Novel lipids and lipid nanoparticle formulations for delivery of nucleic acids

Abstract

To provide improved cationic lipids and lipid nanoparticles for oligonucleotide delivery.SOLUTION: Compounds having the following structure (I), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof are provided. Also provided are use of the compounds as a component of a lipid nanoparticle formulation for delivery of a therapeutic agent, compositions comprising the compounds, and methods for their use and preparation.SELECTED DRAWING: None

Description

Technical Field

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

Background Art

[0002] There are many challenges associated with the delivery of nucleic acids to produce a desired response within a biological system. Nucleic acid-based therapeutics have great potential, but there remains a need for more effective delivery of nucleic acids to appropriate sites within cells or organisms in order to realize this potential. Examples of therapeutic nucleic acids include messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, immunostimulatory nucleic acids, antagomirs, antimir, mimics, supermir, and aptamers. Some nucleic acids, such as mRNA or plasmids, can be used to cause the expression of specific cell products, such as those useful in the treatment of diseases related to a deficiency of a protein or enzyme. The therapeutic use of translatable nucleotide delivery is extremely broad because any selected protein sequence can be generated by synthesizing the construct, regardless of whether it is endogenous to the system. The expression product of the nucleic acid can increase the existing level of a protein, replace a defective or non-functional version of the protein, or introduce new proteins and related functionality within a cell or organism.

[0003] Some nucleic acids, such as miRNA inhibitors, can be used to cause the expression of specific cell products regulated by miRNA, which are useful, for example, in the treatment of diseases related to the lack of proteins or enzymes. By synthesizing constructs, one or more miRNAs can be inhibited, which in turn regulates the expression of mRNA products, so the therapeutic uses of miRNA inhibition are extremely broad. Inhibition of endogenous miRNA can increase the expression of its downstream target endogenous proteins and restore appropriate functions 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, which can in turn downregulate the synthesis of the corresponding proteins through processes such as RNA interference (RNAi) or complementary binding of antisense RNA. Since oligonucleotide constructs can be synthesized using any nucleotide sequence directed against the target mRNA, the therapeutic uses of antisense oligonucleotides and RNAi are also extremely broad. Examples of targets include mRNAs derived from normal cells, mRNAs associated with disease states such as cancer, and mRNAs of infectious agents such as viruses. To date, antisense oligonucleotide constructs have shown the ability to specifically downregulate target proteins through degradation of cognate mRNAs in both in vitro and in vivo models. In addition, antisense oligonucleotide constructs are currently being evaluated in clinical studies.

[0005] However, the use of oligonucleotides in a therapeutic context currently faces two problems. First, free RNA is susceptible to nuclease digestion in plasma. Second, free RNA has limited access to intracellular compartments where the relevant translation machinery exists. Lipid nanoparticles formed from cationic lipids with other lipid components such as neutral lipids, cholesterol, PEG, pegylated lipids, and oligonucleotides are RN It has been used to block the degradation of A and promote the cellular uptake of oligonucleotides.

SUMMARY OF THE INVENTION

MEANS FOR SOLVING THE PROBLEM

[0006] There remains a need for improved cationic lipids and lipid nanoparticles for oligonucleotide delivery. Preferably, these lipid nanoparticles provide an optimal drug:lipid ratio, protect nucleic acids from degradation and clearance in serum, are appropriate for systemic delivery, and provide intracellular delivery of nucleic acids. In addition, these lipid-nucleic acid particles should be fairly well tolerated and provide a sufficient therapeutic index such that treatment of patients at an effective dose of the nucleic acid is not accompanied by unacceptable toxicity and / or risk to the patient. The present invention provides these and related advantages.

[0007] Briefly, the present invention provides lipid compounds, including stereoisomers, pharmaceutically acceptable salts or tautomers thereof, which can form lipid nanoparticles for the delivery of therapeutic agents, either alone or in combination with other lipid components such as neutral lipids, charged lipids, steroids (including all sterols) and / or analogs thereof, and / or polymer-conjugated lipids, etc. In certain cases, lipid nanoparticles are used to deliver nucleic acids such as antisense and / or messenger RNA. Also provided are methods of using such lipid nanoparticles for the treatment of various diseases or conditions, such as those caused by infectious entities and / or protein deficiencies.

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

CHEMICAL

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

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

[0011] In yet further embodiments, the present invention is directed to a pegylated lipid having the following structure (II):

Chemical formula

[0012] These and other aspects of the present invention will become apparent with reference to the following detailed description.

[0013] In the figures, the same reference numbers identify similar elements. The sizes and relative positions of the elements in the figures are not necessarily drawn to scale, and some of these elements are arbitrarily enlarged and arranged to improve the readability of the figures. Further, the specific shapes of the drawn elements are not intended to convey any information regarding the actual shapes of the specific elements, but are merely selected to make the figures easier to recognize.

Brief Description of the Drawings

[0014]

Figure 1

[0015]

Figure 2

[0016]

Figure 3

[0017]

Figure 4

Modes for Carrying Out the Invention

[0018] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the present invention. However, one skilled in the art will understand that the present invention may be practiced without these specific details.

[0019] The present invention is based in part on the discovery of novel cationic (amino) lipids that provide advantages when used in lipid nanoparticles for the in vivo delivery of active agents or therapeutic agents, such as nucleic acids, to mammalian cells. In particular, embodiments of the present invention provide an increase in the activity of nucleic acids and an improvement in the tolerance of the composition in vivo, resulting in a significant increase in the therapeutic index when compared to previously described nucleic acid-lipid nanoparticle compositions, and provide nucleic acid-lipid nanoparticle compositions comprising one or more of the novel cationic lipids described herein.

[0020] 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 proteins encoded by mRNA. In other embodiments, these improved lipid nanoparticle compositions are useful for upregulating the expression of endogenous proteins by delivering miRNA inhibitors that target one target mRNA or a group of one specific miRNA or miRNAs that regulate several mRNAs. In other embodiments, these improved lipid nanoparticle compositions are useful for downregulating (e.g., silencing) the protein level and / or mRNA level of a target gene. In some other embodiments, the lipid nanoparticles are also useful for the delivery of mRNA and plasmids for the expression of transgenes. In yet other embodiments, the lipid nanoparticle compositions are useful for inducing pharmacological effects resulting from protein expression, such as an increase in erythropoiesis via delivery of appropriate erythropoietin mRNA, or protection from infection via delivery of mRNA encoding an appropriate antibody.

[0021] The lipid nanoparticles and compositions of the present invention can be used for various purposes, including delivering encapsulated or associated (e.g., complexed) therapeutic agents, such as nucleic acids, to cells both in vitro and in vivo. Accordingly, embodiments of the present invention are methods of treating or preventing a disease or disorder in a subject in need thereof by contacting the subject with lipid nanoparticles encapsulating or associated with a suitable therapeutic agent, wherein the lipid nanoparticles comprise one or more of the novel cationic lipids described herein.

[0022] As described herein, embodiments of the lipid nanoparticles of the present invention are particularly useful for the delivery of nucleic acids, including, for example, mRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomirs), messenger-RNA-interfering complementary RNA (micRNA), DNA, multivalent RNA, Dicer substrate RNA, complementary DNA (cDNA), and the like. Thus, 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 comprising one or more of the novel cationic lipids described herein, and the lipid nanoparticles encapsulate or are associated with a nucleic acid (e.g., messenger RNA or plasmid encoding the desired protein) that is expressed to produce the desired protein. 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 comprising one or more of the novel cationic lipids described herein, and the lipid nanoparticles encapsulate or are associated with a nucleic acid (e.g., antisense oligonucleotide or small interfering RNA (siRNA)) that reduces the expression of the target gene. The lipid nanoparticles and compositions of the present invention can also be useful for providing an effect that requires the co-localization of different nucleic acids (e.g., mRNA encoding an appropriate gene-modifying enzyme and DNA segment(s) for integration into the host genome), and can be used to co-deliver different nucleic acids (e.g., mRNA and plasmid DNA) separately or in combination.

[0023] Nucleic acids for use in the present invention can be prepared according to any available technique. For mRNA, the main method of preparation is enzymatic synthesis (also called in vitro transcription), which currently represents the most efficient method of generating mRNA specific to a long sequence, although not limited to this. In vitro transcription describes the process of template-directed synthesis of RNA molecules from an engineered DNA template composed of an upstream bacteriophage promoter sequence (including, but not limited to, those from T7, T3, and SP6 coliphages) ligated to a downstream sequence encoding the gene of interest. The template DNA can be prepared for in vitro transcription from several sources using suitable techniques well known in the art, including, but not limited to, plasmid DNA and polymerase chain reaction amplification (see Linpinsel, J. L and Conn, G. L., General protocols for preparation of plasmid DNA template, and Bowman, J.C., Azizi, B., Lenz, T.K., Ray, P. and Williams, L.D., RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods, Vol. 941, Conn G.L. (ed.), New York, N.Y. Humana Press, 2012).

[0024] Transcription of RNA occurs in vitro using a linearized DNA template in the presence of the corresponding RNA polymerase and ribonucleoside triphosphates (rNTPs) of adenosine, guanosine, uridine, and cytidine, under conditions that support polymerase activity while minimizing possible degradation of the generated mRNA transcript. In vitro transcription can be carried out using a variety of commercially available kits including, but not limited to, the RiboMax Large Scale RNA Production System (Promega), the MegaScript Transcription Kit (Life Technologies), as well as commercially available reagents including RNA polymerase and rNTPs. Methods for in vitro transcription of mRNA are well known in the art. (See, for example, Losick, R., 1972, In vitro transcription, Ann Rev Biochem, 41:409-46; Kamakaka, R. T. and Kraus, W. L., 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, 703 (Neilson, H. ed.), New York, N.Y. Humana Press, 2010; Brunelle, J.L. and Green, R., 2013, Chapter 5 In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology, 530:101-114, all of which are incorporated herein by reference).

[0025] Next, the desired, in vitro transcribed mRNA is purified from unwanted components of the transcription or related reactions (including unincorporated rNTPs, protein enzymes, salts, short RNA oligos, etc.). Techniques for the isolation of mRNA transcripts are well known in the art. Well-known procedures include phenol / chloroform extraction or precipitation with alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride. Additional, non-limiting examples of purification procedures that can be used include size exclusion chromatography (Lukavsky, P.J. and Puglisi, J.D., 2004, Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides, RNA, 10, pp. 889-893), silica-based affinity chromatography and polyacrylamide gel electrophoresis (Bowman, J.C., Azizi, B., Lenz, T.K., Ray, P. and Williams, L.D., in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods, 941, Conn G.L. (ed.), New York, N.Y. Humana Press, 2012). Purification can be carried out using a variety of commercially available kits, including but not limited to the S V Total Isolation System (Promega) and the in Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).

[0026] Furthermore, reverse transcription can produce large amounts of mRNA, but the product may contain some abnormal RNA impurities associated with unwanted polymerase activities that may need to be removed from the preparation of full-length mRNA. These include incomplete transcription initiation as well as RNA-dependent RNA polymerase activity, RNA primed transcription from the RNA template, and short RNAs generated from double-stranded RNA (dsRNA) produced by self-complementary 3’ extensions. These contaminants with dsRNA structures have been demonstrated to potentially result in unwanted immune-stimulatory activity through interaction with various innate immune sensors within eukaryotic cells that recognize specific nucleic acid structures and function to induce a strong immune response. Consequently, this can lead to a dramatic decrease in mRNA translation as protein synthesis is decreased during the cell's innate immune response. Thus, additional techniques for removing these dsRNA contaminants, including but not limited to expandable HPLC purification, have been developed and are known in the art (see, for example, Kariko, K., Muramatsu, H., Ludwig, J. and Weissman, D., 2011, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA, Nucl Acid Res, 39:el42; 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, 969 (Rabinovich, P.H. ed.), 2013). HPLC-purified mRNA has been reported to be translated at much higher levels, particularly in primary cells and in vivo.

[0027] A variety of quite diverse modifications are described in the art for altering specific properties of in vitro transcribed mRNA and improving its utility. These include, but are not limited to, modifications to the 5’ and 3’ ends of the mRNA. Endogenous eukaryotic mRNA usually contains a cap structure on the 5’-end of the mature molecule that plays an important role in mediating the binding of mRNA cap-binding protein (CBP), which in turn is involved in enhancing mRNA stability and the efficiency of mRNA translation in the cell. Thus, the highest level of protein expression is achieved using capped mRNA transcripts. The 5’-cap contains a 5’-5’-triphosphate bond between the most 5’-terminal nucleotide and a guanine nucleotide. The conjugated guanine nucleotide is methylated at the N7 position. Additional modifications include methylation of the most 5’-terminal nucleotide and the second nucleotide from the 5’-end on the 2’-hydroxyl group.

[0028] G becomes non-identical to the 5'-cap structure of authentic cellular mRNA, potentially reducing translatability and cellular stability. Instead, synthetic mRNA molecules may also be enzymatically capped post-transcriptionally. These can generate a more authentic 5'-cap structure that more closely structurally or functionally mimics the endogenous 5'-cap with enhanced binding of cap-binding proteins, increased half-life, decreased sensitivity to 5'-endonucleases, and / or decreased 5'-decapping. To enhance mRNA stability and translatability, many synthetic 5'-cap analogs have been developed and are known in the art (see, e.g., Grudzien-Nogalska, E., Kowalska, J., Su, W., Kuhn, A.N., Slepenkov, S.V., Darynkiewicz, E., Sahin, U., Jemielity, J. and Rhoads, R.E., Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology, Vol. 969 (Rabinovich, P.H. ed.), 2013).

[0029] At the 3'-end, a long chain of adenine nucleotides (polyA tail) is usually added to the mRNA molecule during RNA processing. Immediately after transcription, the 3'-end of the transcript is cleaved to release the 3'-hydroxyl, to which, in a process called polyadenylation, polyA polymerase adds a chain of adenine nucleotides to the RNA. The polyA tail has been widely shown to enhance both the translational 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, 14, 373-377; Guhaniyogi, J. and Brewer, G., 2001, Regulation of mRNA stability in mammalian cells, Gene, 265, 11-23; Dreyfus, M. and Regnier, P., 2002, The poly (A) tail of mRNAs: Bodyguard in eukaryotes, scavenger in bacteria, Cell, 111, 611-613).

[0030] Poly(A) tailing of in vitro transcribed mRNA can be achieved using a variety of techniques including, but not limited to, cloning into a DNA template with a poly(T) region or post-transcriptional addition using a poly(A) polymerase. The first case allows for in vitro transcription of mRNA with a defined length of poly(A) tail depending on the size of the poly(T) region, but requires additional manipulation of the template. The latter case involves enzymatically adding a poly(A) tail to in vitro transcribed mRNA using a poly(A) polymerase that catalyzes the incorporation of adenine residues to the 3’ end of the RNA, and does not require additional manipulation of the DNA template, but results in mRNA with a heterogeneous length of poly(A) tail. 5’-capping and 3’-poly(A) tailing can be performed using a variety of commercially available kits including, but not limited to, a 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.

[0031] In addition to 5’ capping and 3’ polyadenylation, other modifications of in vitro transcripts have been reported to provide benefits such as those related to translation efficiency and stability. Pathogenic DNA and RNA are recognized by various sensors within eukaryotic cells and trigger strong innate It is well known in the art that nucleic acids can elicit an immune response. Since most nucleic acids from natural sources contain modified nucleosides, the ability to discriminate between pathogen DNA and RNA and self-DNA and RNA has been shown to be based, at least in part, on structure and nucleoside modifications. In contrast, RNA synthesized in vitro lacks these modifications and is thus immunostimulatory, which can, in turn, inhibit effective mRNA translation as outlined above.Using the introduction of modified nucleosides into in vitro transcribed mRNA, recognition and activation of RNA sensors can be prevented, thus reducing this undesired immunostimulatory activity and enhancing translational capacity (see, for example, Kariko, K. and Weissman, D., 2007, Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: implication for therapeutic RNA development, Curr Opin Drug Discov Devel, 10, pp. 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, 969 (Rabinovich, P.H. ed.), 2013; Kariko, K., Muramatsu, H., Welsh, F.A., 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, 16, pp. 1833-1840). Modified nucleosides and nucleotides used in the synthesis of modified RNA can be prepared, monitored, and utilized using general methods and procedures known in the art. A wide variety of nucleoside modifications are available that can be incorporated to some extent into in vitro 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 decrease the ability to activate immune sensors while enhancing translational ability at the same time.

[0032] Other components of mRNA that can be modified to provide benefits regarding translatability and stability include the 5’ and 3’ untranslated regions (UTRs). Optimizing either or both of the UTRs (preferred 5’ and 3’ UTRs can be obtained from cellular or viral RNA) has been shown to increase mRNA stability and translation efficiency of in vitro transcribed mRNA (see, e.g., Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology, Vol. 969 (ed. Rabinovich, P.H.), 2013).

[0033] In addition to mRNA, other nucleic acid payloads can be used in the present invention. For oligonucleotides, methods of preparation include, but are not limited to, chemical synthesis of long precursors and enzymatic and chemical cleavage, in vitro transcription as described above, etc. Methods of synthesizing DNA and RNA nucleotides are widely used and well-known in the art (See, for example, Gait, M. J. (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, D.C.: IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, Vol. 288 (Clifton, N.J.), Totowa, N.J.: Humana Press, 2005, both of which are incorporated herein by reference).

[0034] Regarding plasmid DNA, preparations for use in the present invention generally utilize, but are not limited to, the in vitro amplification and isolation of plasmid DNA in a liquid culture of bacteria containing the plasmid of interest. The presence of a gene in the plasmid of interest that encodes resistance to a specific antibiotic (such as penicillin, kanamycin, etc.) enables the selective growth of bacteria containing the plasmid of interest in a culture containing the antibiotic. Methods for isolating plasmid DNA are widely used and well-known in the art (see, for example, Heilig, J., Elbing, K. L., and Brent, R (2001) Large-Scale Preparation of Plasmid DNA. Current Protocols in Molecular Biology., Vol. 41: II: 1.7: pp. 1.7.1 - 1.7.16; Rozkov, A., Larsson, B., Gillstrom, S., Bjornestedt, R., and Schmidt, S. R. (2008), Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture. Biotechnol. Bioeng., Vol. 99: 557 - 566; and US6197553B1). Plasmid isolation can be carried out using a variety of commercially available kits, including, but not limited to, Plasmid Plus (Qiagen), GenJET Plasmid MaxiPrep (Thermo), and Pure Yield MaxiPrep (Promega) kits, as well as using commercially available reagents.

[0035] The cationic lipids of the present invention, lipid nanoparticles and compositions containing the same, and various exemplary embodiments of their use for delivering active agents or therapeutic agents such as nucleic acids that modulate gene and protein expression are described in further detail below.

[0036] As used in this specification, unless otherwise specified, the following terms have the meanings ascribed to them.

[0037] Unless the context clearly dictates otherwise, throughout this specification and the claims, the word "comprising" and variations thereof, such as "comprises" and "comprising," are to be construed in an open, inclusive sense, i.e., "including, but not limited to."

[0038] References to "one embodiment" or "an embodiment" throughout this specification mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0039] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. When used in the specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0040] The phrase "inducing the expression of a desired protein" refers to the ability of a nucleic acid to increase the expression of a desired protein. To investigate the degree of protein expression, a test sample (e.g., a cell sample in culture expressing a desired protein) or a test mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model) is contacted with a nucleic acid (e.g., a nucleic acid combined with a lipid of the present invention). The expression of the desired protein in the test sample or test animal is compared with the expression of the desired protein in a control sample (e.g., a cell sample in culture expressing a desired protein) or a control mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model) that has not been contacted with the nucleic acid or to which the nucleic acid has not been administered. 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 the 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 expression of the desired protein in the control sample or control mammal exceeds 1, e.g., is 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 any measurable level of the desired protein is detected in the test sample or test mammal. Those skilled in the art will understand suitable assays for determining the level of protein expression in a sample, such as dot blot, Northern blot, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays, or assays based on a reporter protein that can generate fluorescence or luminescence under appropriate conditions.

[0041] The phrase "inhibiting the expression of a target gene" refers to the ability of a nucleic acid to silence, decrease, or inhibit the expression of a target gene. To investigate the degree of gene silencing, a test sample (e.g., a cell sample in culture that expresses the target gene) or a test mammal (e.g., a mammal, such as a human, or an animal model, such as a rodent (e.g., a mouse), or a non-human primate (e.g., a monkey) model) is contacted with a nucleic acid that silences, decreases, or inhibits the expression of the target gene. The expression of the target gene in the test sample or test mammal is compared to the expression of the target gene in a control sample (e.g., a cell sample in culture that expresses the target gene) or a control mammal (e.g., a mammal, such as a human, or an animal model, such as a rodent (e.g., a mouse), or a non-human primate (e.g., a monkey) model) that has not been contacted with the nucleic acid or to which the nucleic acid has not been administered. A value of 100% can be assigned to the expression of the target gene in the control sample or control mammal. In certain embodiments, silencing, inhibition, or decrease of the expression of the target gene is achieved when the level of expression of the target gene in the test sample or test mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% of the level of expression of the target gene in the control sample or control mammal. In other words, the nucleic acid is capable of silencing, decreasing, or inhibiting the expression of the target gene in the test sample or test mammal by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to the level of expression of the target gene in a control sample or control mammal that has not been contacted with the nucleic acid or to which the nucleic acid has not been administered. Suitable assays for determining the level of expression of the target gene include, but are not limited However, investigations at the protein or mRNA level using techniques known to those skilled in the art, such as dot blot, Northern blot, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those skilled in the art, etc., are included.

[0042] An "effective amount" or "therapeutically effective amount" of an active agent or therapeutic agent, such as a therapeutic nucleic acid, etc., is an amount sufficient to produce an increase or inhibition in the expression of a target sequence as compared to the normal expression level detected in the absence of the nucleic acid, i.e., a desired effect. An increase in the expression of a target sequence is achieved when any measurable level is detected in the case of an expression product that does not exist in the absence of the nucleic acid. In the case where an expression product exists at a certain level prior to contact with the nucleic acid, an increase in expression is achieved when the fold increase in the value obtained using a nucleic acid such as mRNA is about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000-fold or more compared to the control. Inhibition of the expression of a target gene or target sequence is achieved when the value obtained using a nucleic acid such as an antisense oligonucleotide is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% compared to the control. Examples of assays suitable for measuring the expression of a target gene or target sequence include investigations at the protein or RNA level using techniques known to those skilled in the art, such as dot blot, Northern blot, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of an appropriate reporter protein, and phenotypic assays known to those skilled in the art, etc.

[0043] As used herein, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form, including DNA, RNA, and their hybrids. DNA may be in the form of an antisense molecule, plasmid DNA, cDNA, a PCR product, or a 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 are synthetic, natural, and non-natural, and include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages having binding properties similar to those of a reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral methylphosphonate, 2'-O-methyl ribonucleotide, and peptide-nucleic acid (PNA). Unless specifically limited, the term includes nucleic acids containing known analogs of natural nucleotides having binding properties similar to those of a reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences as well as the explicitly recited sequences. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., Vol. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., Vol. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, Vol. 8:91-98 (1994)). A "nucleotide" contains a sugar deoxyribose (for DNA) or ribose (for RNA), a base, and a phosphate group. Nucleotides are linked together via phosphate groups."Base" includes purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, which synthetic derivatives of purines and pyrimidines include, for example, those having novel reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides arranged thereon. including, but not limited to, modifications that place novel reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0044] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that includes a partial or full-length coding sequence necessary for the production of a polypeptide or precursor polypeptide.

[0045] As used herein, the term "gene product" refers to the product of a gene such as an RNA transcript or a polypeptide.

[0046] The term "lipid" refers to a group of organic compounds that generally includes, but is not limited to, esters of fatty acids, are sparingly soluble in water, and are soluble in many organic solvents. Lipids are typically divided into at least three classes: (1) "simple lipids", which include fats and oils and waxes; (2) "compound lipids", which include phospholipids and glycolipids; and (3) "derived lipids", such as, for example, steroids.

[0047] "Steroid" has the following carbon skeleton:

Chemical formula

[0048] The term "cationic lipid" refers to a lipid that can be positively charged. Exemplary cationic lipids contain one or more amine groups that retain a positive charge. Preferred cationic lipids can be ionized such that they are either positively charged or exist in a neutral form depending on the pH. Ionization of the cationic lipid affects the surface charge of the lipid nanoparticles under different pH conditions. This charge state has important implications for plasma protein absorption, blood clearance and tissue distribution (Semple, S.C. et al., Adv. Drug Deliv Rev, 32:3-17 (1998)) and the ability to form an endosome-lytic non-bilayer structure that is important for intracellular delivery of nucleic acids (Hafez, I.M. et al., Gene Ther 8:1188-1196 (2001)).

[0049] The term "lipid nanoparticle" refers to a particle having a size of at least about one nanometer (e.g., 1-1,000 nm) and containing one or more of the compounds of formula (I) or one or more other specified cationic lipids. In some embodiments, the lipid nanoparticles are included in a formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), to a target site of interest (e.g., a cell, tissue, organ, tumor, etc.). In some embodiments, the lipid nanoparticles of the present invention contain a nucleic acid. Such lipid nanoparticles typically contain a compound of formula (I) and one or more excipients selected from neutral lipids, charged lipids, steroids and polymer-conjugated lipids. In some embodiments, the active agent or therapeutic agent, such as a nucleic acid, is encapsulated in an aqueous space enclosed by the lipid portion of the lipid nanoparticle or by a part or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other harmful effects induced by the mechanisms of the host organism or cell, such as a harmful immune response.

[0050] In various embodiments, the lipid nanoparticles are from about 30 nm to about 150 nm, from about 40 nm to about 1 It has an average diameter of 50 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm and is substantially non-toxic. In certain embodiments, the nucleic acid, when present in lipid nanoparticles, is resistant to degradation by nucleases in aqueous solution. Lipid nanoparticles containing nucleic acids and methods for their preparation are disclosed, for example, in U.S. Patent Publications 2004 / 0142025, 2007 / 0042031, and PCT Publications WO2013 / 016058 and WO2013 / 086373, the entire disclosures of which are incorporated herein by reference for all purposes.

[0051] As used herein, "lipid encapsulating" refers to lipid nanoparticles that provide an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), using complete encapsulation, partial encapsulation, or both. In certain embodiments, the nucleic acid (e.g., mRNA) is completely encapsulated within the lipid nanoparticles.

[0052] The term "polymer-conjugated lipid" refers to a molecule that includes both a lipid moiety and a polymer moiety. An example of a polymer-conjugated lipid is a pegylated lipid. The term "pegylated lipid" refers to a molecule that includes both a lipid moiety and a polyethylene glycol moiety. Pegylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-DMG) and the like.

[0053] The term "neutral lipid" refers to any of several lipid species that are either uncharged or in the zwitterionic form at a selected pH. Such lipids at physiological pH include, but are not limited to, phosphatidylcholines such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM), ceramides, steroids such as sterols, and derivatives thereof. Neutral lipids can be synthetic or of natural origin.

[0054] The term "charged lipid" refers to any of several lipid species that exist in a positively or negatively charged form regardless of the pH within a useful physiological range, e.g., from about pH 3 to about pH 9. Charged lipids can be synthetic or of natural origin. Examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyltrimethylammonium-propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, dimethylaminoethanecarbamoylsterol (e.g., DC-Chol).

[0055] As used herein, the term "aqueous solution" refers to a composition that contains water.

[0056] "Serum-stable" in relation to nucleic acid lipid nanoparticles means having free DNA or RNA Means that upon intentional degradation, exposure to serum or nuclease assay, the nucleotides do not degrade significantly. Suitable assays include, for example, standard serum assay, DNAse assay, or RNAse assay.

[0057] "Systemic delivery", as used herein, refers to the delivery of a therapeutic product that can result in widespread exposure of an active agent within an organism. Some administration techniques can result in systemic delivery of a particular agent, while others cannot. Systemic delivery means that a useful, preferably therapeutic, amount of the agent is exposed to most of the body. Systemic delivery of lipid nanoparticles can be by any means known in the art, including, for example, intravenous, intraarterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery.

[0058] "Local delivery", as used herein, refers to the direct delivery of an active agent to a target site within an organism. For example, the agent can be delivered locally by directly injecting it into a diseased site, such as a tumor, another target site, such as an inflammatory site, or a target organ, such as the liver, heart, pancreas, kidney, etc. Local delivery can also include techniques of local application or local injection, such as intramuscular, subcutaneous or intradermal injection. Local delivery does not interfere with systemic pharmacological effects.

[0059] "Alkyl" means saturated or unsaturated (i.e., containing one or more double and / or triple bonds) and having 1 to 24 carbon atoms (C1 - C 24 alkyl), 1 to 12 carbon atoms (C1 - C 12"(alkyl), a linear or branched hydrocarbon chain group 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, 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, penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc. Unless specifically stated otherwise in the specification, the alkyl group is optionally substituted."

[0060] "“Cycloalkyl” or “carbocyclic ring” may include a fused or bridged ring system, has 3 to 15 carbon atoms, preferably 3 to 10 carbon atoms, is saturated or unsaturated, and is a stable non-aromatic monocyclic or polycyclic hydrocarbon group consisting only of carbon and hydrogen atoms bonded to the rest of the molecule by a single bond. Examples of monocyclic groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of polycyclic groups include adamantyl, norbornyl, decalinyl, 7,7-dimethylbicyclo[2.2.1]heptanyl, etc. Unless specifically stated otherwise in the specification, the cycloalkyl group is optionally substituted."

[0061] "Heterocyclyl" or "heterocyclic" refers to a stable 3- to 18-membered non-aromatic ring group consisting of 2 to 12 carbon atoms and 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Unless specifically stated otherwise herein, a heterocyclyl group may be monocyclic, bicyclic, tricyclic, or tetracyclic, may include a fused or bridged ring system, and the nitrogen, carbon, or sulfur atoms of the heterocyclyl group may optionally be oxidized, the nitrogen atoms may optionally be quaternized, and the heterocyclyl group may be partially or fully saturated. Examples of such heterocyclyl groups include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoind yl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless specifically stated otherwise herein, a heterocyclyl group may optionally be substituted.

[0062] As used herein, the term "substituted" means that at least one hydrogen atom is replaced by a non-hydrogen atom, such as, but not limited to, a halogen atom, such as F, Cl, Br, and I; an oxo group (=O); a hydroxyl group (-OH); an alkoxy group (-OR a , wherein R a is C1-C 12 alkyl or cycloalkyl); a carboxyl group (-OC(=O)R a or -C(=O)OR a , wherein R a is H, C1-C 12alkyl or cycloalkyl); an amine group (-NR a R b , wherein R a and R b are each independently H, C1-C 12 alkyl or cycloalkyl); a C1-C 12 alkyl group; and any of the above groups (e.g., alkyl, cycloalkyl or heterocyclyl) replaced by a bond to a cycloalkyl group. In some embodiments, the substituent is a C1-C 12 alkyl group. In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group, e.g., fluoro, etc. 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. In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group.

[0063] "Optional" or "optionally" (e.g., optionally substituted) means that the event or circumstance described subsequently may or may not occur, and the description means both the case where the event or circumstance occurs and the case where it does not occur. For example, "optionally substituted alkyl" means that the alkyl group may or may not be substituted, and the description means that it includes both substituted alkyl groups and alkyl groups having no substitution.

[0064] The term "prodrug" is intended to indicate a compound that can be converted under physiological conditions or by solvolysis to a biologically active compound of the present invention. Thus, the term "prodrug" refers to a metabolically-precursor of a pharmaceutically acceptable compound of the present invention. A prodrug may be inactive when administered to a subject in need thereof, but is converted in vivo to an active compound of the present invention. Prodrugs are usually rapidly converted in vivo to produce the parent compound of the present invention, for example, by hydrolysis in the blood. Prodrug compounds often provide advantages in mammalian organisms such as solubility, tissue compatibility or delayed release (see Bundgard, H., Design of Prodrugs (1985), pages 7-9, 21-24 (Elsevier, Amsterdam)). Discussions of prodrugs are provided in Higuchi, T. et al., A.C.S. Symposium Series, Volume 14 and Bioreversible Carriers in Drug Design, edited by Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

[0065] The term "prodrug" is also intended to include any covalently-bonded carrier that releases an active compound of the present invention in vivo when such prodrug is administered to a mammalian subject. Prodrugs of the compounds of the present invention can be prepared by modifying the functional groups present in the compounds of the present invention such that the modification is cleaved either by a specified manipulation or in vivo to produce the parent compound of the present invention. Prodrugs are A prodrug of a compound of the present invention includes a compound in which a droxy, amino or mercapto group is bonded to an arbitrary group, and when the prodrug of the compound of the present invention is administered to a mammalian subject, this arbitrary group is cleaved to form a free hydroxy, free amino or free mercapto group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohols or amide derivatives of amine functional groups in the compounds of the present invention.

[0066] The present invention disclosed herein also intends to encompass all pharmaceutically acceptable compounds of the compounds of formula (I) or (II) that are isotopically labeled by replacing one or more atoms with atoms having different atomic weights or mass numbers. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, for example, 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 36 Cl, 123 I, and 125 I and the like. These radiolabeled compounds can be useful, for example, in helping to determine or measure the effectiveness of a compound by characterizing the site or mechanism of action, or the binding affinity to a pharmacologically important site of action. Compounds labeled with certain isotopes of structure (I) or (II), such as those incorporating a radioisotope, are useful in drug and / or substrate tissue distribution studies. The radioactive isotope tritium, namely, 3 H, and carbon-14, namely, 14 C are particularly useful for this purpose in view of their ease of incorporation and rapid detection means.

[0067] Substitution with heavier isotopes, such as deuterium, i.e., 2 substitution with H, etc., can result in certain therapeutic advantages due to greater metabolic stability, e.g., an increase in in vivo half-life or a decrease in the required dose, and may thus be preferred in some situations.

[0068] Substitution with positron-emitting isotopes, such as 11 C, 18 F, 15 O and 13 N, etc., can be useful in positron emission tomography (PET) studies for investigating substrate receptor occupancy. The isotopically labeled compounds of structure (I) or (II) can be generally prepared by conventional techniques known to those skilled in the art or by using appropriate isotopically labeled reagents in place of the unlabeled reagents previously utilized, in a process similar to that described in the preparations and examples presented below.

[0069] The present invention disclosed herein also intends to encompass the in vivo metabolites of the disclosed compounds. Such products can arise mainly from enzymatic processes, e.g., oxidation, reduction, hydrolysis, amidation, esterification, etc. of the administered compound. Thus, the present invention includes compounds produced by a process that includes administering a compound of the present invention to a mammal for a period sufficient to produce its metabolites. Such products are usually identified by administering a radiolabeled compound of the present invention in a detectable dose to an animal such as a rat, mouse, guinea pig, monkey, or human, allowing sufficient time for metabolism to occur, and isolating the conversion products from urine, blood, or other biological samples.

[0070] By "stable compound" and "stable structure" is intended a compound strong enough to withstand isolation to a useful degree of purity from the reaction mixture and formulation into an effective therapeutic agent.

[0071] "Mammal" includes both humans and domestic animals such as laboratory animals and household pets (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits), and non-domestic animals such as wildlife, etc.

[0072] "Pharmaceutically acceptable carrier, diluent or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier, which are approved by the US Food and Drug Administration as being acceptable for use in humans or domestic animals.

[0073] "Pharmaceutically acceptable salt" includes both acid addition salts and base addition salts.

[0074] The term "pharmaceutically acceptable acid addition salts" refers to salts formed with inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, caffeic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, etc., which retain the biological effectiveness and properties of the free base and are not biologically or otherwise harmful.

[0075] The term "pharmaceutically acceptable basic addition salt" refers to salts that retain the biological effectiveness and properties of the free acid and are not biologically or otherwise harmful. These salts are prepared by addition of an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including natural substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0076] Crystallization often results in the formation of solvates of the compounds of the present invention. As used herein, the term "solvate" refers to an aggregate that includes one or more molecules of a compound of the present invention and one or more molecules of a solvent. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of the present invention may exist as hydrates including, but not limited to, monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, and the like, as well as corresponding solvated forms. The compounds of the present invention may be true solvates, while in other cases, the compounds of the present invention may simply be adventitious It may hold water or may be a mixture of water and a certain exogenous solvent.

[0077] The "pharmaceutical composition" refers to a formulation of the compound of the present invention and a medium generally recognized in the art for delivering a bioactive compound to a mammal, such as a human. Such media include all pharmaceutically acceptable carriers, diluents or excipients therefor.

[0078] "Effective amount" or "therapeutically effective amount" refers to the amount of the compound of the present invention that is sufficient to effect treatment in a mammal, preferably a human, when administered to the mammal, preferably a human. The amount of the lipid nanoparticles of the present invention constituting the "therapeutically effective amount" will vary depending on the compound, the condition and its severity, the mode of administration, and the age of the mammal to be treated, but those skilled in the art can determine it as prescribed in consideration of their knowledge and the present disclosure.

[0079] As used herein, "treating" or "treatment" encompasses the treatment of a target disease or condition in a mammal, preferably a human, having the target disease or condition, (i) in a mammal, particularly when such a mammal is susceptible to the condition but has not yet been diagnosed as having it, preventing the occurrence of the disease or condition, (ii) inhibiting a disease or condition, i.e., suppressing its onset, (iii) alleviating a disease or condition, i.e., causing regression of the disease or condition, or (iv) alleviating the symptoms caused by a disease or condition, i.e., alleviating pain without addressing the underlying disease or condition It includes. As used herein, the terms "disease" and "condition" may be used interchangeably, or a particular disease or condition may have no known causative agent (therefore, the cause has not yet been resolved), and thus is not yet recognized as a disease, but is only recognized as an undesirable condition or syndrome, and may differ in that a particular set of symptoms has been confirmed by a clinician, although to varying degrees.

[0080] The compounds of the present invention or their pharmaceutically acceptable salts can contain one or more asymmetric centers, and thus can give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined as (R)- or (S)-, or (D)- or (L)- with respect to amino acids from the point of absolute stereochemistry. The present invention is intended to include all such possible isomers, as well as their racemic and optically pure forms. The optically active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers may be prepared using a chiral synthon or chiral reagent, or may be resolved using conventional techniques, such as chromatography and fractional recrystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from an appropriate optically pure precursor, or resolution of a racemate (or a racemate of a salt or derivative) using, for example, chiral high performance liquid chromatography (HPLC). If the compounds described herein contain an olefinic double bond or other geometrically asymmetric center, unless otherwise specified, the compounds are intended to include both the E and Z geometric isomers. Similarly, all tautomeric forms are also intended to be included.

[0081] "Stereoisomer" refers to a compound composed of the same atoms bonded by the same bonds but having different three-dimensional structures that are not interchangeable. The present invention contemplates various stereoisomers and mixtures thereof, including "enantiomers", which refers to two stereoisomers whose molecules are non-superimposable mirror images of each other.

[0082] "Tautomer" refers to the proton transfer from one atom of a molecule to another atom of the same molecule. The present invention includes tautomers of any of the aforementioned compounds.

[0083] Compound In one aspect, the present invention provides a novel lipid compound that can be combined with other lipid components, such as neutral lipids, charged lipids, steroids, and / or polymer-conjugated lipids, etc., to form lipid nanoparticles together with oligonucleotides. 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.

[0084] In one embodiment, the lipid compound has the formula (I):

Chemical formula

[0085] In certain embodiments of the compound of formula (I), R 1a , R 2a , R 3a or R 4a at least one of which is C1-C 12 alkyl, or L 1 or L 2 at least one of which is -O(C=O)- or -(C=O)O-. In other embodiments, R 1a and R 1b are not isopropyl when a is 6 and not n-butyl when a is 8.

[0086] In yet further embodiments, R 1a , R 2a , R 3a or R 4a at least one of which is C1-C 12 alkyl, or L 1 or L 2 at least one of which is -O(C=O)- or -(C=O)O-, R 1a and R 1b are not isopropyl when a is 6 and not n-butyl when a is 8.

[0087] In the compound of formula I, L 1 or L 2 any one of which may be -O(C=O)- or a carbon-carbon double bond. L 1 and L 2 may each be -O(C=O)- or each be a carbon-carbon double bond.

[0088] In some embodiments, one of L 1 or L 2 is -O(C=O)-. In other embodiments, both L 1 and L 2 are -O(C=O)-.

[0089] In some embodiments, one of L 1 or L 2One of them is -(C=O)O-. In other embodiments, L 1 and L 2 are both -(C=O)O-.

[0090] In some embodiments, L 1 or L 2 is a carbon-carbon double bond. In other embodiments, L 1 and L 2 are both carbon-carbon double bonds.

[0091] In yet other embodiments, one of L 1 or L 2 is -O(C=O)-, and one of L 1 or L 2 is -(C=O)O-. In further embodiments, one of L 1 or L 2 is -O(C=O)-, and one of L 1 or L 2 is a carbon-carbon double bond. In still further embodiments, one of L 1 or L 2 is -(C=O)O-, and one of L 1 or L 2 is a carbon-carbon double bond.

[0092] The "carbon-carbon" double bond refers to one of the following structures:

Chemical formula

[0093] In other embodiments, the lipid compound has the following structure (Ia): [Chemistry] has

[0094] In other embodiments, the lipid compound has the following structure (Ib): [Chemistry] has

[0095] In still other embodiments, the lipid compound has the following structure (Ic): [Chemistry] has

[0096] In certain of the aforementioned embodiments, a, b, c, and d are each independently an integer from 2 to 12 or an integer from 4 to 12. In other embodiments, a, b, c, and d are each independently an integer from 8 to 12 or an integer from 5 to 9. In certain specific embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In further embodiments, a is 3. In still further embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In further embodiments, a is 7. In still further embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In further embodiments, a is 11. In still further embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In further embodiments, a is 15. In still further embodiments, a is 16.

[0097] In some embodiments, b is 1. In other embodiments, b is 2. In further embodiments, b is 3. In still other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In further embodiments, b is 7. In still other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In further embodiments, b is 11. In still other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In further embodiments, b is 15. In still other embodiments, b is 16.

[0098] In some embodiments, c is 1. In other embodiments, c is 2. In further embodiments, c is 3. In still other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In further embodiments, c is 7. In still other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In further embodiments, c is 11. In still other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In further embodiments, c is 15. In still other embodiments, c is 16.

[0099] In some certain embodiments, d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In further embodiments, d is 3. In still other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In further embodiments, d is 7. In still other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In further embodiments, d is 11. In still other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In further embodiments, d is 15. In still other embodiments, d is 16.

[0100] In some other various embodiments, a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments, a and d are the same and b and c are the same.

[0101] The sum of a and b and the sum of c and d are factors that can be varied to obtain a lipid having desired properties. In one embodiment, a and b are selected such that their sum is an integer in the range of 14 to 24. In other embodiments, c and d are selected such that their sum is an integer in the range of 14 to 24. In a further embodiment, the sum of a and b and the sum of c and d are the same. For example, in some embodiments, the sum of a and b and the sum of c and d can both be the same integer in the range of 14 to 24. In still further embodiments, a, b, c, and d are selected such that the sum of a and b and the sum of c and d is 12 or more.

[0102] In some embodiments, e is 1. In other embodiments, e is 2.

[0103] R 1a 、R 2a 、R 3a およびR 4a The substituents at are not particularly limited. In certain embodiments, R 1a 、R 2a 、R 3a およびR 4a are H each time they appear. In certain other embodiments, at least one of R 1a 、R 2a 、R 3a およびR 4a is C1-C 12 alkyl. In certain other embodiments, at least one of R 1a 、R 2a 、R 3a およびR 4a is C1-C8 alkyl. In certain other embodiments, at least one of R 1a 、R 2a 、R 3a およびR 4aAt least one of them is C1-C6 alkyl. In some of the foregoing embodiments, C1-C8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl is.

[0104] In certain specific embodiments of the foregoing, R 1a , R 1b , R 4a and R 4b are C1-C 12 alkyl each time they appear.

[0105] In a further embodiment of the foregoing, R 1b , R 2b , R 3b and R 4b At least one of them is H, or R 1b , R 2b , R 3b and R 4b are H each time they appear.

[0106] In certain specific embodiments of the foregoing, R 1b and the carbon atom to which it is attached, together with the carbon atom to which the adjacent R 1b is attached, form a carbon-carbon double bond. In other embodiments of the foregoing, R 4b and the carbon atom to which it is attached, together with the carbon atom to which the adjacent R 4b is attached, form a carbon-carbon double bond.

[0107] R 5 and R 6 The substituents in are not particularly limited in the foregoing embodiments. In certain specific embodiments, one or both of R 5 or R 6 is methyl. In certain other specific embodiments, R 5 or R 6One or both of them are cycloalkyl, such as cyclohexyl. In these embodiments, the cycloalkyl may or may not be substituted. In certain other embodiments, the cycloalkyl is substituted with C1-C 12 alkyl, such as tert-butyl.

[0108] R 7 The substituents at are not particularly limited in the foregoing embodiments. In certain embodiments, at least one R 7 is H. In some other embodiments, R 7 is H each time it appears. In certain other embodiments, R 7 is C1-C 12 alkyl.

[0109] In certain other embodiments of the foregoing embodiments, one of R 8 or R 9 is methyl. In other embodiments, both R 8 and R 9 are methyl.

[0110] In some different embodiments, R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6- or 7-membered heterocyclic ring. In some embodiments of the foregoing, R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-membered heterocyclic ring, such as a pyrrolidinyl ring.

[0111] In various different embodiments, the compound has one of the structures described in Table 1 below.

Table 1

Table 2

Table 3

Table 4

Table 5

Table 6

Table 7

[0112] Any embodiment of the compounds of formula (I) as described above, and any particular substituent and / or variable in the compounds of formula (I) as described above, can, by combination with other embodiments of the compounds of formula (I) and / or independently of substituents and / or variables, form embodiments of the invention not specifically described above. It is understood that. In addition, in certain embodiments and / or claims, if a list of substituents and / or variables is recited for any particular R group, L group or variables a - e, each individual substituent and / or variable may be deleted 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.

[0113] It is understood that in this description, combinations of substituents and / or variables of the formulas shown are only acceptable if such contributions result in stable compounds. In various embodiments, the following compounds:

Chemical formula

[0114] are understood to be outside the scope of the present invention (wherein each R c and R d is H, or R c and R d are joined to form oxo, and x and y are each independently an integer from 0 to 6).

[0115] In various embodiments, pegylated lipids are also provided. For example, in certain embodiments, the pegylated lipid has the following structure (II):

Chemical formula

[0116] In some embodiments of the foregoing embodiment of the pegylated lipid (II), R 10 and R 11 are not both n-octadecyl when z is 42. In some embodiments, R 10 and R 11 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 18 carbon atoms. In some embodiments, R 10 and R 11 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 12 to 16 carbon atoms. In some embodiments, R 10 and R 11 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms. In some embodiments, R 10 and R 11 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms. In other embodiments, R 10 and R 11 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 16 carbon atoms. In still further embodiments, R 10 and R11 is, independently of each other, a linear or branched, saturated or unsaturated alkyl chain containing 18 carbon atoms. In other embodiments, R 10 is a linear or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms, and R 11 is a linear or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms.

[0117] In various embodiments, z ranges from a selected range such that the PEG moiety of (II) has an average molecular weight of about 400 to about 6000 g / mol. In some embodiments, the average of z is about 45.

[0118] In other embodiments, the pegylated lipid has the following structure:

Chemical formula

[0119] Compositions comprising (II) and a cationic lipid are also provided. The cationic lipid can be selected from any cationic lipid. In various embodiments, the cationic lipid is a compound having the structure (I) as described above, including either the substructures of Table 1 or a specific compound.

[0120] In some embodiments, compositions comprising one or more of the compounds of formula (I) are provided. For example, in some embodiments, the composition comprises any one of the compounds of formula (I), a therapeutic agent, and one or more excipients selected from neutral lipids, steroids, and pegylated lipids. Other pharmaceutically acceptable excipients and / or carriers are also included in various embodiments of the composition.

[0121] In certain embodiments, the therapeutic agent comprises a nucleic acid, such as an antisense oligonucleotide or messenger RNA. In some embodiments, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In various embodiments, the molar ratio of the compound to the neutral lipid ranges from about 2:1 to about 8:1.

[0122] In various embodiments, the composition further comprises a steroid or steroid analog. In certain embodiments, the steroid or steroid analog is cholesterol. In some of these embodiments, the molar ratio of the compound to cholesterol ranges from about 2:1 to 1:1.

[0123] In various embodiments, the composition comprises a pegylated lipid. For example, some embodiments include PEG-DMG. In various embodiments, the molar ratio of the compound to the pegylated lipid ranges from about 100:1 to about 25:1.

[0124] In some embodiments, the composition has the following structure (II):

Chemical formula

[0125] In some embodiments, R 10 and R 11 are each independently a linear saturated alkyl chain containing 12 to 16 carbon atoms. In other embodiments, the average of z is about 45.

[0126] In some embodiments of the foregoing compositions, the therapeutic agent comprises a nucleic acid. For example, in some embodiments, the nucleic acid is selected from antisense, plasmid DNA, and messenger RNA.

[0127] For the purposes of administration, the compounds of the invention (usually in the form of lipid nanoparticles combined with a therapeutic agent) may be administered as a raw chemical or formulated as a pharmaceutical composition. The pharmaceutical compositions of the invention comprise a compound of formula (I) and one or more pharmaceutically acceptable carriers, diluents, or excipients. The compound of formula (I) is present in the composition in an amount effective to form lipid nanoparticles and deliver a therapeutic agent, for example, to treat a particular disease or condition of interest. Appropriate concentrations and dosages can be readily determined by those skilled in the art.

[0128] Administration of the compositions of the invention can be effected via any of the recognized modes of administration of agents to achieve a similar utility. The pharmaceutical compositions of the invention can be formulated into preparations in solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes for administering such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. As used herein, the term parenteral includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection, or infusion techniques. The pharmaceutical compositions of the invention are formulated such that the active ingredient contained therein is bioavailable when the composition is administered to a patient. The composition administered to a subject or patient takes the form of one or more dosage units, where, for example, a tablet may be a single dosage unit, and a container of the compound of the invention in aerosol form can hold a plurality of dosage units. The actual methods for preparing such dosage forms are known or will be apparent to those skilled in the art. For example, Remington: The See Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). In any case, the administered composition contains, in accordance with the teachings of the present invention, 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 of interest.

[0129] The pharmaceutical composition of the present invention may be in solid or liquid form. In one aspect, the carrier(s) is / are fine particles, whereby the composition is, for example, in the form of tablets or powders. The carrier(s) may be liquid and the composition is, for example, an oral syrup, an injection solution or an aerosol useful for, for example, inhalation administration.

[0130] When oral administration is intended, the pharmaceutical composition is preferably in either solid or liquid form, and forms such as semi-solid, semi-liquid, suspensions and gels are included herein in the forms considered to be either solid or liquid.

[0131] As a solid composition for oral administration, the pharmaceutical composition can be formulated in the form of powders, granules, compressed tablets, pills, capsules, chewing gums, cachets, etc. Such solid compositions usually contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth gum or gelatin; excipients such as starch, lactose or dextrin; disintegrants such as alginic acid, sodium alginate, Primogel, corn starch; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavoring agents such as peppermint, methyl salicylate or orange flavor; and coloring agents.

[0132] When the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, the pharmaceutical composition may contain, in addition to the above types of materials, a liquid carrier such as polyethylene glycol or oil.

[0133] The pharmaceutical composition may be in liquid form, for example, an elixir, syrup, solution, emulsion or suspension. As two examples, the liquid may be for oral administration or for delivery by injection. When oral administration is intended, the preferred composition contains, in addition to the present compound, one or more of a sweetening agent, a preservative, a dye / colorant and a flavor enhancer. In compositions intended for administration by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer and an isotonic agent may be included.

[0134] The liquid pharmaceutical composition of the present invention may contain one or more of the following adjuvants, whether they are solutions, suspensions or other similar forms: sterile diluents such as water for injection, saline, preferably physiological saline, Ringer's solution, isotonic sodium chloride, etc., non-volatile oils such as synthetic mono- or diglycerides, polyethylene glycol, glycerin, propylene glycol or other solvents that can serve as solvents or suspending media; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate or phosphate, and tonicity regulators such as sodium chloride or dextrose; agents that act as antifreeze agents such as sucrose or trehalose. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes or multi-dose vials. Physiological saline is a preferred adjuvant. The pharmaceutical composition for injection is preferably sterile.

[0135] The 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 so as to obtain an appropriate dosage.

[0136] The pharmaceutical composition of the present invention may be intended for topical administration, in which case the carrier may suitably include a solution, an emulsion, an ointment or a gel base. The base may include, for example, one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. A thickening agent may be present in the pharmaceutical composition for topical administration. When transdermal administration is intended, the composition may include a transdermal patch or an iontophoresis device.

[0137] The pharmaceutical composition of the present invention may be intended for rectal administration, for example, in the form of a suppository that melts in the rectum to release the drug. The composition for rectal administration may contain an oily base as a suitable non-irritating excipient. Such bases include, but are not limited to, lanolin, cocoa butter and polyethylene glycol.

[0138] The pharmaceutical composition of the present invention may include various materials that modify the physical form of the 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 usually inert and can be selected, for example, from sugars, shellac, and other enteric coating agents. Alternatively, the active ingredient can be placed in a gelatin capsule.

[0139] The pharmaceutical composition of the present invention in solid or liquid form may include an agent that binds to the compound of the present invention and thereby aids in the delivery of the compound. Suitable agents that can act in this capacity include monoclonal or polyclonal antibodies, or proteins.

[0140] The pharmaceutical composition of the present invention may consist of dosage units for administration as an aerosol. The term aerosol is used to represent various systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system for dispensing the active ingredient. The aerosol of the compound of the present invention can be delivered in a single-phase, two-phase, or three-phase system for delivering the active ingredient(s). Delivery of the aerosol includes the necessary containers, active agents, valves, sub-containers, etc. which can be put together to form a kit. A person skilled in the art can determine a preferred aerosol without undue experimentation.

[0141] The pharmaceutical composition of the present invention can be prepared by methods well known in the pharmaceutical art. 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. Surfactants can be added to facilitate the formation of a homogeneous solution or suspension. A surfactant is a compound that interacts non-covalently with the compound of the present invention to facilitate dissolution or homogeneous suspension of the compound in an aqueous delivery system.

[0142] The composition of the present invention or its pharmaceutically acceptable salts are administered in a therapeutically effective amount, which will vary depending on various factors including the activity of the particular therapeutic agent utilized, the metabolic stability and duration of action of the therapeutic agent, the age, weight, general health, sex, and diet of the patient, the mode and time of administration, the rate of excretion, drug combinations, the severity of the particular disorder or condition, and the subject being treated.

[0143] The composition of the present invention may also be administered simultaneously with, prior to, or It can also be administered after dosing. Such combination therapies include the administration of a single pharmaceutical dosage formulation of the composition of the present invention and one or more additional active agents, as well as the administration of the composition of the present invention and each active agent in a separate pharmaceutical dosage formulation. For example, the composition of the present invention and the other active agent can be administered together to a patient as a single oral dosage composition, such as a tablet or capsule, or each agent can be administered as a separate oral dosage formulation. When separate dosage formulations are used, the compound of the present invention and one or more additional active agents can be administered essentially at the same time, i.e., simultaneously, or at staggered times, i.e., sequentially, and combination therapies are understood to include all of these regimens.

[0144] Methods for the preparation of the above compounds and compositions are described herein below and / or are known in the art.

[0145] Those skilled in the art will understand that in the processes 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 acid. Appropriate protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, etc. Appropriate protecting groups for amino, amidino and guanidino include t-butoxycarbonyl, benzyloxycarbonyl, etc. Appropriate protecting groups for mercapto include -C(O)-R” (wherein R” is alkyl, aryl or arylalkyl), p-methoxybenzyl, trityl, etc. Appropriate protecting groups for carboxylic acid include alkyl, aryl or arylalkyl esters. Protecting groups are known to those skilled in the art and can be added or removed according to standard techniques as described herein. The use of protecting groups is described in detail in Green, T.W. and P.G.M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd edition, Wiley. As will be understood by those skilled in the art, the protecting group may also be a polymer resin, such as Wang resin, Rink resin or 2-chlorotrityl-chloride resin, etc.

[0146] Such protected derivatives of the compounds of the present invention may not possess pharmacological activity as such, but it is also understood by those skilled in the art that these can be administered to mammals and then metabolized in vivo to form the pharmacologically active compounds of the present invention. Thus, 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.

[0147] Furthermore, all compounds of the present invention that exist in the free base or acid form can be converted into these pharmaceutically acceptable salts by treatment with a suitable inorganic base or organic base or inorganic acid or organic acid by methods known to those skilled in the art. The salts of the compounds of the present invention can be converted into the forms of these free bases or free acids by standard techniques.

[0148] The following reaction scheme illustrates a method for preparing a compound of the present invention, namely, formula (I):

Chemical formula

[0149] [Chemistry] Embodiments of the compound of structure (I) (e.g., compound A-5) can be prepared according to general reaction scheme 1 ("Method A") (wherein R is a saturated or unsaturated C1-C 24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1, and n is an integer from 1 to 24). Referring to general reaction scheme 1, the compound of structure A-1 can be purchased from a commercial source or prepared according to methods well known to those skilled in the art. A mixture of A-1, A-2, and DMAP is treated with DCC to obtain bromide A-3. A mixture of bromide A-3, a base (e.g., Ν,Ν-diisopropylethylamine), and Ν,Ν-dimethyldiamine A-4 is heated at a sufficient temperature and for a sufficient time to produce A-5 after any necessary workup and / or purification steps.

[0150] [Chemistry] Embodiments of the compound of structure (I) (e.g., compound B-5) can be prepared according to general reaction scheme 2 ("Method B") (wherein R is a saturated or unsaturated C1-C 24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1, and n is an integer from 1 to 24). As shown in general reaction scheme 2, the compound of structure B-1 can be purchased from a commercial source or prepared according to methods well known to those skilled in the art. A solution of B-1 (1 equivalent) is treated with acid chloride B-2 (1 equivalent) and a base (e.g., triethylamine). The crude product is treated with an oxidizing agent (e.g., pyridinium chlorochromate) to recover the intermediate product B-3. Then, a solution of crude B-3, an acid (e.g., acetic acid), and Ν,Ν-dimethylaminoamine B-4 are treated with a reducing agent (e.g., sodium triacetoxyborohydride) after any necessary workup and / or purification to obtain B-5.

[0151] Although starting materials A-1 and B-1 are shown above as containing only saturated methylene carbons, it should be noted that starting materials containing a carbon-carbon double bond can also be utilized for the preparation of compounds containing a carbon-carbon double bond.

[0152]

Chemical formula

[0153] The following examples are provided for illustrative purposes only and are not limiting.

Examples

[0154] (Example 1) Synthesis of Compound 1 Compound 1 was prepared as follows according to Method B: A solution of octane-1,8-diol (9.8 g) in methylene chloride (100 mL) and tetrahydrofuran (60 mL) was treated with 2-ethylhexanoyl chloride (10 g). Triethylamine (15 mL) was added slowly and the solution was stirred for 3 days. The reaction mixture was filtered and the filtrate was washed with brine (2×). The organic fraction was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. Using methylene chloride, the crude product was filtered through silica gel (20 g) to give 15.8 g of a crude product. The resulting oily substance was dissolved in methylene chloride (100 mL) and treated with pyridinium chlorochromate (13 g) for 2 h. Diethyl ether (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 passed through a silica gel (77 g) column using an ethyl acetate / hexane (0 - 6%) gradient. 8-O-(2’-ethylhexanoyloxy)octanal (6.7 g) was recovered as an oily substance.

[0155] A solution of 8-O-(2’-ethylhexanoyloxy)octanal (6.7 g), acetic acid (25 drops) and 2-N,N-dimethylaminoethylamine (0.54 g) in methylene chloride (40 mL) was treated with sodium triacetoxyborohydride (1.5 g) overnight. The solution was washed with aqueous sodium hydrogen carbonate followed by brine. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel (75 g) column using a methanol / methylene chloride (0 - 10%) gradient followed by a second column (20 g) to give Compound 1 (1 g) as a colorless oily substance.

[0156] (Example 2) Synthesis of Compound 2 Compound 2 was prepared as follows according to Method A: In an argon atmosphere, dicyclohexylcarbodiimide (908 mg, 4.4 mmol) was added to a round-bottom flask charged with phytol (593 mg, 2 mmol), 6-bromohexanoic acid (780 mg, 4 mmol), and 4-(dimethylamino)pyridine (60 mg) in dichloromethane (20 mL). The precipitate was discarded by filtration. The filtrate was concentrated, and the resulting residue was purified by column chromatography on silica gel eluted with a gradient mixture of ethyl acetate in hexane (0% - 3%). This gave a colorless oily substance of (E)-3,7,11,15-tetramethylhexadec-2-enyl 6-bromohexanoate (0.79 g, 1.67 mmol, 83%).

[0157] (E)-3,7,11,15-Tetramethylhexadec-2-enyl 6-bromohexanoate (0.42 g, 0.887 mmol), N,N-diisopropylethylamine (1.5 molar equivalents, 1.33 mmol, MW 129.25, 171 mg), and N,N-dimethylethylenediamine (39 mg, 0.44 mmol) in DMF (4 mL) were heated at 77 °C for 18 h. The reaction mixture was then cooled and extracted with hexane (3 × 20 mL). The hexane extracts were combined, dried over sodium sulfate, filtered, and concentrated. This was combined with the second reactant (total amount approximately 0.7 g). The crude product was purified several times by column chromatography on silica gel eluted with a gradient mixture of methanol in DCM (0% - 5%). This gave a slightly yellow oily substance of the desired product (39 mg).

Number

[0158] (Example 3) Synthesis of Compound 3 Starting from bromoacetic acid instead of 6-bromohexanoic acid, Compound 3 was prepared in a manner similar to Compound 2, yielding 22 mg of a thick colorless oily substance, 0.029 mmol, 6%.

Number

[0159] (Example 4) Synthesis of Compound 4 Compound 4 was prepared as follows according to Method B: A solution of dodecane-1,12-diol (10 g) in methylene chloride (100 mL) and tetrahydrofuran (50 mL) was treated with 2-ethylhexanoic acid (7.2 g), DCC (10.5 g), DMAP (3.5 g), and triethylamine (10 mL). The solution was stirred for 4 days. The reaction mixture was filtered, and the filtrate was washed with dilute hydrochloric acid. The organic fraction was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was dissolved in methylene chloride (50 mL), allowed to stand overnight, and filtered. The solvent was removed to yield 12.1 g of a crude product.

[0160] The crude product was dissolved in methylene chloride (100 mL) and treated with pyridinium chlorochromate (8 g) overnight. Diethyl ether (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 passed through a silica gel (75 g) column using an ethyl acetate / hexane (0 - 6%) gradient. Crude 12-O-(2'-ethylhexanoyloxy)dodecanal (3.5 g) was recovered as an oily substance.

[0161] A solution of the crude product (3.5 g), acetic acid (60 drops), and 2-N,N-dimethylaminoethylamine (0.30 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (0.86 g) overnight. The solution was washed with aqueous sodium bicarbonate and then brine. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel (20 g) column using a methanol / methylene chloride (0 - 8%) gradient, followed by a second column (20 g) to yield the desired product (0.6 g) as a colorless oily substance.

[0162] (Example 5) Synthesis of Compound 5 Compound 5 was prepared as follows according to Method B: A solution of hexane-1,6-diol (10 g) in methylene chloride (40 mL) and tetrahydrofuran (20 mL) was treated with 2-hexyldecanoyl chloride (10 g) and triethylamine (10 mL). The solution was stirred for 1 hour and the solvent was removed. The reaction mixture was suspended in hexane, filtered, and the filtrate was washed with water. The solvent was removed and the residue was passed through a silica gel (50 g) column using hexane as the eluent, followed by methylene chloride, to produce 6-(2’-hexyldecanoyloxy)hexan-1-ol as an oily substance (7.4 g).

[0163] The purified product (7.4 g) was dissolved in methylene chloride (50 mL) and treated with pyridinium chlorochromate (5.2 g) for 2 hours. Diethyl ether (200 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 passed through a silica gel (50 g) column using an ethyl acetate / hexane (0 - 5%) gradient. 6-(2’-hexyldecanoyloxy)dodecanal (5.4 g) was recovered as an oily substance.

[0164] A solution of the product (4.9 g), acetic acid (0.33 g) and 2-N,N-dimethylaminoethylamine (0.40 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (2.1 g) for 2 hours. The solution was washed with aqueous sodium hydroxide. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel (50 g) column using a methanol / methylene chloride (0 - 8%) gradient to produce the desired product (1.4 g) as a colorless oily substance.

[0165] (Example 6) Synthesis of Compound 6 Compound 6 was prepared as follows according to Method B: A solution of nonane-1,9-diol (12.6 g) in methylene chloride (80 mL) was treated with 2-hexyldecanoic acid (10.0 g), DCC (8.7 g) and DMAP (5.7 g). The solution was stirred for 2 hours. The reaction mixture was filtered and the solvent removed. The residue was dissolved in warm hexane (250 mL) and crystallized. The solution was filtered and the solvent removed. The residue was dissolved in methylene chloride and washed with dilute hydrochloric acid. The organic fraction was dried over anhydrous magnesium sulfate, filtered and the solvent removed. The residue was passed through a silica gel column (75 g) using 0 - 12% ethyl acetate / hexane as eluent to yield 9-(2'-hexyldecanoyloxy)nonan-1-ol (9.5 g) as an oily substance.

[0166] The product was dissolved in methylene chloride (60 mL) and treated with pyridinium chlorochromate (6.4 g) for 2 hours. Diethyl ether (200 mL) was added and the supernatant filtered through a silica gel bed. The solvent was removed from the filtrate and the resulting oily substance was passed through a silica gel column (75 g) using an ethyl acetate / hexane (0 - 12%) gradient to yield 9-(2'-ethylhexanoyloxy)nonanal (6.1 g) as an oily substance.

[0167] A solution of the crude product (6.1 g), acetic acid (0.34 g) and 2-N,N-dimethylaminoethylamine (0.46 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (2.9 g) for 2 hours. The solution was diluted with methylene chloride and washed with aqueous sodium hydroxide followed by water. The organic phase was dried over anhydrous magnesium sulfate, filtered and the solvent removed. The residue was passed through a silica gel column (75 g) using a methanol / methylene chloride (0 - 8%) gradient followed by a second column (20 g) using a methylene chloride / acetic acid / methanol gradient. The purified fraction was dissolved in methylene chloride, washed with dilute aqueous sodium hydroxide, dried over anhydrous magnesium sulfate, filtered and the solvent removed to yield the desired product (1.6 g) as a colorless oily substance.

[0168] (Example 7) Synthesis of Compound 7 According to Method A, Compound 7 was prepared from 3,5,5-trimethylhexyl 10-bromodecanoate and N,N-dimethylethane-1,2-diamine to produce 144 mg of a slightly yellow oily substance, 0.21 mmol, 11%.

Number

[0169] (Example 8) Synthesis of Compound 8 Compound 8 was prepared by Method A in a 15% yield.

Number

[0170] (Example 9) Synthesis of Compound 9 Compound 9 was prepared as follows according to Method B: A solution of nonane-1,9-diol (10.0 g) in methylene chloride (100 mL) was treated with citroneloyl chloride (10.1 g, prepared from citronellic acid and oxalyl chloride) and triethylamine (10 mL) and stirred for 3 days. The reaction mixture was diluted with methylene chloride and washed with dilute hydrochloric acid. The organic fraction was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was dissolved in hexane, filtered, and the solvent was removed. The residue was passed through a series of silica gel columns (60 - 70 g) using hexane followed by methylene chloride as the eluent to produce 9-(citroneloyloxy)nonan-1-ol (7.6 g) as an oily substance. The product was dissolved in methylene chloride (50 mL) and treated with pyridinium chlorochromate (6.4 g) for 90 minutes. Diethyl ether (200 mL) was added and the supernatant was filtered through a silica gel bed. The residue was dissolved in hexane and passed through a silica gel (20 g) column using hexane as the eluent to produce 9-(citroneloyloxy)nonanal (5 g) as an oily substance.

[0171] The product was dissolved in methylene chloride (50 mL) and treated with pyridinium chlorochromate (6.4 g) for 90 minutes. Diethyl ether (200 mL) was added and the supernatant was filtered through a silica gel bed. The residue was dissolved in hexane and passed through a silica gel (20 g) column using hexane as the eluent to produce 9-(citroneloyloxy)nonanal (5 g) as an oily substance.

[0172] A solution of the crude product (5 g), acetic acid (0.33 g), and 2-N,N-dimethylaminoethylamine (0.48 g) in methylene chloride (40 mL) was treated with sodium triacetoxyborohydride (1.2 g) overnight. The solution was diluted with methylene chloride and washed with aqueous sodium hydroxide. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel (50 g) column using a 0 - 12% methanol / methylene chloride gradient, followed by a second silica gel column (20 g) using the same gradient to yield the desired product (0.6 g) as a colorless oil.

[0173] (Example 10) Synthesis of Compound 10 Compound 10 was prepared according to Method A, yielding 147 mg of a colorless oil, 0.23 mmol, 17%.

Number

[0174] (Example 11) Synthesis of Compound 11 Compound 11 was prepared according to Method A, yielding 154 mg of a slightly yellow oil, 0.22 mmol, 14%.

Number

[0175] (Example 12) Synthesis of Compound 12 Compound 12 was prepared according to Method A, yielding 169 mg of a slightly yellow oil, 0.26 mmol, 17%.

[0176]

Number

Number

[0177] (Example 14) Synthesis of Compound 14 Compound 14 was prepared according to Method A to give 111 mg of a colorless oily substance, 0.16 mmol, 11%.

Number

[0178] (Example 15) Synthesis of Compound 15 Compound 15 was prepared according to Method A to give 116 mg of a white paste substance, 0.16 mmol, 10%.

Number

[0179] (Example 16) Synthesis of Compound 16 Compound 16 was prepared according to Method A to give 118 mg of a colorless oily substance, 0.17 mmol, 12%.

Number

[0180] (Example 17) Synthesis of Compound 17 Compound 17 was prepared according to Method A to give 145 mg of a slightly yellow oily substance, 0.21 mmol, 13%.

Number

[0181] (Example 18) Synthesis of Compound 18 Compound 18 was prepared according to Method A to give 111 mg of a colorless oily substance, 0.17 mmol, 14%. [Number]

[0182] (Example 19) Synthesis of Compound 19 Compound 19 was prepared according to Method A to give 76 mg of a colorless oily substance, 0.11 mmol, 6%. [Number]

[0183] (Example 20) Synthesis of Compound 20 Compound 20 was prepared according to General Procedure A to give 157 mg of a colorless oily substance, 0.22 mmol, 14%. [Number]

[0184] (Example 21) Synthesis of Compound 21 Compound 21 was prepared according to General Procedure A to give 164 mg of a colorless oily substance, 0.21 mmol, 14%. [Number]

[0185] (Example 22) Synthesis of Compound 22 Compound 22 was prepared according to Method A as follows: Step 1. A solution of 6-bromohexanoic acid (20 mmol, 3.901 g), 2-hexyl-1-decanol (1.8 equiv, 36 mmol, 8.72 g), and 4-dimethylaminopyridine (DMAP 0.5 equiv, 10 mmol, 1.22 g) in DCM (80 mL) was added with DCC (1.1 equiv, 22 mmol, 4.54 g). The resulting mixture was stirred at room temperature for 16 h. The precipitate was discarded by filtration. The filtrate was concentrated. The residue was purified by column chromatography on silica gel eluted with a gradient mixture of ethyl acetate in hexane (0 - 2%). Thereby, the desired product was obtained as a colorless oily substance (7.88 g, 18.8 mmol, 94%). Step 2.

[0186] A mixture of the bromide obtained in Step 1 (1.34 equiv, 7.88 g, 18.8 mmol), N,N-diisopropylethylamine (1.96 equiv, 27.48 mmol, 4.78 mL), and N,N-dimethylethylenediamine (1 equiv, 14.02 mmol, 1.236 g, 1.531 mL) in acetonitrile (70 mL) was heated at 79 °C (oil bath) for 16 h in a 250 mL flask equipped with a condenser. The reaction mixture was cooled to room temperature and concentrated. The residue was put into a mixture of ethyl acetate and hexane (1:9) and water. The phases were separated and washed with water (100 mL) and brine. Dried over sodium sulfate and concentrated (8.7 g of oily substance). The crude product (8.7 g of oily substance) was purified by column chromatography on silica gel (0 - 3% MeOH in chloroform). The fractions containing the desired product were combined and concentrated. The residue was dissolved in 1 mL of hexane and filtered through a layer of silica gel (3 - 4 mm, washed with 8 mL of hexane). The filtrate was blown dry under an Ar stream and dried well in vacuo overnight (1.30 g, mmol, %, colorless oily substance, desired product).

Number

[0187] (Example 23) Synthesis of Compound 23 Compound 23 was prepared according to General Procedure A to give 200 mg of a colorless oil, 0.24 mmol, 16%.

Number

[0188] (Example 24) Synthesis of Compound 24 Compound 24 was prepared according to General Procedure A to give 138 mg of a colorless oil, 0.18 mmol, 12%.

Number

[0189] (Example 25) Synthesis of Compound 25 Compound 25 was prepared according to General Procedure A to give 214 mg of a colorless oil, 0.24 mmol, 17%.

Number

[0190] (Example 26) Synthesis of Compound 26 Compound 26 was prepared according to General Procedure A to give 170 mg of a colorless oil, 0.21 mmol, 13%.

Number

[0191] (Example 27) Synthesis of Compound 27 Compound 27 was prepared according to General Procedure A to give 255 mg of a colorless oil, 0.29 mmol, 18%.

Number

[0192] (Example 28) Synthesis of Compound 28 Compound 28 was prepared according to General Procedure A to give 248 mg of a colorless oil, 0.27 mmol, 19%.

Number

[0193] (Example 29) Synthesis of Compound 29 Compound 29 was prepared according to General Procedure A to give 181 mg of a colorless oil, 0.23 mmol, 17%.

Number

[0194] (Example 30) Synthesis of Compound 30 Compound 30 was prepared according to General Procedure A to give 88 mg of a colorless oil, 0.11 mmol, 3%.

Number

[0195] (Example 31) Synthesis of Compound 31 Compound 31 was prepared according to General Procedure C to give 275 mg of a slightly yellow oil, 0.30 mmol, 35% overall yield over 3 steps.

Number

[0196] (Example 32) Synthesis of Compound 32 Compound 32 was prepared according to Method C as follows: Step 1. To a solution of 2-aminoethanol (116 mg, 1.9 mmol, 115 μL, MW 61.08, d 1.012) in 15 mL of anhydrous THF, 2-hexyldecyl 6-bromohexanoate (1.9 eq, 1.52 g, 3.62 mmol), potassium carbonate (1.9 eq, 3.62 mmol, 500 mg), cesium carbonate (0.3 eq, 0.57 mmol, 186 mg) and sodium iodide (10 mg) were added, and the mixture was heated to reflux under Ar for 6 days. The solvent was evaporated under reduced pressure, the residue was dissolved in hexane, and washed with water and brine. The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure to give a colorless oily substance. The crude product was purified by flash column chromatography on silica gel (230 - 400 mesh silica gel, MeOH in chloroform, 0 - 4%) to give 936 mg of a colorless oily substance (1.27 mmol, 70%). Step 2.

[0197] To a magnetically stirred, ice-cooled solution of 936 mg (1.27 mmol) of the product obtained in Step 1 in 2 mL of CHCl3, thionyl chloride (2.9 eq, 3.70 mmol, 440 mg, 270 μL) in 15 mL of chloroform was added dropwise under an Ar atmosphere. After completion of the addition of SOCl2, the ice bath was removed and the reaction mixture was stirred at room temperature for 16 h under an Ar atmosphere. CHCl3 and SOCl2 were removed under reduced pressure to give a thick yellow oily substance. Step 3.

[0198] The crude product obtained in Step 2 was dissolved in THF (20 mL). Pyrrolidine (1.6 mL, 1.36 g, 19 mmol) was added to the THF solution. The sealed mixture was heated at 64 °C overnight. The reaction mixture was concentrated (dark brown oily substance). The residue was purified by flash dry column chromatography on silica gel (MeOH in chloroform, 0 - 4%). Thereby, the desired product was obtained as a slightly yellow oily substance (419 mg, 0.53 mmol, 83%).

Number

[0199] (Example 33) Synthesis of Compound 33 Compound 33 was prepared according to General Procedure C to give 419 mg of a slightly yellow oily substance, 0.54 mmol, 60% overall yield for 3 steps.

Number

[0200] (Example 34) Synthesis of Compound 34 Compound 34 was prepared as follows according to Method B: A solution of nonane-1,9-diol (10 g) in methylene chloride (250 mL) was treated with 2-ethylhexanoic acid (4.5 g), DCC (7.7 g) and DMAP (4.2 g). The solution was stirred for 3 days. The reaction mixture was filtered and hexane (200 mL) was added to the filtrate. The mixture was stirred and the precipitate was allowed to settle. The supernatant was decanted and the solvent was removed. The residue was suspended in hexane (70 mL) and allowed to settle. The supernatant was decanted and the solvent was removed. The residue was dissolved in hexane, allowed to stand at room temperature and then filtered. The solvent was removed and the residue was passed through a silica gel column (50 g) using a 0-10% ethyl acetate / hexane gradient followed by a 0-8% methanol / methylene chloride gradient to give 5.6 g of 9-(2’ethylhexanoyloxy)nonan-1-ol as a colorless oily substance.

[0201] The product was dissolved in methylene chloride (70 mL) and treated with pyridinium chlorochromate (5 g) for 2 h. Diethyl ether (250 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 plug and the solvent was removed to give crude 9-(2’ethylhexanoyloxy)nonanal (3.4 g) as an oily substance.

[0202] A solution of the crude product (3.4 g), acetic acid (0.52 g) and 2-N,N-dimethylaminoethylamine (0.33 g) in methylene chloride (50 mL) was treated with sodium triacetoxyborohydride (1.86 g) overnight. The solution was washed with aqueous sodium hydroxide solution. The organic phase was dried over anhydrous magnesium sulfate, filtered and the solvent removed. The residue was passed through a silica gel (50 g) column using an acetic acid / methanol / methylene chloride (2 - 0% / 0 - 12% / 98 - 88%) gradient. The purified fractions were washed with aqueous sodium hydrogen carbonate, dried over magnesium sulfate, filtered and the solvent removed to give compound 34 as an oily substance (0.86 g).

[0203] (Example 35) Synthesis of Compound 35 Compound 35 was prepared as follows according to Method B: A solution of dodecane-1,12-diol (18.1 g) in methylene chloride (90 mL) was treated with citronellic acid (7.5 g), DCC (10.0 g) and DMAP (9.5 g). The solution was stirred overnight. The reaction mixture was filtered and the filtrate washed with dilute hydrochloric acid. The organic fraction was dried over anhydrous magnesium sulfate, filtered and the solvent removed to give 12.2 g of crude 12-citronellyloxidodecan-1-ol.

[0204] The crude product was dissolved in methylene chloride (60 mL) and treated with pyridinium chlorochromate (6.8 g) for 3 h. Diethyl ether (200 mL) was added and the supernatant filtered through a silica gel bed. The solvent was removed from the filtrate and the resulting oily substance passed through a silica gel (75 g) column using an ethyl acetate / hexane (0 - 12%) gradient. Crude 12-citronellyloxidodecanal (6.2 g) was recovered as an oily substance.

[0205] A solution of the crude product (6.2 g), acetic acid (0.44 g) and 2-N,N-dimethylaminoethylamine (0.50 g) in methylene chloride (40 mL) was treated with sodium triacetoxyborohydride (2.9 g) overnight. The solution was washed with aqueous sodium bicarbonate and then with brine. The organic phase was dried over anhydrous magnesium sulfate, filtered and the solvent removed. The residue was passed through a silica gel (75 g) column using an acetic acid / methanol / methylene chloride (2 - 0% / 0 - 12% / 98 - 88%) gradient. The purified fractions were washed with aqueous sodium bicarbonate, dried over magnesium sulfate, filtered and the solvent removed to yield Compound 35 (1.68 g) as an oily substance.

[0206] (Example 36) Synthesis of Compound 36 Compound 36 was prepared according to General Procedure C to give 108 mg of a colorless oily substance (0.14 mmol).

Number

[0207] (Example 37) Synthesis of Compound 37 Compound 37 was prepared according to General Procedure C to give 330 mg of a colorless oily substance (0.40 mmol, 80% overall yield for 3 steps).

Number

[0208] (Example 38) Synthesis of Compound 38 Compound 38 was prepared according to Method B as follows: A solution of nonane-1,9-diol (16 g) in methylene chloride (100 mL) was treated with 2-butyloctanoic acid (10 g), DCC (10.3 g) and DMAP (6.7 g). The solution was stirred for 3 days. The reaction mixture was filtered and hexane (250 mL) was added to the filtrate. The mixture was stirred and the precipitate was allowed to settle. The supernatant was decanted and the solvent was removed. The residue was suspended in hexane and allowed to settle. The supernatant was decanted and the solvent was removed (repeated 2 times). The residue was dissolved in hexane, allowed to stand at room temperature and then filtered. The solvent was removed and the residue was passed through a silica gel column (18 g) using methylene chloride to produce crude 9-(2’-butyloctanoyloxy)nonan-1-ol (17.7 g) as an oily substance.

[0209] The crude product was dissolved in methylene chloride (250 mL) and treated with pyridinium chlorochromate (11.2 g) overnight. Diethyl ether (750 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 (150 mL ). The suspension was filtered through a silica gel plug and the solvent was removed. The crude product was passed through a silica gel (80 g) column using a 0 - 6% ethyl acetate / hexane gradient to produce 9-(2’-butyloctanoyloxy)nonanal (5.3 g) as an oily substance.

[0210] A solution of the product (5.3 g), acetic acid (0.37 g) and 2-N,N-dimethylaminoethylamine (0.47 g) in methylene chloride (50 mL) was treated with sodium triacetoxyborohydride (3.35 g) overnight. The solution was washed with aqueous sodium hydroxide. The organic phase was dried over anhydrous magnesium sulfate, filtered and the solvent removed. The residue was passed through a silica gel (60 g) column using an acetic acid / methanol / methylene chloride (2 - 0% / 0 - 12% / 98 - 88%) gradient. The purified fractions were washed with aqueous sodium bicarbonate, dried over magnesium sulfate, filtered and the solvent removed to produce Compound 38 as an oily substance (2.3 g).

[0211] (Example 39) Synthesis of Compound 39 Compound 39 was prepared as follows according to Method B: A solution of hexane-1,6-diol (12 g) in methylene chloride (250 mL) was treated with 2-decyltetradecanoic acid (17.5 g), DCC (11.3 g) and DMAP (6.8 g). The solution was stirred overnight. The reaction mixture was filtered and hexane was added to the filtrate. The mixture was stirred and the precipitate was allowed to settle. The supernatant was decanted and the solvent was removed. The residue was passed through a silica gel column (80 g) using hexane, followed by 0-1% methanol / methylene chloride to yield crude 6-(2'-decyltetradecanoyloxy)hexan-1-ol (5.8 g) as an oily substance.

[0212] The crude product was dissolved in methylene chloride (70 mL) and treated with pyridinium chlorochromate (2.9 g) for 2 hours. Diethyl ether (250 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 plug and the solvent was removed. The crude product was passed through a silica gel (10 g) column using a 0-5% ethyl acetate / hexane gradient to yield 6-(2'-decyltetradecanoyloxy)hexanal (3.2 g) as an oily substance.

[0213] A solution of the product (3.2 g), acetic acid (0.28 g) and 2-N,N-dimethylaminoethylamine (0.15 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (0.98 g) overnight. The solution was washed with aqueous sodium hydroxide. The organic phase was dried over anhydrous magnesium sulfate, filtered and the solvent was removed. The residue was passed through a silica gel (50 g) column using an acetic acid / methanol / methylene chloride (2-0% / 0-12% / 98-88%) gradient. The purified fractions were washed with aqueous sodium hydrogen carbonate, dried over magnesium sulfate, filtered and the solvent was removed to yield Compound 39 as an oily substance (1.2 g).

[0214] (Example 40) Synthesis of Compound 40 Compound 40 was prepared as follows according to Method B: A solution of nonane-1,9-diol (10.1 g) in methylene chloride (200 mL) was treated with 2-octyldodecanoic acid (10.0 g), DCC (8.3 g), and DMAP (5.0 g). The solution was stirred overnight. The reaction mixture was filtered, and hexane (200 mL) was added to the filtrate. The mixture was stirred, and the precipitate was allowed to settle. The supernatant was decanted, and the solvent was removed. This process was repeated twice. The residue was passed through a silica gel column (75 g) using hexane, followed by 4 - 10% methanol / methylene chloride to give crude 9-(2’-octyl dodecanoyloxy)nonan-1-ol (about 11 g) as an oily substance.

[0215] The crude product was dissolved in methylene chloride (70 mL) and treated with pyridinium chlorochromate (8 g) for 2 hours. Diethyl ether (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 plug, and the solvent was removed to give crude 9-(2’-octyldodecanoyloxy)nonanal (8.4 g) as an oily substance.

[0216] A solution of the product (8.4 g), acetic acid (0.84 g), and 2-N,N-dimethylaminoethylamine (0.55 g) in methylene chloride (60 mL) was treated with sodium triacetoxyborohydride (2.9 g) for 2 hours. The solution was washed with aqueous sodium hydroxide. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel (75 g) column using an acetic acid / methanol / methylene chloride (2 - 0% / 0 - 12% / 98 - 88%) gradient. The purified fractions were washed with aqueous sodium bicarbonate, dried over magnesium sulfate, filtered, and the solvent was removed to give Compound 40 as an oily substance (3.2 g).

[0217] (Example 41) Synthesis of Compound 41 Compound 41 was prepared as follows according to Method B: A solution of nonan-1,9-diol (9.6 g) in methylene chloride (200 mL) was treated with 2-decyltetradecanoic acid (8.4 g), DCC (8.6 g) and DMAP (5.0 g). The solution was stirred overnight. The reaction mixture was filtered and hexane (200 mL) was added to the filtrate. The mixture was stirred and the precipitate was allowed to settle. The supernatant was decanted and the solvent was removed. This process was repeated twice. The residue was passed through a silica gel column (75 g) using hexane followed by 4 - 10% methanol / methylene chloride to yield crude 9-(2'-decyltetradecanoyloxy)nonan-1-ol (6.4 g) as an oily substance.

[0218] The crude product was dissolved in methylene chloride (50 mL) and treated with pyridinium chlorochromate (5.7 g) for 2 hours. Diethyl ether (200 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 plug and the solvent was removed to yield crude 9-(2'-decyltetradecanoyloxy)nonanal (5 g) as an oily substance.

[0219] A solution of the product (5 g), acetic acid (0.45 g) and 2-N,N-dimethylaminoethylamine (0.32 g) in methylene chloride (20 mL) was treated with sodium triacetoxyborohydride (1.6 g) for 2 hours. The solution was washed with aqueous sodium hydroxide. The organic phase was dried over anhydrous magnesium sulfate, filtered and the solvent was removed. The residue was passed through a silica gel (50 g) column using an acetic acid / methanol / methylene chloride (2 - 0% / 0 - 12% / 98 - 88%) gradient. The purified fractions were washed with aqueous sodium hydrogen carbonate, dried over magnesium sulfate, filtered and the solvent was removed to yield Compound 41 as an oily substance (2.2 g).

[0220] (Example 42) Synthesis of PEG Lipid [Chemical formula] PEGylated lipid 42-6 ("PEG-DMA") was prepared according to the above reaction scheme (where n approximates the center of the repeating unit of a series of ethylene oxides in the PEGylated lipid). Synthesis of 42-1 and 42-2

[0221] To a solution of myristic acid (6 g, 26 mmol) in toluene (50 mL) was added oxalyl chloride (39 mmol, 1.5 equiv, 5 g) at room temperature. The resulting mixture was heated at 70 °C for 2 h, after which the mixture was concentrated. The residue was dissolved in toluene and concentrated again. The remaining oily substance was added via syringe to concentrated ammonia solution (20 mL) at 10 °C. The reaction mixture was filtered and washed with water. The white solid was dried in vacuo. The desired product was obtained as a white solid (3.47 g, 15 mmol, 58.7%). Synthesis of 42-3

[0222] To a suspension of 20-2 (3.47 g, 15 mmol) in THF (70 mL) was added lithium aluminum hydride (1.14 g, 30 mmol) portionwise at room temperature over a period of 30 min. The mixture was then gently heated to reflux overnight (in an oil bath at 65 °C). The mixture was cooled to 5 °C and sodium sulfate nonahydrate was added. The mixture was stirred for 2 h, filtered through a layer of celite, and washed with 15% MeOH in DCM (200 mL). The filtrate and washings were combined and concentrated. The remaining solid was dried in vacuo. The desired product was obtained as a white solid (2.86 13.4 mmol, 89.5%). Synthesis of 42-4

[0223] To a solution of myristic acid (3.86 g, 16.9 mmol) in benzene (40 mL) and DMF (1 drop) was added oxalyl chloride (25.35 mmol, 1.5 equiv, 3.22 g) It was added at room temperature. The mixture was stirred at room temperature for 1.5 hours. It was heated at 60 °C for 30 minutes. The mixture was concentrated. The residue was dissolved in toluene and concentrated again. The remaining oily substance (light yellow) was put into 20 mL of benzene and added dropwise via a syringe at 10 °C to a solution of 20-3 (2.86 g, 13.4 mmol) and triethylamine (3.53 mL, 1.5 equiv) in benzene (40 mL). After the addition, the resulting mixture was stirred at room temperature overnight. The reaction mixture was diluted with water and adjusted to pH 6 - 7 with 20% H₂SO₄. The mixture was filtered and washed with water. A light-colored solid was obtained. The crude product was recrystallized from methanol. Thereby, the desired product was obtained as an off-white solid (5.65 g, 13 mmol, 100%). Synthesis of 42-5

[0224] To a suspension of 20-4 (5.65 g, 13 mmol) in THF (60 mL), lithium aluminum hydride (0.99 g, 26 mmol) was added portionwise at room temperature over a period of 30 minutes. Then, the mixture was gently heated to reflux overnight. The mixture was cooled to 0 °C, and sodium sulfate nonahydrate was added. The mixture was stirred for 2 hours and then filtered through a pad of celite and silica gel and first washed with ether. The filtrate became turbid and a precipitate formed. A white solid was obtained by filtration. The solid was recrystallized from MeOH to give a colorless crystalline solid (2.43 g).

[0225] Subsequently, the pads of celite and silica gel were washed with 5% MeOH in DCM (400 mL), then 10% MeOH in DCM containing 1% triethylamine (300 mL). The fractions containing the desired product were combined and concentrated. A white solid was obtained. The solid was recrystallized from MeOH to give a colorless crystalline solid (0.79 g). The above two solids (2.43 g and 0.79 g) were combined and dried in vacuo (3.20 g, 60%).

Number

[0226] To a solution of 20-5 (7 mmol, 2.87 g) and triethylamine (30 mmol, 4.18 mL) in DCM (100 mL) was added a solution of mPEG-NHS (manufactured by NOF, 5.0 mmol, 9.97 g, PEG MW approximately 2,000, n = about 45) in DCM (120 mL). After 24 hours, the reaction solution was washed with water (300 mL). The aqueous phase was extracted twice with DCM (100 mL × 2). The combined DCM extracts were washed with brine (100 mL). The organic phase was dried over sodium sulfate, filtered, and partially concentrated. The concentrated solution (about 300 mL) was cooled to about -15 °C. A white solid was obtained by filtration (1.030 g, unreacted starting amine). To the filtration, Et3N (1.6 mmol, 0.222 mL, 4 equivalents) and acetic anhydride (1.6 mmol, 164 mg) were added. The mixture was stirred at room temperature for 3 hours and then concentrated to a solid. The remaining solid was purified by column chromatography on silica gel (0 - 8% methanol in DCM). Thereby, the desired product was obtained as a white solid (9.211 g). [Number]

[0227] (Example 43) Synthesis of PEG lipid [Chemistry] A suspension of palmitic acid (10 g) in benzene (50 mL) was treated with oxalyl chloride (5 mL) for 3 hours. The solvent was removed and the residue was dissolved in dichloromethane (40 mL). The solution was slowly added to concentrated ammonia (100 mL) with stirring. The resulting suspension was filtered and washed with water. The precipitate was suspended in methanol, warmed to 50 °C to dissolve the solid, and then cooled to room temperature. The recrystallized crude product was filtered and dried to produce hexadecanoylamide as a white solid (8.7 g).

[0228] The crude product was suspended in THF (50 mL) and treated with lithium aluminum hydride (1.1 g, added slowly). The reaction mixture was stirred for 1 hour. Excess methanol was then slowly added, followed by water (2 mL). Dichloromethane (200 mL) was added and the mixture was filtered. The solvent was removed from the filtrate to yield crude hexadecylamine (7 g).

[0229] A solution of hexadecyanoyl chloride (10.5 g) prepared as described above in dichloromethane (40 mL) was slowly added with stirring to a solution of crude hexdecylamine in dichloromethane (40 mL). Triethylamine (15 mL) was added and the solution was stirred at room temperature overnight. The solution was filtered and the collected precipitate (N-(hexadecanoyl)hexadecylamide, about 10.7 g) was dried under vacuum.

[0230] The crude product was suspended in THF (60 mL) and treated with lithium aluminum hydride (1.1 g, added slowly). The reaction mixture was stirred at room temperature overnight. Excess methanol was then slowly added, followed by water (3 mL). Dichloromethane (250 mL) was added and the solution was filtered. The solvent was removed to yield crude dihexadecylamine (7.6 g) as a white powder.

[0231] A solution of dihexadecylamine (3 g) and monomethoxy-PEG-2000-acetoyl N-hydroxysuccinimide ester (10 g) in dichloromethane (60 mL) was treated with triethylamine (3 mL) and stirred overnight. Acetic anhydride (1 mL) was added and the solution was stirred for 30 minutes. The reaction mixture was diluted with dichloromethane and washed with brine. The organic fraction was dried over magnesium sulfate, filtered, and the solvent was removed. The residue was passed through a silica gel (75 g) column using a 0 - 8% methanol / dichloromethane gradient to yield 43-1 as a white powder (5.2 g).

[0232] (Example 44) Synthesis of PEG lipid

Chem.

[0233] The crude product was suspended in THF (80 mL) and treated with excess lithium aluminum hydride (1.6 g, added slowly). The reaction mixture was stirred for 1 hour. Excess methanol was added followed by slow addition of concentrated hydrochloric acid until the precipitate dissolved. The reaction mixture was diluted with water and recrystallized. The solution was filtered and the collected precipitate was dried to yield crude octadecylamine (5.9 g).

[0234] A solution of octadecyanoyl chloride (8 g) prepared as described above in dichloromethane (40 mL) was treated with crude octadecylamine with stirring. Triethylamine (15 mL) and hexane (100 mL) were added and the solution was stirred at 45 °C for 1 hour. The solution was cooled, filtered and the collected precipitate was washed with methanol. The precipitate was recrystallized from dichloromethane to yield N-(octadecanoyl)octadecylamide (6.8 g) as a white powder.

[0235] The product was suspended in THF (150 mL) and treated with lithium aluminum hydride (1.5 g, added slowly). The reaction mixture was refluxed overnight. Excess methanol was added followed by slow addition of concentrated hydrochloric acid until the precipitate dissolved. The solution was diluted with water and crystallized. The solution was filtered and the collected precipitate was washed with water (4×). The precipitate was dried under vacuum to yield dioctadecylamine (6.2 g) as a white powder.

[0236] A solution of dioctadecylamine (4.5 g) and monomethoxy-PEG-2000-acetoil N-hydroxysuccinimide ester (9 g) in chloroform (90 mL) was treated with triethylamine (40 mL) and stirred at 50 °C for 30 minutes. The solution was filtered. Acetic anhydride (1 mL) was added to the filtrate and the solution was stirred for 15 minutes. Ammonia (150 mL), followed by brine (150 mL) were added. The reaction mixture was extracted with dichloromethane and the organic phase was washed with dilute hydrochloric acid. The organic fraction was dried over magnesium sulfate, filtered and the solvent removed. The residue was passed through a silica gel (75 g) column using a 0-8% methanol / dichloromethane gradient to produce 44-1 as a white powder (7.4 g).

[0237] (Example 45) Synthesis of PEG lipid [Chemical formula] A suspension of lauric acid (10 g) in benzene (20 mL) was treated with oxalyl chloride (10 mL) for 1 hour. The solvent was removed and the residue was dissolved in dichloromethane (50 mL). The solution was slowly added to concentrated ammonia (150 mL) with stirring. The reaction mixture was washed with dichloromethane. The organic phase was dried over magnesium sulfate, filtered and the solvent removed to produce crude dodecanoylamide as a white powder (10 g).

[0238] The product was suspended in THF (150 mL) and treated with lithium aluminum hydride (3 g, added slowly). The reaction mixture was stirred for 1 hour. Excess methanol, followed by aqueous sodium hydroxide solution (5 mL) were added slowly. Dichloromethane (100 mL) was added and the resulting suspension was filtered. The solvent was removed from the filtrate and the residue was passed through a silica gel (80 g) column using a methanol / dichloromethane gradient to produce dodecylamine (4.5 g). chloromethane gradient to produce dodecylamine (4.5 g).

[0239] A solution of lauric acid (7.3 g), crude tetradecylamine (4.5 g) and N-hydroxysuccinimide (4.2 g) in dichloromethane (200 mL) was treated with EDC (7.0 g), followed by triethylamine (15 mL), and the solution was stirred overnight at room temperature. The solution was filtered to produce crude N-dodecanoyldodecylamide (1 g) as a powder. The solvent was removed from the filtrate, and the residue was suspended in methanol and filtered to produce an additional 2 g of crude N-dodecanoyldodecylamide.

[0240] The crude product (3 g) was suspended in THF (60 mL) and treated with lithium aluminum hydride (added slowly in an excess amount) for 2 hours. Excess methanol, followed by water (1 mL), was added slowly. Dichloromethane (100 mL) was added, and the suspension was filtered. The solvent was removed from the filtrate, and the residue was passed through a silica gel column (20 g) using a 0-12% methanol / dichloromethane gradient to produce N-dodecanoyldodecylamine (1.4 g) as a waxy solid.

[0241] A solution of N-dodecanylamine (0.52 g) and monomethoxy-PEG-2000-acetoil N-hydroxysuccinimide ester (1.5 g) in dichloromethane (10 mL) was treated with triethylamine (0.2 mL) and stirred overnight. The solvent was removed, and the product was passed through a silica gel column (20 g) using a 0-6% methanol / dichloromethane gradient. Anhydrous acetic acid (5 drops) and trimethylamine (10 drops) were added to the solution of the recovered product in dichloromethane and stirred for 1 hour. The solvent was removed, and the residue was passed through a silica gel (20 g) column using a 0-4% methanol / dichloromethane gradient to produce MePEGA-2000-DLA as a white powder (0.44 g). (Example 46) Synthesis of PEG Lipids [Chemical formula]

[0242] A suspension of myristic acid (30 g) in benzene (100 mL) was treated with oxalyl chloride (15 mL) overnight. The solvent was removed, and the residue was dissolved in dichloromethane (100 mL). The solution was slowly added to concentrated ammonia (70 mL) with stirring. The resulting suspension was filtered and washed with water. The precipitate was dried to yield crude tetradecanoylamide as a white solid (27 g).

[0243] The product was suspended in THF (200 mL) and treated with lithium aluminum hydride (4.5 g, added slowly). The reaction mixture was stirred for 1 hour. Excess methanol was then slowly added, followed by water (10 mL). Dichloromethane (250 mL) was added, and the resulting suspension was filtered. The solvent was removed from the filtrate to yield crude tetradecylamine (17.6 g).

[0244] A solution of lauric acid (3.5 g), crude tetradecylamine (3 g), and N-hydroxysuccinimide (1.9 g) in dichloromethane (40 mL) was treated with EDC (3.3 g), followed by triethylamine (4 mL), and the solution was stirred at room temperature for 3 days. The solution was filtered and the collected precipitate was dried to yield crude N-lauroyltetradecylamine (2.6 g) as a powder.

[0245] The crude product was suspended in THF (60 mL) and treated with lithium aluminum hydride (0.8 g, added slowly) for 1 hour. Excess methanol was then slowly added, followed by water (2 mL). Dichloromethane was added, and the suspension was filtered. The solvent was removed, and the residue was dissolved in hot methanol (100 mL). The solution was cooled and filtered to yield 0.5 g of N-dodecyltetradecylamine. The solvent was removed from the filtrate, and the process was repeated using 20 mL of methanol to yield a second harvest of N-dodecyltetradecylamine (0.9 g).

[0246] A solution of N-dodecyltetradecylamine (0.5 g) and monomethoxy-PEG-2000-acetoil N-hydroxysuccinimide ester (1.5 g) in dichloromethane (10 mL) was treated with triethylamine (0.2 mL) and stirred overnight. The solvent was removed and the product was passed through a silica gel column (20 g) using a 0-6% methanol / dichloromethane gradient. Anhydrous acetic acid (5 drops) and trimethylamine (10 drops) were added to the solution of the recovered product in dichloromethane and stirred for 1 hour. The solvent was removed and the residue was passed through a silica gel (20 g) column using a 0-4% methanol / dichloromethane gradient to produce MePEGA-2000-LMA as a white powder (0.76 g).

[0247] (Example 47) In vivo evaluation of luciferase mRNA using lipid nanoparticle compositions Cationic lipid (MC3), DSPC, cholesterol and PEG-lipid were solubilized in ethanol at a molar ratio of 50:10:38.5:1.5. Lipid nanoparticles (LNP) were prepared at a weight ratio of total lipid to mRNA of about 10:1 to 30:1. Briefly, mRNA was diluted to 0.2 mg / mL at pH 4 in 10-50 mM citrate buffer. Using a syringe pump, the ethanolic lipid solution and the aqueous mRNA solution were mixed at a ratio of about 1:5 to 1:3 (vol / vol) at a total flow rate above 15 ml / min. Ethanol was then removed and the external buffer was replaced with PBS by dialysis. Finally, the lipid nanoparticles were filtered through a sterile filter with 0.2 μm pores. The particle size of the lipid nanoparticles was 70-90 nm in diameter as determined by quasi-elastic light scattering using a Nicomp 370 submicron particle sizer (Santa Barbara, CA).

[0248] Studies were performed in 6 - 8 week - old female C57BL / 6 mice (Charles River) in accordance with the guidelines established by the institutional animal care committee (ACC) and the Canadian Council on Animal Care (CCAC). Different doses of mRNA - lipid nanoparticles were administered systemically via tail vein injection, and the animals were euthanized at specific time points (1, 2, 4, 8, and 24 hours) after administration. The liver and spleen were collected into pre - weighed tubes, weighed, immediately snap - frozen in liquid nitrogen, and stored at - 80°C until processed for analysis.

[0249] For the liver, approximately 50 mg was dissected for analysis and placed into 2 mL FastPrep tubes (MP Biomedicals, Solon OH). 1 / 4 - inch ceramic beads (MP Biomedicals) were 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 using a FastPrep24 device (MP Biomedicals) at 2×6.0 m / sec for 15 seconds. The homogenate was incubated at room temperature for 5 minutes, then diluted 1:4 in GLB and evaluated using the SteadyGlo Luciferase assay system (Promega). Specifically, 50 μL of the diluted tissue homogenate was reacted with 50 μL of the SteadyGlo substrate, shaken for 10 seconds, followed by a 5 - minute incubation, and then, CentroXS 3Quantification was performed using an LB 960 illuminometer (Berthold Technologies, Germany). The amount of protein assayed was determined using a BCA protein assay kit (Pierce, Rockford IL). Relative light units (RLU) were then normalized to the total protein (μg) assayed. A standard curve was generated using QuantiLum Recombinant Luciferase (Promega) to convert RLU to luciferase (ng). Based on the data provided in Figure 1, the 4-hour time point was selected for the evaluation of the efficacy of the lipid formulation (see Example 48).

[0250] FLuc mRNA (L-6107) from Trilink Biotechnologies expresses the luciferase protein originally isolated from the firefly, Photinus pyralis. FLuc is commonly used in mammalian cell cultures to measure both gene expression and cell viability. It emits bioluminescence in the presence of the substrate, luciferin. This capped and polyadenylated mRNA is completely substituted with 5-methylcytidine and pseudouridine.

[0251] (Example 48) Determination of the efficacy of lipid nanoparticle formulations containing various cationic lipids using a luciferase mRNA-expressing rodent model in vivo The cationic lipids shown in Table 2 were previously tested using nucleic acids. For comparison purposes, using these lipids, lipid nanoparticles containing FLuc mRNA (L-6107) were also formulated using the strain mixing method as described in Example 47 and PCT / US10 / 22614, the entirety of which is incorporated herein by reference. The lipid nanoparticles were formulated using the following molar ratio: 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid (“PEG-DMG”, i.e., (1-(methoxy-polyethylene glycol)-2,3-dimyristoyl glycerol, average PEG molecular weight 2000). Four hours after administration via tail vein injection as described in Example 47, relative activity was determined by measuring luciferase expression in the liver. The activities at doses of 0.3 and 1.0 mg of mRNA / kg were compared and expressed as luciferase (ng) / liver (g) measured 4 hours after administration as described in Example 47.

Table 8

[0252] The novel lipids of the present invention shown in Table 3 were formulated using the following molar ratio: 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid (“PEG-DMA” compound 42-6). Four hours after administration via tail vein injection as described in Example 47, relative activity was determined by measuring luciferase expression in the liver. The activities at doses of 0.3 and 1.0 mg of mRNA / kg were compared and expressed as luciferase (ng) / liver (g) measured 4 hours after administration as described in Example 47.

Table 9

Table 10

Table 11

Table 12

[0253] (Example 49) Determination of the pKa of the formulated lipid As described elsewhere, the pKa of the formulated cationic lipid correlates with the efficacy of the LNP 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)). The preferred range of pKa is from about 5 to about 7. Using an assay based on the fluorescence of 2-(p-toluidino)-6-naphthalene sulfonic acid (TNS), the pK a of each cationic lipid was determined in lipid nanoparticles. Lipid nanoparticles containing cationic lipid / DSPC / cholesterol / PEG-lipid (50 / 10 / 38.5 / 1.5 mol%) at a total lipid concentration of 0.4 mM in PBS were prepared using an inline process as described in Example 47. TNS was prepared as a 100 μΜ stock solution in distilled water. Vesicles were diluted to 24 μΜ lipid in 2 mL of buffer containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, 130 mM NaCl (the pH of which ranges from 2.5 to 11). An aliquot of the TNS solution was added to a final concentration of 1 μΜ, vortexed, and then the fluorescence intensity was measured at room temperature using an SLM Aminco Series 2 Luminescence Spectrophotometer with excitation and emission wavelengths of 321 nm and 445 nm. A sigmoid best-fit analysis was applied to the fluorescence data, and the pK a was measured as the pH that causes the maximum half-maximal fluorescence intensity (see Figure 2).

[0254] (Example 50) Comparison of the activities of amino lipids and the effect of hydrocarbon chain structure The cationic lipids shown in Table 5 were formulated using the following molar ratios: 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG-lipid (42-6). Relative activity was determined by measuring luciferase expression in the liver 4 hours after administration via tail vein injection as described in Example 48. The activities at doses of 0.1, 0.3, and 1.0 mg of mRNA / kg were compared and expressed as luciferase (ng) / liver (g) measured 4 hours after administration as described in Example 48. The data are plotted in Figure 3 (highest to lowest: diamond = compound 6; square = compound 5; triangle = MC3; and circle = compound A). Compounds A, 5, and 6 have a common end group but different hydrocarbon chain structures.

Table 13

[0255] (Example 51) Activity comparison of cationic lipids and effect of end group chain length The cationic lipids shown in Table 6 were formulated using the following molar ratios: 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG-lipid (42-6). Relative activity was determined by measuring luciferase expression in the liver 4 hours after administration via tail vein injection as described in Example 48. The activities at doses of 0.1, 0.3, and 1.0 mg of mRNA / kg were compared and expressed as luciferase (ng) / liver (g) measured 4 hours after administration as described in Example 48. Compounds A, B, and C have a common hydrocarbon chain structure but different end group chain lengths. Compound 6 shares a preferred end group with compound A and demonstrates an unexpected advantage in the combination of end group and hydrocarbon chain structure.

Table 14

[0256] (Example 52) Activity Comparison of PEG-DMG and PEG-DMA Lipids The activity comparison of PEG-DMG and PEG-DMA lipids is shown in Figure 4. LNPs were formulated using the following molar ratios: 50% MC3 lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG-lipid (PEG-DMG or PEG-DMA). Relative activity was determined by measuring luciferase expression in the liver 4 hours after administration via tail vein injection as described in Example 48. Activity at doses of 0.1, 0.3, and 1.0 mg of mRNA / kg was compared and expressed as luciferase (ng) / liver (g) measured 4 hours after administration as described in Example 48.

[0257] The data are presented in bar graph form in Figure 4.

[0258] (Example 53) LNP Activity Using a Red Fluorescent Protein Called Cherry Red (CR) mRNA LNPs using Compound 6 are formulated as described in Example 47 using mRNA encoding a red fluorescent protein called Cherry Red (CR, e.g., product L-6113 from TriLink Biotechnologies). An in vivo study is performed as described in Example 47, and sections of liver tissue are processed and observed by confocal fluorescence microscopy at 4, 6, and 24 hours after administration. The expression level reached a peak at approximately 6 hours and was maintained for at least 24 hours. The tissue sections observed demonstrate homogeneous expression throughout the liver.

[0259] (Example 54) In Vivo Delivery of Human FIX mRNA to Hepatocytes by LNPs Results in Therapeutic Levels of hFIX Protein in Mouse Plasma The LNP having the cationic lipid shown in Table 7 is formulated using the mRNA encoding human FIX (e.g., product L-6110 from TriLink Biotechnologies) as described in Example 47, with the following lipid molar ratio: 50% lipid / 10% distearoyl phosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG-lipid. Plasma proteins are analyzed by ELISA using a commercially available kit (e.g., Abcam ab108831) according to the manufacturer's instructions. The measured levels of hFIX expression given in Table 7 for the LNP of Compound 5 and Compound 6 are clinically relevant, and when these hFIX mRNA LNPs are administered at a dose of 1 mg / kg, a hFIX protein concentration is produced that is sufficient to change patients with severe disease to a mild disease state. The duration of these levels of hFIX is about 15 hours or longer.

Table 15

[0260] Exemplary embodiments include the following: Embodiment 1. Formula I:

Chemical formula

[0261] Embodiment 2. A compound according to Embodiment 1, wherein one of L 1 or L 2 is -O(C=O)-.

[0262] Embodiment 3. A compound according to any one of Embodiments 1 or 2, wherein one of L 1 or L 2 is -(C=O)O-.

[0263] Embodiment 4. A compound according to any one of Embodiments 1 to 3, wherein one of L 1 or L 2 is a carbon-carbon double bond.

[0264] Embodiment 5. The following structure (Ia), (Ib) or (Ic):

Chemical formula

[0265] Embodiment 6. A compound according to any one of Embodiments 1 to 5, wherein a, b, c and d are each independently an integer from 2 to 12.

[0266] A compound according to any one of Embodiments 1 to 5, wherein Embodiments 7.a, b, c, and d are each independently an integer from 5 to 9.

[0267] Embodiment 8. R 1a , R 2a , R 3a and R 4a of a compound according to any one of Embodiments 1 to 7, wherein at least one of them is H.

[0268] Embodiment 9. R 1a , R 2a , R 3a and R 4a of a compound according to any one of Embodiments 1 to 7, wherein each occurrence of them is H.

[0269] Embodiment 10. R 1a , R 2a , R 3a and R 4a of a compound according to any one of Embodiments 1 to 7, wherein at least one of them is C1 - C8 alkyl.

[0270] Embodiment 11. The compound according to Embodiment 10, wherein the C1 - C8 alkyl is methyl, ethyl, n - propyl, iso - propyl, n - butyl, iso - butyl, tert - butyl, n - hexyl, or n - octyl.

[0271] Embodiment 12. R 1b , R 2b , R 3b and R 4b of a compound according to any one of Embodiments 1 to 11, wherein at least one of them is H.

[0272] Embodiment 13. R 1b , R 2b , R 3b and R 4b of a compound according to any one of Embodiments 1 to 11, wherein each occurrence of them is H.

[0273] Embodiment 14. R 1b and the carbon atom to which it is attached are adjacent to R 1bA compound according to any one of Embodiments 1 to 11, which together with the carbon atom to which it is attached forms a carbon-carbon double bond.

[0274] Embodiment 15.R 4b wherein the carbon atom to which it is attached is adjacent to R 4b A compound according to any one of Embodiments 1 to 11 or 14, which together with the carbon atom to which it is attached forms a carbon-carbon double bond.

[0275] Embodiment 16.R 5 or R 6 A compound according to any one of Embodiments 1 to 15, wherein one of them is methyl.

[0276] Embodiment 17.R 5 and R 6 A compound according to any one of Embodiments 1 to 15, wherein each of them is methyl.

[0277] Embodiment 18.R 5 or R 6 A compound according to any one of Embodiments 1 to 15, wherein one of them is cycloalkyl.

[0278] Embodiment 19.R 5 and R 6 A compound according to any one of Embodiments 1 to 15, wherein each of them is cycloalkyl.

[0279] Embodiment 20. A compound according to any one of Embodiments 18 or 19, wherein the cycloalkyl is unsubstituted.

[0280] Embodiment 21. A compound according to any one of Embodiments 18 or 19, wherein the cycloalkyl is substituted.

[0281] Embodiment 22. A compound according to any one of Embodiments 18 or 19, wherein the cycloalkyl is substituted with C1-C6 alkyl.

[0282] The compound of Embodiment 22, wherein the C1-C6 alkyl is tert-butyl.

[0283] The compound according to any one of Embodiments 18 to 23, wherein the cycloalkyl is cyclohexyl.

[0284] Embodiment 25. At least one R 7 is H, and the compound according to any one of Embodiments 1 to 24.

[0285] Embodiment 26. Each R 7 is H, and the compound of Embodiment 25.

[0286] Embodiment 27. At least one R 7 is C1-C6 alkyl, and the compound according to any one of Embodiments 1 to 25.

[0287] Embodiment 28. The compound according to any one of Embodiments 1 to 27, wherein e is 2.

[0288] Embodiment 29. At least one of R 8 or R 9 is methyl, and the compound according to any one of Embodiments 1 to 28.

[0289] Embodiment 30. Each of R 8 and R 9 is methyl, and the compound of Embodiments 1 to 29.

[0290] Embodiment 31. The compound of Embodiment 1, selected from the compounds in Table 1.

[0291] Embodiment 32. A composition comprising the compound according to any one of Embodiments 1 to 31 and a therapeutic agent.

[0292] Embodiment 33. The composition of Embodiment 32, further comprising one or more excipients selected from neutral lipids, steroids, and polymer-conjugated lipids.

[0293] The composition of Embodiment 33, comprising one or more neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

[0294] Embodiment 35. The composition of Embodiment 34, wherein the neutral lipid is DSPC.

[0295] Embodiment 36. The composition of any one of Embodiments 33 to 35, wherein the molar ratio of the compound to the neutral lipid is in the range of about 2:1 to about 8:1.

[0296] Embodiment 37. The composition of any one of Embodiments 32 to 36, wherein the steroid is cholesterol.

[0297] Embodiment 38. The composition of Embodiment 37, wherein the molar ratio of the compound to cholesterol is in the range of about 2:1 to 1:1.

[0298] Embodiment 39. The composition of any one of Embodiments 32 to 38, wherein the polymer-conjugated lipid is a pegylated lipid.

[0299] Embodiment 40. The composition of Embodiment 39, wherein the molar ratio of the compound to the pegylated lipid is in the range of about 100:1 to about 25:1.

[0300] Embodiment 41. The composition of any one of Embodiments 39 or 40, wherein the pegylated lipid is PEG-DMG.

[0301] Embodiment 42. The pegylated lipid has the following structure (II):

Chemical formula

[0302] Embodiment 43.R 10 and R 11 A composition according to embodiment 42, wherein each of and R is independently a straight-chain saturated alkyl chain containing 12 to 16 carbon atoms.

[0303] A composition according to any one of embodiments 42 or 43, wherein the average of z is about 45.

[0304] A composition according to any one of embodiments 32 to 44, wherein the therapeutic agent comprises a nucleic acid.

[0305] A composition according to embodiment 45, wherein the nucleic acid is selected from antisense and messenger RNA.

[0306] A method for administering a therapeutic agent to a patient in need thereof, the method comprising the steps of preparing or providing a composition according to any one of embodiments 32 to 46, and administering the composition to the patient.

[0307] Embodiment 48. The following structure (II):

Chemical formula

[0308] Embodiment 49.R 10 and R 11 is each independently a straight-chain saturated alkyl chain containing 12 to 16 carbon atoms, the pegylated lipid of Embodiment 48.

[0309] Embodiment 50. The pegylated lipid of any one of Embodiments 48 or 49, wherein z is about 45.

[0310] Embodiment 51. The following structure:

Chemical formula

[0311] Embodiment 52. A composition comprising the pegylated lipid of any one of Embodiments 48 to  51 and a cationic lipid.

[0312] Embodiment 53. The composition of Embodiment 52, wherein the cationic lipid is a compound of any one of Embodiments 1 to 31.

[0313] The various embodiments described above can, in combination, provide further embodiments. Without limitation, all of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referenced herein and / or listed in the Application Data Sheet, including U.S. Provisional Application No. 62 / 016,839, filed Jun. 25, 2014, are hereby incorporated by reference in their entirety. Aspects of the embodiments can be modified as necessary to utilize the concepts of the various patents, applications, and publications and can provide further embodiments. These and other changes can be made to the embodiments in view of the foregoing detailed description. Generally, in the following claims, the terms used should not be construed as limiting the claims to the specific embodiments disclosed herein and in the claims, but rather should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the present disclosure.

Claims

1. Formula I: 【Chemical 1】 (wherein, L １ and L ２ each independently represents -O(C=O)-, -(C=O)O- or a carbon-carbon double bond, R １ａ and R １ｂ each occur independently and are (a) H or C １ to C １２ alkyl, or (b) R １ａ is H or C １ to C １２ alkyl, and the carbon atom to which R １ｂ is attached, together with the carbon atom to which the adjacent R １ｂ is attached, forms a carbon-carbon double bond, R ２ａ and R ２ｂ each independently, upon occurrence, is (a) H or C １ -C １２ alkyl, or (b) R ２ａ is H or C １ -C １２ alkyl, and the carbon atom to which R ２ｂ is attached, together with the carbon atom to which it is attached and the adjacent R ２ｂ forms a carbon-carbon double bond, R ３ａ and R ３ｂ each independently, upon occurrence, is (a) H or C １ -C １２ alkyl, or (b) R ３ａ is H or C １ -C １２ alkyl, and the carbon atom to which R ３ｂ is attached, together with the carbon atom to which the adjacent R ３ｂ is attached, forms a carbon-carbon double bond, R ４ａ and R ４ｂ each independently, upon occurrence, is (a) H or C １ to C １２ alkyl, or (b) R ４ａ is H or C １ to C １２ alkyl, and the carbon atom to which R ４ｂ is attached, together with the carbon atom to which the adjacent R ４ｂ is attached, forms a carbon-carbon double bond, R ５ and R ６ are each independently methyl or cycloalkyl, R ７ is, independently for each occurrence, H or C １ to C １２ alkyl, R ８ and R ９ are each independently unsubstituted C １ -C １２ alkyl, or R ８ and R ９ together with the nitrogen atom to which they are attached form a 5-, 6- or 7-membered heterocycle containing one nitrogen atom, a and d are each independently an integer from 0 to 24, b and c are each independently an integer from 1 to 24, e is 1 or 2, provided that R １ａ 、R ２ａ 、R ３ａ or R ４ａ at least one of which is C １ ~C １２ alkyl, or at least one of L １ or L ２ is -O(C=O)- or -(C=O)O-, R １ａ and R １ｂ is not isopropyl when a is 6 and is not n-butyl when a is 8) a compound having the structure of or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

2. L １ or L ２ The compound according to claim 1, wherein one of them is -O(C=O)-

3. L １ or L ２ The compound according to claim 1, wherein one of them is -(C=O)O-.

4. L １ or L ２ The compound according to claim 1, wherein one of them is a carbon-carbon double bond.

5. having one of the following structures (Ia), (Ib) or (Ic): 【Chemical 2】 The compound according to claim 1.

6. The compound according to claim 1, wherein a, b, c and d are each independently an integer from 2 to 12.

7. The compound according to claim 1, wherein a, b, c and d are each independently an integer from 5 to 9.

8. R １ａ 、 R ２ａ 、 R ３ａ and R ４ａ The compound according to claim 1, wherein at least one of

9. R １ａ 、 R ２ａ 、 R ３ａ and R ４ａ is H each time it appears, the compound according to claim 1.

10. R １ａ 、 R ２ａ 、 R ３ａ and R ４ａ at least one of which is C １ to C ８ alkyl, the compound according to claim 1.

11. C １ ~C ８ The compound according to claim 10, wherein the alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

12. R １ｂ 、R ２ｂ 、R ３ｂ and R ４ｂ The compound according to claim 1, wherein at least one of

13. R １ｂ 、 R ２ｂ 、 R ３ｂ and R ４ｂ is H each time it appears, the compound according to claim 1.

14. R １ｂ and the carbon atom to which it is attached, together with the adjacent R １ｂ and the carbon atom to which it is attached, form a carbon-carbon double bond, the compound according to claim 1.

15. R ４ｂ and the carbon atom to which it is attached, together with the adjacent R ４ｂ and the carbon atom to which it is attached, form a carbon-carbon double bond, the compound according to claim 1.

16. R ５ or R ６ The compound according to claim 1, wherein one of them is methyl.

17. R ５ and R ６ each of which is methyl, the compound according to claim 1.

18. R ５ or R ６ The compound according to claim 1, wherein one of them is cycloalkyl.

19. R ５ and R ６ The compound according to claim 1, wherein each of them is cycloalkyl.

20. The compound according to claim 18, wherein the cycloalkyl is unsubstituted.

21. The compound according to claim 18, wherein the cycloalkyl is substituted.

22. wherein the cycloalkyl is C １ -C ６ alkyl-substituted compound according to claim 18.

23. C １ -C ６ The compound according to claim 22, wherein the alkyl is tert-butyl.

24. The compound according to claim 18, wherein the cycloalkyl is cyclohexyl.

25. At least one R ７ The compound according to claim 1, wherein said at least one R is H.

26. Each R ７ The compound according to claim 25, wherein R is H.

27. At least one R ７ is C １ -C ６ alkyl, and the compound according to claim 1.

28. The compound according to claim 1, wherein e is 2.

29. R ８ or R ９ The compound according to claim 1, wherein at least one of them is methyl.

30. R ８ and R ９ The compound according to claim 1, wherein each of them is methyl.

31. having one of the following structures: [Chemical Formula 3] 【Chemical Formula 4】 【Chemical Formula 5】 ​ 【Chemical Formula 7】 【Chemical Formula 8】 【Chemical Formula 9】 The compound according to claim 1.

32. A composition comprising the compound according to any one of claims 1 to 31 and a therapeutic agent.

33. The composition according to claim 32, further comprising one or more excipients selected from neutral lipids, steroids and polymer-conjugated lipids.

34. The composition according to claim 33, comprising one or more neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.

35. The composition according to claim 34, wherein the neutral lipid is DSPC.

36. The composition according to claim 33, wherein the molar ratio of the compound to the neutral lipid is in the range of about 2:1 to about 8:

1.

37. The composition according to claim 32, wherein the steroid is cholesterol.

38. The composition according to claim 37, wherein the molar ratio of the compound to cholesterol is in the range of about 2:1 to 1:

1.

39. The composition according to claim 32, wherein the polymeric conjugate lipid is a pegylated lipid.

40. The composition according to claim 39, wherein the molar ratio of the compound to the pegylated lipid is in the range of about 100:1 to about 25:

1.

41. The composition according to claim 39, wherein the pegylated lipid is PEG-DMG.

42. The pegylated lipid has the following structure (II): 【Chemical 10】 (wherein, R １０ and R １１ are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, and the alkyl chain is optionally interrupted by one or more ester bonds, z has an average value in the range of 30 to 60) or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, of the composition according to claim 39.

43. R １０ and R １１ The composition according to claim 42, wherein each of them is independently a linear saturated alkyl chain containing 12 to 16 carbon atoms.

44. The composition according to claim 42, wherein the average of z is about 45.

45. The composition according to claim 32, wherein the therapeutic agent comprises a nucleic acid.

46. The composition according to claim 45, wherein the nucleic acid is selected from antisense and messenger RNA.

47. A method for administering a therapeutic agent to a patient in need thereof, the method comprising the step of preparing or providing the composition according to claim 32, and the step of administering the composition to the patient.

48. The following structure (II): 【Chemical 11】 (wherein, R １０ and R １１ are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, and the alkyl chain is optionally interrupted by one or more ester bonds, z has an average value in the range of 30 to 60, However, R １０ and R １１ are not both n-octadecyl when z is 42) and having a pegylated lipid, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

49. R １０ and R １１ are each independently a linear saturated alkyl chain containing 12 to 16 carbon atoms, the pegylated lipid according to claim 48.

50. The pegylated lipid according to claim 48, wherein z is about 45.

51. The following structure: 【Chemical 12】 (wherein n has an average value in the range of 40 to 50) One of which has the pegylated lipid according to claim 48.

52. A composition comprising the pegylated lipid according to any one of claims 48 to 51 and a cationic lipid.

53. The composition according to claim 52, wherein the cationic lipid is the compound according to claim 1.

Images