Lipids for lipid nanoparticles

Abstract

Compounds are provided having the following formula (I): (I) or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein R1, R2, R3, G1, G2, L1, and L2 are as defined herein. Uses of the compounds as components of lipid nanoparticle formulations for the delivery of therapeutic agents, compositions comprising the compounds, and methods of their use and preparation are also provided.

Description

Background Technology Technical Field

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

[0003] Related technical descriptions

[0004] Nucleic acid delivery presents numerous challenges that impact the desired response in biological systems. Nucleic acid-based therapies hold immense potential, but the need for more efficient delivery of nucleic acids to appropriate sites within cells or organisms remains to realize this potential. Therapeutic nucleic acids include messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNases, plasmids, immunostimulatory nucleic acids, antagomir, antimir, mimics, supermir, and aptamers. Some nucleic acids, such as mRNA or plasmids, can be used to achieve the expression of specific cellular products, which would be useful in treating diseases, for example, those related to protein or enzyme deficiencies. Therapeutic applications of translatable nucleotide delivery are extremely broad because constructs can be synthesized to produce any chosen protein sequence, whether or not the sequence is inherent to the system. The expression products of nucleic acids can increase existing protein levels, replace missing or nonfunctional forms of proteins, or introduce new proteins and associated functions into cells or organisms.

[0005] Some nucleic acids, such as miRNA inhibitors, can be used to achieve the expression of specific cellular products regulated by miRNAs, which will be useful in treating diseases such as those related to protein or enzyme deficiencies. The therapeutic applications of miRNA inhibition are extremely broad because constructs can be synthesized to inhibit one or more miRNAs, which in turn regulate the expression of mRNA products. Inhibition of endogenous miRNAs can increase the expression of their downstream target endogenous proteins and restore appropriate function in cells or organisms, serving as a means of treating diseases associated with specific miRNAs or groups of miRNAs.

[0006] Other nucleic acids can downregulate the intracellular levels of specific mRNAs, and therefore the synthesis of the corresponding proteins can be downregulated through processes such as RNA interference (RNAi) or complementary binding of antisense RNA. The therapeutic applications of antisense oligonucleotides and RNAi are also very broad because oligonucleotide constructs with any nucleotide sequence targeting the target mRNA can be synthesized. Targets can include mRNAs from normal cells, mRNAs associated with disease states (e.g., cancer), and mRNAs from infectious agents (e.g., viruses). To date, antisense oligonucleotide constructs have shown the ability to specifically downregulate target proteins by degrading homologous mRNAs in both in vitro and in vivo models. Furthermore, antisense oligonucleotide constructs are currently being evaluated in clinical studies.

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

[0008] There remains a need for modified cationic lipids and lipid nanoparticles for delivering oligonucleotides. Preferably, these lipid nanoparticles will provide an optimal drug-to-lipid ratio, protect nucleic acids from degradation and clearance in serum, be suitable for systemic delivery, and provide intracellular delivery of nucleic acids. Additionally, these lipid-nucleic acid particles should be well-tolerated and provide a sufficient therapeutic index so that treating patients with an effective dose of nucleic acid is not associated with unacceptable toxicity and / or risk to the patient. This disclosure provides for these and related advantages.

[0009] Brief Overview

[0010] In summary, this disclosure provides lipid compounds (including their stereoisomers, pharmaceutically acceptable salts, or tautomers) that can be used alone or in combination with other lipid components (e.g., neutral lipids, charged lipids, steroids (including, for example, all steroids) and / or their analogues, and / or polymerically conjugated lipids) to form lipid nanoparticles for delivering therapeutic agents. In some cases, the lipid nanoparticles are used to deliver nucleic acids such as antisense RNA and / or messenger RNA. Methods for treating various diseases or conditions (e.g., those caused by infectious agents and / or protein deficiency) using such lipid nanoparticles are also provided.

[0011] In one embodiment, a compound having the structure of formula (I) is provided:

[0012] (I)

[0013] Or its pharmaceutically acceptable salt, tautomer or stereoisomer, wherein R 1 R 2 R 3 G 1 G 2 L 1 and L 2 As defined in this article.

[0014] Pharmaceutical compositions comprising one or more compounds of formula (I) above and therapeutic agents are also provided. In some embodiments, the pharmaceutical composition further comprises one or more components selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. Such compositions can be used to form lipid nanoparticles for delivery of therapeutic agents.

[0015] In other embodiments, this disclosure provides a method for administering a therapeutic agent to a patient in need, the method comprising preparing a composition of lipid nanoparticles comprising a compound of formula (I) and a therapeutic agent, and delivering the composition to the patient. Such methods can be used to induce protein expression in an individual, for example, to express an antigen for the purpose of vaccination or gene editing of proteins.

[0016] These and other aspects of this disclosure will become apparent from the following detailed description.

[0017] Detailed description

[0018] In the following description, certain specific details are set forth to provide a thorough understanding of various embodiments of this disclosure. However, those skilled in the art will understand that embodiments of this disclosure can be practiced without these details.

[0019] This disclosure is based in part on the discovery of novel cationic lipids that offer a number of advantages when used in lipid nanoparticles to deliver active agents or therapeutic agents, such as nucleic acids, in vivo to mammalian cells. Embodiments of this disclosure provide nucleic acid-lipid nanoparticle compositions comprising one or more of the novel cationic lipids described herein, which provide increased nucleic acid activity and improved composition tolerability in vivo compared to previously described nucleic acid-lipid nanoparticle compositions, resulting in a significant improvement in the therapeutic index.

[0020] In some embodiments, this disclosure provides novel cationic lipids capable of being formulated into modified compositions for the in vitro and in vivo delivery of mRNA and / or other oligonucleotides. In some embodiments, these modified lipid nanoparticle compositions can be used to express proteins encoded by mRNA. In other embodiments, these modified lipid nanoparticle compositions can be used to upregulate the expression of one or more target mRNAs by delivering miRNA inhibitors targeting a specific miRNA or a group of miRNAs. In other embodiments, these modified lipid nanoparticle compositions can be used to downregulate (e.g., silence) protein and / or mRNA levels of target genes. In some other embodiments, the lipid nanoparticles can also be used to deliver mRNA and plasmids to express transgenes. In other embodiments, the lipid nanoparticle compositions can be used to induce pharmacological effects resulting from protein expression, such as increasing red blood cell production by delivering suitable erythropoietin mRNA, or protecting against infection by delivering mRNA encoding suitable antibodies.

[0021] The lipid nanoparticles and compositions disclosed herein can be used for a variety of purposes, including the in vitro and in vivo delivery of encapsulated or associated (e.g., complexed) therapeutic agents such as nucleic acids to cells. Therefore, embodiments of this disclosure provide methods for treating or preventing diseases or conditions in an individual by contacting an individual in need with lipid nanoparticles encapsulating a suitable therapeutic agent or lipid nanoparticles associated with a suitable therapeutic agent, wherein the lipid nanoparticles comprise one or more novel cationic lipids as described herein.

[0022] As described herein, embodiments of the lipid nanoparticles disclosed herein are particularly suitable for the delivery of nucleic acids, including, for example, mRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomir / antimir), complementary RNA to interfering messenger RNA (micRNA), DNA, multivalent RNA, Dicer substrate RNA, complementary DNA (cDNA), etc. Therefore, the lipid nanoparticles and compositions of this disclosure can be used to induce the expression of desired proteins in vivo and in vitro by contacting cells with lipid nanoparticles comprising one or more novel cationic lipids described herein, wherein the lipid nanoparticles encapsulate or associate with nucleic acids, which are expressed to produce desired proteins (e.g., messenger RNA or plasmids encoding the desired protein). Alternatively, the lipid nanoparticles and compositions of this disclosure can be used to reduce the expression of target genes and proteins in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more novel cationic lipids described herein, wherein the lipid nanoparticles encapsulate or associate with nucleic acids, which reduce the expression of target genes (e.g., antisense oligonucleotides or small interfering RNA (siRNA)). The lipid nanoparticles and compositions disclosed herein can also be used to co-deliver different nucleic acids (e.g., mRNA and plasmid DNA) separately or in combination, such as to provide co-localization of different nucleic acids (e.g., mRNA encoding suitable gene-modifying enzymes and DNA segments for incorporation into the host genome).

[0023] Nucleic acids used in conjunction with this disclosure can be prepared using any available technology. For mRNA, the primary preparation method is, but is not limited to, enzymatic synthesis (also known as in vitro transcription), which represents the most efficient method currently available for producing long sequence-specific mRNAs. In vitro transcription describes the process of template-guided RNA molecule synthesis from an engineered DNA template containing an upstream phage promoter sequence (e.g., including but not limited to promoter sequences from T7, T3, and SP6 E. coli phages) linked to a downstream sequence encoding a target gene. Template DNA for in vitro transcription can be prepared from a variety of sources using appropriate techniques well known in the art (including, but not limited to, plasmid DNA and polymerase chain reaction amplification) (see Linpinsel, JL and Conn, GL, General protocols for preparation of plasmid DNA template, and Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LD in 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, NY Humana Press, 2012).

[0024] RNA transcription is performed in vitro using a linearized DNA template in the presence of the appropriate RNA polymerase and adenosine, guanosine, uridine, and cytidine ribonucleoside triphosphates (rNTPs), while supporting polymerase activity and minimizing potential degradation of the resulting mRNA transcript. In vitro transcription can be performed using a variety of commercially available kits and reagents (including RNA polymerases and rNTPs), including but not limited to the RiboMax large-scale RNA production system (Promega) and the MegaScript transcription kit (Life Technologies). Methods for in vitro transcription of mRNA are well known in the art. (See, for example, Losick, R., 1972, In vitro transcription, Ann RevBiochem, Vol. 41, pp. 409-46; Kamakaka, RT and Kraus, WL 2001. In Vitro Transcription. Current Protocols in Cell Biology. 2:11.6:11.6.1–11.6.17; Beckert, B. and Masquida, B., (2010) Synthesis of RNA by In Vitro Transcription in RNA in Methods in Molecular Biology, Vol. 703 (edited by Neilson, H.), New York, NY Humana Press, 2010; Brunelle, JL and Green, R., 2013, Chapter 5 – In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology, Vol. 530, pp. 101-114; all are incorporated herein by reference).

[0025] The desired mRNA transcribed in vitro is then purified from undesirable components of transcription or related reactions, including unincorporated rNTPs, proteases, salts, short RNA oligomers, etc. Techniques for isolating mRNA transcripts are well-known in the art. Well-known procedures include phenol / chloroform extraction or precipitation with alcohols (ethanol, isopropanol) (in the presence of monovalent cations or lithium chloride). Other non-limiting examples of purification procedures that may be used include size exclusion chromatography (Lukavsky, PJ and Puglisi, JD, 2004, Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides, RNA v.10, 889-893), silica-based affinity chromatography and polyacrylamide gel electrophoresis (Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LD in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941 ConnG.L. (ed.), New York, NY Humana Press, 2012). Purification can be performed using a variety of commercially available kits, including but not limited to the SV Total Isolation System (Promega) and the In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).

[0026] Furthermore, although reverse transcription can produce large amounts of mRNA, the product may contain several aberrant RNA impurities associated with undesirable polymerase activity, which may need to be removed from the full-length mRNA preparation. These include short RNAs generated by abortion transcription initiation, and double-stranded RNAs (dsRNAs) generated by RNA-dependent RNA polymerase activity, RNA-induced transcription from an RNA template, and self-complementary 3' elongation. These contaminants with dsRNA structures have been shown to lead to undesirable immunostimulatory activity by interacting with various innate immune sensors in eukaryotic cells that function to recognize specific nucleic acid structures and induce effective immune responses. This can then significantly reduce mRNA translation due to decreased protein synthesis during the innate cellular immune response. Therefore, additional techniques for removing these dsRNA contaminants have been developed, and are known in the art, including but not limited to scalable HPLC purification (see, for example, Kariko, K., Muramatsu, H., Ludwig, J., and Weissman, D., 2011, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA, Nucl Acid Res, v. 39 e142; Weissman, D., Pardi, N., Muramatsu, H., and Kariko, K., HPLC Purification of invitro transcribed long RNA in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH ed.), 2013). HPLC-purified mRNA has been reported to be translated at much higher levels, particularly in primary cells and in vivo.

[0027] Various modifications have been described in this art to alter the specificity and improve the usability of in vitro transcribed mRNA. These include, but are not limited to, modifications to the 5′ and 3′ ends of mRNA. Endogenous eukaryotic mRNA typically contains a cap structure at the 5′ end of mature molecules, which plays a crucial role in mediating the binding of mRNA cap-binding proteins (CBPs), which in turn are responsible for enhancing mRNA stability and the efficiency of mRNA translation in cells. Therefore, capped mRNA transcripts achieve the highest levels of protein expression. The 5′ cap contains a 5′-5′-triphosphate bond between the 5′-most nucleotide and the guanine nucleotide. The conjugated guanine nucleotide is methylated at the N7 position. Additional modifications include methylation of the penultimate and penultimate most 5′ nucleotides on the 2′-hydroxyl group.

[0028] A variety of different cap structures can be used to generate the 5'-cap of synthetic mRNA transcribed in vitro. 5'-capping of synthetic mRNA can be performed using co-transcription with chemical cap analogs (i.e., capping during in vitro transcription). For example, the anti-reverse cap analog (ARCA) contains a 5'-5'-guanine triphosphate-guanine bond, where one guanine contains an N7 methyl group and a 3'-O-methyl group. However, during this co-transcription, up to 20% of the transcript remains uncapped, and the synthetic cap analogs differ from the 5'-cap structure of real cellular mRNA, potentially reducing translatability and cellular stability. Alternatively, synthetic mRNA molecules can also be enzymatically capped post-transcriptionally. These can generate more realistic 5'-cap structures that more closely mimic the endogenous 5'-cap structurally or functionally, with enhanced cap-binding protein binding, prolonged half-life, reduced sensitivity to 5' endonucleases, and / or reduced 5' uncapping. Numerous synthetic 5'-cap analogues have been developed, and it is known in the art that said synthetic 5'-cap analogues enhance mRNA stability and translateability (see, e.g., Grudzien-Nogalska, E., Kowalska, J., Su, W., Kuhn, AN, Slepenkov, SV, Darynkiewicz, E., Sahin, U., Jemielity, J. and Rhoads, RE, Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH ed.), 2013).

[0029] At the 3' end, long-chain adenine nucleotides (poly-A tails) are typically added to the mRNA molecule during RNA processing. Immediately after transcription, the 3' end of the transcript is cleaved to release the 3' hydroxyl group, and during a process called polyadenylation, poly-A polymerase adds a chain of adenine nucleotides to the 3' hydroxyl group into the RNA. Poly(A) tails have been widely shown to enhance mRNA translation efficiency and stability (see Bernstein, P. and Ross, J., 1989, Poly(A), poly(A) binding protein and the regulation of mRNA stability, Trends Bio Sci v. 14 373-377; Guhaniyogi, J. and Brewer, G., 2001, Regulation of mRNA stability in mammalian cells, Gene, v. 265, 11-23; Dreyfus, M. and Regnier, P., 2002, The poly(A) tail of mRNAs: Bodyguard in eukaryotes, scavenger in bacteria, Cell, v.111, 611-613).

[0030] Poly(A) tailing of in vitro transcribed mRNA can be achieved using various methods, including but not limited to cloning poly(T) tracts into a DNA template or adding them post-transcriptionally using a poly(A) polymerase. The first approach allows for the in vitro transcription of mRNA with a defined poly(A) tail, depending on the size of the poly(T) tract, but requires additional manipulation of the template. The latter approach involves enzymatically adding the poly(A) tail to in vitro transcribed mRNA using a poly(A) polymerase that catalyzes the incorporation of adenine residues into the 3' end of the RNA. This does not require additional manipulation of the DNA template but produces mRNA with poly(A) tails of uneven length. A variety of commercially available kits and reagents, various ARCA caps, poly(A) polymerases, etc., can be used for 5'-capping and 3'-poly(A) tailing. The kits include, but are not limited to, the Poly(A) Polymerase Tailing kit (EpiCenter), the mMESSAGE mMACHINE T7 Ultra kit, and the Poly(A) Tailing kit (Life Technologies).

[0031] It has been reported that, in addition to 5' caps and 3' polyadenylation, other modifications to in vitro transcripts provide benefits related to translation efficiency and stability. It is well known in the art that pathogenic DNA and RNA can be recognized by a variety of sensors within eukaryotes and trigger an effective innate immune response. The ability to distinguish pathogenicity from self-DNA and RNA has been shown to be based at least in part on structural and nucleoside modifications, as most nucleic acids of natural origin contain modified nucleosides. In contrast, in vitro synthesized RNA lacks these modifications, thus making it immunostimulatory and subsequently capable of inhibiting the effective mRNA translation outlined above. Introducing modified nucleosides into in vitro transcribed mRNA can prevent recognition and activation by RNA sensors, thereby mitigating this undesirable immunostimulatory activity and enhancing translational capacity (see Kariko, K. and Weissman, D. 2007, Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: implication for therapeutic RNA development, Curr Opin DrugDiscov Devel, v.10 523-532; Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH edited), 2013; Kariko, K., Muramatsu, H., Welsh, FA, Ludwig, J., Kato, H., Akira, S., Weissman, D., 2008, Incorporation of Pseudouridine Into mRNA Yields Superior NonimmunogenicVector With Increased Translational Capacity and Biological Stability, MolTher v.16, 1833-1840).Modified nucleosides and nucleotides used in modified RNA synthesis can be prepared, monitored, and utilized using general methods and procedures known in the art. A wide variety of nucleoside modifications are available, which can be incorporated, alone or in combination with other modified nucleosides, to a certain extent into in vitro transcribed mRNA (see, for example, U.S. Publication No. 2012 / 0251618). It has been reported that the in vitro synthesis of nucleoside-modified mRNA reduces the ability to activate immune sensors, while simultaneously enhancing translational capacity.

[0032] Other components of mRNA that can be modified to provide benefits in terms of translatability and stability include the 5' and 3' untranslated regions (UTRs). Optimization of the UTR (which can yield favorable 5' and 3' UTRs from cellular or viral RNA) has been shown to improve mRNA stability and translation efficiency of in vitro transcribed mRNA, whether performed simultaneously or alone (see, for example, Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH editor), 2013).

[0033] In addition to mRNA, other nucleic acid payloads can also be used in this disclosure. For oligonucleotides, preparation methods include, but are not limited to, chemical synthesis and enzymatic catalysis, chemical cleavage of longer precursors, and in vitro transcription as described above. Methods for synthesizing DNA and RNA nucleotides are widely used and well known in the art (see, for example, Gait, MJ (edit) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, DC: IRL Press, 1984; and Herdewijn, P. (edit) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, NJ) Totowa, NJ: Humana Press, 2005; both of which are incorporated herein by reference).

[0034] For plasmid DNA, preparations used with this disclosure typically utilize, but are not limited to, in vitro amplification and isolation of plasmid DNA in liquid cultures of bacteria containing the target plasmid. The presence of genes encoding resistance to specific antibiotics (penicillin, kanamycin, etc.) in the target plasmid allows those bacteria containing the target plasmid to selectively grow in cultures containing antibiotics. Methods for isolating plasmid DNA are widely used and well known in the art (see, for example, Heilig, J., Elbing, KL and Brent, R (2001) Large-Scale Preparation of Plasmid DNA. Current Protocols in Molecular Biology. 41:II:1.7:1.7.1–1.7.16; Rozkov, A., Larsson, B., Gillström, S., Björnestedt, R. and Schmidt, SR (2008), Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture. Biotechnol. Bioeng., 99: 557–566; and U.S. Patent No. 6,197,553). Plasmid isolation can be performed using a variety of commercially available kits and reagents, including but not limited to Plasmid Plus (Qiagen), GenJET Plasmid MaxiPrep (Thermo), and PureYield MaxiPrep (Promega) kits.

[0035] The following provides a more detailed description of various exemplary embodiments of the compounds, lipid nanoparticles, and compositions comprising the present disclosure, as well as their use in delivering active agents or therapeutic agents such as nucleic acids to regulate gene and protein expression.

[0036] As used herein, unless otherwise stated, the following terms have the meanings assigned to them.

[0037] Unless the context otherwise requires, throughout the specification and claims, the word “comprise” and its variations (e.g., “comprises” and “comprising”) shall be interpreted in an open and inclusive sense, meaning “including but not limited to”.

[0038] Throughout this specification, references to "an embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of this disclosure. Therefore, the phrases "in an embodiment" or "in an embodiment" appearing in various places throughout this specification do not necessarily refer to the same embodiment. Furthermore, 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 herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. As used in the specification and claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise.

[0040] The phrase "inducing the expression of a desired protein" means that nucleic acids can increase the expression of a desired protein. To examine the extent of protein expression, a test sample (e.g., a cultured cell sample expressing the desired protein) or a test mammal (e.g., a mammalian, 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) can be contacted with nucleic acids (e.g., nucleic acids combined with the lipids of this disclosure). The expression of the desired protein in the test sample or test animal is compared with the expression of the desired protein in a control sample (e.g., a cultured cell sample expressing the desired protein) or a control mammal (e.g., a mammalian, 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 nucleic acids or has not been treated with nucleic acids. When the desired protein is present in the control sample or control mammal, the expression of the desired protein in the control sample or control mammal can be assigned a value of 1.0. In some embodiments, the expression of a desired protein can be induced when the ratio of the expression level of the desired protein in the test sample or test mammal to the expression level of the desired protein in the control sample or control mammal is greater than 1, for example, about 1.1, 1.5, 2.0, 5.0, or 10.0. The expression of a desired protein is also induced when the desired protein is absent in the control sample or control mammal, or when any measurable level of the desired protein is detected in the test sample or test mammal. Those skilled in the art will understand that appropriate assays for determining protein expression levels in a sample, such as dot blots, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme function and phenotypic assays, or assays based on reporter proteins that may produce fluorescence or luminescence under appropriate conditions, are suitable.

[0041] The phrase "inhibits target gene expression" means that nucleic acids can silence, reduce, or inhibit the expression of a target gene. To examine the degree of gene silencing, a test sample (e.g., a cultured cell sample expressing the target gene) or a test mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model) is exposed to nucleic acids that silence, reduce, or inhibit the expression of the target gene. The expression of the target gene in the test sample or test animal is compared to the expression of the target gene in a control sample (e.g., a cultured cell sample expressing the target gene) or a control mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model) that has not been exposed to or treated with nucleic acids. The expression of the target gene in the control sample or control mammal can be assigned a value of 100%. In a specific implementation, target gene expression is silenced, suppressed, or reduced when the target gene expression level in the test sample or test mammal is approximately 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% relative to the target gene expression level in a control sample or control mammal that has not been exposed to or administered nucleic acid. In other words, the nucleic acid is capable of silencing, reducing, or suppressing the expression of the target gene in the test sample or test mammal by at least approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to the target gene expression level in a control sample or control mammal that has not been exposed to or administered nucleic acid. Suitable assays for determining target gene expression levels include, but are not limited to, the use of techniques known to those skilled in the art to examine protein or mRNA levels, such as dot blot, Northern blot, in situ hybridization, ELISA, immunoprecipitation, enzyme function assays, and phenotypic assays known to those skilled in the art.

[0042] The "effective amount" or "therapeutic effective amount" of an active agent or therapeutic agent, such as a therapeutic nucleic acid, is an amount sufficient to produce the desired effect (e.g., an increase or inhibition of target sequence expression compared to normal expression levels detected in the absence of nucleic acid). An increase in target sequence expression is achieved when any measurable level is detected in the absence of the expression product in the absence of nucleic acid. An increase in expression is achieved when, prior to contact with nucleic acid, the expression product is present at a certain level, and the value obtained with nucleic acid, such as mRNA, represents an increase of approximately 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000, or greater than that of the control. When, relative to a control, values ​​obtained using nucleic acids such as antisense oligonucleotides are approximately 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%, inhibition of target gene or target sequence expression is achieved. Suitable assays for measuring target gene or target sequence expression include examination of protein or RNA levels using techniques known to those skilled in the art, such as dot blot, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of suitable reporter proteins, and phenotypic assays known to those skilled in the art.

[0043] As used herein, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form, and includes DNA, RNA, and their hybrids. DNA can be in the form of antisense molecules, plasmid DNA, cDNA, PCR products, or vectors. RNA can be in the form of hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA, Dicer substrate RNA, or viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or bonds, which are synthetic, naturally occurring, or non-natural and have similar binding properties to a reference nucleic acid. Examples of such analogs include, but are not limited to, phosphate thioesters, aminophosphate esters, methylphosphonates, chiral methylphosphonates, 2'-O-methylribonucleotides, and peptide-nucleic acids (PNAs). Unless specifically defined, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to a reference nucleic acid. Unless otherwise stated, a particular nucleic acid sequence also implicitly encompasses variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences, as well as explicitly stated sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). A “nucleotide” contains the following sugars: deoxyribose (DNA) or ribose (RNA); a base; and a phosphate group. Nucleotides are linked together by phosphate groups. "Base" includes purines and pyrimidines, and further includes natural compounds such as adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, including but not limited to modifications that place new reactive groups (e.g., but not limited to amines, alcohols, thiols, carboxylates, and hydrocarbon halides).

[0044] The term "gene" refers to a nucleic acid (such as DNA or RNA) sequence that contains a coding sequence of partial or full length necessary to produce a polypeptide or precursor polypeptide.

[0045] As used in this article, “gene product” refers to gene products such as RNA transcripts or polypeptides.

[0046] The term “lipid” refers to a group of organic compounds, including but not limited to esters of fatty acids, and is generally characterized by being poorly soluble in water but soluble in many organic solvents. They are generally classified into at least three categories: (1) “simple lipids”, including fats and oils as well as waxes; (2) “complex lipids”, including phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

[0047] "Steroids" are compounds that contain the following carbon skeleton: .

[0048] Unrestricted examples of steroids include cholesterol.

[0049] "Cationic lipids" refer to lipids capable of carrying a positive charge. Exemplary cationic lipids include one or more amine groups carrying a positive charge. Preferred cationic lipids are ionizable, such that they exist in a positively charged or neutral form depending on the pH. Under different pH conditions, the ionization of cationic lipids affects the surface charge of lipid nanoparticles. This charge state can affect plasma protein uptake, blood clearance, and tissue distribution (Semple, SC, et al., Adv. Drug Deliv Rev 32:3-17 (1998)), which are crucial for intracellular nucleic acid delivery, as well as the ability to form non-bilayer endosomal structures (Hafez, IM, et al., Gene Ther 8:1188-1196 (2001)).

[0050] The term "lipid nanoparticle" refers to particles having at least one nanometer-scale size (e.g., 1-1,000 nm) comprising one or more or other specified components of a compound of formula (I). In some embodiments, lipid nanoparticles are included in formulations that can be used to deliver active agents or therapeutic agents (such as nucleic acids (e.g., mRNA)) to target sites (e.g., cells, tissues, organs, tumors, etc.). In some embodiments, the lipid nanoparticles of this disclosure comprise nucleic acids. Such lipid nanoparticles typically comprise 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, may be encapsulated in the lipid portion of the lipid nanoparticle or in an aqueous space encapsulated by some or all of the lipid portions of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by host organism or cellular mechanisms, such as adverse immune responses.

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

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

[0053] The term "polymer-conjugated lipid" refers to a molecule comprising both a lipid moiety and a polymer moiety. An example of a polymer-conjugated lipid is a polyethylene glycol-modified lipid. The term "polyethylene glycol-modified lipid" refers to a molecule comprising both a lipid moiety and a polyethylene glycol moiety. Polyethylene glycol-modified lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), etc.

[0054] The term "neutral lipid" refers to any of a number of lipid substances that exist as uncharged or neutral zwitterions at a selected pH. At physiological pH, such lipids include, but are not limited to, phosphatidylcholine such as 1,2-distearate- sn -Glyceryl-3-phosphocholine (DSPC), 1,2-dipalmitoyl- sn -glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl- sn-Glyceryl-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC), 1,2-dioleoyl- sn 3-glycero-3-phosphocholine (DOPC), phosphatidylethanolamine such as 1,2-dioleoyl- sn 3-glycero-3-phosphate ethanolamine (DOPE), sphingomyelin (SM), ceramides, steroids such as sterols and their derivatives. Neutral lipids can be synthetic or of natural origin.

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

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

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

[0058] As used herein, “systemic delivery” refers to the delivery of a therapeutic product that results in widespread exposure of the active agent within the body. Some administration techniques can result in systemic delivery of certain agents without the systemic delivery of others. 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 performed by any means known in the art, including, for example, intravenous, intra-arterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is performed via intravenous delivery.

[0059] As used herein, “local delivery” refers to the direct delivery of an active agent to a target site within an organism. For example, agents can be delivered locally by direct injection into a site of disease (e.g., a tumor), other target sites (e.g., a site of inflammation), or target organs (e.g., the liver, heart, pancreas, kidneys, etc.). Local delivery can also include local application or injection techniques, such as intramuscular, subcutaneous, or intradermal injection. Local delivery does not preclude systemic pharmacological effects.

[0060] "Alkyl" refers to a straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms. It is saturated (i.e., does not contain double and / or triple bonds) and has one to twenty-four carbon atoms (C1-C2). 24 Alkyl groups, with one to sixteen carbon atoms (C1-C6). 16 Alkyl groups, with one to twelve carbon atoms (C1-C2). 12 Alkyl groups, and six to twenty-four carbon atoms (C6-C6). 24 Alkyl groups (one to eight carbon atoms (C1-C8 alkyl) or one to six carbon atoms (C1-C6 alkyl) are attached to the rest of the molecule by single bonds, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, etc. Unless otherwise specified in this specification, alkyl groups are optionally substituted.

[0061] "Alkenyl" refers to a straight-chain or branched hydrocarbon chain group composed only of carbon and hydrogen atoms, containing at least one carbon-carbon double bond and having one to twenty-four carbon atoms (C2-C4). 24 alkenyl), one to twelve carbon atoms (C2-C) 12 alkenyl), six to twenty-four carbon atoms (C6-C) 24 alkenyl), two to sixteen carbon atoms (C2-C) 16 alkenyl), four to twelve carbon atoms (C4-C) 12 The alkenyl group (e.g., vinyl, propenyl, 1-methylvinyl, n-butenyl, n-pentenyl, 1,1-dimethylvinyl, 3-methylhexenyl, 2-methylhexenyl, etc.) has one to eight carbon atoms (C2-C8 alkenyl) or one to six carbon atoms (C2-C6 alkenyl) and is connected to the rest of the molecule by a single bond. Unless otherwise specified in this specification, the alkenyl group is optionally substituted.

[0062] "Alkyne" refers to a straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, containing at least one carbon-carbon triple bond and having one to twenty-four carbon atoms (C2-C4). 24 alkynyl group), one to twelve carbon atoms (C2-C) 12 The alkynyl group (containing one to eight carbon atoms (C2-C8 alkynyl) or one to six carbon atoms (C2-C6 alkynyl) is attached to the rest of the molecule by a single bond, for example, ethynyl, n-propynyl, 1-methylethynyl, n-butynyl, n-pentynyl, 1,1-dimethylethynyl, 3-methylhexynyl, 2-methylhexynyl, etc. Unless otherwise specified in this specification, the alkynyl group is optionally substituted.

[0063] "alkylene" or "alkylene chain" refers to a straight-chain or branched divalent saturated hydrocarbon chain consisting only of carbon and hydrogen, which links the rest of the molecule to a group. In some embodiments, the alkylene chain has one to twenty-four carbon atoms (C1-C2). 24 Alkylenes, one to fifteen carbon atoms (C1-C5) 15 Alkylene), one to twelve carbon atoms (C1-C2) 12 Alkylenes (C1-C8 alkylenes), one to eight carbon atoms (C1-C6 alkylenes), one to six carbon atoms (C4-C6 alkylenes), four to six carbon atoms (C2-C4 alkylenes), two to four carbon atoms (C1-C2 alkylenes), one to two carbon atoms (C1-C2 alkylenes), such as methylene, ethylene, propylene, n-butylene, etc. The alkylene chain is attached to the rest of the molecule by a single bond and to a functional group by a single bond. The connection point between the alkylene chain and the rest of the molecule, as well as the connection point with the functional group, can be one carbon or any two carbons within the chain. Unless otherwise specified in the specification, the alkylene chain is optionally substituted.

[0064] "Alkenyl" or "alkenyl chain" refers to a straight-chain or branched divalent hydrocarbon chain consisting only of carbon and hydrogen and containing at least one carbon-carbon double bond, which connects the rest of the molecule to a group. In some embodiments, the alkenyl chain has two to twenty-four carbon atoms (C2-C4). 24 (alkenyl), two to fifteen carbon atoms (C2-C) 15 (alkenyl), two to twelve carbon atoms (C2-C) 12 The alkenyl chain comprises two to eight carbon atoms (C2-C8 alkenyl), two to six carbon atoms (C2-C6 alkenyl), four to six carbon atoms (C4-C6 alkenyl), and two to four carbon atoms (C2-C4 alkenyl), such as vinylidene, propenylidene, n-butenylidene, etc. The alkenyl chain is attached to the rest of the molecule by a single bond and to a group by a single bond. The attachment points of the alkenyl chain to the rest of the molecule and to the group can be one carbon or any two carbons within the chain. Unless otherwise specifically stated in the specification, the alkenyl chain is optionally substituted.

[0065] "Cyclic hydrocarbon group" or "carbocyclic ring" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon group composed only of carbon and hydrogen atoms. It can include fused or bridged ring systems with three to fifteen ring carbon atoms (C3-C4). 15 ), three to ten ring carbon atoms (C3-C 10The cyclic group consists of three to eight carbon atoms (C3-C8), and is either saturated or unsaturated and is connected to the rest of the molecule by single bonds. Monocyclic groups include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic groups include, for example, adamantyl, norbornyl, decahydronaphthyl, 7,7-dimethylbicyclo[2.2.1]heptyl, etc. Unless otherwise specifically stated in the specification, the cyclic hydrocarbon groups are optionally substituted.

[0066] "Aryl" refers to a carbocyclic group comprising hydrogen, 6 to 18 carbon atoms, and at least one aromatic ring. For the purposes of this disclosure, an aryl group is a monocyclic, bicyclic, tricyclic, or tetracyclic system, which may comprise fused or bridged ring systems. Aryl groups include, but are not limited to, aryl groups derived from acetane, acenaphthene, phenanthrene, anthracene, chamomile, benzene, phenanthrene, fluorene, asymmetric indole, symmetric indole, indene, indene, naphthalene, phenanthracene, heptamethrin, pyrene, and benzo[a]phenanthrene.

[0067] "Aromatic hydrocarbon group" refers to the formula -R b -R c The group, wherein R b It is an alkylene or alkenylene group as defined above and R c It can be one or more aryl groups as defined above, such as benzyl, diphenylmethyl, etc. Unless otherwise specified in the specification, the aryl hydrocarbon group is optionally substituted.

[0068] "Heterocyclic group" or "heterocycle" refers to a stable 3- to 18-membered non-aromatic cyclic group having one to twelve cyclic carbon atoms (e.g., two to twelve) and one to six cyclic heteroatoms selected from nitrogen, oxygen, and sulfur. Unless otherwise specified in the specification, the heterocyclic group is a monocyclic, bicyclic, tricyclic, or tetracyclic system, which may include fused, spirocyclic ("spiro-heterocyclic group"), and / or bridged ring systems; and the nitrogen, carbon, or sulfur atom in the heterocyclic group is optionally oxidized; the nitrogen atom is optionally quaternized; and the heterocyclic group is partially or fully saturated. Examples of such heterocyclic groups include, but are not limited to, dioxolane, thienyl[1,3]dithiaalkyl, decahydroisoquinolinyl, imidazolinyl, imidazoalkyl, isothiazolyl, isoxazolyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopiperidinyl, oxazolyl, piperidinyl, piperazinyl, 4-piperidinoneyl, pyrrolyl, pyrazolyl, quininecycloyl, thiazoalkyl, tetrahydrofuranyl, trithiaalkyl, tetrahydropyranyl, thiomorpholinyl, thio-morpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless otherwise specifically stated in the specification, the heterocyclic group is optionally substituted.

[0069] "Heteroaryl" refers to a 5- to 14-membered ring system group comprising a hydrogen atom, one to thirteen carbon atoms, one to six heteroatoms selected from nitrogen, oxygen, and sulfur, and at least one aromatic ring. For the purposes of this disclosure, the heteroaryl group can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon, or sulfur atom in the heteroaryl group may optionally be oxidized; the nitrogen atom may optionally be quaternized. Examples include, but are not limited to, azirrolyl, acridinel, benzimidazolyl, benzothiazolyl, benzoindolyl, benzodioxacyclopentenyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[ b [1,4]dioxane-heptenyl, 1,4-benzodioxane, benzonaphthylfuranyl, benzooxazolyl, benzodioxane-pentenyl, benzodioxane-hexenyl, benzopyranyl, benzopyranoneyl, benzofuranyl, benzofuranoneyl, benzothienyl / benzothiophenyl, benzotriazolyl, benzo[4,6]imidazo[1,2- a Pyridyl, benzoxazolinone, benzimidazolylthionyl, carbazole, cenyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanoneyl, isothiazolyl, imidazolyl, indazole, indolyl, indazole, isoindolyl, dihydroindolyl, isodihydroindolyl, isoquinolinyl, inazinyl, isoxazolyl, naphridyl, oxadiazolyl, 2-oxozazironeyl, oxazolyl, ethylene oxide, 1-pyridyl oxide, 1-pyrimidinyl oxide, 1-pyrazinyl oxide 1-Oxyridazinyl, 1-Phenyrrolyl, Phenazinyl, Phenthiazinyl, Phenoxazinyl, Phtharazinyl, Pteridinyl, Pteridinoneyl, Puryl, Pyrroleyl, Pyrazolyl, Pyridinyl, Pyridinoneyl, Pyrazinyl, Pyrimidinyl, Pyrimidinoneyl, Pyridazinyl, Pyrroleyl, Pyridoboroneyl, Quinazolinyl, Quinazolinoneyl, Quinoxolinyl, Quinoxolinoneyl, Quinolinyl, Isoquinolinyl, Tetrahydroquinolinyl, Thiazolyl, Thiadiazolyl, Thiophene[3,2- d ]Pyrimidine-4-keto, thiophene[2,3- d The aryl group may be substituted with pyrimidin-4-one, triazolyl, tetrazolyl, triazine, or thiophenyl / thienyl. Unless otherwise specified in this specification, the heteroaryl group may be optionally substituted.

[0070] As used herein, the term "substituted" refers to any of the aforementioned groups (e.g., alkyl, alkenyl, alkynyl, alkylene, cycloalkyl, aryl, heteroaryl, or heterocyclic) in which at least one hydrogen atom is bonded to a non-hydrogen atom, said non-hydrogen atom being, for example, but not limited to: halogen atoms, such as F, Cl, Br, and I; oxo groups (=O); hydroxyl groups (-OH); alkyloxy groups (-OR). a , where R aIt is C1-C 12 Alkyl or cyclic hydrocarbon groups); carboxyl groups (-OC(=O)R) a or -C(=O)OR a , where R a It is H, C1-C 12 Alkyl or cyclic hydrocarbon groups); amine groups (-NR) a R b , where R a and R b Each independently constitutes H, C1-C 12 Alkyl or cyclic hydrocarbon groups); C1-C 12 Alkyl groups; and cyclic hydrocarbon groups. In some embodiments, the substituents are C1-C. 12 Alkyl group. In other embodiments, the substituent is a cyclic alkyl group. In other embodiments, the substituent is a halogen group, such as fluorine. 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 alkyloxy group. In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group.

[0071] The terms "optional" or "optionally" (e.g., optionally substituted) mean that the event described below may or may not occur, and the description includes both cases where the event or condition occurs and cases where the event or condition does not occur. For example, "optionally substituted alkyl" means that the alkyl group may or may not be substituted, and the description includes both substituted and unsubstituted alkyl groups. In some embodiments, "optionally substituted" means that a particular group is substituted by one or more substituents selected from: halogens (e.g., F, Cl, Br, and I), oxo (=O), hydroxyl (-OH), alkoxy (-OR). a , where R a It is C1-C 12 Alkyl), cyclic alkyl group (-OR) a , where R a It is a C3-C8 cyclic hydrocarbon group, a carboxyl group (-OC(=O)R) a or -C(=O)OR a , where R a It is H, C1-C 12 Alkyl or C3-C8 cyclic hydrocarbon groups), amines (-NR) a R b , where R a and R b Each independently constitutes H, C1-C 12 Alkyl or C3-C8 cyclic hydrocarbon groups), C1-C 12 Alkyl and C3-C8 cyclic hydrocarbon groups.

[0072] In some embodiments, "optionally substituted" means substituted with one or more halogen substituents. In some embodiments, "optionally substituted" means substituted with one or more oxo substituents. In some embodiments, "optionally substituted" means substituted with one or more hydroxyl substituents. In some embodiments, "optionally substituted" means substituted with one or more alkoxy substituents. In some embodiments, "optionally substituted" means substituted with one or more cycloalkoxy substituents. In some embodiments, "optionally substituted" means substituted with one or more carboxyl substituents. In some embodiments, "optionally substituted" means substituted with one or more amine substituents. In some embodiments, "optionally substituted" means substituted with one or more C1-C1 substituents. 12 Alkyl substituent substitution. In some embodiments, "optionally substituted" means substituted with one or more C3-C8 cyclic alkyl substituents.

[0073] When the functional group is described as "optionally substituted" and, in turn, the substituents on the functional group are also "optionally substituted," for the purposes of this disclosure, such iteration is limited to five times, preferably limited to two times. In some embodiments, such iteration is limited to one time. In some embodiments, such iteration is limited to zero times.

[0074] This disclosure is also intended to cover all pharmaceutically acceptable compounds of formula (I) that are isotopically labeled by replacing one or more atoms with atoms having different atomic masses 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, such as... 2 H, 3 H, 11 C 13 C 14 C 13 N、 15 N、 15 O、 17 O、 18 O、 31 P, 32 P, 35 S, 18 F, 36 Cl、 123 I and 125 I. These radiolabeled compounds can be used to help determine or measure the effectiveness of a compound by characterizing, for example, its site of action or mode of action, or its binding affinity to pharmacologically important sites of action. Some isotope-labeled compounds of formula (I), such as those incorporating a radioactive isotope, can be used for drug and / or substrate tissue distribution studies. Given that the radioactive isotope tritium is... 3 H and carbon-14, i.e. 14C doping is easy and detection methods are readily available, which can be specifically used for this purpose.

[0075] Using heavier isotopes such as deuterium (i.e., 2 H) substitution may offer certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dose requirements, and may therefore be preferred in some cases.

[0076] Using positron emission isotopes, such as 11 C 18 F, 15 O and 13 N-substitution can be used in positron emission tomography (PET) studies to examine substrate acceptor occupancy. Isotopically labeled compounds of formula (I) can generally be prepared using conventional techniques known to those skilled in the art or by a process similar to those described in the preparations and examples set forth below, using a suitable isotopically labeled reagent in place of the previously used unlabeled reagent.

[0077] This disclosure is also intended to cover in vivo metabolites of the disclosed compounds. Such products can be generated, for example, by oxidation, reduction, hydrolysis, amidation, esterification, etc., of the applied compound, primarily due to enzymatic processes. Therefore, this disclosure includes compounds produced by a method comprising administering a compound of this disclosure to a mammal for a period sufficient to produce its metabolites. Such products are typically identified by administering a detectable dose of the radiolabeled compound of this disclosure to an animal (e.g., rat, mouse, guinea pig, monkey, or human), allowing sufficient time for metabolism, and separating its metabolites from urine, blood, or other biological samples.

[0078] "Stable compound" and "stable structure" are intended to refer to compounds that are robust enough to withstand separation from the reaction mixture to a useful level of purity and formulation into an effective therapeutic agent.

[0079] "Mammals" include humans as well as domesticated animals such as laboratory animals and pets (e.g., cats, dogs, pigs, cattle, sheep, goats, horses, rabbits) and non-domesticated animals (e.g., wild animals).

[0080] "Pharmaceutically acceptable carriers, diluents, or excipients" include, but are not limited to, any excipients, carriers, excipients, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers that have been approved by the U.S. Food and Drug Administration for use in humans or domestic animals and are acceptable for human or domestic animal use.

[0081] "Pharmaceutical acceptable salts" include acid addition salts and base addition salts.

[0082] "Pharmaceutically acceptable acid addition salts" refer to those salts that retain the bioavailability and properties of the free base, are not biologically or otherwise undesirable, and form with inorganic or organic acids, such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., and such organic acids as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetaminophen, camphoric acid, camphor-10-sulfonic acid, decanoic acid, hexanoic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclohexane, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactoside. (acids), gentian acid, glucoheponic acid, gluconic acid, glucuronic acid, glutamic acid, glutamate, 2-oxoglutamate, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucoic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, dihydroxynaphthalic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanate, p-toluenesulfonic acid, trifluoroacetic acid, undecenoic acid, etc.).

[0083] "Pharmaceutically acceptable base addition salts" refer to salts that retain the bioavailability and properties of the free acid, and are not those that are biologically or otherwise undesirable. These salts are prepared by the addition of an inorganic or organic base to a free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, and salts from basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dianophenoxylate, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, heparin, choline, betaine, benzylamine, benzathine, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, and others. N -Ethylpiperidine, polyamine resins, etc. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0084] Typically, crystallization produces solvates of the compounds disclosed herein. As used herein, the term "solvate" refers to an aggregate comprising one or more molecules of the disclosed compound and one or more solvent molecules. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of this disclosure may exist in hydrated forms (including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc.) and corresponding solvated forms. The compounds of this disclosure may be true solvates, while in other cases, the compounds of this disclosure may contain only external water or be a mixture of water and some external solvent.

[0085] "Pharmaceutical composition" means the compounds of this disclosure and formulations generally accepted in the art for delivering biologically active compounds to mammals such as humans. Such media include all pharmaceutically acceptable carriers, diluents, or excipients.

[0086] "Effective amount" or "therapeutic effective amount" means the amount of the compound of this disclosure that is sufficient to achieve a therapeutic effect in a preferred mammal when administered to a preferred mammal. The amount of lipid nanoparticles of this disclosure constituting a "therapeutic effective amount" will vary depending on the compound, the condition and its severity, the method of administration, and the age of the mammal to be treated, but can be determined by a person skilled in the art based on their knowledge and the conventions of this disclosure.

[0087] As used herein, “treating” or “treatment” encompasses treatment of a target disease or condition in mammals, preferably humans, and includes: (i) To prevent the occurrence of the disease or condition in mammals, especially when such mammals are susceptible to the condition but have not yet been diagnosed with it; (ii) To suppress the disease or condition, that is, to prevent its development; (iii) To alleviate the disease or condition, that is, to cause the disease or condition to subside; or (iv) Relieve the symptoms caused by the disease or condition, i.e., relieve pain without addressing the underlying disease or condition. As used herein, the terms “disease” and “condition” may be used interchangeably or differently, because a disease or condition may not have a known causative agent (making the cause unknown), and therefore it is not recognized as a disease but is only considered an undesirable condition or syndrome in which a clinician has identified more or less a specific group of symptoms.

[0088] The compounds disclosed herein, or pharmaceutically acceptable salts thereof, may contain one or more asymmetric centers, thus yielding enantiomers, diastereomers, and other stereoisomers, which may be defined in absolute stereochemistry as (R )-or( S ()-, or (D)- or (L)- for amino acids. This disclosure is intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), ( R )-and( S (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques such as chromatography and stepwise crystallization. Conventional techniques for the preparation / separation of individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of racemic mixtures (or racemic mixtures of salts or derivatives) using, for example, chiral high-performance liquid chromatography (HPLC). When the compounds described herein contain an alkene double bond or other geometrically asymmetric center, and unless otherwise stated, the compounds are intended to include E and Z geometric isomers. Similarly, all tautomeric forms are intended to be included.

[0089] "Stereoisomers" are compounds composed of identical atoms bonded by the same bonds but having different three-dimensional structures that are not interchangeable. This disclosure considers various stereoisomers and mixtures thereof, and includes "enantiomers," which are two stereoisomers whose molecules are non-overlapping mirror images of each other.

[0090] "Tautomerism" refers to the transfer of a proton from one atom of a molecule to another atom of the same molecule. This disclosure includes tautomerisms of any of the said compounds.

[0091] compound

[0092] In one aspect, this disclosure provides novel lipid compounds that can be combined with other lipid components such as neutral lipids, charged lipids, steroids, and / or polymer-conjugated lipids to form lipid nanoparticles with oligonucleotides. Without being bound by theory, it is believed that these lipid nanoparticles protect oligonucleotides from degradation in serum and provide efficient delivery of oligonucleotides to cells both in vitro and in vivo.

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

[0094] (I)

[0095] Or its pharmaceutically acceptable salts, tautomers or stereoisomers, wherein: G 1 It is N or CH; When G 1 When it is N, G 2 It's a direct-connect key, or when G... 1 When it is CH, G2 Yes -NR 1a -; R 1a It is C4-C 12 Alkyl, the C4-C 12 The alkyl group is optionally substituted by one or more substituents selected from oxo, -OH and NH2; R 1 It is a C1-C8 alkyl group, wherein the C1-C8 alkyl group is optionally radically oxidized by one or more radicals selected from oxo, -OH, -N(R) 1b )R 1c Substitution of substituents in cyclic hydrocarbon groups or heteroaryl groups; R 1b and R 1c Each is independently hydrogen or C1-C4 alkyl; or R 1b and R 1c They combine with the nitrogen atoms to which they are attached to form heterocyclic groups; R 2 It is -OC(=O)OR 2a -OC(=O)R 2b or -C(=O)OR 2c ; R 2a It is C4-C 24 Alkyl, C4-C 24 alkenyl or C4-C 24 alkynyl group; R 2b and R 2c It has the following structure: ; R 2d and R 2e Each is independently C4-C 12 Alkyl, C4-C 12 alkenyl or C4-C 12 alkynyl group; R 3 It is -C(=O)N(R) 3a )R 3b or -NR 3a -C(=O)R 3b ; R 3a and R 3b Each is independently C6-C 24 Alkyl, C6-C 24 alkenyl or C6-C 24 acetylinyl group; and L 1 and L 2 Each is independently C4-C12 Alkylene Each of the alkyl, alkenyl, alkynyl, alkylene, cycloalkyl, heterocyclic and heteroaryl groups is optionally substituted with one or more fluorine molecules.

[0096] In some implementation schemes, R 1b and R 1c They combine with the nitrogen atoms to which they are attached to form 3 to 12-membered heterocyclic groups. In some embodiments, R 1b and R 1c They combine with the nitrogen atoms to which they are attached to form 3 to 10-membered heterocyclic groups.

[0097] In some embodiments, the compound has the following formula (Ia):

[0098] (Ia)

[0099] Or its pharmaceutically acceptable salt, tautomer or stereoisomer.

[0100] In some embodiments, the compound has the following formula (Ib):

[0101] (Ib)

[0102] Or its pharmaceutically acceptable salt, tautomer or stereoisomer.

[0103] In some implementation schemes, R 1a It is C8-C that is optionally substituted with oxygen. 12 Alkyl group. In some more specific embodiments, R 1a It has one of the following structures: or .

[0104] In some implementations, R 1 Optionally substituted with one or two substituents selected from -OH, oxo, fluorine, -N(CH3)2 and the following structures: .

[0105] In some implementation schemes, R 1 Replaced by -OH. In some embodiments, R 1 It is -CH3. In some other implementations, R 1 It has one of the following structures: : : or .

[0106] In some implementation schemes, R 1 It has one of the following structures: : : : : or .

[0107] In some implementation schemes, R 2 It is -OC(=O)OR 2a In some implementations, R 2a It is C4-C 24 Alkyl group. In some embodiments, R 2a It is unsubstituted C4-C 24 Alkyl group. In some embodiments, R 2a It is an unbranched C4-C 24 Alkyl group. In some embodiments, R 2a It is a C4-C branch. 24 alkyl.

[0108] In some implementations, R 2 It is -OC(=O)R 2b In some implementations, R 2 It is -C(=O)OR 2c In some implementations, R 2b Or R 2c It has the following structure: .

[0109] In some implementation schemes, R 2b Or R 2c It has the following structure: .

[0110] In some implementations, R 2d It is an unbranched C4-C8 alkyl group. In some embodiments, R 2e It is an unbranched C4-C8 alkyl group. In some embodiments, R 2d It is an unbranched C4 alkyl group. In some embodiments, R 2e It is an unbranched C4 alkyl group. In some embodiments, R 2d It is an unbranched C6 alkyl group. In some embodiments, R 2e It is an unbranched C6 alkyl group. In some embodiments, R 2d It is an unbranched C8 alkyl group. In some embodiments, R 2e It is an unbranched C8 alkyl group.

[0111] In some implementations, R 2 It has one of the following structures: ; ; ; or .

[0112] In some implementations, R 2 It has one of the following structures: ; ; ; ; ; ; ; ; ; ; ; ; ; or .

[0113] In some implementations, R 3 It is -C(=O)N(R) 3a )R 3b In some implementations, R 3 Yes -NR 3a -C(=O)R 3b In some implementations, R 3a and R 3b Each is independently C6-C 24 Alkyl group. In some embodiments, R 3a and R 3b Each is independently C6-C 12 Alkyl group. In some embodiments, R 3a and R 3b Each is independently C8-C 10 Alkyl group. In some embodiments, R 3a and R 3b All are C8 alkyl groups. In some embodiments, R 3a and R 3b All are C 10 Alkyl group. In some embodiments, R 3a and R 3b All are C 12 alkyl.

[0114] In some implementations, L 1 and L 2Each is independently a C4-C8 alkylene group. In some embodiments, L 1 and L 2 Each is independently C4-C 10 Alkylene. In some embodiments, L 1 and L 2 Each is independently a C5 alkylene, C7 alkylene, or C8 alkylene. In some embodiments, L 1 and L 2 Each is independently a C4 alkylene, C5 alkylene, C6 alkylene, C7 alkylene, C8 alkylene, or C9 alkylene. In some embodiments, L 1 and L 2 Same. In some implementations, L 1 and L 2 Different. In some implementations, L 1 and L 2 It is not replaced. In some implementations, L 1 L 2 Or both may be substituted by one or more fluorine substituents.

[0115] In various embodiments, the compound has one of the structures listed in Table 1 below (or its pharmaceutically acceptable salt, tautomer or stereoisomer).

[0116] Table 1. Representative compounds of formula (I)

[0117] It should be understood that any embodiment of the compound of formula (I) as described above, and any particular substituent and / or variable of the compound of formula (I) as described above, can be independently combined with other embodiments and / or substituents and / or variables of the compound of formula (I) to form embodiments of this disclosure not specifically set forth above. Furthermore, where a list of substituents and / or variables of any specific R, G, or L groups is provided in the embodiments and / or claims, it should be understood that each individual substituent and / or variable may be removed from the embodiments and / or claims, and the remaining list of substituents and / or variables will be considered to be within the scope of embodiments of this disclosure.

[0118] It should be understood that the combination of substituents and / or variables in the general formulas described in this specification is permitted only if such contributions produce stable compounds.

[0119] For application purposes, the compounds of this disclosure (typically in the form of lipid nanoparticles in combination with therapeutic agents) can be applied as chemical raw materials or formulated into pharmaceutical compositions. The pharmaceutical compositions of this disclosure 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 that effectively forms lipid nanoparticles and delivers a therapeutic agent, such as a therapeutic agent for treating a specific target disease or condition. Appropriate concentrations and dosages can be readily determined by those skilled in the art.

[0120] The embodiments provide compositions comprising a compound of formula (I) and a therapeutic agent (e.g., lipid nanoparticles). In some embodiments, the composition (e.g., lipid nanoparticles) further comprises one or more excipients selected from neutral lipids, steroids, and polymer-conjugated lipids.

[0121] In some implementations, the therapeutic agent comprises a nucleic acid. In some implementations, the nucleic acid is selected from antisense RNA and messenger RNA.

[0122] In some embodiments, the composition (e.g., lipid nanoparticles) comprises one or more neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the neutral lipid is DSPC. In some embodiments, the molar ratio of the compound to the neutral lipid is from about 2:1 to about 8:1. In some embodiments, the steroid is cholesterol. In some embodiments, the molar ratio of the compound to cholesterol is from about 2:1 to 1:1. In some embodiments, the molar ratio of the compound to cholesterol is from 5:1 to 1:1 or from 2:1 to 1:1.

[0123] In some embodiments, the polymer-conjugated lipid is a polyethylene glycol-modified lipid. In various embodiments, the polymer-conjugated lipid is a polyethylene glycol-modified lipid. For example, some embodiments include PEGylated diacylglycerols (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG); PEGylated phosphatidylethanolamine (PEG-PE); PEGylated succinate diacylglycerols (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(g-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG); PEGylated ceramides (PEG-cer); or PEGylated dialkoxypropyl carbamates, such as g-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoyloxy)propyl)carbamate or 2,3-di(tetradecanoyloxy)propyl-N-(g-methoxy(polyethoxy)ethyl)carbamate.

[0124] In some embodiments, the molar ratio of the compound to the PEGylated lipid is about 100:1 to about 10:1 or about 100:1 to about 25:1. In some embodiments, the molar ratio of the compound to the PEGylated lipid is about 100:1 to about 20:1 or about 100:1 to about 10:1.

[0125] In some embodiments, the polyethylene glycol-modified liposome has the following formula (II):

[0126] (II)

[0127] Or its pharmaceutically acceptable salts, tautomers or stereoisomers, wherein: R 10 and R 11 Each of the following is independently a straight-chain or branched alkyl, alkenyl, or ynyl group having 10 to 30 carbon atoms, wherein the alkyl, alkenyl, or ynyl group is optionally interrupted by one or more ester bonds; and w has an average value of 30 to 60.

[0128] In some implementations, R 10 and R 11 Each is independently a straight-chain alkyl chain containing 12 to 16 carbon atoms. In some embodiments, the average value of w is about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55. In some embodiments, the average value of w is about 49. In some embodiments, w has a value of 30 to 60. In some embodiments, w is 40 to 50. In some embodiments, w is 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0129] In some embodiments, the lipid nanoparticles or composition comprise polyethylene glycol-modified lipids of various formulas (II), and the average value of w for the various polyethylene glycol-modified lipids is 40 to 50. In some embodiments, the average value of w is 43, 44, 45, 46, 47, or 48.

[0130] The synthesis of PEGylated lipids can be found in U.S. Patent No. 9,738,593, the disclosure of which is incorporated herein by reference.

[0131] The compositions of this disclosure can be administered via any acceptable method of administration of the pharmaceutical agent to achieve similar efficacy. The pharmaceutical compositions of this disclosure can be formulated into formulations in solid, semi-solid, liquid, or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalers, gels, microspheres, and aerosols. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal administration. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intradermal, intrasternal injection, or infusion techniques. The pharmaceutical compositions of this disclosure are formulated such that the active ingredient contained therein is bioavailable after administration of the composition to a patient. The composition to be administered to an individual or patient is in the form of one or more dose units, wherein, for example, a tablet may be a single dose unit, and a container of the compounds of this disclosure in aerosol form may contain multiple dose units. Practical methods for preparing such dosage forms are known to those skilled in the art or will be apparent; see, for example, [link to relevant documentation]. Remington: The Science and Practice of Pharmacy , 20th edition (Philadelphia College of Pharmacy and Science, 2000). In accordance with the teachings of this disclosure, in any case, the composition to be administered will contain a therapeutically effective amount of the compound of this disclosure or a pharmaceutically acceptable salt thereof for the treatment of a target disease or condition.

[0132] The pharmaceutical compositions disclosed herein can be in solid or liquid form. In one aspect, the carrier is granular, such that the composition is in the form of, for example, tablets or powders. The carrier can be liquid, wherein the composition is, for example, an oral syrup, an injectable liquid, or an aerosol, which can be used for, for example, inhalation administration.

[0133] When intended for oral administration, the pharmaceutical composition is preferably in solid or liquid form, wherein semi-solid, semi-liquid, suspension and gel forms are included in the forms that are considered solid or liquid herein.

[0134] As solid compositions for oral administration, pharmaceutical compositions can be formulated into powders, granules, compressed tablets, pills, capsules, chewing gum, wafers, etc. Such solid compositions typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch, lactose, or dextrin; disintegrants such as alginate, sodium alginate, Primogel, corn starch, etc.; lubricants such as magnesium stearate or Sterotex; gliding agents such as colloidal silica; sweeteners such as sucrose or saccharin; flavoring agents such as peppermint oil, methyl salicylate, or orange flavoring; and coloring agents.

[0135] When the pharmaceutical composition is in the form of capsules, such as gelatin capsules, it may contain liquid carriers such as polyethylene glycol or oil, in addition to the materials described above.

[0136] Pharmaceutical compositions may be in liquid form, such as elixirs, syrups, solutions, emulsions, or suspensions. As two examples, the liquid may be intended for oral administration or for delivery by injection. When intended for oral administration, preferred compositions, in addition to the compounds of this application, may contain one or more of sweeteners, preservatives, dyes / colorants, and flavor enhancers. Compositions intended for injection may contain one or more of surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents.

[0137] The liquid pharmaceutical compositions disclosed herein, whether in solution, suspension, or other similar form, may comprise one or more of the following excipients: sterile diluents such as water for injection, saline solution (preferably physiological saline), Ringer's solution, isotonic sodium chloride, non-volatile oils such as synthetic monoglycerides or diglycerides, polyethylene glycol, glycerol, propylene glycol, or other solvents that can be used as solvents or suspension media; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and agents for adjusting tension such as sodium chloride or dextrose; and agents used as cryoprotectants such as sucrose or trehalose. Parenteral preparations may be packaged in glass or plastic ampoules, disposable syringes, or multi-dose vials. Physiological saline is a preferred excipient. Injectable pharmaceutical compositions are preferably sterile.

[0138] Liquid pharmaceutical compositions of this disclosure intended for parenteral or oral administration shall contain an amount of the compounds of this disclosure to obtain a suitable dosage form.

[0139] The pharmaceutical compositions disclosed herein may be intended for topical application, in which case the carrier may suitably comprise a solution, emulsion, ointment, or gel matrix. The matrix may, for example, comprise one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents (such as water and alcohol), and emulsifiers and stabilizers. A thickener may be present in the pharmaceutical composition for topical application. If intended for transdermal application, the composition may comprise a transdermal patch or an iontophoresis device.

[0140] The pharmaceutical compositions disclosed herein are intended for rectal administration, for example, in the form of suppositories, which melt in the rectum and release the drug. Compositions for rectal administration may contain an oily matrix as a suitable, non-irritating excipient. Such matrices include, but are not limited to, lanolin, cocoa butter, and polyethylene glycol.

[0141] The pharmaceutical compositions disclosed herein can comprise a variety of materials in physical form that alters the dosage unit of a solid or liquid. For example, the composition can comprise a material that forms a coating around the active ingredient. The material forming the coating is typically inert and can be selected from, for example, sugars, shellac, and other enteric coating agents. Optionally, the active ingredient can be encapsulated in a gelatin capsule.

[0142] The pharmaceutical compositions of this disclosure, in solid or liquid form, may contain an agent that binds to the compound of this disclosure to facilitate delivery of the compound. Suitable agents that can function in this way include monoclonal or polyclonal antibodies or proteins.

[0143] The pharmaceutical compositions disclosed herein can consist of dosage units that can be administered as aerosols. The term "aerosol" is used to refer to a range of systems, from those of colloidal nature to those consisting of pressurized packaging. Delivery can be made by liquefied or compressed gas or by a suitable pump system dispensing the active ingredient. Aerosols of the compounds of this disclosure can be delivered in single-phase, two-phase, or three-phase systems to deliver the active ingredient. Aerosol delivery includes the necessary containers, activators, valves, sub-containers, etc., which together can form a kit. Preferred aerosols can be determined by those skilled in the art without excessive experimentation.

[0144] The pharmaceutical compositions disclosed herein can be prepared using methods well known in the pharmaceutical industry. For example, a pharmaceutical composition intended for injection can be prepared by combining the lipid nanoparticles of this disclosure with sterile distilled water or other carriers to form a solution. Surfactants can be added to promote the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the compounds of this disclosure to promote the dissolution or homogeneous suspension of the compounds in an aqueous delivery system.

[0145] The compositions disclosed herein, or pharmaceutically acceptable salts thereof, are administered in therapeutically effective amounts, said amounts which will vary depending on a number of factors, including the activity of the specific therapeutic agent used; the metabolic stability and duration of action of the therapeutic agent; the patient's age, weight, general health, sex, and diet; the method and timing of administration; the excretion rate; the combination of drugs; the severity of the specific condition or disease; and the individual being treated.

[0146] The compositions of this disclosure may also be administered concurrently with, before, or after the administration of one or more other therapeutic agents. Such combination therapies include single-dose formulations of the compositions of this disclosure and one or more additional active agents, as well as separate drug-dose formulations of the compositions of this disclosure and each active agent. For example, the compositions of this disclosure and other active agents may be administered to a patient together as a single oral dose composition (such as tablets or capsules), or as each agent in separate oral dose formulations. When using separate dose formulations, the compounds of this disclosure and one or more additional active agents may be administered at substantially the same time, i.e., simultaneously, or at separately staggered times, i.e., sequentially; combination therapy is understood to include all of these regimens.

[0147] The methods for preparing the above compounds and compositions are described below and / or are known in the art.

[0148] Those skilled in the art will understand that, in the methods described herein, the functional groups of the intermediate compounds may require protection by suitable protecting groups. Such functional groups include hydroxyl, amino, mercapto, and carboxylic acids. Suitable protecting groups for hydroxyl groups include trialkylsilyl or diarylalkylsilyl (e.g., tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or trimethylsilyl), tetrahydropyranyl, benzyl, etc. Suitable protecting groups for amino, amidine, and guanidine groups include tert-butyloxycarbonyl, benzyloxycarbonyl, etc. Suitable protecting groups for mercapto groups include -C(O)-R″ (where R″ is alkyl, aryl, or arylalkyl), p-methoxybenzyl, triphenylmethyl, etc. Suitable protecting groups for carboxylic acids include alkyl esters, aryl esters, or arylalkyl esters. Protecting groups may be added or removed according to techniques known to those skilled in the art and as described herein, as well as standard techniques. The use of protecting groups is described in detail in Green, TW, and PGM Wutz. Protective Groups in Organic Synthesis (1999), 3rd edition, Wiley. As those skilled in the art will understand, the protecting group may also be a polymeric resin, such as Wang resin, Rink resin, or 2-chlorotriphenylmethyl-chloride resin.

[0149] Those skilled in the art will also understand that although such protected derivatives of the compounds of this disclosure may not be pharmacologically active on their own, they can be administered to mammals and subsequently metabolized in vivo to form pharmacologically active compounds of this disclosure. Therefore, such derivatives can be described as “prodrugs.” All prodrugs of the compounds of this disclosure are included within the scope of this disclosure.

[0150] Furthermore, all compounds of this disclosure, existing in free base or acid form, can be converted into their pharmaceutically acceptable salts by methods known to those skilled in the art, through treatment with suitable inorganic or organic bases or acids. Salts of the compounds of this disclosure can be converted into their free base or acid forms using standard techniques.

[0151] The following reaction scheme illustrates a method for preparing the compounds disclosed herein, namely compounds of formula (I):

[0152] (I)

[0153] Or its pharmaceutically acceptable salt, tautomer or stereoisomer, wherein R 1 R 2 R 3 G 1 G 2 L 1 and L 2 As defined herein. It should be understood that those skilled in the art can prepare these compounds by similar methods or by combining other methods known to them. It should also be understood that those skilled in the art will be able to prepare other compounds of formula (I) not specifically exemplified below in a similar manner by using appropriate starting components and varying the synthetic parameters as needed. Typically, starting components can be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, or synthesized from sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Edition (Wiley, December 2000)) or prepared as described in this disclosure.

[0154] General Reaction Scheme 1

[0155] The embodiments of compounds of formula (I) (e.g., compounds I-1 and I-2) can be prepared according to general reaction scheme 1, wherein the variable (e.g., R) 1 R 2 R 3 G 1 G 2 L 1 L 2 R 2a R 3a and R 3b As defined in this article.

[0156] Referring to General Reaction Scheme 1, reagents and starting materials (e.g., compounds 1A, 1B, 1D, 1F, and 1H) can be purchased from commercial sources or prepared according to methods familiar to those skilled in the art. A mixture of 1A and 1B is combined under suitable reaction conditions to promote a coupling reaction (e.g., (COCl)₂, DMF, triethylamine, DMAP). The resulting product (1C) can be combined with amine 1D and suitable reagents and reaction conditions (e.g., acetonitrile, under reflux) to give the desired product (1E). In a parallel pathway, compound 1F is reacted with a suitable reagent (e.g., p-nitrobenzene chloroformate, pyridine, in DCM) to give compound 1G. Compound 1G is then reacted with compound 1H under the desired reaction conditions (e.g., DMAP, pyridine, in DCM) to give compound 1I. Compound 1I is then reacted with compound 1E under suitable conditions (e.g., DIPEA, acetonitrile, under reflux) to give the compound of formula (I).

[0157] General Reaction Scheme 2

[0158] The embodiments of compounds of formula (I) (e.g., compound I-8) can be prepared according to general reaction scheme 2, wherein the variable (e.g., R) 1 R 2 R 3 G 1 G 2 L 1 L 2 R 2c R 3a R 1a and R 3b As defined in this article.

[0159] Referring to General Reaction Scheme 2, the starting materials and other reagents (e.g., compounds 2A, 2B, 2D, 2F, and 2H) can be purchased from commercial sources or prepared according to methods familiar to those skilled in the art. As a first step, a mixture of 2A and 2B is combined under suitable reaction conditions to promote a coupling reaction (e.g., DIPEA, HATU, in a DCM) and yield the desired product shown. Compound 2C is then reacted with compound 2D under suitable conditions (e.g., DCC, DMAP, in a DCM) to give compound 2E. This reaction product is then reacted with compound 2F under suitable conditions (e.g., NaBH(AcO)3, acetic acid, and DCE) to give compound 2G. Compound 2G and compound 2H are then combined under suitable conditions (e.g., DIPEA, HATU, in a DCM) to produce a compound of formula (I).

[0160] It should be understood that those skilled in the art can prepare compounds of formula (I) by similar methods or by combining other methods known to them. It should also be understood that those skilled in the art can prepare other compounds of formula (I) not specifically exemplified below as needed, using appropriate starting components and varying synthetic parameters, in a similar manner as described below. Generally, starting components can be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, or synthesized from sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Edition, (Wiley, December 2000)), or prepared as described in this disclosure.

[0161] Example 1

[0162] In vivo evaluation of luciferase mRNA using lipid nanoparticle compositions

[0163] The lipids of formula (I), DSPC, cholesterol, and the PEG-lipids of formula (II) were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 or 47.5:10:40.7:1.8. Lipid nanoparticles (LNPs) were prepared at a total lipid to mRNA weight ratio of approximately 10:1 to 40:1. Briefly, the mRNA was diluted to 0.2 mg / mL in 10 to 50 mM citrate buffer (pH 4–6) or 10 to 25 mM acetate buffer (pH 4–6). The ethanol lipid solution was mixed with the aqueous mRNA solution using a syringe pump at a ratio of approximately 1:5 to 1:3 (vol / vol) at a total flow rate greater than 15 mL / min. The ethanol was then removed, and PBS was used instead of the external buffer for dialysis. Finally, the lipid nanoparticles were filtered through a 0.2 μm pore sterile filter.

[0164] The study was conducted in 6-8 week old female C57BL / 6 mice (Charles River) or 8-10 week old CD-1 mice (Charles River or Inotiv), following guidelines established by the Institutional Animal Care Committee (ACC) and the Canadian Animal Care Council (CCAC). Various doses of mRNA-lipid nanoparticles were administered systemically via tail vein injection, and the animals were euthanized at specific time points (e.g., 4 hours) after administration. Liver and spleen were collected in pre-weighed tubes, weighed, immediately flash-frozen in liquid nitrogen, and stored at -80°C until processing for analysis.

[0165] For the liver, approximately 50 mg was excised and placed in a 2 mL FastPrep tube (MP Biomedicals, Solon OH) for analysis. Add ¼” ceramic balls (MP Biomedicals) to each tube and add 500 to 750 µL of Glo Lysis Buffer-GLB (Promega, Madison WI) equilibrated to room temperature to the liver tissue. Homogenize the liver tissue for 15 seconds at 2 × 6.0 m / s using a FastPrep24 instrument (MP Biomedicals). Incubate the homogenate at room temperature for 5 minutes, then dilute it in GLB at a ratio of 1:4 to 1:6 and evaluate it using the SteadyGlo luciferase assay system (Promega). Specifically, react 50 µL of diluted tissue homogenate with 50 µL of SteadyGlo substrate, vortex for 10 seconds, incubate for 5 minutes, and then quantify the luminescence using a CentroXS³ LB 960 spectrophotometer (Berthold Technologies, Germany) or a Filter Max F5 Microplate Reader (Molecular Devices, USA). Determine the amount of protein measured using a BCA protein assay kit (Pierce, Rockford, IL). (Assembled). The relative luminescent units (RLU) were then normalized to the total µg protein or tissue weight (g) measured. To convert RLU to ng luciferase, a standard curve was generated using QuantiLum recombinant luciferase (Promega).

[0166] FLuc mRNA (7202) from Trilink Biotechnologies will express the original expression from the firefly *Fireflyia davidii* (also known as the Big Dipper firefly). photinus pyralis The luciferase protein isolated from [a specific organism / organism] is commonly used in mammalian cell culture to measure gene expression and cell viability. It emits bioluminescence in the presence of its substrate, luciferin. This capped and polyadenylated mRNA is modified with 5-methoxyuridine and optimized for mammalian systems.

[0167] Example 2

[0168] In vivo evaluation of immunoglobulin G (IgG) mRNA using lipid nanoparticle compositions

[0169] The lipids of formula (I), DSPC, cholesterol, and the PEG-lipids of formula (II) were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 or 47.5:10:40.7:1.8. Lipid nanoparticles (LNPs) were prepared at a total lipid to mRNA weight ratio of approximately 10:1 to 40:1. Briefly, the mRNA was diluted to 0.2 mg / mL in 10 to 50 mM citrate buffer (pH 4–6) or 10 to 25 mM acetate buffer (pH 4–6). The ethanol lipid solution was mixed with the aqueous mRNA solution using a syringe pump at a ratio of approximately 1:5 to 1:3 (vol / vol) at a total flow rate greater than 15 mL / min. The ethanol was then removed, and the external buffer was replaced with PBS by dialysis. Finally, the lipid nanoparticles were filtered through a 0.2 μm pore sterile filter.

[0170] The study was conducted in 6- to 8-week-old CD-1 / ICR mice (Charles River, or Inotiv) following guidelines established by the Institutional Animal Care Committee (ACC) and the Canadian Animal Care Council (CCAC). Different doses of mRNA-lipid nanoparticles were administered systemically via tail vein injection, and the animals were euthanized at specific time points after administration (e.g., 24 hours). Whole blood was collected, and serum was subsequently separated by centrifugation of the whole blood at 2000 xg for 10 minutes at 4°C and stored at -80°C until analysis.

[0171] For the immunoglobulin G (IgG) ELISA (Life Diagnostics Human IgG ELISA kit), serum samples were diluted 100 to 20,000 times with 1× diluent solution. 100 μL of diluted serum was dispensed into duplicate into anti-human IgG-coated 96-well plates, along with human IgG standards, and incubated at 25°C for 45 minutes at 150 rpm in a plate shaker. The wells were washed 5 times with 1× washing solution using a plate washer (400 μL / well). 100 μL of HRP conjugate was added to each well, and the plates were incubated under the same conditions as above in a plate shaker. The wells were washed again 5 times with 1× washing solution using a plate washer (400 μL / well). 100 μL of TMB reagent was added to each well, and the plates were incubated under the same conditions as above in a plate shaker. The reaction was terminated by adding 100 μL of stop solution to each well. The absorbance was read at 450 nm (A450) using a microplate reader. The amount of human IgG in mouse serum was determined by plotting the A450 values ​​of the assay standards against the concentration of human IgG.

[0172] Example 3

[0173] pK of the formulated lipidsa Measurement

[0174] As described elsewhere, the pK of the formulated lipids a This is related to the efficacy of LNP in delivering nucleic acids (see Jayaraman et al., Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semil et al., Nature Biotechnology 28, 172–176 (2010)). In some implementations, pK a The preferred range is ~5 to ~7. The pK of each lipid in the lipid nanoparticles can be determined using a fluorescence assay based on 2-(p-toluidine)-6-naphthalenesulfonic acid (TNS). a Lipid nanoparticles containing compound (I) / DSPC / cholesterol / PEG-lipid (50:10:38.5:1.5 or 47.5:10:40.7:1.8 mol%) of formula (I) in PBS were prepared using the in-line process described in Example 1. TNS was prepared as a stock solution of 100 μM in distilled water. The vesicles were diluted to 24 μM lipid in a 2 mL buffer solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl, with a pH range of 2.5 to 11. Aliquots of the TNS solution were added to obtain a final concentration of 1 µM, and after vortex mixing, 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. S-shaped best-fit analysis was applied to the fluorescence data, and pK... a The pH value is measured at half the level that produces the maximum fluorescence intensity.

[0175] Example 4

[0176] The efficacy of lipid nanoparticle formulations containing various cationic lipids was determined using a rodent model expressing IgG mRNA.

[0177] The representative compounds of this disclosure shown in Table 2 were prepared using the following molar ratios: 50% cationic lipid / 10% distearate phosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid 2-[2-(g-methoxy(polyethylene glycol)] 2000[Ethoxy]-N,N-bistetradecylacetamide or 47.5% cationic lipid / 10% DSPC / 40.7% cholesterol / 1.8% PEG lipid. Relative activity was determined by measuring the amount of human IgG in mouse serum as described in Example 1. Activity was compared at doses of 1.0 or 0.3 mg mRNA / kg and expressed as ng luciferase / g liver measured 4 hours after administration, as described in Example 1, or as µg IgG / mL serum measured 24 hours after administration, as described in Example 2. The compound numbers in Table 2 refer to the compound numbers in Table 1.

[0178] Table 2. Novel cationic lipids and their related activities

[0179] Synthesis example 1

[0180] Synthetic routes of compounds I-1 and I-2

[0181] Synthesis of 8-bromo-N,N-didecyloctamide

[0182] A catalytic amount of DMF and oxaloyl chloride (3.0 equivalents) was added to a solution of 8-bromooctanoic acid (1.0 equivalent) in DCM (2 mL / mmol). The reaction mixture was stirred at room temperature under N2 until complete conversion. Excess oxaloyl chloride and DCM were then evaporated under vacuum. The resulting acyl chloride solution in anhydrous DCM (1 mL / mmol) was slowly added to a solution of didecylamine (1.1 equivalent), triethylamine (6.0 equivalent), and DMAP (catalytic amount) in anhydrous DCM (3 mL / mmol). The mixture was then stirred at room temperature under N2 for 16 hours. After concentration under reduced pressure, the residue was partitioned between H2O (10 mL / mmol) and ethyl acetate (10 mL / mmol). The aqueous phase was extracted with ethyl acetate, and the combined organic phases were dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography (hexane / ethyl acetate, 100:0 to 80:20). The product obtained was a pale yellow oil (15 g, 30 mmol, 66%).

[0183] 1H NMR (400 MHz, CDCl3) δ 3.39 (t, J = 6.8 Hz, 2H), 3.32 – 3.24 (m,2H), 3.23 – 3.14 (m, 2H), 2.33 – 2.22 (m, 2H), 1.92 – 1.80 (m, 2H), 1.63 (m,5H), 1.57 – 1.39 (m, 8H), 1.26 (m, 40H), 0.88 (td, J = 6.7, 3.2 Hz, 7H). ESI-MS:C 28 H 56 The calculated MW value for BrNONa [M+Na]+ is 524.34, and the measured value is 525.82.

[0184]

[0185] Synthesis of N,N-didecyl-8-(methylamino)octamide

[0186] A solution of 8-bromo-N,N-didecyloctamide (1 equivalent) and methylamine (5 equivalents) in acetonitrile (10 mL / mmol) was heated under reflux overnight. The reaction mixture was concentrated, and the crude product was purified by column chromatography (Hex / EtOAc, 95:5 to 0:100, followed by DCM / MeOH containing 3% NH3, 100:0 to 90:10). The product was given as a pale yellow oil (1.4 g, 3.09 mmol, 77%). ESI-MS: C 29 H 60 The calculated MW value for N2O [M+H]+ is 453.48; the measured value is 453.67.

[0187]

[0188] Synthesis of N,N-didecyl-8-((5-hydroxypentyl)amino)octamide

[0189] N,N-Dicedecyl-8-((5-hydroxypentyl)amino)octamide was prepared from 8-bromo-N,N-dicecyloctamide (2 g, 3.99 mmol) and 5-amino-1-pentanol (2.05 g, 19.89 mmol) in a similar manner to that used for N,N-dicecyl-8-(methylamino)octamide. The product was a pale yellow oil (1.2 g, 2.28 mmol, 57%). ESI-MS: C 33 H 68 N₂O₂ [M+H] + The calculated MW value is 525.54; the measured value is 526.64.

[0190]

[0191] Synthesis of 8-bromooctyl(4-nitrophenyl) carbonate

[0192] At room temperature under N2, p-nitrobenzene chloroformate (1.2 equivalents, 5.78 g, 28.69 mmol) and pyridine (dropwise, 1.3 equivalents, 2.46 g, 31.08 mmol) were added to a solution of 8-bromo-1-octanol (1.0 equivalent, 5 g, 23.91 mmol) in DCM (3 mL / mmol). The resulting mixture was stirred overnight at room temperature. Subsequently, the reaction mixture was diluted with water and dichloromethane (DCM) to separate the aqueous and organic phases. The aqueous phase was extracted twice with DCM, dried over Na2SO4, and concentrated under reduced pressure. The crude product was purified by column chromatography (Hex / EtOAc, 100:0 to 80:20). The product was given as a colorless oil (7.5 g, 20 mmol, 84%). ESI-MS: C 15 H 20 BrNO5 [M+Na] + The calculated m / z values ​​are 396.04 & 398.04, and the measured values ​​are 396.08 & 398.09.

[0193]

[0194] Synthesis of 8-bromooctyldecyl carbonate

[0195] 1-Decanol (4 equivalents, 8.45 g, 53.44 mmol), DMAP (0.2 equivalents, 0.33 g, 2.67 mmol), and pyridine (dropwise, 1.3 equivalents, 1.37 g, 17.37 mmol) were added to a solution of 8-bromooctyl(4-nitrophenyl) carbonate (1 equivalent, 5 g, 13.36 mmol) in DCM (3.5 mL / mmol) at room temperature under N2. After stirring overnight at room temperature under N2, the reaction mixture was diluted with water and DCM. The resulting aqueous and organic phases were separated, and the aqueous phase was extracted twice with DCM, dried over Na2SO4, and concentrated under reduced pressure. The crude product was purified by column chromatography (Hex / EtOAc, 100:0 to 80:20). The product was obtained as a colorless oil (3.47 g, 8.85 mmol, 66%).

[0196] 1 H NMR (400 MHz, CDCl3) δ 4.12 (t, J = 6.7 Hz, 4H), 3.40 (t, J= 6.8Hz, 2H), 1.89-1.78 (m, 2H), 1.66 (p, J = 6.7 Hz, 4H), 1.48-1.18 (m, 22H), 0.94-0.81 (m, 3H), ESI-MS: C 19 H 37 BrO3 [M+H] + The calculated m / z values ​​are 393.20 & 395.20, and the measured values ​​are 393.30 & 395.32.

[0197]

[0198] Synthesis of decyl(8-((8-(didecylamino)-8-oxooctyl)(5-hydroxypentyl)amino)octyl) carbonate (compound I-1)

[0199] DIPEA (4 equivalents, 0.39 g, 3.05 mmol) and N,N-didecyl-8-((5-hydroxypentyl)amino)octylamide (1.0 equivalent, 0.4 g, 0.76 mmol) were added to a solution of 8-bromooctyldecyl carbonate (1.2 equivalents, 0.36 g, 0.91 mmol) in acetonitrile (5.5 mL / mmol). The reaction was carried out in a sealed tube at 80 °C for 24 h. The reaction mixture was then cooled to room temperature and concentrated under reduced pressure. The crude product was purified by column chromatography (Hex / EtOAc containing 1% Et3N, 95:5 to 0:100). The product was obtained as a colorless oil (0.275 g, 0.328 mmol, 33%).

[0200] 1 H NMR (400 MHz, CDCl3) δ 4.11 (t, J = 6.7 Hz, 4H), 3.64 (t, J = 6.5Hz, 2H), 3.38 – 3.23 (m, 2H), 3.22 – 3.07 (m, 2H), 2.38 (m, 6H), 2.30 – 2.11(m, 2H), 1.84 – 1.07 (m, 77H), 0.98 – 0.81 (m, 9H), ESI-MS: C 52 H 104 N₂O₅ [M+H] + The calculated m / z value is 837.79, and the measured value is 837.49.

[0201]

[0202] Synthesis of decyl(8-((8-(didecylamino)-8-oxooctyl)(methyl)amino)octyl) carbonate (compound I-2)

[0203] Compound I-2 was prepared in a similar manner to compound I-1 from 8-bromooctyldecyl carbonate (0.417 g, 1.06 mmol), N,N-decyl-8-(methylamino)octylamide (0.4 g, 0.88 mmol), and DIPEA (0.46 g, 3.53 mmol). The product was obtained as a colorless oil (0.262 g, 0.342 mmol, 34%).

[0204] 1 H NMR (400 MHz, CDCl3) δ 4.11 (t, J = 6.7 Hz, 4H), 3.33 – 3.23 (m,2H), 3.23 – 3.14 (m, 2H), 2.27 (m, 6H), 2.19 (s, 3H), 1.73 – 1.58 (m, 8H), 1.58 – 1.39 (m, 9H), 1.28 (d, J = 8.7 Hz, 57H), 0.88 (t, J = 6.6 Hz, 9H), ESI-MS: C 48 H 96 N₂O₄ [M+H] + The calculated m / z value is 765.75, and the measured value is 765.40.

[0205] Synthesis example 2

[0206] Synthetic routes of compounds I-3 and I-4

[0207] Synthesis of 4-nitrophenylpentadecan-8-yl carbonate

[0208] 4-Nitrophenylpentadecan-8-yl carbonate was prepared from pentadecane-8-ol (2 g, 8.75 mmol), p-nitrobenzene chloroformate (2.12 g, 10.50 mmol), and pyridine (0.98 g, 11.38 mmol) according to the general procedure of Synthetic Example 1. The product was obtained as a colorless oil (2.75 g, 6.99 mmol, 80%). ESI-MS: C 22 H 35 NO5 [M+H] + The calculated m / z value is 394.53, and the measured value is 394.90.

[0209]

[0210] Synthesis of 8-bromooctylpentadecan-8-yl carbonate

[0211] Following the general procedure of Synthetic Example 1, 8-bromooctylpentadecan-8-yl carbonate was prepared from 4-nitrophenylpentadecan-8-yl carbonate (2.5 g, 6.35 mmol), 8-bromo-1-octanol (5.31 g, 25.41 mmol), DMAP (0.155 g, 1.27 mmol), and pyridine (0.98 g, 8.25 mmol). The product was obtained as a colorless solid (1.62 g, 3.49 mmol, 55%).

[0212] 1 H NMR (400 MHz, CDCl3) δ 4.75–4.61 (m, 1H), 4.11 (t, J = 6.7 Hz, 2H), 3.40 (t, J = 6.8 Hz, 2H), 1.85 (m, 2H), 1.67 (m, 2H), 1.55 (m, 2H), 1.47-1.20(m, 30H), 0.92-0.83 (m, 6H), ESI-MS: C 24 H 47 BrO3 [M+Na] + The calculated m / z values ​​are 485.26 & 487.26, and the measured values ​​are 485.03 & 487.49.

[0213]

[0214] Synthesis of 8-((8-(decylamino)-8-oxooctyl)(5-hydroxypentyl)amino)octylpentadecan-8-yl carbonate (compound I-3)

[0215] Compound I-3 was prepared from 8-bromooctylpentadecan-8-yl carbonate (0.423 g, 0.914 mmol), N,N-decyl-8-((5-hydroxypentyl)amino)octamide (0.4 g, 0.76 mmol), and DIPEA (0.39 g, 3.05 mmol) according to the general procedure of Synthetic Example 1. The product was obtained as a colorless oil (0.274 g, 0.302 mmol, 35%).

[0216] 1H NMR (400 MHz, CDCl3) δ 4.76 - 4.63 (m, 1H), 4.11 (t, J = 6.7 Hz, 2H), 3.64 (t, J = 6.5 Hz, 2H), 3.32 – 3.23 (m, 2H), 3.22 – 3.14 (m, 2H), 2.38(m, 6H), 2.32 – 2.21 (m, 2H), 1.86 – 1.14 (m, 83H), 0.99 – 0.76 (m, 12H),ESI-MS:C 57 H 114 N₂O₅ [M+H] + The calculated m / z value is 907.88, and the measured value is 907.95.

[0217]

[0218] Synthesis of 8-((8-(decylamino)-8-oxooctyl)(methyl)amino)octylpentadecan-8-yl carbonate (compound I-4)

[0219] Compound I-4 was prepared from 8-bromooctylpentadecan-8-yl carbonate (0.491 g, 1.06 mmol), N,N-decyl-8-(methylamino)octamide (0.4 g, 0.88 mmol), and DIPEA (0.46 g, 3.53 mmol) according to the general procedure of Synthetic Example 1. The product was obtained as a colorless oil (0.253 g, 0.302 mmol, 32.6%).

[0220] 1 H NMR (400 MHz, CDCl3) δ 4.68 (p, J = 7.1, 6.3 Hz, 1H), 4.11 (t, J =6.7 Hz, 2H), 3.32 – 3.23 (m, 2H), 3.23 – 3.13 (m, 2H), 2.27 (m, 6H), 2.19 (s,3H), 1.66 (m, 7H), 1.61 – 1.40 (m, 12H), 1.40 – 1.08 (m, 58H), 1.03 – 0.57(m, 12H), ESI-MS: C 53 H 106 N₂O₄ [M+H] + The calculated value of m / z is 835.83, and the measured value is 835.72.

[0221] Synthesis example 3

[0222] 10-(N-decyl-3-(dimethylamino)propionylamino)-19-(didecylamino)-19-oxononadecanoic acid 2-butyloctyl ester (compound I-8)

[0223] Synthesis of 19-(dicepanoylamino)-10,19-dioxononadecanoic acid

[0224] HATU (7.6 mmol, 2.9 g) was added to a mixture of 10-oxononadecanedioic acid (5.8 mmol, 2.0 g), didecylamine (5.8 mmol, 1.7 g), and DIPEA (17.5 mmol, 3.05 mL) in DCM (29 mL), and the reaction mixture was stirred at room temperature for 50 minutes. The reaction mixture was concentrated, and the crude product was partitioned between EtOAc and 1 M HCl. The organic layers were separated, dried over Na2SO4, and concentrated. The crude product was purified by automated rapid chromatography (DCM containing 1% to 10% MeOH). The separated product was ground in hexane and filtered. The filtrate was further purified by automated rapid chromatography (hexane containing 10% to 70% EtOAc) to give the desired product (500 mg, 14%).

[0225]

[0226] Synthesis of 2-butyloctyl 19-(decylamino)-10,19-dioxononadecanoic acid

[0227] A mixture of 19-(decylamino)-10,19-dioxonadecanoic acid (0.8 mmol, 0.5 g), 2-butyloctyl-1-ol (1.4 mmol, 262 mg), 4-dimethylaminopyridine (31.2 mmol, 147 mg), and DCC (1.6 mmol, 332 mg) in DCM (8 mL) was stirred overnight at room temperature. The reaction was concentrated, and the crude product was suspended in hexane and filtered. The filtrate was purified by automated rapid chromatography (hexane containing 1% to 15% EtOAc) to give the desired product (396 mg, 62%).

[0228]

[0229] Synthesis of 2-butyloctyl 10-(decylamino)-19-(dicepylamino)-19-oxononadecanoic acid

[0230] Sodium triacetoxyborohydride (1.0 mmol, 215 mg) was added to a mixture of 2-butyloctyl 19-(decylamino)-10,19-dioxonadecanoate (0.51 mmol, 400 mg), decylamine (0.76 mmol, 119 mg), and acetic acid (0.76 mmol, 0.043 mL) in dichloroethane (3 mL). The reaction mixture was stirred at room temperature for 22 hours. An additional 1.0 mmol, 215 mg of sodium triacetoxyborohydride was added, and the reaction mixture was stirred again for 24 hours. The reaction mixture was concentrated, and the crude product was partitioned between EtOAc and saturated NaHCO3. The organic layer was separated, dried over Na2SO4, and concentrated. Purification was performed by automated rapid chromatography (hexane containing 5% to 100% EtOAc) to give the desired product (420 mg, 89%).

[0231]

[0232] Synthesis of 2-butyloctyl 10-(N-decyl-3-(dimethylamino)propionylamino)-19-(didecylamino)-19-oxononadecanoic acid (compound I-8)

[0233] HATU (0.16 mmol, 61 mg) was added to a mixture of 2-butyloctyl 10-(decylamino)-19-(decylamino)-19-oxononadecanoate (0.11 mmol, 100 mg), 3-(dimethylamino)propionate (0.14 mmol, 21 mg), and DIPEA (0.43 mmol, 0.075 mL) in DCM (1.1 mL). The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was partitioned between EtOAc and saturated NaHCO3. The organic layer was separated, dried over Na2SO4, and concentrated. Purification by automated rapid chromatography (hexane containing 5% to 65% EtOAc, doped with 1% Et3N) gave compound I-8 (69 mg, 62%).

[0234] 1¹H NMR (400 MHz, CDCl₃) δ 3.96 (m, 2H), 3.65 – 3.55 (m, 1H), 3.32 – 3.24 (m, 2H), 3.23 – 3.15 (m, 2H), 3.07 – 2.99 (m, 2H), 2.65 (m, 2H), 2.53 – 2.43 (m, 2H), 2.33 – 2.21 (m, 10H), 1.71 – 1.37 (m, 19H), 1.28 (m, 78H), 0.93– 0.83 (m, 15H). Chemical formula C 66 H 131 N3O4 m / z Calculated value = 1030.0. Measured value [M+H] + = 1031.3

[0235] Synthesis example 4

[0236] 8-((8-(decylamino)-8-oxooctyl)(5-hydroxypentyl)amino)octyl(2-hexyldecyl)carbonate (compound I-19)

[0237] Synthesis of 1H-imidazolium-1-carboxylic acid 2-hexyldecyl ester

[0238] A solution of commercially available 2-hexyldec-1-ol (7.9 mmol, 2.3 mL), CDI (7.9 mmol, 1.3 g), and potassium hydroxide (20 mg) in DCM (15 mL) was stirred at 60 °C for 6 hours in a sealed flask. The reaction mixture was washed with water, dried over Na₂SO₄, and concentrated. The crude product was purified by rapid chromatography (hexane containing 0% to 20% EtOAc) to give 1H-imidazolium-1-carboxylic acid 2-hexyldecyl ester (1.73 g, 46%).

[0239] Synthesis of 8-bromooctyl(2-hexyldecyl) carbonate

[0240] A solution of 2-hexyldecyl 1H-imidazolium-1-carboxylic acid (5.1 mmol, 1.73 g), commercially available 8-bromooct-1-ol (5.1 mmol, 0.9 mL), and potassium hydroxide (10 mg) in DCM (15 mL) was stirred at 60 °C for 6 hours in a sealed flask. The crude product was purified by rapid chromatography (hexane containing 0% to 20% EtOAc) to give 8-bromooctyl(2-hexyldecyl) carbonate (1.16 g, 48%).

[0241] Synthesis of 8-((8-(decylamino)-8-oxooctyl)(5-hydroxypentyl)amino)octyl(2-hexyldecyl)carbonate

[0242] A mixture of 8-bromooctyl(2-hexyldecyl) carbonate (0.71 mmol, 339 mg), N,N-decyl-8-((5-hydroxypentyl)amino)octamide (0.65 mmol, 339 mg), KI (1.9 mmol, 321 mg), and DIEA (2.6 mmol, 0.45 mL) in acetonitrile (2 mL) was heated by microwave at 140 °C for 30 min. The reaction mixture was concentrated, and the resulting crude product was suspended in hexane and filtered. The filtrate was purified by rapid chromatography to give the desired product (109 mg, 18%).

[0243] 1 H NMR (400 MHz, MeOD) δ 4.15 (t, J = 6.5 Hz, 2H), 4.07 (d, J = 5.7Hz, 2H), 3.59 (t, J = 6.6 Hz, 2H), 2.57 – 2.50 (m, 6H), 2.39 (t, 3H), 1.72 –1.49 (m, 18H), 1.47 – 1.22 (m, 53H), 0.98 – 0.90 (m, 12H). ESI-MS:C 58 H 116 N2O5 m / z Calculated value = 920.89, measured value [M+H] + = 922.17.

[0244] Synthesis example 5

[0245] 7-((8-(decylamino)-8-oxooctyl)(5-hydroxypentyl)amino)heptylheptadecane-9-yl carbonate (compound I-18)

[0246] Synthesis of 1H-imidazol-1-carboxylic acid heptadecan-9-yl ester

[0247] Heptadecanyl ester of 1H-imidazolium-1-carboxylic acid was prepared from commercially available heptadecanyl-9-ol according to the procedure outlined in the literature. Yield (1.85 g, 70%).

[0248] Synthesis of 7-bromoheptylheptadecane-9-yl carbonate

[0249] 7-Bromoheptylheptadecyl carbonate was prepared from commercially available 7-bromoheptylheptadecyl-9-yl carbonate following the procedure outlined in the section on 8-bromooctyl(2-hexyldecyl) carbonate. Yield (1.91 g, 76%).

[0250] Synthesis of compound I-18

[0251] Compound I-18 was prepared according to the detailed procedure described in this article, based on the reaction scheme described above. Yield (119 mg, 23%).

[0252] 1 H NMR (400 MHz, MeOD) δ 4.78 – 4.60 (m, 1H), 4.13 (t, J = 6.5 Hz, 2H), 3.57 (t, J = 6.6 Hz, 2H), 2.54 – 2.45 (m, 6H), 2.37 (t, J = 7.5 Hz, 2H), 1.72 – 1.46 (m, 9H), 1.44 – 1.30 (m, 72H), 0.96 – 0.88 (m, 12H). ESI-MS:C 58 H 116 N2O5 m / z Calculated value = 920.89, measured value [M+H] + = 922.18.

[0253] Synthesis example 6

[0254] 9-((8-(decylamino)-8-oxooctyl)(5-hydroxypentyl)amino)nonyltridecane-7-yl carbonate (compound I-17)

[0255] Synthesis of 1H-imidazolium-1-carboxylic acid tridecane-7-yl ester

[0256] Following the detailed procedure described in this article, 1H-imidazol-1-carboxylic acid tridecane-7-yl ester was prepared from commercially available tridecane-7-ol. Yield (559 mg, 76%).

[0257] Synthesis of 9-bromononyltridecane-7-yl carbonate

[0258] The desired product was prepared from commercially available 9-bromonon-1-ol and 1H-imidazol-1-carboxylic acid tridecane-7-yl ester, following the detailed procedure described in this article. Yield (716 mg, 84%).

[0259] Synthesis of compound I-17

[0260] Compound I-17 was prepared according to the detailed procedure described in this article and the reaction scheme described above. Yield (156 mg, 28%).

[0261] 1 H NMR (400 MHz, MeOD) δ 4.76 – 4.65 (m, 1H), 4.15 (t, J = 6.5 Hz, 2H), 3.59 (t, J = 6.6 Hz, 2H), 2.55 – 2.47 (m, 7H), 2.39 (t, J = 7.5 Hz, 2H), 1.77 – 1.22 (m, 82H), 0.99 – 0.91 (m, 12H). ESI-MS:C 56 H 112 N2O5 m / z Calculated value = 892.86, measured value [M+H] + = 894.15.

[0262] Synthesis Example 7

[0263] 5-((8-(decylamino)-8-oxooctyl)(5-hydroxypentyl)amino)pentylnon-5-yl carbonate (compound I-16)

[0264] Synthesis of 1H-imidazolium-1-carboxylic acid non-5-yl ester

[0265] Following the procedure outlined in this article, non-5-yl 1H-imidazolium-1-carboxylic acid was prepared from commercially available non-5-ol. Yield (256 mg, 31%).

[0266] Synthesis of 5-bromopentylnon-5-yl carbonate

[0267] Following the procedure outlined herein, 5-bromopentylnon-5-yl carbonate was prepared from commercially available 5-bromopent-1-ol and non-5-yl 1H-imidazolium-1-carboxylate. Yield (105 mg, 29%). Synthesis of compound I-16 Compound I-16 was prepared according to the detailed procedure described in this article and the reaction scheme described above. Yield (105 mg, 33%).

[0268] 1H NMR (400 MHz, MeOD) δ 4.71 – 4.62 (m, 1H), 4.12 (t, J = 6.5 Hz, 2H), 3.55 (t, J = 6.6 Hz, 2H), 2.52 – 2.42 (m, 6H), 2.35 (t, J = 7.5 Hz, 2H), 1.77 – 1.22 (m, 57H), 0.97 – 0.83 (m, 12H). ESI-MS:C 48 H 96 N2O5 m / z Calculated value = 780.73, measured value [M+H] + = 781.89.

[0269] Synthesis example 8

[0270] 5-((8-(decylamino)-8-oxooctyl)(5-hydroxypentyl)amino)pentylicoseno-11-yl carbonate (compound I-15)

[0271] Synthesis of 1H-imidazolium-1-carboxylic acid dodecane-11-yl ester

[0272] Following the procedure outlined herein, 1H-imidazol-1-carboxylic acid dodecano-11-yl ester was prepared from commercially available dodecano-11-ol. Yield (357 mg, 55%).

[0273] Synthesis of 5-bromopentyl-11-yl carbonate

[0274] 5-Bromopentyl-1-alkyl carbonate was prepared from commercially available 5-bromopentan-1-ol and 1H-imidazol-1-carboxylic acid dodecyl-11-yl ester, following the procedure outlined herein. Yield (198 mg, 45%).

[0275] Synthesis of compound I-15

[0276] Compound I-15 was prepared according to the detailed procedure described in this article and the reaction scheme described above. Yield (63.5 mg, 19%).

[0277] 1 H NMR (400 MHz, MeOD) δ 4.73 – 4.62 (m, 1H), 4.13 (t, J = 6.4 Hz, 2H), 3.55 (t, J= 6.6 Hz, 2H), 2.53 – 2.41 (m, 6H), 2.35 (t, J = 7.5 Hz, 2H), 1.77 – 1.18 (m, 100H), 0.97 – 0.83 (m, 12H). ESI-MS:C 60 H 120 N2O5 m / z Calculated value = 948.92, measured value [M+H] + = 950.22.

[0278] Synthesis example 9

[0279] 9-((8-(decylamino)-8-oxooctyl)(5-hydroxypentyl)amino)nonylnon-5-yl carbonate (compound I-14)

[0280] Synthesis of 1H-imidazolium-1-carboxylic acid non-5-yl ester

[0281] Following the procedure outlined in this article, non-5-yl 1H-imidazolium-1-carboxylic acid was prepared from commercially available non-5-ol. Yield (268 mg, 73%).

[0282] Synthesis of 9-bromononylnon-5-yl carbonate

[0283] 9-Bromononylnon-5-yl carbonate was prepared from commercially available 9-bromonon-1-ol and 1H-imidazolium-1-carboxylic acid non-5-yl ester, following the procedure outlined herein. Yield (240 mg, 55%).

[0284] Synthesis of compound I-14

[0285] Compound I-14 was prepared according to the detailed procedure described in this article, based on the reaction scheme described above. Yield (89.3 mg, 19%).

[0286] 1 H NMR (400 MHz, MeOD) δ 4.73 – 4.62 (m, 1H), 4.11 (t, J = 6.5 Hz, 2H), 3.55 (t, J = 6.6 Hz, 2H), 2.55 – 2.42 (m, 6H), 2.35 (t, J= 7.5 Hz, 2H), 1.72 – 1.19 (m, 68H), 0.97 – 0.84 (m, 12H). ESI-MS:C 52 H 104 N2O5 m / z Calculated value = 836.79, measured value [M+H] + = 838.07.

[0287] Synthesis example 10

[0288] 9-((8-(decylamino)-8-oxooctyl)(5-hydroxypentyl)amino)nonyldocoane-11-yl carbonate (compound I-13)

[0289] Synthesis of 9-bromononyldocoane-11-yl carbonate

[0290] 9-Bromononyl-1-ol and 1H-imidazol-1-carboxylic acid dodecyl-11-yl carbonate were prepared from commercially available 9-bromononyl-1-ol and 1H-imidazol-1-carboxylic acid dodecyl-11-yl carbonate according to the procedure outlined in this article. Yield (269 mg, 40%).

[0291] Synthesis of compound I-13

[0292] Compound I-13 was prepared according to the detailed procedure described in this article, based on the reaction scheme described above. Yield (59.7 mg, 27%).

[0293] 1 H NMR (400 MHz, MeOD) δ 4.73 – 4.62 (m, 1H), 4.12 (t, J = 6.4 Hz, 2H), 3.55 (t, J = 6.6 Hz, 2H), 2.54 – 2.43 (m, 6H), 2.35 (t, J = 7.5 Hz, 2H), 1.71 – 1.21 (m, 94H), 0.95 – 0.85 (m, 12H). ESI-MS:C 64 H 128 N2O5 m / z Calculated value = 1004.98, measured value [M+H] + = 1006.20.

[0294] Synthesis example 11

[0295] 2-Butyloctyl(8-((8-(decylamino)-8-oxooctyl)(5-hydroxypentyl)amino)octyl)carbonate (compound I-12)

[0296] Synthesis of 1H-imidazolium-1-carboxylic acid 2-butyloctyl ester

[0297] Following the procedure outlined in this article, 1H-imidazol-1-carboxylic acid 2-butyloctyl ester was prepared from commercially available 2-butyloct-1-ol. Yield (743 mg, 43%).

[0298] Synthesis of 8-bromooctyl(2-butyloctyl) carbonate

[0299] 8-Bromooctyl (2-Butyloctyl) carbonate was prepared from commercially available 8-bromooct-1-ol and 2-butyloctyl 1H-imidazolium-1-carboxylate, following the procedure outlined herein. Yield (488 mg, 44%).

[0300] Synthesis of compound I-12

[0301] Compound I-12 was prepared according to the detailed procedure described in this article and the reaction scheme described above. Yield (72.7 mg, 25%).

[0302] 1 H NMR (400 MHz, MeOD) δ 4.11 (t, J = 6.5 Hz, 2H), 4.03 (d, J = 5.7Hz, 2H), 3.55 (t, J = 6.6 Hz, 2H), 2.53 – 2.43 (m, 6H), 2.35 (t, J = 7.5 Hz, 2H), 1.72 – 1.23 (m, 82H), 0.96 – 0.85 (m, 12H). ESI-MS:C 54 H 108 N2O5 m / z Calculated value = 864.83, measured value [M+H] + = 866.11.

[0303] Synthesis example 12

[0304] 8-((8-(decylamino)-8-oxooctyl)(5-hydroxypentyl)amino)octyl(2-ethylhexyl)carbonate (compound I-11)

[0305] Synthesis of 1H-imidazol-1-carboxylic acid 2-ethylhexyl ester

[0306] Following the procedure outlined in this article, 2-ethylhexyl 1H-imidazol-1-carboxylic acid was prepared from commercially available 2-ethylhexyl-1-ol. Yield (802 mg, 58%).

[0307] Synthesis of 8-bromooctyl(2-ethylhexyl) carbonate

[0308] 8-Bromooctyl (2-ethylhexyl) carbonate was prepared from commercially available 8-bromooct-1-ol and 2-ethylhexyl 1H-imidazol-1-carboxylic acid, following the procedure outlined herein. Yield (718 mg, 55%).

[0309] Synthesis of compound I-11

[0310] Compound I-11 was prepared according to the detailed procedure described in this article and the reaction scheme described above. Yield (79.7 mg, 24%).

[0311] 1 H NMR (400 MHz, MeOD) δ 4.11 (t, J = 6.6 Hz, 2H), 4.04 (d, J = 5.7Hz, 2H), 3.55 (t, J = 6.6 Hz, 2H), 2.52 – 2.43 (m, 7H), 2.35 (t, J = 7.5 Hz, 2H), 1.71 – 1.22 (m, 68H), 0.96 – 0.84 (m, 12H). ESI-MS:C 50 H 100 N2O5 m / z Calculated value = 808.76, measured value [M+H] + = 809.99.

[0312] Synthesis example 13

[0313] 7-((8-(dioctylamino)-8-oxooctyl)(5-hydroxypentyl)amino)heptylheptadecane-9-yl carbonate (compound I-23)

[0314] Synthesis of 8-bromo-N,N-dioctyloctamide

[0315] 8-Bromo-N,N-Dioctyloctamide was prepared from commercially available dioctylamine and 8-bromooctyl chloride according to the procedure outlined in this article. Yield (642 mg, 35%).

[0316] Synthesis of 8-((5-hydroxypentyl)amino)-N,N-dioctyloctamide

[0317] Following the procedure outlined herein, 8-((5-hydroxypentyl)amino)-N,N-dioctyloctamide was prepared from commercially available 5-aminopentan-1-ol and 8-bromo-N,N-dioctyloctamide. Yield (294 mg, 88%).

[0318] Synthesis of compound I-23

[0319] Compound I-23 was prepared according to the detailed procedure described in this article and the reaction scheme described above. Yield (79.7 mg, 24%).

[0320] 1 H NMR (400 MHz, MeOD) δ 4.72 – 4.62 (m, 1H), 4.11 (t, J = 6.5 Hz, 2H), 3.55 (t, J = 6.5 Hz, 2H), 2.53 – 2.42 (m, 6H), 2.35 (t, J = 7.5 Hz, 2H), 1.72 – 1.21 (m, 76H), 0.95 – 0.85 (m, 12H). ESI-MS:C 54 H 108 N2O5 m / z Calculated value = 864.83, measured value [M+H] + = 866.09.

[0321] Synthesis example 14

[0322] 7-((8-(bis(dodecylamino)-8-oxooctyl)(5-hydroxypentyl)amino)heptylheptadecane-9-yl carbonate (compound I-22)

[0323] Synthesis of 8-bromo-N,N-bisdodecyloctamide

[0324] 8-Bromo-N,N-bisdodecyloctylamide was prepared from commercially available dodecylamine and 8-bromooctyl chloride according to the procedure outlined in this article. Yield (663 mg, 29%).

[0325] Synthesis of N,N-bis(dodecyl-8-((5-hydroxypentyl)amino)octamide

[0326] Following the procedure outlined herein, N,N-bisdodecyl-8-((5-hydroxypentyl)amino)octamide was prepared from commercially available 5-aminopentan-1-ol and 8-bromo-N,N-bisdodecyloctamide. Yield (220 mg, 64%).

[0327] Synthesis of compound I-22

[0328] Compound I-22 was prepared according to the detailed procedure described in this article and the reaction scheme described above. Yield (79.1 mg, 19%).

[0329] 1 H NMR (400 MHz, MeOD) δ 4.73 – 4.63 (m, 1H), 4.11 (t, J = 6.5 Hz, 2H), 3.55 (t, J = 6.5 Hz, 2H), 2.58 – 2.45 (m, 6H), 2.35 (t, J = 7.5 Hz, 2H), 1.71 – 1.20 (m, 101H), 0.96 – 0.85 (m, 12H). ESI-MS:C 62 H 124 N2O5 m / z Calculated value = 976.95, measured value [M+H] + = 978.20.

[0330] Synthesis Example 15

[0331] 7-((6-(bis(dodecylamino)-6-oxohexyl)(5-hydroxypentyl)amino)heptylheptadecane-9-yl carbonate (compound I-21)

[0332] Synthesis of 6-bromohexanoyl chloride

[0333] 6-Bromhexanoyl chloride was prepared from commercially available 6-bromohexanoic acid according to the procedure outlined in this article.

[0334] Synthesis of 6-bromo-N,N-bisdodecylhexamide

[0335] 6-Bromo-N,N-bisdodecylhexanoamide was prepared from commercially available dodecylamine and 6-bromohexanoyl chloride according to the procedure outlined herein. Yield (1.08 g, 44%).

[0336] Synthesis of N,N-bis(dodecyl-6-((5-hydroxypentyl)amino)hexamamide

[0337] Following the procedure outlined herein, N,N-bisdodecyl-6-((5-hydroxypentyl)amino)hexamamide was prepared from commercially available 5-aminopentan-1-ol and 6-bromo-N,N-bisdodecylhexamamide. Yield (246 mg, 47%).

[0338] Preparation of compound I-21

[0339] Compound I-21 was prepared according to the detailed procedure described in this article and the reaction scheme described above. Yield (53.8 mg, 14%).

[0340] 1 H NMR (400 MHz, MeOD) δ 4.73 – 4.62 (m, 1H), 4.11 (t, J = 6.5 Hz, 3H), 3.55 (t, J = 6.5 Hz, 2H), 2.57 – 2.44 (m, 7H), 2.36 (t, J = 7.4 Hz, 3H), 1.71 – 1.19 (m, 106H), 0.96 – 0.85 (m, 12H). ESI-MS:C 60 H 120 N2O5 m / z Calculated value = 948.92, measured value [M+H] + = 950.15.

[0341] Synthesis example 16

[0342] 7-((10-(dioctylamino)-10-oxodecyl)(5-hydroxypentyl)amino)heptylheptadecane-9-yl carbonate (compound I-20)

[0343] Synthesis of 10-bromodecanoyl chloride

[0344] 10-Bromodecanoyl chloride was prepared from commercially available 10-bromodecanoic acid according to the procedure outlined in this article.

[0345] Synthesis of 10-bromo-N,N-dioctyldecanoamide

[0346] 10-Bromo-N,N-Dioctyldecanoamide was prepared from commercially available dioctylamine and 10-bromodecanoyl chloride according to the procedure outlined herein. Yield (1.01 g, 58%).

[0347] Synthesis of 10-((5-hydroxypentyl)amino)-N,N-dioctyldecanoamide

[0348] Following the procedure outlined herein, 10-((5-hydroxypentyl)amino)-N,N-dioctyldecanoamide was prepared from commercially available 5-aminopentan-1-ol and 10-bromo-N,N-dioctyldecanoamide. Yield (208 mg, 40%).

[0349] Synthesis of compound I-20

[0350] Compound I-20 was prepared according to the detailed procedure described in this article, based on the reaction scheme described above. Yield (21 mg, 5.6%).

[0351] 1 H NMR (400 MHz, CDCl3) δ 4.74 – 4.61 (m, 1H), 4.11 (t, J = 6.8 Hz, 2H), 3.64 (t, J = 6.5 Hz, 2H), 3.33 – 3.13 (m, 4H), 2.46 – 2.32 (m, 6H), 2.30 – 2.22 (m, 2H), 1.72 – 1.17 (m, 79H), 0.93 – 0.79 (m, 12H). ESI-MS:C 56 H 112 N2O5 m / z Calculated value = 892.86, measured value [M+H] + = 894.13.

[0352] Synthesis Example 17

[0353] 10-(N-decyl-4-(dimethylamino)butyrylamino)-19-(dicepylamino)-19-oxononadecanoic acid 2-butyloctyl ester (compound I-25)

[0354] Synthesis of compound I-25

[0355] Compound I-25 was prepared from 2-butyloctyl 10-(decylamino)-19-(dicepylamino)-19-oxononadecanoate and 4-(dimethylamino)butyric acid, according to the procedure outlined in Synthetic Example 3. Yield (41 mg, 37%).

[0356] 1 H NMR (400 MHz, CDCl3) δ 3.99 (dd, J= 5.8, 1.4 Hz, 2H), 3.70 - 3.60(m, 1H), 3.34 - 3.26 (m, 2H), 3.25 - 3.17 (m, 2H), 3.10 - 3.01 (m, 2H), 2.39- 2.27 (m, 8H), 2.24 (s, 6H), 1.91 – 1.77 (m, 2H), 1.70 – 1.06 (m, 99H), 0.95- 0.86 (m, 15H). ESI-MS:C 67 H 133 N3O4 m / z Calculated value = 1044.0, Measured value [M+H] + = 1045.3.

[0357] Synthesis Example 18

[0358] 19-(decylamino)-10-(N-octyl-4-(pyrrolidone-1-yl)butyrylamino)-19-oxononadecanoic acid 2-butyloctyl ester (compound I-24)

[0359] Synthesis of 2-butyloctyl 19-(decylamino)-10-(octylamino)-19-oxononadecanoic acid

[0360] 19-(dicepamino)-10-(octylamino)-19-oxononadecanoic acid 2-butyloctyl ester was prepared from 19-(dicepamino)-10,19-dioxonadecanoic acid 2-butyloctyl ester and oct-1-amine, according to the procedure outlined in Synthetic Example 3. Yield (440 mg, 85%).

[0361] Synthesis of 2-butyloctyl 10-(4-chloro-N-octylbutyrylamino)-19-(didecylamino)-19-oxononadecanoic acid

[0362] A mixture of 19-(decylamino)-10-(octylamino)-19-oxononadecanoic acid 2-butyloctyl ester (0.18 mmol, 160 mg), triethylamine (0.18 mmol, 0.050 mL), and 4-chlorobutyryl chloride (0.18 mmol, 43 mg) in dichloromethane (1.8 mL) was stirred at room temperature for 1 hour. The reaction mixture was concentrated, and the resulting crude product was purified (in hexane containing 0% to 20% EtOAc) to give 10-(4-chloro-N-octylbutyrylamino)-19-(decylamino)-19-oxononadecanoic acid 2-butyloctyl ester (130 mg, 73%).

[0363]

[0364] Synthesis of compound I-24

[0365] A mixture of 2-butyloctyl 10-(4-chloro-N-octylbutyrylamino)-19-(decylamino)-19-oxononadecanoate (0.13 mmol, 130 mg), pyrrolidine (0.77 mmol, 0.065 mL), DIEA (0.39 mmol, 0.067 mL), and potassium iodide (0.39 mmol, 65 mg) in acetonitrile (0.4 mL) was heated by microwave at 140 °C for 40 min. The reaction mixture was concentrated, and the resulting crude product was suspended in EtOAc:hexane:Et3N (5:95:1) and filtered. The filtrate was purified by rapid chromatography (hexane containing 5% to 65% EtOAc, doped with 1% Et3N) to give compound I-24 (106 mg, 79%).

[0366] 1 H NMR (400 MHz, CDCl3) δ 3.96 (dd, J = 5.8, 0.9 Hz, 2H), 3.62 - 3.45(m, 1H), 3.31 - 3.23 (m, 2H), 3.23 - 3.14 (m, 2H), 3.12 - 2.66 (m, 6H), 2.49- 2.35 (m, 2H), 2.33 - 2.21 (m, 4H), 2.15 - 1.91 (m, 6H), 1.75 - 1.07 (m, 94H), 0.93 - 0.83 (m, 15H). ESI-MS: m / z calcd for C 67 H 131 N3O4 = 1042.0, measured value [M+H] + = 1043.3.

[0367] The various embodiments described above can be combined to provide other embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referenced in this specification, including but not limited to U.S. Provisional Application Serial No. 63 / 508,772, filed June 16, 2023, are incorporated herein by reference in their entirety. If necessary, aspects of the embodiments can be modified to provide other embodiments by employing the concepts of various patents, applications and publications.

[0368] These and other changes to the embodiments can be considered in light of the detailed description above. Generally, the terminology used in the appended claims should not be construed as limiting the claims to the specific embodiments disclosed in this specification and claims, but rather as encompassing all possible embodiments and the full scope of equivalents conferred by such claims. Therefore, the claims are not limited by this disclosure.

Claims

1. A compound having the structure of Formula (I): or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein: (I) wherein each alkyl, alkenyl, alkynyl, alkylene, cycloalkyl, heterocyclyl, and heteroaryl is optionally substituted with one or more fluorines. G 1 is N or CH; G is -N= when G 1 is N, G 2 is a direct bond, or G 1 is -CH when G 2 is -NR 1a -; R 1a is C4-C 12 alkyl, said C4-C 12 alkyl is optionally substituted with one or more substituents selected from the group consisting of oxo, -OH, and NH2; R 1 is C1-C8alkyl optionally substituted with one or more substituents selected from oxo, -OH, -N(R 1b )R 1c , cycloalkyl, or heteroaryl; R 1b and R 1c each independently is hydrogen or C1-C4alkyl; or R 1b and R 1c together with the nitrogen to which they are attached to form a heterocyclyl; R 2 is -OC(=O)OR 2a , -OC(=O)R 2b or -C(=O)OR 2c ; R 2a is C4-C 24 alkyl, C4-C 24 alkenyl or C4-C 24 alkynyl; R 2b and R 2c having the structure: ； R 2d and R 2e each independently is C4-C 12 alkyl, C4-C 12 alkenyl or C4-C 12 alkynyl; R 3 is -C(=O)N(R 3a )R 3b or -NR 3a -C(=O)R 3b ; R 3a and R 3b each independently is C6-C 24 alkyl, C6-C 24 alkenyl or C6-C 24 alkynyl; and L 1 and L 2 each independently is C4-C 12 alkylene, 2. The compound of claim 1, wherein the compound has the following Formula (la): (Ia) or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

3. The compound of claim 1, wherein the compound has the following Formula (lb): (Ib) or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

33. The compound of any one of claims 1 to 32, wherein the compound has one of the following structures:

4. The compound of any one of claims 1 to 3, wherein R 1a is C8-C 12 alkyl optionally substituted with oxo.

5. The compound of any one of claims 1 to 4, wherein R 1a one of the following structures: or .

6. The compound of any one of claims 1 to 5, wherein R 1 optionally substituted with one or two substituents selected from -OH, oxo, fluoro, -N(CH3)2, and the following structures: 。 7. The compound of any one of claims 1 to 6, wherein R 1 is substituted with -OH.

8. The compound of any one of claims 1 to 5, wherein R 1 is -CH3.

9. The compound of any one of claims 1 to 8, wherein R 1 one of the following structures: ; ; ; ; or .

10. The compound of any one of claims 1 to 9, wherein R 2 is -OC(=0)OR 2a .

11. The compound of any one of claims 1 to 10, wherein R 2a is C4-C 24 alkyl.

12. The compound of any one of claims 1 to 11, wherein R 2a is unsubstituted C4-C 24 alkyl.

13. The compound of any one of claims 1 to 12, wherein R 2a is unbranched C4-C 24 alkyl.

14. The compound of any one of claims 1 to 12, wherein R 2a is branched C4-C 24 alkyl.

15. The compound of any one of claims 1 to 9, wherein R 2 is -OC(=0)R 2b .

16. The compound of any one of claims 1 to 9, wherein R 2 is -C(=0)OR 2c .

17. The compound of any one of claims 1 to 16, wherein R 2b or R 2c has the structure: 。 18. The compound of any one of claims 1 to 16, wherein R 2b or R 2c has the structure: 。 19. The compound of any one of claims 1 to 18, wherein R 2 one of the following structures: ; ; ; ; ; ; ; ; ; ; ; ; ; ; or .

20. The compound of any one of claims 1 to 19, wherein R 3 is -C(=0)N(R 3a )R 3b .

21. The compound of any one of claims 1 to 19, wherein R 3 is -NR 3a -C(=O)R 3b .

22. The compound of any one of claims 1 to 21, wherein R 3a and R 3b are each independently C6-C 24 alkyl.

23. The compound of any one of claims 1 to 22, wherein R 3a and R 3b each independently is C6-C 12 alkyl.

24. The compound of any one of claims 1 to 23, wherein R 3a and R 3b are each independently C8-C 10 alkyl.

25. The compound of any one of claims 1 to 23, wherein R 3a and R 3b are each C 12 alkyl.

26. The compound of any one of claims 1 to 23, wherein R 3a and R 3b are each C8alkyl.

27. The compound of any one of claims 1 to 23, wherein R 3a and R 3b are each C 10 alkyl.

28. The compound of any one of claims 1 to 27, wherein L 1 and L 2 each independently is C4-C 10 alkylene.

29. The compound of any one of claims 1 to 28, wherein L 1 and L 2 each independently is C4alkylene, C5alkylene, C6alkylene, C7alkylene, C8alkylene, or C9alkylene.

30. The compound of any one of claims 1 to 29, wherein L 1 and L 2 are the same.

31. The compound of any one of claims 1 to 30, wherein L 1 and L 2 is unsubstituted.

32. The compound of any one of claims 1 to 30, wherein L 1 , L 2 or both are substituted with one or more fluoro substituents. or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ; ; ; ; or 34. A lipid nanoparticle comprising the compound of any one of claims 1 to 33 and a therapeutic agent.

35. A composition comprising the compound of any one of claims 1 to 33 and a therapeutic agent.

36. The lipid nanoparticle or composition of any one of claims 34 or 35, further comprising one or more excipients selected from neutral lipids, sterols, and polymer-conjugated lipids.

37. The lipid nanoparticle or composition of claim 36, comprising one or more neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

38. The lipid nanoparticle or composition of claim 36 or 37, wherein the neutral lipid is DSPC.

39. The lipid nanoparticle or composition of any one of claims 36 to 38, wherein the molar ratio of the compound to the neutral lipid is about 2: 1 to about 8:

1.

40. The lipid nanoparticle or composition of any one of claims 36 to 39, wherein the sterol is cholesterol.

41. The lipid nanoparticle or composition of claim 40, wherein the molar ratio of the compound to cholesterol is 5: 1 to 1: 1 or 2: 1 to 1:

1.

42. The lipid nanoparticle or composition of any one of claims 36 to 41, wherein the polymer-conjugated lipid is a pegylated lipid.

43. The lipid nanoparticle or composition of claim 42, wherein the molar ratio of the compound to pegylated lipid is about 100: 1 to about 20: 1 or about 100: 1 to about 10:

1.

44. The lipid nanoparticle or composition of claim 43, wherein the pegylated lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, or PEG dialkoxylpropyl carbamate.

45. The lipid nanoparticle or composition of claim 43, wherein the pegylated lipid has the following Formula (II): (II) or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein: w has a value of 30 to 60. R 10 and R 11 each independently is a linear or branched alkyl, alkenyl, or alkinyl of 10 to 30 carbon atoms, wherein the alkyl, alkenyl, or alkinyl is optionally interrupted by one or more ester bonds; and 47. The lipid nanoparticle or composition of claim 46, wherein w is 40 to 50.

46. The lipid nanoparticle or composition of claim 45, wherein R 10 and R 11 each independently is a straight alkyl chain containing 12 to 16 carbon atoms. ​ 48. The lipid nanoparticle or composition of any one of claims 36-47, wherein the therapeutic agent comprises a nucleic acid.

49. The lipid nanoparticle or composition of claim 48, wherein the nucleic acid is selected from the group consisting of an antisense RNA and a messenger RNA.

50. A method for inducing expression of a desired protein in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of the lipid nanoparticle or pharmaceutical composition of any one of claims 34-49, wherein the therapeutic agent comprises a nucleic acid.

51. The method of claim 50, wherein the nucleic acid is an antisense RNA, an mRNA, or a Cas9 mRNA.

52. The method of claim 51, wherein the mRNA encodes an antigen.

53. The method of any one of claims 50-52, wherein the method is for vaccination against a viral pathogen.

54. The method of claim 51, wherein the nucleic acid is a Cas9 mRNA, and the method is for editing a gene of interest.

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