 44H), 0.93-0.87 (m, 12H).

[0201] The various embodiments described above can be combined to provide further embodiments. Unless inconsistent with certain teachings and definitions herein, U.S. Patent Applications No. 62 / 247,616 (filed October 28, 2015) and No. 62 / 328,244 (filed April 27, 2016); all U.S. patents, U.S. patent publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications, all of which are referenced herein and / or enumerated in the application datasheet, are incorporated herein by reference in their entirety. The aspects of the embodiments may be modified, as necessary, to use various patent, patent application, and patent publication concepts to provide yet another embodiment. These and other modifications may be made to the embodiments in light of the detailed description above. In general, the terms used in the following claims should not be construed as limiting the claims to any specific embodiment disclosed in the specification and claims, but rather as encompassing the entire scope of all possible embodiments and equivalents to which such claims are entitled. Therefore, the claims are not limited by this disclosure.

Claims

1. The following structural formula (I): 【Chemistry 1】 (I) [In the formula, L 1 or L 2 One of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -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 the other of L 1 or L 2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -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- or a direct bond; G 1 and G 2 Each of these is independently of the non-substituted C 1 -C 12 Alkylene or C 1 -C 12 It is alkenylene; G 3 C 1 -C 24 Alkylene, C 1 -C 24 Alkenylene, C 3 -C 8 Cycloalkylene, C 3 -C 8 It is a cycloalkenylene; R a is H or C 1 -C 12 It is alkyl; R 1 and R 2 Each is independent of C 6 -C 24 Alkyl or C 6 -C 24 It is Alkenil; R 3 H, OR 5 , CN, -C(=O)OR 4 -OC(=O)R 4 or -NR 5 C(=O)R 4 It is; R 4 C 1 -C 12 It is alkyl; R 5 is H or C 1 -C 6 It is alkyl; and x is 0, 1, or 2. Compounds having, or pharmaceutically acceptable salts thereof, tautomers, prodrugs, or stereoisomers thereof.

2. The following structural formula (IA) or (IB): 【Chemistry 2】 (IA) or 【Transformation 3】 (IB) [In the formula, A is a 3- to 8-membered cycloalkyl or cycloalkylene ring; R 6 Each instance is independently of H, OH, or C. 1 -C 24 It is alkyl; n is an integer between 1 and 15. The compound according to claim 1, having one of the following.

3. The following structural formula (IC) or (ID): 【Chemistry 4】 (I C) or 【Transformation 5】 (ID) [In the formula, y and z are each independent integers between 1 and 12. The compound according to claim 2, having one of the above.

4. L 1 and L 2 The compound according to any one of claims 1 to 3, wherein each of them is independently -(C=O)O- or -O(C=O)-.

5. The following structural formula (IE) or (IF): 【Transformation 6】 (IE) or 【Transformation 7】 (IF) The compound according to claim 4, having one of the above.

6. The following structural formulas: (IG), (IH), (II), or (IJ): 【Transformation 8】 (IG); 【Chemistry 9】 (IH) 【Chemistry 10】 (II) or 【Chemistry 11】 (IJ) A compound according to any one of claims 2 to 5, having one of the above.

7. The compound according to any one of claims 2 to 6, wherein n is an integer from 2 to 12.

8. The compound according to claim 7, wherein n is 3, 4, 5, or 6.

9. The compound according to any one of claims 3 to 8, wherein y and z are each independently integers from 2 to 10.

10. The compound according to any one of claims 3 to 8, wherein y and z are each independently integers from 4 to 9.

11. R 1 or R 2 , or both, C 6 -C 24 The compound according to any one of claims 1 to 10, wherein it is an alkenyl.

12. R 1 or R 2 , or both, C 6 -C 24 The compound according to any one of claims 1 to 10, wherein it is alkyl.

13. R 1 and R 2 Each of these independently forms the following structural formula: 【Chemistry 12】 [In the formula, R 7a and R 7b Each instance of H or C is independent. 1 -C 12 It is alkyl; and a is an integer between 2 and 12; Here, R 7a , R 7b and a are R 1 and R 2 Each of these is independently selected to contain between 6 and 20 carbon atoms. The compound according to claim 12, having the following characteristics.

14. The compound according to claim 13, wherein a is an integer between 8 and 12.

15. R 7a The compound according to any one of claims 13 to 14, wherein the presence of at least one of is H.

16. R 7a The compound according to any one of claims 13 to 14, wherein each instance is H.

17. R 7b The existence of at least one of C 1 -C 8 The compound according to any one of claims 13 to 16, wherein it is alkyl.

18. C 1 -C 8 The compound according to claim 17, wherein the alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

19. R 1 or R 2 , or both, have the following structural formula: 【Chemistry 13】 A compound according to any one of claims 12 to 18, having one of the above.

20. R 3 The compound according to any one of claims 1 to 19, wherein the compound is an OH group.

21. R 3 The compound according to any one of claims 1 to 19, wherein the compound is CN.

22. R 3 is -C(=O)OR 4 -OC(=O)R 4 or -NHC(=O)R 4 and is the compound according to any one of claims 1 to 19.

23. R 4 The compound according to claim 22, wherein the compound is methyl or ethyl.

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

25. A composition comprising the compound and therapeutic agent according to any one of claims 1 to 24.

26. The composition according to claim 25, further comprising one or more excipients selected from neutral lipids, steroids, and polymer complex lipids.

27. The composition according to claim 26, wherein the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

28. The composition according to claim 27, wherein the neutral lipid is DSPC.

29. The composition according to any one of claims 25 to 28, wherein the molar ratio of the compound to the neutral lipid is in the range of about 2:1 to about 8:

1.

30. The composition according to any one of claims 26 to 29, wherein the steroid is cholesterol.

31. The composition according to claim 30, wherein the molar ratio of the compound to cholesterol is in the range of 5:1 to 1:

1.

32. The composition according to any one of claims 26 to 31, wherein the polymer composite lipid is a pegylated lipid.

33. The composition according to claim 32, wherein the molar ratio of the compound to the pegylated lipid is in the range of about 100:1 to about 20:

1.

34. The composition according to any one of claim 32 or 33, wherein the pegylated lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, or PEG-dialkoxypropyl carbamate.

35. The pegylated lipid has the following structural formula (II): 【Chemistry 14】 (II) [In the formula, R 8 and R 9 Each of these is independently a linear or branched saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, where the alkyl chain is optionally interrupted by one or more ester bonds; and w has an average value in the range of 30 to 60. A composition according to any one of claims 32 or 33, having the following:

36. R 8 and R 9 The composition according to claim 35, wherein each of them is independently a straight-chain saturated alkyl chain containing 12 to 16 carbon atoms.

37. The composition according to any one of claims 35 or 36, wherein the average w is approximately 49.

38. The composition according to any one of claims 25 to 37, wherein the therapeutic agent comprises nucleic acid.

39. The composition according to claim 38, wherein the nucleic acid is selected from antisense RNA and messenger RNA.

40. A method for administering a therapeutic agent to a patient in need thereof, comprising preparing or preparing one of the compositions described in any one of claims 25 to 39, and administering the composition to the patient.

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