Lipids for use in lipid nanoparticle formulations

Abstract

Structure (I): TIFF2026516015000166.tif2055(I) We provide compounds having or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof, wherein R 1 , R 1 , L 1 , L 2 , L 2a , L 2b And A are as defined herein. The use of the compound as a component of lipid nanoparticle formulations for the delivery of therapeutic agents, compositions containing the compound, and methods for the use and manufacture thereof are also provided.

Description

[Technical Field]

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

[0002] Many challenges exist in the delivery of nucleic acids to influence desired responses in biological systems. Nucleic acid-based therapies hold immense potential, but realizing this potential still requires more effective delivery of nucleic acids to the appropriate sites within cells or organisms. Examples of therapeutic nucleic acids include messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, immunostimulatory nucleic acids, antagonists, antimyls, mimics, supermyls, and aptamers. Some nucleic acids, such as mRNA or plasmids, can be used to induce the expression of specific cell products, for example, in the treatment of diseases associated with protein or enzyme deficiencies. The therapeutic applications of translatable nucleotide delivery are extremely broad, as constructs can be synthesized to produce any selected protein sequence, whether system-specific or not. Nucleic acid expression products can increase existing protein levels, replace missing or non-functional versions of proteins, or introduce novel proteins and associated functionalities in cells or organisms.

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

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

[0005] However, there are currently two problems with using oligonucleotides in therapeutic settings. First, free RNA is susceptible to nuclease digestion in plasma. Second, free RNA has a limited ability to access intracellular compartments where the relevant translation mechanisms reside. Lipid nanoparticles, formed from cationic lipids and other lipid components such as neutral lipids, cholesterol, PEG, PEGylated lipids, and oligonucleotides, are used to block the degradation of RNA in plasma and promote the cellular uptake of oligonucleotides.

[0006] Improvements to cationic lipids and lipid nanoparticles for oligonucleotide delivery remain necessary. Preferably, these lipid nanoparticles provide an optimal drug-to-lipid ratio, protect nucleic acids from degradation and clearance in serum, are suitable for systemic delivery, and provide intracellular delivery of nucleic acids. Furthermore, these lipid-nucleic acid particles must be well-tolerated and provide an appropriate therapeutic index so that treatment of a patient with an effective dose of nucleic acid does not involve unacceptable toxicity and / or risk to the patient. This disclosure provides these and related advantages. [Overview of the project]

[0007] In short, this disclosure provides lipid compounds, including their stereoisomers, pharmaceutically acceptable salts, or tautomers, which can be used alone or in combination with other lipid components, such as neutral lipids, charged lipids, steroids (e.g., all sterols) and / or their analogues, and / or polymer-conjugate lipids, to form lipid nanoparticles for the delivery of therapeutic agents. In some examples, lipid nanoparticles are used to deliver nucleic acids, such as antisense and / or messenger RNs. Methods of using such lipid nanoparticles for the treatment of various diseases or conditions, such as those caused by infectious entities and / or protein dysfunction, are also provided.

[0008] In one embodiment, the following structure (I): [ka] (I) We provide compounds having or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof, wherein R 1 , R 2 , L 1 , L 2 , L 2a , L 2b And A are as defined herein.

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

[0010] In other embodiments, the Disclosure provides a method for administering a therapeutic agent to a patient in need thereof, comprising preparing a composition of lipid nanoparticles containing a compound of structure (I) and the therapeutic agent, and delivering the composition to the patient. Such a method is useful for inducing the expression of a protein in a target body, for example, to express an antigen or gene-editing protein for the purpose of vaccination.

[0011] These and other aspects of this disclosure will become apparent from the following detailed description. [Modes for carrying out the invention]

[0012] The following description includes certain specific details in order to provide a complete understanding of the various embodiments of this disclosure. However, those skilled in the art will understand that embodiments of this disclosure can be carried out without these details.

[0013] This disclosure is partly based on the discovery of novel cationic (amino) lipids that offer advantages when used in lipid nanoparticles for in vivo delivery of active substances or therapeutic agents, such as nucleic acids, to mammalian cells. In particular, embodiments of this disclosure provide nucleic acid-lipid nanoparticle compositions comprising one or more of the novel cationic lipids described herein that provide increased nucleic acid activity and improved in vivo tolerability of the composition, resulting in a significant increase in therapeutic metrics compared to previously described nucleic acid-lipid nanoparticle compositions.

[0014] In certain embodiments, the disclosure provides novel cationic lipids that enable the formulation of improved compositions for the in vitro and in vivo delivery of mRNA and / or other oligonucleotides. In some embodiments, these improved lipid nanoparticle compositions are useful for the expression of mRNA-encoded proteins. In other embodiments, these improved lipid nanoparticle compositions are useful for the upregulation of endogenous protein expression by delivering miRNA inhibitors that target one specific miRNA or group of miRNAs that modulate one target mRNA or several mRNAs. In other embodiments, these improved lipid nanoparticle compositions are useful for downregulating (e.g., silencing) the protein and / or mRNA levels of a target gene. In some other embodiments, lipid nanoparticles are also useful for the delivery of mRNA and plasmids for the expression of transgenes. In yet another embodiment, lipid nanoparticle compositions are useful for inducing pharmacological effects resulting from protein expression, e.g., increased red blood cell production by delivery of appropriate erythropoietin mRNA, or protection against infection by delivery of mRNA encoding an appropriate antibody.

[0015] The lipid nanoparticles and compositions of this disclosure may be used for a variety of purposes, both in vitro and in vivo, including the delivery of encapsulated or bound (e.g., complexed) therapeutic agents, such as nucleic acids, to cells. Accordingly, embodiments of this disclosure provide a method for treating or preventing a disease or disorder in an object requiring treatment or prevention by contacting the object with lipid nanoparticles encapsulating or bound to a suitable therapeutic agent, wherein the lipid nanoparticles comprise one or more of the novel cationic lipids described herein.

[0016] As described herein, embodiments of the lipid nanoparticles of this disclosure are particularly useful for the delivery of nucleic acids, including, for example, mRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomyl / antimyl), messenger RNA interference complementary RNA (micRNA), DNA, multivalent RNA, Dicer substrate RNA, and complementary DNA (cDNA). Accordingly, the lipid nanoparticles and compositions of this disclosure may be used to induce the expression of a desired protein both in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more novel cationic lipids described herein, wherein the lipid nanoparticles encapsulate or bind to nucleic acids (e.g., messenger RNA or plasmid encoding the desired protein) to be expressed in order to produce the desired protein. Alternatively, the lipid nanoparticles and compositions of this disclosure may be used to reduce the expression of target genes and proteins both in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more novel cationic lipids described herein, wherein the lipid nanoparticles encapsulate or bind to nucleic acids (e.g., antisense oligonucleotides or small interfering RNA (siRNA)) that reduce the expression of the target gene. The lipid nanoparticles and compositions of this disclosure may also be used separately or in combination for the co-delivery of different nucleic acids (e.g., mRNA and plasmid DNA), which may be useful, for example, to provide effects requiring the co-localization of different nucleic acids (e.g., mRNA encoding a suitable gene-modifying enzyme and a DNA segment for integration into the host genome).

[0017] Nucleic acids for use in this disclosure may be prepared according to any available technology. For mRNA, the primary method of preparation is, but is not limited to, enzymatic synthesis (also known as in vitro transcription), which is currently the most efficient method for producing long sequence-specific mRNA. In vitro transcription describes the process of template-directed synthesis of an RNA molecule from an engineered DNA template consisting of an upstream bacteriophage promoter sequence (including, but not limited to, those derived from T7, T3, and SP6 Escherichia coli phages) ligated with a downstream sequence encoding the gene of interest. Template DNA can be prepared for in vitro transcription from a number 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 v. 941 Conn GL (ed), New York, NY Humana Press, 2012).

[0018] RNA transcription occurs in vitro using a linearized DNA template under conditions that support polymerase activity but minimize potential degradation of the resulting mRNA transcript, in the presence of the corresponding RNA polymerase and adenosine, guanosine, uridine, and cytidine ribonucleoside triphosphates (rNTPs). In vitro transcription can be performed using a variety of commercially available kits, including but not limited to the RiboMax Large Scale RNA Production System (Promega) and the MegaScript Transcription Kit (Life Technologies), as well as using commercially available reagents containing RNA polymerase and rNTPs. Methods for in vitro transcription of mRNA are well known in the art (e.g., Losick, R., 1972, In vitro transcription, Ann Rev Biochem v.41 409-46; Kamakaka, RT and Kraus, WL 2001. In Vitro Transcription. Current Protocols in Cell Biology. 2:11.6:11.6.1-11.6.17; Beckert, B. And Masquida, B., (2010) Synthesis of RNA by In Vitro Transcription in RNA in Methods in Molecular Biology v. 703 (Neilson, H. Ed), New York, NY Humana Press, 2010; Brunelle, JL and Green, R., 2013, Chapter Five - In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology v. 530, 101-114). (All of these are incorporated with proper attribution. See reference).

[0019] Next, the desired in vitro transcribed mRNA is purified from undesirable components of the transcription or related reaction (including non-integrated rNTPs, protein enzymes, salts, short-chain RNA oligos, etc.). Techniques for isolating mRNA transcripts are well known in the art. Known procedures include phenol / chloroform extraction or precipitation with either a monovalent cation or an alcohol (ethanol, isopropanol) in the presence of lithium chloride. Further non-limiting examples of possible purification procedures 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 Conn GL (ed), New York, NY Humana Press, 2012). Purification can be carried out 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).

[0020] Furthermore, while reverse transcription can produce large amounts of mRNA, the product may contain numerous abnormal RNA impurities associated with undesirable polymerase activity that may need to be removed from the full-length mRNA preparation. These include short-chain RNA resulting from incomplete transcription initiation, as well as double-stranded RNA (dsRNA) generated by RNA-dependent RNA polymerase activity, RNA-primed transcription from RNA templates, and self-complementary 3' extension. It has been demonstrated that these impurities with dsRNA structures can lead to undesirable immunostimulatory activity through interaction with various innate immune sensors in eukaryotic cells that recognize specific nucleic acid structures and function to induce a potent immune response. This, in turn, can dramatically reduce mRNA translation, as protein synthesis is reduced during the innate cellular immune response. Therefore, further techniques for removing these dsRNA contaminants have been developed and are known in the art, including, but not limited to, scalable HPLC purification (see, for example, Kariko, K., Muramatsu, H., Ludwig, J. And Weissman, D., 2011, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA, Nucl Acid Res, v. 39 e142; Weissman, D., Pardi, N., Muramatsu, H., and Kariko, K., HPLC Purification of in vitro transcribed long RNA in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH Ed), 2013). HPLC-purified mRNA has been reported to be translated at much higher levels, particularly in primary cells and in vivo.

[0021] A considerable variety of modifications used to alter specific properties of in vitro transcribed mRNA and improve its utility have been described in the art. These include, but are not limited to, modifications to the 5' and 3' ends of mRNA. Endogenous eukaryotic mRNA typically contains a cap structure at the 5' end of the mature molecule, which plays a crucial role in mediating the binding of mRNA cap-binding proteins (CBPs), and this, in turn, contributes to improving intracellular mRNA stability and the efficiency of mRNA translation. Thus, the highest levels of protein expression are achieved in capped mRNA transcripts. The 5'-cap contains a 5'-5'-triphosphate bond between the 5'-terminal nucleotide and the guanine nucleotide. The conjugated guanine nucleotide is methylated at the N7 position. Further modifications include methylation of the last and second-to-last 5'-nucleotides on the 2'-hydroxyl group.

[0022] Multiple different cap structures can be used to generate the 5' cap of in vitro transcribed synthetic mRNA. 5' capping of synthetic mRNA can be carried out by co-transcription with chemical cap analogs (i.e., capping during in vitro transcription). For example, anti-reverse cap analog (ARCA) caps contain a 5'-5'-triphosphate guanine-guanine bond, where one guanine contains an N7 methyl group and a 3'-O-methyl group. However, up to 20% of the transcript remains uncapped during this co-transcription process, and synthetic cap analogs are not identical to the 5' cap structure of true cellular mRNA, potentially reducing translatability and cellular stability. Alternatively, synthetic mRNA molecules can also be enzymatically capped after transcription. These may generate more authentic 5' cap structures that more closely mimic the endogenous 5' cap structurally or functionally, enhancing the binding of cap-binding proteins, increasing half-life, reducing sensitivity to 5' endonucleases, and / or reducing 5' decapping. Numerous synthetic 5'-cap analogs have been developed and are known in the field to improve mRNA stability and translatability (see, for example, Grudzien-Nogalska, E., Kowalska, J., Su, W., Kuhn, AN, Slepenkov, SV, Darynkiewicz, E., Sahin, U., Jemielity, J., and Rhoads, RE, Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH Ed), 2013).

[0023] At the 3' end, a long chain of adenine nucleotides (poly-A tail) is usually attached to the mRNA molecule during RNA processing. Immediately after transcription, the 3' end of the transcript is cleaved, releasing a 3' hydroxyl group, to which poly-A polymerase attaches the adenine nucleotide chain to the RNA in a process called polyadenylation. Poly(A) tails have been widely shown to improve both the translation efficiency and stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly(A), poly(A) binding protein and the regulation of mRNA stability, Trends Bio Sci v. 14 373-377; Guhaniyogi, J. And Brewer, G., 2001, Regulation of mRNA stability in mammalian cells, Gene, v. 265, 11-23; Dreyfus, M. And Regnier, P., 2002, The poly(A) tail of mRNAs: Bodyguard in eukaryotes, scavenger in bacteria, Cell, v.111, 611-613).

[0024] Poly(A) tailing of in vitro transcribed mRNA can be achieved using a variety of approaches, including, but not limited to, cloning of poly(T) tracts to a DNA template or post-transcriptional addition using poly(A) polymerase. The first case allows in vitro transcription of mRNA with a poly(A) tail of a predetermined length, depending on the size of the poly(T) tract, but requires further manipulation of the template. The latter case involves enzymatically adding a poly(A) tail to in vitro transcribed mRNA using poly(A) polymerase that catalyzes the incorporation of an adenine residue into the 3' end of the RNA, and does not require further manipulation of the DNA template, but results in mRNA with poly(A) tails of non-uniform length. 5'-capping and 3'-poly(A) tailing can be performed using a variety of commercially available kits, including but not limited to the Poly(A) Polymerase Tailing Kit (EpiCenter), mMESSAGE mMACHINE T7 Ultra Kit, and Poly(A) Tailing Kit (Life Technologies), as well as using commercially available reagents, various ARCA caps, poly(A) polymerases, etc.

[0025] In addition to 5' capping and 3' polyadenylation, other modifications of in vitro transcripts have been reported to offer advantages related to translation efficiency and stability. It is well known in the art that pathogenic DNA and RNA can be recognized by various sensors within eukaryotes and can trigger a potent innate immune response. Since most nucleic acids from natural sources contain modified nucleosides, the ability to distinguish between pathogenic and self DNA and RNA has been shown to be based, at least in part, on structural and nucleoside modifications. In contrast, RNA synthesized in vitro lacks these modifications and is therefore immunostimulant, which can consequently inhibit effective mRNA translation as described above.The introduction of modified nucleosides into in vitro transcribed mRNA can prevent the recognition and activation of RNA sensors, thereby mitigating this undesirable immunostimulatory activity and improving translational capacity (e.g., Kariko, K. And Weissman, D. 2007, Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: implication for therapeutic RNA development, Curr Opin Drug Discov Devel, v.10 523-532; Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH Ed), 2013); Kariko, K., Muramatsu, H., Welsh, FA, Ludwig, J., Kato, H., Akira, S., Weissman, (See D., 2008, Incorporation of Pseudouridine Into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability, Mol Ther v.16, 1833-1840). Modified nucleosides and nucleotides used in the synthesis of modified RNA can be prepared, monitored, and utilized using common methods and procedures known in the art. A wide variety of nucleoside modifications are available that can be incorporated to some extent into in vitro transcribed mRNA, either alone or in combination with other modified nucleosides (see, for example, U.S. Patent Application Publication 2012 / 0251618).It has been reported that in vitro synthesis of nucleoside-modified mRNA reduces its ability to activate immune sensors while simultaneously improving its translational capacity.

[0026] Other components of mRNA that can be modified to provide benefits in terms of translationability and stability include the 5' and 3' untranslated regions (UTRs). Optimization of the UTR (preferred 5' and 3' UTRs can be obtained from cellular or viral RNA) has been shown to increase mRNA stability and translational efficiency of in vitro transcribed mRNA, both or independently (see, for example, Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH Ed), 2013)).

[0027] In addition to mRNA, other nucleic acid payloads may be used in this disclosure. For oligonucleotides, preparation methods include, but are not limited to, chemical synthesis and enzymatic methods, chemical cleavage of longer precursors, and in vitro transcription as described above. Methods for synthesizing DNA and RNA nucleotides are widely used and well known in the art (see, for example, Gait, MJ (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, DC: IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, NJ) Totowa, NJ: Humana Press, 2005 (both incorporated with attribution)).

[0028] With respect to plasmid DNA, the preparation for use in this disclosure generally involves, but is not limited to, the in vitro growth and isolation of plasmid DNA in a liquid culture of bacteria containing the plasmid of interest. The presence of genes in the plasmid of interest that encode resistance to a specific antibiotic (such as penicillin or kanamycin) allows bacteria containing the plasmid of interest to selectively grow in an antibiotic-containing culture. Methods for isolating plasmid DNA are widely used and well-known in the art (e.g., 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., Gillstrom, S., Bjoornestedt, R. and Schmidt, SR (2008), Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture. Biotechnol. Bioeng., 99: 557-566; see U.S. Patent No. 6,197,553). Plasmid isolation can be performed using a variety of commercially available kits, including but not limited to Plasmid Plus (Qiagen), GenJET plasmid MaxiPrep (Thermo), and PureYield MaxiPrep (Promega) kits, as well as with commercially available reagents.

[0029] The cationic lipids, lipid nanoparticles and compositions comprising them, as well as various exemplary embodiments of their use for delivering active substances or therapeutic agents such as nucleic acids for regulating gene and protein expression, are described in further detail below.

[0030] In this specification, the following terms have the meanings of those terms unless otherwise specified.

[0031] Unless the context requires otherwise, the word “comprise” and its variations, such as “comprises” and “comprising,” throughout this specification and the claims should be interpreted in an open and comprehensive sense, that is, “including, but not limited to.”

[0032] Throughout this specification, any reference to “one embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described in relation to an embodiment is included in at least one embodiment of this disclosure. Therefore, even if the phrases “in one embodiment” or “in one embodiment” appear in various places throughout this specification, they do not necessarily all refer to the same embodiment. Furthermore, a particular feature, structure, or characteristic may be combined in any suitable way in one or more embodiments.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure pertains. In this specification and in the claims, the singular forms “a,” “an,” and “the” include plural references unless otherwise explicitly indicated by the context.

[0034] The phrase "induces the expression of a desired protein" refers to the ability of nucleic acids to increase the expression of a desired protein. To examine the degree of protein expression, a test sample (e.g., a sample of cells in a culture expressing the desired protein) or a test mammal (e.g., a mammal, e.g., human, or an animal model, e.g., a rodent (e.g., mouse), or a non-human primate (e.g., monkey) model) is brought into contact 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 to the expression of the desired protein in a control sample (e.g., a sample of cells in a culture expressing the desired protein) or a control mammal (e.g., a mammal, e.g., human, or an animal model, e.g., a rodent (e.g., mouse), or a non-human primate (e.g., monkey) model) that has not been brought into contact with or administered nucleic acids. If the desired protein is present in the control sample or control mammal, a value of 1.0 may be assigned to the expression of the desired protein in the control sample or control mammal. In certain embodiments, induction of desired protein expression is achieved when the ratio of the desired protein expression level in the test sample or test mammal to the desired protein expression level in the control sample or control mammal is greater than 1, for example, about 1.1, 1.5, 2.0, 5.0, or 10.0. If the desired protein is not present in the control sample or control mammal, induction of desired protein expression is achieved when any measurable level of the desired protein is detected in the test sample or test mammal. Those skilled in the art will understand appropriate assays for determining the level of protein expression in a sample, such as dot blotting, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays, or assays based on reporter proteins that can produce fluorescence or luminescence under appropriate conditions.

[0035] The phrase "inhibits target gene expression" refers to the ability of nucleic acids to silence, reduce, or inhibit the expression of a target gene. To investigate the degree of gene silencing, a test sample (e.g., a sample of cells in a culture expressing the target gene) or a test mammal (e.g., a mammal, e.g., human, or an animal model, e.g., a rodent (e.g., mouse), or a non-human primate (e.g., monkey) model) is exposed to the nucleic acid that silences, reduces, or inhibits the expression of the target gene. The expression of the target gene in the test sample or test animal is compared to the expression of the target gene in a control sample (e.g., a sample of cells in a culture expressing the target gene) or a control mammal (e.g., a mammal, e.g., human, or an animal model, e.g., a rodent (e.g., mouse), or a non-human primate (e.g., monkey) model) that has not been exposed to or administered nucleic acids. The expression of the target gene in the control sample or control mammal may be assigned a value of 100%. In certain embodiments, silencing, inhibition, or reduction of target gene expression is achieved when the expression level of the target gene in the test sample or test mammal is approximately 995%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% relative to the expression level of the target gene in the control sample or control mammal. In other words, nucleic acids can silence, reduce, or inhibit the expression of a target gene in a test sample or test mammal by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the expression level of the target gene in a control sample or control mammal that has not been in contact with or administered nucleic acids. Suitable assays for determining the level of target gene expression include, but are not limited to, tests at the protein or mRNA level using techniques known to those skilled in the art, such as dot blotting, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays.

[0036] The “effective dose” or “therapeutic effective dose” of an active substance or therapeutic agent, such as a therapeutic nucleic acid, is an amount sufficient to produce the desired effect, for example, an increase or inhibition of the expression of a target sequence compared to a normal expression level detected in the absence of the nucleic acid. An increase in the expression of a target sequence is achieved when any measurable level is detected in the case of an expression product that does not exist in the absence of the nucleic acid. If the expression product is present at a certain level before contact with the nucleic acid, an increase in expression is achieved when the increase factor of the value obtained with the nucleic acid, such as mRNA, compared to the control is approximately 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000 or more. Inhibition of the expression of a target gene or target sequence is achieved when the value obtained using nucleic acids such as antisense oligonucleotides is approximately 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% compared to the control. Suitable assays for measuring the expression of a target gene or target sequence include protein or RNA-level tests using techniques known to those skilled in the art, such as dot blotting, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and fluorescence or luminescence and phenotypic assays of a suitable reporter protein.

[0037] As used herein, the term “nucleic acid” refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form, and includes DNA, RNA, and hybrids thereof. DNA may be in the form of an antisense molecule, plasmid DNA, cDNA, PCR product, or vector. RNA may be in the form of small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA, dicer substrate RNA, or viral RNA (vRNA), and combinations thereof. Nucleic acids include synthetic, natural, and unnatural nucleic acids containing known nucleotide analogs or modified backbone residues or bindings that have similar binding properties to a reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2'-O-methylribonucleotides, and peptide-nucleic acids (PNAs). Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to a reference nucleic acid. Unless otherwise specified, a given nucleic acid sequence implicitly includes not only the explicitly indicated sequence but also its conserved modified variants (e.g., degenerate codon substitutions), alleles, orthologues, single nucleotide polymorphisms, and complementary 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 substituted with a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked to each other by phosphate groups."Bases" include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications that arrange novel reactive groups such as amines, alcohols, thiols, carboxylates, and alkyl halides.

[0038] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that contains a coding sequence of partial or full length necessary to produce a polypeptide or precursor polypeptide.

[0039] As used herein, "gene product" refers to the product of a gene, such as an RNA transcript or polypeptide.

[0040] The term "lipid" refers to a group of organic compounds that include, but are not limited to, fatty acid esters, and is generally characterized by being poorly soluble in water but soluble in many organic solvents. These are typically classified into at least three classes: (1) "simple lipids" including fats, oils, and waxes; (2) "complex lipids" including phospholipids and glycolipids; and (3) "derived lipids" such as steroids.

[0041] A "steroid" is a compound containing the following carbon skeleton: [ka]

[0042] Non-specific examples of steroids include cholesterol.

[0043] "Cationic lipids" refer to lipids that can be positively charged. Exemplary cationic lipids include those with one or more positively charged amine groups. Preferred cationic lipids are ionizable and can exist in a pH-dependently positively charged or neutral form. Ionization of cationic lipids affects the surface charge of lipid nanoparticles under different pH conditions. This charge state can influence plasma protein absorption, blood clearance and tissue distribution (Semple, SC, et al., Adv. Drug Deliv Rev 32:3-17 (1998)), as well as the ability to form endosomal soluble non-bilayer structures important for intracellular delivery of nucleic acids (Hafez, IM, et al., Gene Ther 8:1188-1196 (2001)).

[0044] The term “lipid nanoparticles” refers to particles having at least one dimension on the order of nanometers (e.g., 1 to 1,000 nm), which include one or more compounds of formula (I) or other specific cationic lipids. In some embodiments, lipid nanoparticles are included in formulations that can be used to deliver active substances or therapeutic agents, such as nucleic acids (e.g., mRNA), to a target site of interest (e.g., cells, tissues, organs, tumors, etc.). In some embodiments, the lipid nanoparticles of this disclosure include nucleic acids. Such lipid nanoparticles typically include a compound of structure (I) and one or more additives selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. In some embodiments, active substances or therapeutic agents, such as nucleic acids, are encapsulated in the lipid portion of the lipid nanoparticles, or in an aqueous space enclosed by some or all of the lipid portion of the lipid nanoparticles, thereby protecting them from enzymatic degradation or other undesirable effects induced by host organism or cellular mechanisms, such as adverse immune responses.

[0045] In various embodiments, the lipid nanoparticles are approximately 30nm to 150nm, approximately 40nm to 150nm, approximately 50nm to 150nm, approximately 60nm to 130nm, approximately 70nm to 110nm, approximately 70nm to 100nm, approximately 80nm to 100nm, approximately 90nm to 100nm, approximately 70nm to 90nm, approximately 80nm to 90nm, approximately 70nm to 80nm, and They have an average diameter of approximately 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially nontoxic. In certain embodiments, when nucleic acids are present in lipid nanoparticles, they are resistant to degradation by nucleases in aqueous solutions. Lipid nanoparticles containing nucleic acids and methods for producing them are disclosed, for example, in U.S. Patent Publications 2004 / 0142025, 2007 / 0042031, and PCT Publications WO2013 / 016058 and WO2013 / 086373, all of which are incorporated herein by attribution for all purposes.

[0046] As used herein, “lipid encapsulation” refers to lipid nanoparticles that provide an active substance or therapeutic agent, such as a nucleic acid (e.g., mRNA), having complete encapsulation, partial encapsulation, or both. In certain embodiments, the nucleic acid (e.g., mRNA) is completely encapsulated within the lipid nanoparticles.

[0047] The term "polymer-bound lipid" refers to a molecule that contains both a lipid portion and a polymer portion. An example of a polymer-bound lipid is a PEGylated lipid. The term "PEGylated lipid" refers to a molecule that contains both a lipid portion and a polyethylene glycol portion. PEGylated lipids are well known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG).

[0048] The term "neutral lipid" refers to any of the many lipid species that exist in either an uncharged or neutral zwitterionic form at a given pH. At physiological pH, such lipids include, but are not limited to, phosphatidylcholine, e.g., 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylethanolamine, e.g., 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM), ceramides, steroids, e.g., sterols, and their derivatives. Neutral lipids may be synthetic or naturally occurring.

[0049] The term "charged lipid" refers to any of the many lipid species that exist in either a positively or negatively charged form, independent of the useful physiological pH range, e.g., pH approximately 3 to pH approximately 9. Charged lipids can be synthetic or naturally occurring. Examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyltrimethylammonium propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, and dimethylaminoethanecarbamoylsterol (e.g., DC-Chol).

[0050] As used herein, the term "aqueous solution" refers to a composition containing water.

[0051] With respect to nucleic acid-lipid nanoparticles, "serum stability" means that the nucleotides are not significantly degraded after exposure to serum or nuclease assays that significantly degrade free DNA or RNA. Suitable assays include, for example, standard serum assays, DNAse assays, or RNAse assays.

[0052] As used herein, "systemic delivery" refers to the delivery of therapeutic products that can result in widespread exposure of an active substance within an organism. Some techniques of administration can result in systemic delivery of a particular agent, while others cannot. Systemic delivery means that a useful, preferably therapeutic amount of the agent is exposed to most parts of the body. Systemic delivery of lipid nanoparticles can be by any means known in the art, including, for example, intravenous, intra-arterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery.

[0053] As used herein, "local delivery" refers to the direct delivery of an active substance to a target site within an organism. For example, a drug can be delivered locally by direct injection to a diseased site such as a tumor, other target sites such as an inflammatory site, or a target organ such as the liver, heart, pancreas, kidney, etc. Local delivery can also include local application or injection techniques, such as intramuscular, subcutaneous, or intradermal injection. Local delivery does not exclude systemic pharmacological effects.

[0054] "Alkyl" consists of only saturated (i.e., containing no double bonds and / or triple bonds) carbon atoms and hydrogen atoms, and has 1 to 24 carbon atoms (C1-C 24 alkyl), 1 to 16 carbon atoms (C1-C 16 alkyl), 1 to 12 carbon atoms (C1-C 12 alkyl), 6 to 24 carbon atoms (C6-C 24 alkyl), 1 to 8 carbon atoms (C1-C8 alkyl) or 1 to 6 carbon atoms (C1-C6 alkyl), and is a straight-chain or branched-chain hydrocarbon radical bonded to the rest of the molecule by a single bond, for example, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, etc. Unless otherwise specifically stated herein, alkyl groups are optionally substituted.

[0055] "Alkylhydroxyl" refers to an alkyl group as defined herein, which contains at least one hydroxyl (OH) substituent.

[0056] "Alkenyl" consists only of carbon and hydrogen atoms containing at least one carbon-carbon double bond, and has 1 to 24 carbon atoms (C2-C 24 Alkenyl), 1 to 12 carbon atoms (C2-C 12 Alkenyl), 6-24 carbon atoms (C6-C6) 24 Alkenyl), 2-16 carbon atoms (C2-C 16 Alkenyl), 4-12 carbon atoms (C4-C 12 Alkenyls refer to linear or branched hydrocarbon chain radicals having 1 to 8 carbon atoms (C2-C8 alkenyls) or 1 to 6 carbon atoms (C2-C6 alkenyls) bonded to the rest of the molecule by single bonds, such as ethenyl, n-propenyl, 1-methylethenyl, n-butenyl, n-pentenyl, 1,1-dimethylethenyl, 3-methylhexenyl, 2-methylhexenyl, etc. Unless otherwise specified herein, alkenyl groups may be substituted as desired.

[0057] "Alkynnyl" consists only of carbon and hydrogen atoms containing at least one carbon-carbon triple bond, and has 1 to 24 carbon atoms (C2-C 24 Alkynyl), 1 to 12 carbon atoms (C2-C 12 Alkynyl refers to a linear or branched hydrocarbon chain radical having 1 to 8 carbon atoms (C2-C8 alkynyl) or 1 to 6 carbon atoms (C2-C6 alkynyl) bonded to the rest of the molecule by a single bond, such as ethynyl, n-propynyl, 1-methylethynyl, n-butynyl, n-pentynyl, 1,1-dimethylethynyl, 3-methylhexynyl, 2-methylhexynyl, etc. Unless otherwise specified herein, the alkynyl group may be substituted as desired.

[0058] "Alkylene" or "alkylene chain" refers to a straight or branched divalent saturated hydrocarbon chain consisting only of carbon and hydrogen, with the rest of the molecule linked to a radical group. In some embodiments, an alkylene chain consists of 1 to 24 carbon atoms (C1-C1). 24 Alkylene), 1 to 15 carbon atoms (C1-C 15 Alkylene), 1 to 12 carbon atoms (C1-C 12 Alkylenes have 1 to 8 carbon atoms (C1-C8 alkylenes), 1 to 6 carbon atoms (C1-C6 alkylenes), 4 to 6 carbon atoms (C4-C6 alkylenes), 2 to 4 carbon atoms (C2-C4 alkylenes), and 1 to 2 carbon atoms (C1-C2 alkylenes), such as methylene, ethylene, propylene, and n-butylene. The alkylene chain is bonded to the rest of the molecule via single bonds and to the radical group via single bonds. The bonding sites of the alkylene chain to the rest of the molecule and to the radical group can be via one carbon or any two carbons in the chain. Unless otherwise specified herein, alkylene chains are optionally substituted. "Cycloalkyl" or "carbocyclic ring" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting only of carbon and hydrogen atoms, which has 3 to 15 ring carbon atoms (C3-C6 15 ), 3 to 10 ring carbon atoms (C3-C 10 ) or may include a condensed or crosslinked ring system having 3 to 8 ring carbon atoms (C3-C8), which may be saturated or unsaturated and bonded to the rest of the molecule by single bonds. Examples of monocyclic radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of polycyclic radicals include adamantyl, norbornyl, dekalinyl, and 7,7-dimethylbicyclo[2.2.1]heptanyl. Unless otherwise specified herein, cycloalkyl groups may be substituted as desired.

[0059] "Aryl" refers to a carbocyclic cyclic radical comprising hydrogen, 6 to 18 carbon atoms, and at least one aromatic ring. For the purposes of this disclosure, an aryl radical is a monocyclic, bicyclic, tricyclic, or tetracyclic cyclic system, which may include condensed or bridging cyclic systems. Examples of aryl groups include, but are not limited to, aryl radicals derived from acetantrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluorantene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene.

[0060] "Arylalkyl" is defined by formula -R b -R c This refers to the radical shown by, where R b R is an alkylene or alkenylene as defined above, c This is one or more aryl radicals as defined above, such as benzyl, diphenylmethyl, etc. Unless otherwise specified herein, arylalkyl groups may be substituted as desired.

[0061] A “heterocyclyl” or “heterocyclic ring” refers to a stable 3-18 member non-aromatic ring radical having 1-12 ring carbon atoms (e.g., 2-12) and 1-6 ring heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Unless otherwise specified herein, heterocyclyl radicals are monocyclic, dicyclic, tricyclic, or tetracyclic ring systems and may include condensed, spirocyclic ("spiro-heterocyclyl") ring systems and / or bridging ring systems; and the nitrogen, carbon, or sulfur atoms in the heterocyclyl radical may be oxidized as desired; the nitrogen atom may be quaternized as desired; and the heterocyclyl radical may be partially or completely saturated. Examples of such heterocyclyl groups include, but are not limited to, dioxolanil, thienyl[1,3]dithianil, decahydroisoquinolyl, imidazolinil, imidazolidinil, isothiazolidinil, isoxazolidinil, morpholinil, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinil, 2-oxopiperidinil, 2-oxopyrrolidinil, oxazolidinil, piperidinil, piperazinil, 4-piperidonil, pyrrolidinil, pyrazolidinil, quinuclidinil, thiazolidinil, tetrahydrofuryl, trithianil, tetrahydropyranil, thiomorpholinil, thiamorpholinil, 1-oxo-thiomorpholinil, and 1,1-dioxo-thiomorpholinil. Unless otherwise specified herein, heterocyclyl groups may be substituted as desired.

[0062] As used herein, the term "substituted" means that at least one hydrogen atom is replaced by a non-hydrogen atom, for example, but not limited to: halogen atoms, e.g., F, Cl, Br, and I; oxo groups (=O); hydroxyl groups (-OH); alkoxy groups (-OR). a , where R a is C1-C 12 Alkyl or cycloalkyl group; carboxyl group (-OC(=O)R a Or -C (=O) OR a , where R a H, C1-C 12 Alkyl or cycloalkyl; amine group (-NR) aR b , where R a and R b Each is independent of H, C1-C 12 Alkyl or cycloalkyl); C1-C 12 Alkyl group; and any of the above groups (e.g., alkyl, alkylhydroxyl, alkenyl, alkynyl, alkylene, cycloalkyl, aryl, aralkyl or heterocyclyl) that are replaced by bonding to a cycloalkyl group. In some embodiments, the substituent is C1-C 12 In other embodiments, the substituent is an alkyl group. In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group, for example, an fluoro group. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group. In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group.

[0063] "Optional" or "optional" (e.g., may be substituted) means that the event of the situation described thereafter may or may not occur, and that the description includes both cases in which such event or situation occurs and cases in which it does not occur. For example, "optionally substituted alkyl" means that the alkyl radical may or may not be substituted, and that the description includes both substituted and unsubstituted alkyl radicals. In some embodiments, "optionally substituted" means that a particular radical is a halo (e.g., F, Cl, Br, and I), oxo (=O), hydroxyl (-OH), alkoxy (-OR) a , where R a is C1-C 12 Alkyl), cycloalkoxy (-OR a , where R a (C3-C8 cycloalkyl), carboxyl (-OC(=O)R a Or -C (=O) OR a , where R a H, C1-C12 Alkyl or C3-C8 cycloalkyl, amine (-NR) a R b , where R a and R b Each is independent of H, C1-C 12 (Alkyl or C3-C8 cycloalkyl), C1-C 12 This means that it is substituted with one or more substituents selected from the group consisting of alkyl and C3-C8 cycloalkyl groups.

[0064] In some embodiments, "may be substituted" means substituted with one or more halo substituents. In some embodiments, "may be substituted" means substituted with one or more oxo substituents. In some embodiments, "may be substituted" means substituted with one or more hydroxyl substituents. In certain embodiments, "may be substituted" means substituted with one or more alkoxy substituents. In some embodiments, "may be substituted" means substituted with one or more cycloalkoxy substituents. In certain embodiments, "may be substituted" means substituted with one or more carboxy substituents. In some embodiments, "may be substituted" means substituted with one or more amine substituents. In certain embodiments, "may be substituted" means one or more C1-C 12 This means that the substituent is substituted with an alkyl substituent. In some embodiments, "may be substituted" means that the substituent is substituted with one or more C3-C8 cycloalkyl substituents.

[0065] Where it is stated that a functional group "may be substituted," and that substituents on the functional group "may also be substituted," for the purposes of this disclosure, such iterations are limited to five, preferably two. In some embodiments, such iterations are limited to one. In some embodiments, such iterations are limited to zero.

[0066] This disclosure also means to encompass all pharmaceutically acceptable compounds of the compound of structure (I) that isotope-labeled by replacing one or more atoms with atoms having different atomic masses or mass numbers. Examples of isotopes that may be incorporated into the compound include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, for example, respectively. 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 36 Cl, 123 I and 125 I is one example. These radiolabeled compounds may be useful in helping to determine or measure the efficacy of a compound by characterizing, for example, the site or mode of action, or the binding affinity to a pharmacologically important site of action. Specific isotope-labeled compounds of structure (I), for example, those incorporating radioisotopes, are useful in drug and / or substrate tissue distribution studies. Radioisotope tritium, i.e. 3 H, and carbon-14, i.e. 14 C is particularly useful for this purpose, considering the ease of their incorporation and the ease of detection.

[0067] Heavier isotopes, such as deuterium, 2 Substitution with H may offer certain therapeutic benefits resulting from greater metabolic stability, such as an increased in vivo half-life or a reduced required dose, and is therefore preferable in some situations.

[0068] Positron-emitting isotopes, for example 11 C, 18 F, 15 O and 13Substitution with N may be useful in positron emission topography (PET) studies to investigate substrate receptor occupancy. The isotope-labeled compound of structure (I) can generally be prepared by conventional techniques known to those skilled in the art, or by processes similar to those described below in the preparations and examples, using appropriate isotope-labeling reagents instead of previously used unlabeled reagents.

[0069] This disclosure also means that it encompasses in vivo metabolites of the disclosed compounds. Such products may arise primarily from enzymatic processes, such as oxidation, reduction, hydrolysis, amidation, or esterification of the administered compound. Thus, this disclosure includes compounds produced by processes that involve administering the compounds of this disclosure to mammals for a period of time sufficient to obtain their metabolites. Such products are typically identified by administering a radiolabeled compound of this disclosure to an animal, such as a rat, mouse, guinea pig, monkey, or human, in a detectable dose, allowing sufficient time for metabolism to occur, and then isolating the conversion products from urine, blood, or other biological samples.

[0070] "Stable compound" and "stable structure" refer to a compound that is robust enough to survive isolation from a reaction mixture to a useful degree of purity and formulation into an effective therapeutic agent.

[0071] "Mammals" include both humans and livestock, such as laboratory animals and domestic pets (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits), as well as non-livestock, such as wild animals.

[0072] "Medically acceptable carriers, diluents or additives" include, but are not limited to, any adjuvants, carriers, additives, flow enhancers, sweeteners, diluents, preservatives, colorants, flavor enhancers, surfactants, humectants, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers approved by the U.S. Food and Drug Administration as acceptable for use in human or animal products.

[0073] "Medically acceptable salts" include both acid addition salts and base addition salts.

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

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

[0076] In many cases, crystallization produces solvates of the compounds of this disclosure. As used herein, the term “solvate” refers to an aggregate containing one or more molecules of the compounds of this disclosure together with one or more molecules of a solvent. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of this disclosure may exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, and the like, as well as the corresponding solvated forms. The compounds of this disclosure may be true solvates, or in other cases, they may simply hold incidental water, or they may be a mixture of water and some incidental solvents.

[0077] "Pharmaceutical composition" refers to a formulation of the compounds of this disclosure and a medium generally accepted in the art for delivering the biologically active compounds to a mammal, such as a human. Such a medium includes all pharmaceutically acceptable carriers, diluents, or additives for that purpose.

[0078] “Effective dose” or “therapeutic effective dose” means an amount of the compound of the Disclosure sufficient to produce a treatment in a mammal, preferably a human, when administered to a mammal, preferably a human. The amount of lipid nanoparticles of the Disclosure constituting a “therapeutic effective dose” varies depending on the compound, its state and its severity, the mode of administration, and the age of the mammal being treated, but can be routinely determined by a person skilled in the art considering their knowledge and the Disclosure.

[0079] As used herein, “treat” or “treat” encompasses treatment of the disease or condition in a mammal, preferably a human, having the disease or condition of interest, and includes: (i) In mammals, in particular, to prevent the development of a disease or condition in mammals that are susceptible to the condition but have not yet been diagnosed with it; (ii) To inhibit a disease or condition, that is, to stop its onset; (iii) to alleviate a disease or condition, i.e., to cause a regression of the disease or condition; or (iv) Relieving symptoms arising from a disease or condition, that is, reducing pain without addressing the underlying disease or condition. The terms “disease” and “condition” as used herein may be interchangeable, or they may differ in that a particular disease or condition may not have known causative factors (and therefore its etiology is not yet understood), and thus may not yet be recognized as a disease, but only as an undesirable condition or syndrome, and more or less a specific set of symptoms may be identified by a clinician.

[0080] The compounds of this disclosure or their pharmaceutically acceptable salts may contain one or more chiral centers, and thus give rise to enantiomers, diastereomers and other stereoisomers that can be defined as (R)- or (S)- from the viewpoint of absolute stereochemistry, or as (D)- or (L)- for amino acids. This disclosure includes all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)- or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or they may be resolved using conventional techniques, e.g., chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from suitable optically pure precursors, or resolution of racemics (or racemics of salts or derivatives) using, for example, chiral high-pressure liquid chromatography (HPLC). Where a compound described herein contains an olefin double bond or other geometrically asymmetric center, unless otherwise specified, the compound is intended to include both E and Z geometric isomers. Similarly, all tautomer forms are also intended to be included.

[0081] "Stereoisomers" refer to compounds that are composed of the same atoms connected by the same bonds but have different three-dimensional structures that are not interchangeable. This disclosure contemplates various stereoisomers and mixtures thereof, including "enantiomers," which refer to two stereoisomers that are mirror images of each other and whose molecules cannot be superimposed on one another.

[0082] "Tautomers" refer to the transfer of a proton from one atom of a molecule to another atom of the same molecule. This disclosure includes tautomers of any such compound.

[0083] Compound In one aspect, this disclosure provides novel lipid compounds that can form lipid nanoparticles having oligonucleotides in combination with other lipid components such as neutral lipids, charged lipids, steroids, and / or polymer-conjugated lipids. Without wishing to be bound by theory, these lipid nanoparticles are thought to protect oligonucleotides from degradation in serum and provide effective delivery of oligonucleotides to cells in vitro and in vivo.

[0084] One embodiment is the following structure (I):

Chemical formula

[0085] One embodiment is the following structure (I):

Chemical formula

[0086] In some embodiments, R 1 is -C(=O)NR b R c In a particular embodiment, R 1 -NR a C(=O)R 3 In some embodiments, R 2 is -C(=O)NR e R f In a particular embodiment, -NR d C(=O)R 4 In a specific embodiment, -C(=O)NR b Rc and R 2 is -C(=O)NR e R f In a particular embodiment, R 1 -NR a C(=O)R 3 And R 2 -NR d C(=O)R 4 In a particular embodiment, R 1 -NR a C(=O)R 3 And R 2 is -C(=O)NR e R f That is the case.

[0087] In some embodiments, R a C8-C 12 It is alkyl. In a particular embodiment, R a is C8, C9 or C 10 It is alkyl. In some embodiments, R b C8-C 12 It is alkyl. In a particular embodiment, R b is C8, C9 or C 10 It is alkyl. In some embodiments, R c C8-C 12 It is alkyl. In a particular embodiment, R c is C8, C9 or C 10 It is alkyl. In some embodiments, R d C8-C 12 It is alkyl. In a particular embodiment, R d is C8, C9 or C 10 It is alkyl. In some embodiments, R e C8-C 12 It is alkyl. In a particular embodiment, R e is C8, C9 or C 10 It is alkyl. In some embodiments, R f C8-C 12 It is alkyl. In a particular embodiment, R fis C8, C9 or C 10 It is alkyl.

[0088] In some embodiments, R a , R b , R c , R d , R e and R f Each is independent of C1-C 20 It is alkyl. In a particular embodiment, R a , R b , R c , R d , R e and R f Each is independent of C8-C 19 It is alkyl. In some embodiments, R a , R b , R c , R d , R e and R f These are C8, C9, and C, each operating independently. 10 or C 19 It is alkyl. In a particular embodiment, R 3 and R 4 Each is independent of C6-C 19 It is alkyl.

[0089] In some embodiments, R 3 C6-C 12 It is alkyl. In a particular embodiment, R 3 C8-C 10 It is alkyl. In a particular embodiment, R 3 is C8, C9 or C 10 It is alkyl. In some embodiments, R 4 C6-C 12 It is alkyl. In a particular embodiment, R 4 C8-C 10 It is alkyl. In a particular embodiment, R 4 is C8, C9 or C 10 It is alkyl.

[0090] In some embodiments, L 1 is a direct bond. In certain embodiments, L 1 is C1-C6 alkylene. In certain embodiments, L 1 is C1 or C2 alkylene. In some embodiments, L 2 is a direct bond. In certain embodiments, L 2 is C1-C6 alkylene. In certain embodiments, L 2 is C1 or C2 alkylene.

[0091] In some embodiments, L 2a is C6-C 10 alkylene. In certain embodiments, L 2a is C6, C7, C8, C9 or C 10 alkylene. In some embodiments, L 2b is C6-C 10 alkylene. In certain embodiments, L 2b is C6, C7, C8, C9 or C 10 alkylene.

[0092] In some embodiments, A is a C3-C 10 carbocyclic ring optionally substituted with one or more hydroxyl and / or C1-C6 alkylhydroxyl substituents. In certain embodiments, A is monocyclic. In certain embodiments, A is bicyclic. In certain embodiments, A is spirocyclic.

[0093] In some embodiments, A is a C3-C8 carbocyclic ring optionally substituted with one or more hydroxyl and / or C1-C6 alkylhydroxyl substituents. In certain embodiments, A is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl.

[0094] In some embodiments, A is a 3- to 10-membered oxygen-containing heterocyclic ring which may be substituted with one or more hydroxyl and / or C1-C6 alkylhydroxyl substituents. A is a 3- to 6-membered oxygen-containing heterocyclic ring which may be substituted with one or more hydroxyl and / or C1-C6 alkylhydroxyl substituents. In certain embodiments, A is oxyranil, oxetanil, tetrahydrofuranil, or tetrahydropyranil.

[0095] In some embodiments, A is unsubstituted. In certain embodiments, A is substituted with a hydroxyl group.

[0096] In some embodiments, [ka] The structure is as follows: [ka] It has one of the following or a stereoisomer thereof.

[0097] In a particular embodiment, [ka] The structure is as follows: [ka] [ka] It has one of the following.

[0098] In some embodiments, R a is substituted with one or more fluorine atoms. In some embodiments, R b is substituted with one or more fluorine atoms. In some embodiments, R c is substituted with one or more fluorine atoms. In some embodiments, R dis substituted with one or more fluorine atoms. In some embodiments, R e is substituted with one or more fluorine atoms. In some embodiments, R f is substituted with one or more fluorine atoms. In some embodiments, L 1 is substituted with one or more fluorine atoms. In some embodiments, L 2 is substituted with one or more fluorine atoms. In some embodiments, L 2a is substituted with one or more fluorine atoms. In some embodiments, L 2b is substituted with one or more fluorine atoms.

[0099] In various different embodiments, the compound has one of the structures described in Table 1 below.

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

[0100] One embodiment involves the following enantiomer pair of structure (I): [ka] Provides one of the racemic mixtures.

[0101] One embodiment is the following enantiomer pair or combination of structure (I): [ka] [ka] Provides one of the racemic mixtures.

[0102] It is understood that any embodiment of the compound of structure (I) above, and any particular substituent and / or variable in the compound of structure (I) above, may independently be combined with other embodiments and / or substituents and / or variables of the compound of structure (I) above to form embodiments of the present disclosure not specifically described above. Furthermore, if a list of substituents and / or variables is enumerated for any particular A, R, or L group in a particular embodiment and / or claim, it is understood that individual substituents and / or variables may be removed from the particular embodiment and / or claim, and the remaining list of substituents and / or variables is considered to be within the scope of the embodiments of the present disclosure.

[0103] In this specification, it is understood that combinations of substituents and / or variables in the given formulas are permissible only if such contributions result in a stable compound.

[0104] For the purpose of administration, the compounds of this disclosure (typically in the form of lipid nanoparticles combined with a therapeutic agent) may be administered as raw chemicals or formulated as pharmaceutical compositions. The pharmaceutical compositions of this disclosure comprise the compound of structure (I) and one or more pharmaceutically acceptable carriers, diluents, or additives. The compound of structure (I) forms lipid nanoparticles and is present in the composition in an amount effective to deliver, for example, a therapeutic agent for treating a particular disease or condition of interest. Appropriate concentrations and doses can be readily determined by those skilled in the art.

[0105] One embodiment provides a composition comprising a compound of structure (I) and a therapeutic agent. In some embodiments, the composition further comprises one or more additives selected from neutral lipids, steroids, and polymer-conjugated lipids.

[0106] In some embodiments, the therapeutic agent comprises nucleic acid. In certain embodiments, the nucleic acid is selected from antisense and messenger RNA.

[0107] In certain embodiments, the composition 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 in the range of about 2:1 to about 8:1. In certain embodiments, the steroid is D cholesterol. In some embodiments, the molar ratio of the compound to cholesterol is in the range of about 2:1 to 1:1. In certain embodiments, the polymer conjugate lipid is D pegylated lipid. In various embodiments, the polymer conjugate lipid is D pegylated lipid. For example, some embodiments include pegylated diacylglycerol (PEG-DAG), e.g., 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), pegylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG), e.g., 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanediate (PEG-S-DMG), pegylated ceramide (PEG-cer), or PE The present invention includes G-dialkoxypropyl carbamates, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecaneoxy)propyl)carbamate or 2,3-di(tetradecaneoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. In some embodiments, the molar ratio of the compound to the pegylated lipid is in the range of about 100:1 to about 10:1 or about 100:1 to about 25:1. In some embodiments, the pegylated lipid is PEG-DMG. In certain embodiments, the pegylated lipid has the following structure (II): [ka] (II) [In the formula, R10 and R 11 Each is independently a linear or branched alkyl, alkenyl, or alkynyl containing 10 to 30 carbon atoms, and the alkyl, alkenyl, or alkynyl may be cleaved by one or more ester bonds; and w has a value in the range of 30 to 60. It has pharmaceutically acceptable salts, tautomers, or stereoisomers thereof.

[0108] In some embodiments, R 10 and R 11 Each of these is an unbranched alkyl chain containing 12 to 16 carbon atoms.

[0109] In certain embodiments, the lipid nanoparticles or composition comprises compounds of multiple structure(II) forms, and the average value of w for the plurality is about 49. In some embodiments, the compounds of multiple structure(II) forms have an average value of w in the range of 40 to 55. In some embodiments, the compounds of multiple structure(II) forms have an average value of w in the range of 40 to 50 or 42 to 48. In some embodiments, the compounds of multiple structure(II) forms have an average value of w in the range of 30 to 55, 30 to 50, 30 to 45, 30 to 40, or 30 to 35. In some embodiments, the compounds of multiple structure(II) forms have an average value of w in the range of 35 to 55, 40 to 55, 42 to 55, 45 to 55, or 48 to 55. In some embodiments, the average w is about 45 (e.g., 43, 44, 45, 46, or 47). In some embodiments, the average w is about 43 to 47. In some embodiments, the average w is about 40 to 50. In some embodiments, the average w is approximately 48-55.

[0110] The synthesis of pegylated lipids can be found in U.S. Patent No. 9,738,593, which is incorporated herein by attribution.

[0111] Method of administration The compositions of this disclosure may be administered by any acceptable mode of administration of a drug to provide similar utility. The pharmaceutical compositions of the present invention may be formulated in solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes for administering such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, non-enteral, sublingual, oral cavity, rectum, vagina, and nasal cavity. As used herein, the term non-enteral includes subcutaneous, intravenous, intramuscular, intradermal, intrasternal, intrasternal injection, or infusion techniques. The pharmaceutical compositions of this disclosure are formulated so that the active ingredients contained therein are bioavailable upon administration of the composition to a patient. The subject or composition administered to a patient may take the form of one or more dose units; for example, a tablet may be a single dose unit, and a container of the compound of this disclosure in aerosol form may hold multiple dose units. Practical methods for manufacturing such dosage forms are known or obvious to those skilled in the art; for example, Remington: The Science and Practice of Pharmacy, 20 th See Edition (Philadelphia College of Pharmacy and Science, 2000). The administered composition in any case contains a therapeutically effective amount of the compound of the Disclosure or a pharmaceutically acceptable salt thereof for treating the disease or condition of interest in accordance with the teachings of the Disclosure.

[0112] One embodiment provides a method for administering a therapeutic agent to a patient in need thereof, the method comprising preparing and providing lipid nanoparticles or compositions of the present disclosure, and administering the compositions to a patient.

[0113] One embodiment provides a pharmaceutical composition comprising lipid nanoparticles of the present disclosure and a pharmaceutically acceptable diluent or additive.

[0114] Another embodiment provides a method for inducing protein expression in a patient in need thereof, the method comprising administering a pharmaceutical composition of the present disclosure to the patient, wherein lipid nanoparticles contain mRNA encoding the protein. In some embodiments, the protein is an antigen, and the method is for inducing an immune response in the patient. In certain embodiments, the protein is an antigen, and the method is for vaccinating the patient against a pathogen. In some embodiments, the protein is for gene editing.

[0115] The pharmaceutical compositions of this disclosure may be in solid or liquid form. In one embodiment, the carrier is particulate, and therefore the composition may be, for example, in tablet or powder form. The carrier may also be liquid, and the composition may be, for example, an oral syrup, an injection, or an aerosol useful for, for example, inhalation administration.

[0116] When intended for oral administration, the pharmaceutical composition is preferably in either a solid or liquid form, where semi-solid, semi-liquid, suspension, and gel forms are included within the forms considered herein as either solid or liquid.

[0117] As solid compositions for oral administration, pharmaceutical compositions can be formulated in the form of powders, granules, compressed tablets, pills, capsules, chewing gum, wafers, etc. Such solid compositions typically contain one or more inert diluents or food carriers. In addition, one or more of the following may be present: binders, e.g., carboxymethylcellulose, ethylcellulose, crystalline cellulose, tragacanth gum, or gelatin; additives, e.g., starch, lactose, or dextrin; disintegrants, e.g., alginic acid, sodium alginate, Primogel, corn starch, etc.; lubricants, e.g., magnesium stearate, or Sterotex; flow enhancers, e.g., colloidal silicon dioxide; sweeteners, e.g., sucrose, or saccharin; flavorings, e.g., peppermint, methyl salicylate, or orange flavoring; and colorants.

[0118] If the pharmaceutical composition is in the form of a capsule, for example a gelatin capsule, it may contain a liquid carrier, such as polyethylene glycol or oil, in addition to the above-mentioned types of materials.

[0119] Pharmaceutical compositions may be in the form of liquids, such as elixirs, syrups, solutions, emulsions, or suspensions. Liquids may, as two examples, be for oral administration or for delivery by injection. When intended for oral administration, preferred compositions include, in addition to the compounds of the present invention, one or more sweeteners, preservatives, colorants, and flavor enhancers. Compositions intended for administration by injection may include one or more surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents.

[0120] The liquid pharmaceutical compositions of this disclosure, whether in solution, suspension or other similar form, may comprise one or more of the following adjuvants: sterile diluents, e.g., water for injection, physiological saline, preferably physiological saline, Ringer's solution, isotonic sodium chloride; fixing oils, e.g., synthetic mono or diglycerides that can function as a solvent or suspension medium, polyethylene glycol, glycerin, propylene glycol or other solvents; antimicrobial agents, e.g., benzyl alcohol or methylparaben; antioxidants, e.g., ascorbic acid or sodium bisulfite; chelating agents, e.g., ethylenediaminetetraacetic acid; buffers, e.g., acetates, citrates or phosphates and substances for adjusting tonicity, e.g., sodium chloride or dextrose; substances acting as antifreeze, e.g., sucrose or trehalose. Non-enteral formulations may be enclosed in glass or plastic ampoules, disposable syringes or multi-dose vials. Physiological saline is a preferred adjuvant. The injectable pharmaceutical compositions are preferably sterile.

[0121] Liquid pharmaceutical compositions of the present disclosure intended for either enteral or oral administration should contain an amount of the compound of the present disclosure such that an appropriate dose is obtained.

[0122] The pharmaceutical compositions of this disclosure may be intended for topical administration, in which case the carrier may appropriately comprise a solution, emulsion, ointment, or gel base. The base may comprise, for example, one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickeners may be present in the pharmaceutical compositions for topical administration. If intended for transdermal administration, the composition may comprise a transdermal patch or an iontophoresis device.

[0123] The pharmaceutical compositions of this disclosure may be intended for rectal administration, for example, in the form of suppositories that dissolve in the rectum to release the drug. Compositions for rectal administration may include an oily base as a suitable non-irritating additive. Examples of such bases include, but are not limited to, lanolin, cocoa butter, and polyethylene glycol.

[0124] The pharmaceutical compositions of this disclosure may include a variety of materials that alter the physical form of a solid or liquid dosage unit. For example, the composition may include a material that forms a coating shell around the active ingredient. The material forming the coating shell is typically inert and may be selected from, for example, sugars, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule.

[0125] Pharmaceutical compositions of the present disclosure, in solid or liquid form, may include substances that bind to the compounds of the present disclosure, thereby assisting in the delivery of the compounds. Suitable substances that may act in this capacity include monoclonal or polyclonal antibodies or proteins.

[0126] The pharmaceutical compositions of this disclosure may consist of dosing units that can be administered as aerosols. The term aerosol is used to refer to a variety of systems, ranging from colloidal in nature to systems consisting of pressurized packages. Delivery may be by liquefaction or compressed gas, or by a suitable pump system for distributing the active ingredient. Aerosols of the compounds of this disclosure may be delivered in single-phase, two-phase, or three-phase systems for delivering the active ingredient. Delivery of the aerosol may involve necessary containers, activators, valves, sub-containers, etc., which together may form a kit. Those skilled in the art can determine a preferred aerosol without excessive experimentation.

[0127] The pharmaceutical compositions of this disclosure may be manufactured by methods well known in the pharmaceutical field. For example, a pharmaceutical composition intended to be administered by injection may be manufactured by combining the lipid nanoparticles of this disclosure with sterile distilled water or other carriers to form a solution. Surfactants may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that interact non-covalently with the compounds of this disclosure to promote the dissolution or homogeneous suspension of the compounds in an aqueous delivery system.

[0128] The compositions of this disclosure or pharmaceutically acceptable salts thereof are administered in therapeutically effective doses, which vary depending on various factors including the activity of the particular 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 mode and timing of administration; the rate of excretion; the combination of drugs; the severity of a particular disorder or condition; and the subject being treated.

[0129] The compositions of the present disclosure may also be administered simultaneously with, before, or after the administration of one or more other therapeutic agents. Such combination therapy includes the administration of a single drug formulation of the compositions of the present disclosure and one or more additional active substances, and the administration of the compositions of the present disclosure and each active substance in its own separate drug formulation. For example, the compositions of the present disclosure and the other active substances may be administered together to a patient in a single oral formulation such as a tablet or capsule, or each substance may be administered in a separate oral formulation. When separate formulations are used, the compounds of the present disclosure and one or more additional active substances may be administered at essentially the same time, i.e., simultaneously, or separately at staggered times, i.e., sequentially, and combination therapy is understood to include all such regimens.

[0130] Methods for producing the above compounds and compositions are described below herein and / or are known in the art.

[0131] It will be understood by those skilled in the art that in the methods described herein, the functional groups of the intermediate compounds may need to be protected by appropriate protecting groups. Such functional groups include hydroxy, amino, mercapto, and carboxylic acids. Suitable protecting groups for hydroxyls include trialkylsilyl or diarylalkylsilyl (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl, or trimethylsilyl), tetrahydropyranyl, and benzyl. Suitable protecting groups for amino, amidino, and guanidinos include t-butoxycarbonyl and benzyloxycarbonyl. Suitable protecting groups for mercaptos include -C(O)-R'' (wherein R'' is alkyl, aryl, or arylalkyl), p-methoxybenzyl, and trityl. Suitable protecting groups for carboxylic acids include alkyl, aryl, or arylalkyl esters. Protecting groups are known to those skilled in the art and can be added or removed according to standard techniques such as those described herein. For the use of protecting groups, see Green, TW and PGM Wutz, Protective Groups in Organic Synthesis (1999), 3 rd This is described in detail in Ed., Wiley. As those skilled in the art will understand, the protecting group may also be a polymer resin, such as Wang resin, Rink resin, or 2-chlorotrityl chloride resin.

[0132] Such protected derivatives of the compounds of this disclosure may not have pharmacological activity on their own, but it will be understood by those skilled in the art that they may be administered to mammals and subsequently metabolized in the body to form pharmacologically active compounds of this disclosure. Therefore, such derivatives may be described as “prodrugs.” All prodrugs of the compounds of this disclosure are included within the scope of this disclosure.

[0133] Furthermore, all compounds of the present disclosure, existing in the form of free bases or acids, can be converted to their pharmaceutically acceptable salts by treatment with a suitable inorganic or organic base or acid using methods known to those skilled in the art. Salts of the compounds of the present disclosure can be converted to their free base or acid forms by standard techniques.

[0134] The following reaction scheme applies to the compound of this disclosure, i.e., structure (I): [ka] (I) [In the formula, R 1 , R 1 , L 1 , L 2 , L 2a , L 2b And A is as defined in the specification. The following illustrates a method for producing the compounds shown. Those skilled in the art will understand that these compounds can be produced by similar methods or by combining them with other methods known to those skilled in the art. Those skilled in the art will also understand that other compounds of structure (I) not specifically shown below can be produced in a similar manner to that described below, by using appropriate starting components and, as necessary, modifying the parameters of the synthesis. Generally, starting components may be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, or synthesized according to sources known to those skilled in the art (e.g., Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5 th They may be manufactured as described in this disclosure (see edition (Wiley, December 2000)) or as described herein.

[0135] [ka] Embodiments of the compound of structure (I) can be prepared according to general reaction scheme 1 ("Procedure A").

[0136] Procedure A Regarding general reaction scheme 1, compound starting material A1 can be purchased from a commercially available source or prepared according to methods well known to those skilled in the art. A2 was added to the mixture of starting material A1, and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated and used in the next step without further purification. A2 was added to the mixture of starting material A1, and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated and used in the next step without further purification. A3 was added to the mixture, and the reaction mixture was stirred at room temperature for 17 hours. The reaction mixture was concentrated, and the crude substance was resuspended in hexane. The resulting solid was filtered, and the filtrate was purified by autoflash chromatography to obtain A4. A mixture of A4 and a suitable amine was heated at 75°C for 24-72 hours. The reaction mixture was concentrated, the resulting solid was filtered, and the filtrate was purified by autoflash chromatography to obtain the compound of the desired structure (I).

[0137] It should be noted that various alternative strategies for the preparation of compounds of structure (I) are available to those skilled in the art. For example, other compounds of structure (I) may be prepared using suitable starting materials according to similar methods, as shown in the following examples. The use of protecting groups as needed and other modifications to the general reaction scheme described above will be readily apparent to those skilled in the art. The following examples are provided for illustrative purposes only and are not limiting. [Examples]

[0138] Example 1 In vivo evaluation of luciferase mRNA using lipid nanoparticle compositions The lipids of structure (I), DSPC, cholesterol, and PEG-lipids are solubilized in ethanol at a molar ratio of 50:10:38.5:1.5 or 47.5:10:40.7:1.8. Lipid nanoparticles (LNPs) are prepared with a total lipid to mRNA weight ratio of approximately 10:1 to 40:1. Briefly, mRNA is diluted to 0.2 mg / mL in 10-50 mM citrate buffer at pH 4-6 or 10-25 mM acetate buffer at pH 4. Using a syringe pump, the ethanol-soluble lipid solution is mixed with the mRNA aqueous solution at a ratio of approximately 1:5 to 1:3 (vol / vol) at a total flow rate of more than 15 mL / min. Then, the ethanol is removed and the external buffer is replaced with PBS by dialysis. Finally, the lipid nanoparticles are filtered through a sterile filter with a 0.2 μm pore size.

[0139] The experiments will be conducted in 6-8 week old female C57BL / 6 mice (Charles River) and 8-10 week old CD-1 mice (Charles River or Inotiv), following guidelines established by the Institutional Animal Care Committee (ACC) and the Canadian Council on Animal Care (CCAC). Various doses of mRNA-lipid nanoparticles will be administered systemically by tail vein injection, and the animals will be euthanized at a specific time point after administration (e.g., 4 hours). The liver and spleen will be collected in pre-weighed tubes, weighed, immediately rapid-frozen in liquid nitrogen, and stored at -80°C until processing for analysis.

[0140] For liver tissue, approximately 50 mg is cut into 2 mL FastPrep tubes (MP Biomedicals, Solon OH) for analysis. A 1 / 4" ceramic sphere (MP Biomedicals) is added to each tube, and 500-750 μL of Glo Lysis Buffer-GLB (Promega, Madison WI), equilibrated to room temperature, is added to the liver tissue. The liver tissue is homogenized using a FastPrep24 instrument (MP Biomedicals) at 2 × 6.0 m / s for 15 seconds. After incubating the homogenate at room temperature for 5 minutes, it is diluted 1:4-1:6 in GLB and evaluated using the SteadyGlo Luciferase assay system (Promega). Specifically, 50 μL of the diluted tissue homogenate is reacted with 50 μL of SteadyGlo substrate, shaken for 10 seconds, incubated for 5 minutes, and then tested on a FilterMax F5 Microplate Reader (Molecular Luminescence was quantified using a device (USA). The amount of protein being assayed was determined using the BCA protein assay kit (Pierce, Rockford, IL). Then, relative luminescence units (RLU) were normalized to the total μg protein or weight (g) of the assayed tissue. A standard curve was created using QuantiLum Recombinant Luciferase (Promega) to convert RLU to ng luciferase.

[0141] FLuc mRNA (L-7202) (Trilink Biotechnologies) expresses the luciferase protein originally isolated from the firefly, *Photinus pyralis*. Fluc is commonly used in mammalian cell culture to measure both gene expression and cell viability. It emits bioluminescence in the presence of its substrate, luciferin. This capped, polyadenylated mRNA is modified with 5-methoxyuridine and optimized for mammalian systems.

[0142] Example 2 In vivo evaluation of immunoglobulin G (IgG) mRNA using lipid nanoparticle compositions The lipids of structure (I), DSPC, cholesterol, and PEG-lipids are solubilized in ethanol at a molar ratio of 50:10:38.5:1.5 or 47.5:10:40.7:1.8. Lipid nanoparticles (LNPs) are prepared with a total lipid to mRNA weight ratio of approximately 10:1 to 40:1. Briefly, mRNA is diluted to 0.2 mg / mL in 10-50 mM citrate buffer at pH 4-6 or 10-25 mM acetate buffer at pH 4-6. Using a syringe pump, the ethanol-soluble lipid solution is mixed with the mRNA aqueous solution at a ratio of approximately 1:5 to 1:3 (vol / vol) at a total flow rate of more than 15 mL / min. Then, the ethanol is removed and the external buffer is replaced with PBS by dialysis. Finally, the lipid nanoparticles are filtered through a sterile filter with a 0.2 μm pore size.

[0143] The experiments are conducted in 6-8 week old CD-1 / ICR mice (Envigo, Charles River, or Inotiv) according to guidelines set forth by the Institutional Animal Care Committee (ACC) and the Canadian Council on Animal Care (CCAC). Various doses of mRNA-lipid nanoparticles are administered systemically by tail vein injection, and the animals are euthanized at a specific time point (e.g., 24 hours) after administration. Whole blood is collected, and serum is separated by centrifugation of the whole blood tube at 2000×g for 10 minutes at 4°C, and stored at -80°C until ready for analysis.

[0144] For immunoglobulin G (IgG) ELISA (Life Diagnostics Human IgG ELISA kit), dilute the serum sample 100 to 20,000 times with 1× diluent. Dispense 100 μL of diluted serum, along with the human IgG standard, into two duplicates in a 96-well plate coated with anti-human IgG, and incubate in a plate shaker at 150 rpm at 25°C for 45 minutes. Wash the wells five times with 1× washing solution using a plate washer (400 μL / well). Add 100 μL of HRP conjugate to each well and incubate in a plate shaker under the same conditions as above. Wash the wells again five times with 1× washing solution using a plate washer (400 μL / well). Add 100 μL of TMB reagent to each well and incubate in a plate shaker under the same conditions as above. Stop the reaction by adding 100 μL of stop solution to each well. Absorbance is read at 450 nm (A450) using a microplate reader. The amount of human IgG in mouse serum is determined by plotting the A450 value of the assay standard against the human IgG concentration.

[0145] Example 3 Determination of pKa of formulated lipids As described elsewhere, the pKa of formulated lipids correlates with the efficacy of LNPs for nucleic acid delivery (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176 (2010)). In some embodiments, the preferred range of pKa is about 5 to about 7. The pKa of each lipid can be determined in lipid nanoparticles using a fluorescence-based assay of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). Lipid nanoparticles containing compound / DSPC / cholesterol / PEG-lipid of structure (I) (50 / 10 / 38.5 / 1.5 or 47.5:10:40.7:1.8 mol%) in PBS at a total lipid concentration of 0.4 mM are prepared using the in-line process described in Example 1. TNS is prepared as a 100 μM stock solution in distilled water. The vesicles are diluted to 24 μM lipid in 2 mL of buffer solution (pH in the range of 2.5 to 11) containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl. Aliquots of the TNS solution are added to a final concentration of 1 μM, and after vortex mixing, the fluorescence intensity is measured at room temperature using an SLM Aminco Series 2 Luminescence Spectrophotometer with excitation and emission wavelengths of 321 nm and 445 nm. Sigmoid best fit analysis is applied to the fluorescence data, and the pKa is measured at the pH that produces the maximum half of the fluorescence intensity.

[0146] Example 4 Determination of the efficacy of lipid nanoparticle formulations containing various cationic lipids using an in vivo luciferase / IgG mRNA expression rodent model. The representative compounds of this disclosure shown in Table 2 were formulated using the following molar ratios: 50% cationic lipid / 10% distearoyl phosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid 2-[2-(ω-methoxy(polyethylene glycol)] 2000(ethoxy)-N,N-ditetradecylacetamide) or 47.5% cationic lipid / 10% DSPC / 40.7% cholesterol / 1.8% PEG lipid. Relative activity was determined by measuring luciferase expression in the liver 4 hours after administration by tail vein injection as described in Example 1, or by measuring the amount of human IgG in mouse serum as described in Example 2. Activity was compared at doses of 1.0, 0.5, or 0.3 mg mRNA / kg and expressed as ng luciferase / g liver measured 4 hours after administration as described in Example 1, or as μg IgG / mL serum measured 24 hours after administration as described in Example 2. Compound numbers in Table 2 refer to compound numbers in Table 1. [Table 2-1] [Table 2-2]

[0147] Example 5 Synthesis routes of compounds I-1 to I-18 [ka]

[0148] Synthesis of 8-bromo-N,N-didecyloctanamide (intermediate 1) To a mixture of 8-bromooctanoic acid (47.3 mmol, 10.5 g) and DMF (cat.) in DCM (100 mL), oxalyl chloride (142 mmol, 18.0 g) was added, and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated to obtain 8-bromooctanoyl chloride, which was used in the next step without further purification.

[0149] A mixture of didecylamine (69.1 mmol, 20.6 g), triethylamine (377 mmol, 52.5 mL), and N,N-dimethylpyridine-4-amine (catalytic amount 8.0 mg) in 100 mL of DCM was mixed with 8-bromooctanoyl chloride (62.8 mmol, 15.2 g) in 60 mL of DCM, and the reaction mixture was stirred at room temperature for 17 hours. The reaction mixture was concentrated, and the crude product was resuspended in hexane. The resulting solid was filtered, and the filtrate was purified by auto-flash chromatography (5% to 25% siRNA in hexane) to obtain 8-bromo-N,N-didecyloctanamide (26 g, 82%).

[0150] General Procedure A A mixture of 8-bromo-N,N-didecyloctanamide (1.0-2.0 equivalents), a suitable amine (1.0 equivalent), diisopropylethylamine ("DIEA", 3.0-5.0 equivalents), and potassium iodide (0.0-3.0 equivalents) in ACN (0.1M) was heated at 75°C for 24-72 hours. The reaction mixture was concentrated, and the crude product was suspended in a mixture of hexane, ethyl acetate, and Et3N (95:5:1). The resulting solid was filtered, and the filtrate was purified by automated flash chromatography to obtain the desired compound.

[0151] General Procedure B A mixture of 8-bromo-N,N-didecyloctanamide (1.0-2.0 equivalents), a suitable amine (1.0 equivalent), DIEA (3.0-5.0 equivalents), and potassium iodide (3.0-4.0 equivalents) in ACN (0.5M) was heated by microwave irradiation at 140-160°C for 30 minutes to 3 hours. The reaction mixture was concentrated, and the crude product was suspended in a mixture of hexane:siRNA:Et3N (95:5:1). The resulting solid was filtered, and the filtrate was purified by automated flash chromatography to obtain the desired compound.

[0152] Example 6 8,8'-((2-((1s,3r)-3-hydroxycyclobutyl)ethyl)azandiyl)bis(N,N-didecyloctanamide)(compound (1s,3r)-I-1) [ka]

[0153] Synthesis of (1S,3R)-I-1 Compound (1S,3R)-I-1 was synthesized using (1r,3s)-3-(2-aminoethyl)cyclobutan-1-ol according to general procedure A. The title compound was obtained by purification using automated flash chromatography (5-65% ethyl hexane containing 1% Et3N) (yield 12%). 1 H NMR(400 MHz, CDCl3)δ 4.-3-4.03(m, 1H), 3.32-3.23(m, 4H), 3.23-3.15(m, 4H), 2.52-2.41(m, 2H), 2.39-2.22(m, 10H), 1.72-1.45(m, 22H), 1.44-1.16(m, 72H), 0.95-0.83(m, 12H). Chemical formula:C 62 H 123 Calculated m / z value for N3O3 = 958.0. Measured value [M+H] + =959.1.

[0154] Example 7 8,8'-((((1s,3s)-3-hydroxycyclobutyl)methyl)azandiyl)bis(N,N-didecyloctanamide)(compound(1s,3s)-I-2) [ka]

[0155] (1S,3S)-I-2 synthesis Compound (1S,3S)-I-2 was synthesized using (1s,3s)-3-(aminomethyl)cyclobutan-1-ol according to general procedure A. The compound was purified by automated flash chromatography (5-65% ELISA in hexane containing 1% Et3N) to obtain the title compound (yield 44%). 1H NMR(600 MHz, CDCl3)δ 4.18-4.10(m, 1H), 3.33-3.27(m, 4H), 3.24-3.19(m, 4H), 2.52-2.44(m, 4H), 2.40-2.34(m, 4H), 2.32-2.26(m, 4H), 1.96-1.88(m, 1H), 1.69-1.60(m, 5H), 1.60-1.48(m, 11H), 1.46-1.38(m, 4H), 1.38-1.19(m, 69H), 0.93-0.87(m, 12H). Chemical formula:C 61 H 121 Calculated m / z value for N3O3 = 943.9. Measured value [M+H] + =945.0.

[0156] Example 8 8,8'-((((1r,3r)-3-hydroxycyclobutyl)methyl)azandiyl)bis(N,N-didecyloctanamide)(compound (1r,3r)-I-2) [ka]

[0157] (1R,3R)-I-2 synthesis Compound (1R,3R)-I-2 was synthesized using (1s,3s)-3-(aminomethyl)cyclobutan-1-ol according to general procedure A. The compound was purified by automated flash chromatography (5-65% ELISA in hexane containing 1% Et3N) to obtain the title compound (yield 40%). 1 H NMR(600 MHz, CDCl3)δ 4.44-4.36(m, 1H), 3.33-3.27(m, 4H), 3.24-3.19(m, 4H), 2.41(s, 3H), 2.39-2.33(m, 4H), 2.32-2.26(m, 4H), 2.13-1.99(m, 4H), 1.69-1.61(m, 7H), 1.60-1.48(m, 8H), 1.45-1.20(m, 74H), 0.94-0.87(m, 12H). Chemical formula:C 61 H 121Calculated m / z value for N3O3 = 943.9. Measured value [M+H] + =944.9.

[0158] Example 9 8,8'-((((1r,3s)-3-hydroxycyclohexyl)methyl)azandiyl)bis(N,N-didecyloctanamide) 8,8'-((((1s,3r)-3-hydroxycyclohexyl)methyl)azandiyl)bis(N,N-didecyloctanamide) (Compound (rac)-(1rs,2sr)-I-3) [ka]

[0159] (rac)-(1RS,2SR)-I-3 synthesis A mixture of compound I-3 enantiomers was synthesized according to general procedure A using a mixture of (1R,3S)-3-(aminomethyl)cyclohexane-1-ol and (1S,3R)-3-(aminomethyl)cyclohexane-1-ol. The mixture was purified by automated flash chromatography (5-65% ethyl phosphate in hexane containing 1% Et3N), followed by a second purification by automated flash chromatography (10-100% ethyl phosphate in hexane) to obtain the title compound (yield 27%). 1 H NMR(400 MHz, CDCl3)δ 3.63-3.51(m, 1H), 3.33-3.24(m, 4H), 3.25-3.14(m, 4H), 2.40-2.21(m, 8H), 2.20-2.08(m, 3H), 2.01-1.93(m, 1H), 1.77(dt, J= 13.0, 3.4 Hz, 1H), 1.71-1.59(m, 10H), 1.57-1.44(m, 10H), 1.42-1.06(m, 80H), 0.97-0.64(m, 15H). Chemical formula:C 63 H 125 Calculated m / z value for N3O3 = 972.0. Measured value [M+H] + =973.1.

[0160] Example 10 8,8'-(((3-hydroxycyclopentyl)methyl)azandiyl)bis(N,N-didecyloctanamide)(Compound I-4) [ka]

[0161] I-4 Synthesis Compound I-4 was synthesized using 3-(aminomethyl)cyclopentan-1-ol according to general procedure A. It was purified by automated flash chromatography (5-65% ethylethanol in hexane containing 1% Et3N), followed by a second purification by automated flash chromatography (5-100% ethylethanol in hexane) to obtain the title compound (yield 39%). 1 H NMR(400 MHz, CDCl3)δ 4.39-4.31(m, 1H), 4.29-4.20(m, 1H), 3.31-3.24(m, 4H), 3.23-3.14(m, 4H), 2.45-2.30(m, 6H), 2.29-2.21(m, 5H), 2.18-1.97(m, 1H), 1.97-1.84(m, 1H), 1.86-1.08(m, 96H), 0.94-0.83(m, 13H). Chemical formula:C 62 H 123 Calculated m / z value for N3O3 = 958.0. Measured value [M+H] + =959.1.

[0162] Example 11 8,8'-(((1r,3r)-3-hydroxycyclobutyl)azandiyl)bis(N,N-didecyloctanamide)(compound (1r,3r)-I-5) [ka]

[0163] (1R,3R)-I-5 synthesis Compound (1R,3R)-I-5 was synthesized using (1r,3r)-3-aminocyclobutan-1-ol hydrochloride according to general procedure A. It was purified by automated flash chromatography (5-65% ethyl acetate in hexane containing 1% Et3N), followed by a second purification by automated flash chromatography (5-100% ethyl acetate in hexane) to obtain the title compound (yield 10%). 1 H NMR(400 MHz, CDCl3)δ 4.43-4.34(m, 1H), 3.49-3.37(m, 1H), 3.32-3.23(m, 4H), 3.23-3.15(m, 4H), 2.30-2.22(m, 4H), 2.22-2.12(m, 2H), 2.08-1.97(m, 2H), 1.79-1.69(m, 1H), 1.68-1.56(m, 7H), 1.57-1.43(m, 8H), 1.44-1.14(m, 75H), 0.92-0.83(m, 12H). Chemical formula:C 60 H 119 Calculated m / z value for N3O3 = 929.9. Measured value [M+H] + =931.1.

[0164] Example 12 8,8'-((((1S,2S)-2-hydroxycyclohexyl)methyl)azandiyl)bis(N,N-didecyloctanamide) 8,8'-((((1R,2R)-2-hydroxycyclohexyl)methyl)azandiyl)bis(N,N-didecyloctanamide) (Compound (rac)-(1RS,2RS)-I-6) [ka]

[0165] (rac)-(1RS,2RS)-I-6 synthesis A mixture of compound I-6 enantiomers was synthesized according to general procedure A using a mixture of (1R,2R)-2-(aminomethyl)cyclohexane-1-ol hydrochloride and (1S,2S)-2-(aminomethyl)cyclohexane-1-ol hydrochloride. The mixture was purified by automated flash chromatography (10% to 100% butyl in hexane). Secondary purification was performed by automated flash chromatography (5% to 100% butyl in hexane) to obtain the title compound (yield 11%). 1 H NMR(400 MHz, CDCl3)δ 3.84-3.75(m, 1H), 3.32-3.23(m, 4H), 3.23-3.15(m, 4H), 2.96(dd, J= 13.1, 10.5 Hz, 1H), 2.60-2.48(m, 2H), 2.31-2.20(m, 7H), 2.17-2.07(m, 1H), 1.78-1.39(m, 24H), 1.36-1.18(m, 74H), 0.95-0.83(m, 12H). Chemical formula:C 63 H 125 N3O 3- Calculated m / z value = 972.0. Measured value [M+H] + =973.1.

[0166] Example 13 8,8'-((((1r,4r)-4-hydroxycyclohexyl)methyl)azandiyl)bis(N,N-didecyloctanamide)(compound (1r,4r)-I-7) [ka]

[0167] (1R,4R)-I-7 synthesis Compound (1R,4R)-I-7 was synthesized using (1r,4r)-4-(aminomethyl)cyclohexane-1-ol hydrochloride according to general procedure A. It was purified by automated flash chromatography (5% to 100% ethylethanol in hexane). Secondary purification was performed by automated flash chromatography (2% to 10% MeOH in DCM) to obtain the title compound (yield 28%). 1H NMR(400 MHz, CDCl3)δ 3.59-3.49(m, 1H), 3.32-3.23(m, 4H), 3.23-3.15(m, 4H), 2.35-2.23(m, 8H), 2.11(d, J= 6.9 Hz, 2H), 2.03-1.92(m, 2H), 1.89-1.79(m, 2H), 1.69-1.59(m, 7H), 1.57-1.46(m, 8H), 1.45-1.14(m, 79H), 0.94-0.81(m, 15H). Chemical formula:C 63 H 125 Calculated m / z value for N3O3 = 972.0. Measured value [M+H] + =973.0.

[0168] Example 14 8,8'-((((1s,4s)-4-hydroxycyclohexyl)methyl)azandiyl)bis(N,N-didecyloctanamide)(compound(1s,4s)-I-7) [ka]

[0169] (1S,4S)-I-7 synthesis Compound (1S,4S)-I-7 was synthesized using (1s,4s)-4-(aminomethyl)cyclohexane-1-ol hydrochloride according to general procedure A. The compound was purified by automated flash chromatography (5% to 100% ethylethanol in hexane) to obtain the title compound (yield 26%). 1 H NMR(400 MHz, CDCl3)δ 3.95(s, 1H), 3.32-3.23(m, 4H), 3.23-3.15(m, 4H), 2.36-2.22(m, 8H), 2.17(d, J= 6.8 Hz, 2H), 1.73-1.43(m, 24H), 1.43-1.15(m, 81H), 0.94-0.82(m, 14H). Chemical formula:C 63 H 125 Calculated m / z value for N3O3 = 972.0. Measured value [M+H] + =973.1.

[0170] Example 15 8,8'-((((1R,2S)-2-hydroxycyclohexyl)methyl)azandiyl)bis(N,N-didecyloctanamide)(compound(1R,2R)-I-8) [ka]

[0171] (1R,2S)-I-8 synthesis Compound (1R,2S)-I-8 was synthesized using (1S,2R)-2-(aminomethyl)cyclohexane-1-ol hydrochloride according to general procedure A. It was purified by automated flash chromatography (5-65% ethyl phosphate in hexane containing 1% Et3N). Secondary purification was performed by automated flash chromatography (5-100% ethyl phosphate in hexane) to obtain the title compound (yield 22%). 1 H NMR(400 MHz, CDCl3)δ 3.41-3.32(m, 1H), 3.32-3.24(m, 4H), 3.23-3.15(m, 4H), 2.64-2.54(m, 2H), 2.49-2.40(m, 1H), 2.33-2.23(m, 5H), 2.22-2.11(m, 2H), 1.96-1.87(m, 1H), 1.74-1.57(m, 8H), 1.57-1.38(m, 15H), 1.37-1.16(m, 75H), 0.90-0.9(m, 15H). Chemical formula:C 63 H 125 Calculated m / z value for N3O3 = 972.0. Measured value [M+H] + =973.1.

[0172] Example 16 8,8'-(((1r,4r)-4-hydroxycyclohexyl)azandiyl)bis(N,N-didecyloctanamide)(compound (1r,4r)-I-9) [ka]

[0173] (1R,4R)-I-9 synthesis Compound (1R,4R)-I-9 was synthesized using (1r,4r)-4-aminocyclohexane-1-ol according to general procedure A. The title compound was obtained by purification by automated flash chromatography (5% to 100% ethyl acetate in hexane) (yield 42%). 1 H NMR(400 MHz, CDCl3)δ 3.60-3.49(m, 1H), 3.32-3.24(m, 4H), 3.22-3.14(m, 4H), 2.49-2.40(m, 1H), 2.40-2.33(m, 4H), 2.31-2.22(m, 4H), 2.05-1.94(m, 2H), 1.81-1.71(m, 2H), 1.68-1.58(m, 6H), 1.57-1.45(m, 9H), 1.44-1.19(m, 82H), 0.94-0.81(m, 13H). Chemical formula:C 62 H 123 Calculated m / z value for N3O3 = 958.0. Measured value [M+H] + =959.0.

[0174] Example 17 8,8'-(((1s,4s)-4-hydroxycyclohexyl)azandiyl)bis(N,N-didecyloctanamide)(compound (1s,4s)-I-9) [ka]

[0175] (1S,4S)-I-9 synthesis Compound (1S,4S)-I-9 was synthesized using (1s,4s)-4-aminocyclohexane-1-ol according to general procedure A. The title compound was obtained by purification by automated flash chromatography (5% to 100% ethyl acetate in hexane) (yield 42%). 1H NMR(400 MHz, CDCl3)δ 4.01-3.95(m, 1H), 3.32-3.23(m, 4H), 3.23-3.15(m, 4H), 2.49-2.38(m, 5H), 2.26(t, J= 7.6 Hz, 4H), 1.89-1.77(m, 2H), 1.71-1.58(m, 8H), 1.57-1.44(m, 13H), 1.44-1.17(m, 77H), 0.92-0.82(m, 13H). Chemical formula:C 62 H 123 Calculated m / z value for N3O3 = 958.0. Measured value [M+H] + =959.0.

[0176] Example 18 8,8'-(((1s,3s)-3-hydroxycyclobutyl)azandiyl)bis(N,N-didecyloctanamide)(compound (1s,3s)-I-10) [ka]

[0177] (1S,3S)-I-10 synthesis Compound (1S,3S)-I-10 was synthesized using (1s,3s)-3-aminocyclobutan-1-ol hydrochloride according to general procedure A. The compound was purified by automated flash chromatography (5% to 100% ELISA in hexane) to obtain the title compound (yield 47%). 1 H NMR(400 MHz, CDCl3)δ 4.00-3.88(m, 1H), 3.32-3.24(m, 4H), 3.22-3.15(m, 4H), 2.64-2.45(m, 3H), 2.40-2.32(m, 4H), 2.31-2.22(s, 4H), 1.95-1.84(m, 1H), 1.82-1.58(m, 8H), 1.58-1.43(m, 8H), 1.43-1.18(m, 74H), 0.94-0.81(m, 12H). Chemical formula:C 60 H 119 Calculated m / z value for N3O3 = 929.9. Measured value [M+H] + =931.0.

[0178] Example 19 8,8'-(((1-hydroxycyclobutyl)methyl)azandiyl)bis(N,N-didecyloctanamide)(Compound I-11) [ka]

[0179] I-11 synthesis Compound I-11 was synthesized using 1-(aminomethyl)cyclobutan-1-ol according to general procedure A. The crude product was purified by automated reverse-phase flash chromatography (10% to 100% MeOH in water containing 0.1% TFA). The recovered fraction was concentrated and partitioned between ethyl acetate and saturated NaHCO3. Purification by automated flash chromatography (0% to 10% MeOH in DCM) yielded the title compound (yield 28%). 1 H NMR(400 MHz, CDCl3)δ 3.32-3.24(m, 4H), 3.23-3.15(m, 4H), 2.57(s, 2H), 2.52-2.42(m, 4H), 2.31-2.21(m, 4H), 2.20-2.09(m, 2H), 2.04-1.92(m, 2H), 1.86-1.72(m, 1H), 1.69-1.36(m, 19H), 1.36-1.17(m, 67H), 0.92-0.84(m, 12H). Chemical formula:C 61 H 121 Calculated m / z value for N3O3 = 943.9. Measured value [M+H] + =945.0.

[0180] Example 20 8,8'-(((3-hydroxyoxetan-3-yl)methyl)azandiyl)bis(N,N-didecyloctanamide)(Compound I-12) [ka]

[0181] I-12 synthesis Compound I-12 was synthesized using 1-(aminomethyl)cyclobutan-1-ol according to general procedure A. It was purified by automated flash chromatography (0% to 10% MeOH in DCM) to obtain the title compound (yield 54%). 1 H NMR(400 MHz, CDCl3)δ 4.72(d, J= 6.6 Hz, 2H), 4.47(d, J= 6.7 Hz, 2H), 3.32-3.25(m, 4H), 3.22-3.15(m, 4H), 2.82(s, 2H), 2.39(t, J= 7.5 Hz, 4H), 2.26(t, J= 7.6 Hz, 4H), 1.68-1.59(m, 4H), 1.57-1.46(m, 8H), 1.44-1.36(m, 4H), 1.36-1.18(m, 67H), 0.92-0.83(m, 12H). Chemical formula:C 60 H 119 Calculated m / z value for N3O4 = 945.9. Measured value [M+H] + = 946.9.

[0182] Example 21 8,8'-((2-(1-hydroxycyclobutyl)ethyl)azandiyl)bis(N,N-didecyloctanamide)(Compound I-13) [ka]

[0183] I-13 synthesis Compound I-13 was synthesized using 1-(2-aminoethyl)cyclobutan-1-ol according to general procedure A. It was purified by automated flash chromatography (0% to 10% MeOH in DCM) to obtain the title compound (yield 98%). 1 H NMR(400 MHz, CDCl3)δ 3.31-3.23(m, 4H), 3.22-3.14(m, 4H), 2.27(t, J= 7.4 Hz, 4H), 2.15-2.01(m, 6H), 1.81-1.44(m, 20H), 1.40-1.17(m, 71H), 0.94-0.81(m, 12H). Chemical formula:C 62 H123 Calculated m / z value for N3O3 = 958.0. Measured value [M+H] + = 958.9.

[0184] Example 22 8,8'-(((1R,3S)-3-hydroxycyclopentyl)azandiyl)bis(N,N-didecyloctanamide) 8,8'-(((1S,3R)-3-hydroxycyclopentyl)azandiyl)bis(N,N-didecyloctanamide) (Compound (rac)-(1RS,3SR)-I-14) [ka]

[0185] (rac)-(1RS,3SR)-I-14 synthesis A mixture of compound I-14 enantiomers was synthesized according to general procedure A using a mixture of (1S,3R)-3-aminocyclopentan-1-ol hydrochloride and (1R,3S)-3-aminocyclopentan-1-ol hydrochloride. The compound was purified by automated flash chromatography (5-65% toluene in hexane containing 1% Et3N) to obtain the title compound (yield 28%). 1 H NMR(400 MHz, CDCl3)δ 4.24-4.17(m, 1H), 3.32-3.23(m, 4H), 3.23-3.15(m, 4H), 3.07-3.00(m, 1H), 2.55-2.41(m, 4H), 2.26(t, J= 7.6 Hz, 4H), 1.92-1.05(m, 92H), 0.93-0.80(m, 12H). Chemical formula:C 61 H 121 Calculated m / z value for N3O3 = 943.9. Measured value [M+H] + =945.2.

[0186] Example 23 8,8'-(((1R,3R)-3-hydroxycyclopentyl)azandiyl)bis(N,N-didecyloctanamide) 8,8'-(((1S,3S)-3-hydroxycyclopentyl)azandiyl)bis(N,N-didecyloctanamide) (Compound (rac)-(1RS,3RS)-I-14) [ka]

[0187] (rac)-(1RS,3RS)-I-14 synthesis A mixture of compound I-14 enantiomers was synthesized according to general procedure A using a mixture of (1R,3R)-3-aminocyclopentan-1-ol hydrochloride and (1S,3S)-3-aminocyclopentan-1-ol hydrochloride. The compound was purified by automated flash chromatography (5-65% toluene in hexane containing 1% Et3N) to obtain the title compound (yield 33%). 1 H NMR(400 MHz, CDCl3)δ 4.39-4.32(m, 1H), 3.43-3.24(m, 5H), 3.23-3.15(m, 4H), 2.48-2.40(m, 4H), 2.31-2.22(m, 4H), 2.07-1.89(m, 2H), 1.88-1.77(m, 1H), 1.70-1.37(m, 23H), 1.37-1.16(m, 68H), 0.93-0.83(m, 12H). Chemical formula:C 61 H 121 Calculated m / z value for N3O3 = 943.9. Measured value [M+H] + =945.3.

[0188] Example 24 8,8'-(((1S,2S)-2-hydroxycyclopentyl)azandiyl)bis(N,N-didecyloctanamide)(compound (1S,2S)-I-15) [ka]

[0189] (1S,2S)-I-15 synthesis Compound (1S,2S)-I-15 was synthesized using (1S,2S)-2-aminocyclopentane-1-ol hydrochloride according to general procedure A. It was purified by automated flash chromatography (5-65% ethyl acetate in hexane containing 1% Et3N), followed by a second automated flash chromatography (10-100% ethyl acetate in hexane) to obtain the title compound (yield 12%). 1 H NMR(400 MHz, CDCl3)δ 3.94-3.83(m, 1H), 3.32-3.23(m, 4H), 3.23-3.15(m, 4H), 2.87-2.76(m, 1H), 2.52-2.33(m, 4H), 2.27(t, J= 7.6 Hz, 4H), 1.95-1.03(m, 94H), 0.96-0.81(m, 12H). Chemical formula:C 61 H 121 Calculated m / z value for N3O3 = 943.9. Measured value [M+H] + =945.3.

[0190] Example 25 8,8'-(((1S,2R)-2-hydroxycyclopentyl)azandiyl)bis(N,N-didecyloctanamide)(compound (1S,2R)-I-15) [ka]

[0191] (1S,2R)-I-15 synthesis Compound (1S,2R)-I-15 was synthesized using (1R,2S)-2-aminocyclopentane-1-ol hydrochloride according to general procedure A. It was purified by automated flash chromatography (5-65% ethyl acetate in hexane containing 1% Et3N), followed by a second automated flash chromatography (100% ethyl acetate) to obtain the title compound (yield 16%). 1H NMR(400 MHz, CDCl3)δ 4.01-3.97(m, 1H), 3.32-3.24(m, 4H), 3.23-3.15(m, 4H), 2.77-2.63(m, 1H), 2.62-2.49(m, 4H), 2.31-2.23(m, 4H), 1.92-1.05(m, 94H), 0.92-0.81(m, 12H). Chemical formula:C 61 H 121 Calculated m / z value for N3O3 = 943.9. Measured value [M+H] + =945.3.

[0192] Example 26 8,8'-(((1S,2S)-2-hydroxycyclohexyl)azandiyl)bis(N,N-didecyloctanamide)(compound (1S,2S)-I-16) [ka]

[0193] (1S,2S)-I-16 synthesis Compound (1S,2S)-I-16 was synthesized using (1S,2S)-2-aminocyclohexane-1-ol according to general procedure A. It was purified by automated flash chromatography (5-65% ethyl phosphate in hexane containing 1% Et3N), followed by a second automated flash chromatography (0-100% ethyl phosphate), and then by a third automated flash chromatography (0-10% MeOH in DCM) to obtain the title compound (yield 13%). 1 H NMR (400 MHz, CDCl3)δ 3.80-3.02(m, 10H), 2.61-2.18(m, 8H), 2.17-2.05(m, 2H), 2.02-1.00(m, 96H), 0.92-0.83(m, 12H). Chemical formula:C 62 H 123 Calculated m / z value for N3O3 = 958.0. Measured value [M+H] + =959.3.

[0194] Example 27 8,8'-(((1S,2R)-2-hydroxycyclohexyl)azandiyl)bis(N,N-didecyloctanamide)(compound (1S,2R)-I-16) [ka]

[0195] (1S,2R)-I-16 synthesis Compound (1S,2R)-I-16 was synthesized using (1R,2S)-2-aminocyclohexane-1-ol hydrochloride according to general procedure A. It was purified by automated flash chromatography (10% to 100% ethyl phosphate in hexane) and then by a second automated flash chromatography (5% to 65% ethyl phosphate in hexane containing 1% Et3N) to obtain the title compound (yield 17%). 1 H NMR(400 MHz, CDCl3)δ 3.94-3.86(m, 1H), 3.32-3.24(m, 4H), 3.23-3.15(m, 4H), 2.61-2.47(m, 4H), 2.37-2.31(m, 1H), 2.30-2.23(m, 4H), 2.05-1.97(m, 1H), 1.80-1.02(m, 97H), 0.94-0.84(m, 12H). Chemical formula:C 62 H 123 Calculated m / z value for N3O3 = 958.0. Measured value [M+H] + =959.3.

[0196] Example 28 8,8'-(((1R,3R)-3-hydroxycyclohexyl)azandiyl)bis(N,N-didecyloctanamide)(compound (1R,3R)-I-17) [ka]

[0197] (1R,3R)-I-17 synthesis Compound (1R,3R)-I-17 was synthesized using (1R,3R)-3-aminocyclohexane-1-ol hydrochloride according to general procedure A. The compound was purified by automated flash chromatography (5-65% ethylethanol in hexane containing 1% Et3N) to obtain the title compound (yield 29%). 1 H NMR(400 MHz, CDCl3)δ 4.27-4.19(m, 1H), 3.32-3.23(m, 4H), 3.23-3.15(m, 4H), 3.01-2.86(m, 1H), 2.38(t, J= 7.4 Hz, 4H), 2.31-2.22(m, 4H), 1.88-1.05(m, 97H), 0.92-0.83(m, 12H). Chemical formula:C 62 H 123 Calculated m / z value for N3O3 = 958.0. Measured value [M+H] + =959.4.

[0198] Example 29 8,8'-(((1S,3R)-3-hydroxycyclohexyl)azandiyl)bis(N,N-didecyloctanamide)(compound (1S,3R)-I-17) [ka]

[0199] (1S,3R)-I-17 synthesis Compound (1S,3R)-I-17 was synthesized using (1R,3S)-3-aminocyclohexane-1-ol hydrochloride according to general procedure A. The compound was purified by automated flash chromatography (5-65% ethylethanol in hexane containing 1% Et3N) to obtain the title compound (yield 36%). 1H NMR(400 MHz, CDCl3)δ 3.70-3.60(m, 1H), 3.32-3.24(m, 4H), 3.22-3.14(m, 4H), 2.61-2.51(m, 1H), 2.48-2.38(m, 4H), 2.31-2.22(m, 4H), 2.00-1.73(m, 3H), 1.70-1.01(m, 89H), 0.93-0.80(m, 12H). Chemical formula:C 62 H 123 Calculated m / z value for N3O3 = 958.0. Measured value [M+H] + = 959.2.

[0200] Example 30 8,8'-(((1R,2R)-2-hydroxycyclobutyl)azandiyl)bis(N,N-didecyloctanamide) 8,8'-(((1S,2S)-2-hydroxycyclobutyl)azandiyl)bis(N,N-didecyloctanamide) (Compound (rac)-(1RS,2RS)-I-18) [ka]

[0201] (rac)-(1RS,2RS)-I-18 synthesis A mixture of enantiomers of compound I-18 was synthesized according to general procedure A using a mixture of (1R,2R)-2-aminocyclobutan-1-ol hydrochloride and (1S,2S)-2-aminocyclobutan-1-ol hydrochloride. The compound was purified by automated flash chromatography (5-65% ethyl acetate in hexane containing 1% Et3N) to obtain the title compound (yield 30%). 1H NMR(400 MHz, CDCl3)δ 4.02-3.93(m, 1H), 3.32-3.22(m, 4H), 3.23-3.13(m, 4H), 2.90(q, J=8.0 Hz, 1H), 2.60-2.35(m, 4H), 2.26(t, J= 7.5 Hz, 4H), 2.16-2.06(m, 1H), 1.80(q, J=9.2 Hz, 1H), 1.74-1.05(m, 90H), 0.94-0.80(m, 12H). Chemical formula:C 60 H 119 Calculated m / z value for N3O3 = 929.9. Measured value [M+H] + =931.2.

[0202] Example 31 8,8'-(((1S,2R)-2-hydroxycyclobutyl)azandiyl)bis(N,N-didecyloctanamide) 8,8'-(((1R,2S)-2-hydroxycyclobutyl)azandiyl)bis(N,N-didecyloctanamide) (Compound (rac)-(1RS,2SR)-I-18) [ka]

[0203] (rac)-(1RS,2SR)-I-18 synthesis A mixture of enantiomers of compound I-18 was synthesized according to general procedure A using a mixture of (1R,2S)-2-aminocyclobutan-1-ol hydrochloride and (1S,2R)-2-aminocyclobutan-1-ol hydrochloride. The mixture was purified by automated flash chromatography (5-65% ethyl acetate in hexane containing 1% Et3N), followed by a second purification by reverse-phase flash chromatography (50-100% MeOH in water containing 0.1% TFA), and then a third purification by automated flash chromatography (5-65% ethyl acetate in hexane containing 1% Et3N) to obtain the title compound (yield 15%). 1H NMR(400 MHz, CDCl3)δ 4.08-4.00(m, 1H), 3.32-3.23(m, 4H), 3.23-3.15(m, 4H), 3.12-3.02(m, 1H), 2.51-2.30(m, 4H), 2.30-2.22(m, 4H), 2.12(p, J=10.0 Hz, 1H), 2.01-1.89(m, 1H), 1.87-1.76(m, 1H), 1.72-1.05(m, 90H), 0.92-0.83(m, 12H). Chemical formula:C 60 H 119 Calculated m / z value for N3O3 = 929.9. Measured value [M+H] + =931.2.

[0204] Example 32 8,8'-((2-((1s,3r)-3-hydroxycyclobutyl)ethyl)azandiyl)bis(N,N-didecyl-2-fluorooctanamide)(compound (1s,3r)-I-19) [ka]

[0205] Synthesis of Intermediate 2 A mixture of diethyl 2-fluoromalonate (56.1 mmol, 10.0 g), 1,6-dibromohexane (168 mmol, 26 mL), and sodium methoxide (61.7 mmol, 3.3 g) in EtOH (110 mL) was stirred at room temperature for 24 hours. The reaction mixture was concentrated, and the crude product was partitioned between DCM and water. The organic layer was separated, dried over Na2SO4, filtered, and concentrated. It was purified by automated flash chromatography (0% to 25% siRNA in hexane) to obtain intermediate 2 (11.7 g, 61%).

[0206] Synthesis of Intermediate 3 A mixture of intermediate 2 (5.9 mmol, 2.0 g) and KOH (11.8 mmol, 660 mg) in MeOH (20 mL), THF (2 mL), and water (4 mL) was stirred at room temperature for 3 hours. The reaction mixture was concentrated, and the crude product was diluted with 0.1 M NaOH (20 mL). The aqueous layer was washed with DCM (3 × 10 mL), acidified with 1 M HCl, and then extracted with HCl (2 × 20 mL). The combined HCl layers were dried over Na₂SO₄, filtered, and concentrated to obtain intermediate 3 (1.57 g, 94%), which was used in the next step without further purification.

[0207] Synthesis of Intermediate 4 The mixture of intermediate 3 (4.1 mmol, 1.2 g) and DMAP (cat.) in DMF (3 mL) was heated at 180°C for 12 minutes. The reaction mixture was partitioned between SiO2 and 1 M HCl. The organic layer was separated, dried over Na2SO4, filtered, and concentrated to obtain intermediate 4 (960 mg, 98%), which was used in the next step without further purification.

[0208] Synthesis of Intermediate 5 The mixture of intermediate 4 (4.0 mmol, 960 mg), oxalyl chloride (12 mmol, 1.0 mL), and DMF (cat.) in DCM (10 mL) was stirred at room temperature for 20 minutes. The reaction mixture was concentrated to obtain intermediate 5, which was used in the next step without further purification.

[0209] Synthesis of Intermediate 6 A mixture of didecylamine (4.0 mmol, 1.2 g), triethylamine (24 mmol, 3.4 mL), and DMAP (cat.) in DCM (10 mL) was mixed with a solution of crude intermediate 5 (4.0 mmol) in DCM (5 mL). The reaction mixture was stirred at room temperature for 1 hour. The reaction product was purified by automated flash chromatography (5% to 25% SiO in hexane) to obtain intermediate 6 (2 steps, 1.2 g, 58%).

[0210] (1S,3R)-I-19 synthesis Compound (1S,3R)-I-19 was synthesized according to general procedure A using intermediate 6 and (1R,3S)-3-(2-aminoethyl)cyclobutan-1-ol hydrochloride. The title compound was obtained by purification by automated flash chromatography (5-65% ELISA in hexane containing 1% Et3N) (yield 63%). 1 H NMR(400 MHz, CDCl3)δ 5.03(ddd, J= 49.4, 8.5, 4.3 Hz, 2H), 4.14-4.04(m, 1H), 3.38-3.12(m, 8H), 2.51-2.42(m, 2H), 2.39-2.25(m, 5H), 2.01-1.15(m, 87H), 0.92-0.83(m, 12H). Chemical formula:C 62 H 121 Calculated m / z value for F2N3O3 = 993.9. Measured value [M+H] + =995.0.

[0211] Example 33 Synthetic routes of compounds (1r,3r)-I-22 and (1s,3s)-I-22 [ka]

[0212] General procedure for manufacturing Int1 To a solution of 8-aminooctanol (1.0 equivalent) in ACN (7 mL / mmol), an appropriate bromide (1.0 equivalent) was added, and the reaction mixture was refluxed under N2 overnight. The reaction mixture was then concentrated to obtain the crude product. The solid crude product was purified by column chromatography (DCM in MeOH / 3% NH3, 100:0~80:20).

[0213] General procedure for manufacturing Int2 To a solution of acid (0.9 equivalents) in DCM (2.5 mL / mmol), N-hydroxysuccinimide (0.9 equivalents), 4-dimethylaminopyridine (0.9 equivalents), and dicyclohexylcarbodiimide (0.9 equivalents) were added under N2 conditions at room temperature. The resulting mixture was stirred at room temperature until the conversion was complete. The precipitate was filtered off, and the filtrate was added dropwise to a solution of Int1 (1.0 equivalent) in DCM (2.3 mL / mmol) at room temperature. The reaction mixture was stirred overnight under N2 conditions at room temperature. The organic phase was then washed with HCl (aq., 1 mol / L) and Na2CO3 (aq.) and dried over Na2SO4. The organic phase was concentrated. The pale yellow solid was purified by column chromatography (Hex / siRNA, 100:0~0:100).

[0214] General procedure for manufacturing Int4 To a solution of Int2 (1.0 equivalent) in diethyl ether (8.0 mL / mmol), PBr3 (2-3 equivalents) was added dropwise at 0°C under N2. The reaction mixture was slowly warmed to room temperature and stirred under N2 at room temperature until the reaction was complete. Then, ice-cold water was slowly added until a clear solution was obtained. The aqueous phase was extracted three times with ethyl acetate, dried over Na2SO4, and concentrated under reduced pressure. The crude product was purified by column chromatography (Hex / SiO4, 100:0-90:10).

[0215] [ka] Synthesis of N-(8-bromooctyl)-N-decyldecanamide (Int4-1) Int4-1 was manufactured according to the general procedure for manufacturing Int4.

[0216] General procedure for the preparation of compounds (1R,3R)-I-22 and (1S,3S)-I-22 To a solution of Int4-1 (1.5 equivalents) in ACN (5.5 mL / mmol), DIPEA (3.8 equivalents) and the desired alkylating reagent (1.0 equivalent) were added. Alkylation was carried out in a sealed tube at 80°C for 24 hours. Subsequently, Int4-1 (0.5 equivalents) in an additional 2.0 mL / mmol of ACN was added to the reaction mixture, and stirring was continued at 80°C for another 24 hours. The reaction mixture was then cooled to room temperature and concentrated under reduced pressure. The crude product was purified by column chromatography (Hex in SiO / 1% NEt3, 95:5~0:100).

[0217] Synthesis of N,N'-(((((1S,3S)-3-hydroxycyclobutyl)methyl)azandiyl)bis(octane-8,1-diyl))bis(N-decyldecanamide)((1S,3S)-I-22) Compound (1S,3S)-I-22 was prepared according to a general procedure from Int4-1 (0.5 g, 0.994 mmol), (1S,3S)-3-(aminomethyl)cyclobutan-1-ol hydrochloride (0.047 g, 0.338 mmol), and DIPEA (0.83 mL, 5.97 mmol). The product was obtained as a colorless oil (0.2 g, 0.211 mmol, 21.3%). 1 H NMR(600 MHz, chloroform-d)δ 4.12(p, J=7.4 Hz, 1H), 3.31-3.24(t, J= 8.5 Hz, 4H), 3.19(t, J= 6.3 Hz, 4H), 2.45(m, 4H), 2.35(m, 4H), 2.29-2.23(t, J= 7.1 Hz, 4H), 1.89(m, 1H), 1.63(m, 4H), 1.51(m, 10H), 1.39(m, 4H), 1.35-1.19(m, 70H), 0.87(t, J= 6.0 Hz, 12H). ESI-MS:C 61 H 121 N3O3[M+H] + The calculated MW value was 944.95, and the measured value was 945.09.

[0218] Synthesis of N,N'-(((((1R,3R)-3-hydroxycyclobutyl)methyl)azandiyl)bis(octane-8,1-diyl))bis(N-decyldecanamide)((1R,3R)-I-22) Compound (1R,3R)-I-22 was prepared according to a general procedure from Int4-1 (0.5 g, 0.994 mmol), (1R,3R)-3-(aminomethyl)cyclobutan-1-ol hydrochloride (0.047 g, 0.338 mmol), and DIPEA (0.83 mL, 5.97 mmol). The product was obtained as a colorless oil (0.22 g, 0.233 mmol, 23.4%). 1 H NMR(600 MHz, CDCl3)δ 4.41-4.33(p, J=7.4 Hz, 1H), 3.28(t, J= 8.5 Hz, 4H), 3.19(t, J= 6.3 Hz, 4H), 2.39(m, 3H), 2.37-2.30(m, 4H), 2.26(t, J= 7.1 Hz, 4H), 2.07(m, 2H), 2.03(m, 2H), 1.70-1.57(m, 7H), 1.57-1.45(m, 8H), 1.37(m, 4H), 1.34-1.21(m, 70H), 0.90-0.85(t, J= 6.0 Hz, 12H). ESI-MS:C 61 H 121 N3O3[M+H] + The calculated MW value was 944.95, and the measured value was 945.02.

[0219] Example 34 N-decyl-N-(8-((8-(didecylamino)-8-oxooctyl)(((1S,3S)-3-hydroxycyclobutyl)methyl)amino)octyl)decanamide (compound (1s,3s)-I-21)

[0220] [ka] Synthesis of N,N-didecyl-8-((((1S,3S)-3-hydroxycyclobutyl)methyl)amino)octanamide (Int-16) N,N-didecyl-8-((((1S,3S)-3-hydroxycyclobutyl)methyl)amino)octanamide was prepared from 8-bromo-N,N-didecyloctanamide (Example 5, 0.4 g, 0.796 mmol), (1S,3S)-3-(aminomethyl)cyclobutan-1-ol hydrochloride (0.384 g, 2.785 mmol), and DIPEA (0.44 mL, 3.183 mmol). The product was obtained as a colorless oil (0.3 g, 0.574 mmol, 72%). ESI-MS:C 33 H 66 N2O2[M+H] + The calculated MW value was 523.52, and the measured value was 523.61.

[0221] [ka] Synthesis of N-decyl-N-(8-((8-(didecylamino)-8-oxooctyl)(((1S,3S)-3-hydroxycyclobutyl)methyl)amino)octyl)decanamide((1S,3S)-I-21) Compound (1S,3S)-I-21 was prepared from Int4-1 (0.32 g, 0.631 mmol), Int16 (0.3 g, 0.574 mmol), and DIPEA (0.4 mL, 2.296 mmol). The product was obtained as a colorless oil (0.19 g, 0.201 mmol, 35%). 1 H NMR(600 MHz, CDCl3)δ 4.25-4.15(p, J=7.4 Hz, 1H), 3.31-3.23(t, J= 8.5 Hz, 4H), 3.19(t, J= 6.3 Hz, 4H), 3.05(m, 1H), 2.88(m, 3H), 2.60(m, 2H), 2.30-2.23(m, 4H), 1.75(s, 5H), 1.68-1.56(m, 8H), 1.50(m, 9H), 1.40-1.18(m, 70H), 0.88(t, J= 6.0 Hz, 12H). ESI-MS:C 61 H 121 N3O3[M+H] + The calculated MW value was 944.95, and the measured value was 945.09.

[0222] Example 35 Synthetic routes of compounds (1r,3r)-I-20 and (1s,3s)-I-20 [ka]

[0223] Synthesis of 8-bromo-N,N-dioctyloctanamide 8-Bromo-N,N-dioctyloctanamide was prepared according to the procedure described in Example 5 from 8-bromooctanoic acid (2.00 g, 9.0 mmol), DMF (1 drop), oxalyl chloride (2.30 mL, 27 mmol), dioctylamine (2.39 g, 9.9 mmol), triethylamine (7.50 mL, 54 mmol), DMAP (0.02 g, 0.16 mmol), and DCM (50 mL). The product 8-bromo-N,N-dioctyloctanamide was obtained as a colorless oil (3.12 g, 7.0 mmol, 78%). ESI-MS:C 24 H 49 BrNO [M+H] + The calculated values ​​for MW were 446.30 and 448.29, while the measured values ​​were 446.36 and 448.38 (bromine pattern).

[0224] Synthesis of 8,8'-((((1S,3S)-3-hydroxycyclobutyl)methyl)azandiyl)bis(N,N-dioctyloctanamide)((1S,3S)-I-20) Compound (1S,3S)-I-20 was prepared from 8-bromo-N,N-dioctyloctanamide (0.447 g, 1.0 mmol), (1S,3S)-3-(aminomethyl)cyclobutan-1-ol hydrochloride (0.050 g, 0.36 mmol), and DIPEA (1 mL, 6 mmol). The product (117 mg, 0.14 mmol, 39%) was obtained as a yellowish oil. 1H NMR(600 MHz, CDCl3)δ 4.11(p, J=7.4 Hz, 1H), 3.31-3.25(m, 4H), 3.22-3.16(m, 4H), 2.50-2.42(m, 4H), 2.39-2.31(m, 4H), 2.29-2.24(m, 4H), 1.95-1.85(m, 1H), 1.73-1.58(m, 7H), 1.57-1.45(m, 11H), 1.43-1.36(m, 4H), 1.35-1.20(m, 55H), 0.88(dt, J= 9.4, 7.0 Hz, 12H). ESI-MS:C 53 H 105 N3O3[M+H] + The calculated MW value was 832.83, and the measured value was 832.85.

[0225] Synthesis of 8,8'-((((1R,3R)-3-hydroxycyclobutyl)methyl)azandiyl)bis(N,N-dioctyloctanamide)((1R,3R)-I-20) Compound (1R,3R)-I-20 was prepared from 8-bromo-N,N-dioctyloctanamide (0.447 g, 1.0 mmol), (1R,3R)-3-(aminomethyl)cyclobutan-1-ol hydrochloride (0.050 g, 0.36 mmol), and DIPEA (1 mL, 6 mmol). The product (182 mg, 0.22 mmol, 60%) was obtained as a yellowish oil. 1 H NMR(600 MHz, CDCl3)δ 4.37(p, J=7.4 Hz, 1H), 3.32-3.24(m, 4H), 3.21-3.15(m, 4H), 2.38(s, 3H), 2.36-2.31(m, 4H), 2.30-2.23(m, 4H), 2.10-2.04(m, 2H), 2.04-1.96(m, 3H), 1.69-1.57(m, 8H), 1.56-1.46(m, 8H), 1.38(dt, J= 14.7, 7.5 Hz, 4H), 1.35-1.19(m, 54H), 0.88(dt, J= 9.2, 7.0 Hz, 12H). ESI-MS:C 53 H 105 N3O3[M+H] +The calculated MW value was 832.83, and the measured value was 832.85.

[0226] Example 36 8,8'-(((1S,4S)-4-(hydroxymethyl)cyclohexyl)azandiyl)bis(N,N-didecyloctanamide)(compound(1s,4s)-I-23) [ka]

[0227] (1S,4S)-I-23 synthesis Compound (1S,4S)-I-23 was synthesized using ((1s,4s)-4-aminocyclohexyl)methanol according to general procedure A. It was purified by automated flash chromatography (5-65% ethylethanol in hexane containing 1% Et3N) to obtain the title compound (55 mg, 21%). 1 H NMR(400 MHz, CDCl3)δ 3.60(d, J= 7.0 Hz, 2H), 3.32-3.23(m, 4H), 3.23-3.15(m, 4H), 2.50-2.37(m, 5H), 2.27-2.26(m, 4H), 1.83-1.05(m, 99H), 0.92-0.83(m, 12H). ESI-MS:C 63 H 125 Calculated m / z value for N3O3 = 972.0, measured value [M+H] + =973.2.

[0228] Example 37 8,8'-(((1r,4r)-4-(hydroxymethyl)cyclohexyl)azandiyl)bis(N,N-didecyloctanamide)(compound (1r,4r)-I-23) [ka]

[0229] (1R,4R)-I-23 synthesis Compound (1R,4R)-I-23 was synthesized using ((1r,4r)-4-aminocyclohexyl)methanol according to general procedure A. The title compound was obtained by purification by automated flash chromatography (5-65% ethylethanol in hexane containing 1% Et3N) (165 mg, 63%). 1 H NMR(400 MHz, CDCl3)δ 3.48-3.41(m, 2H), 3.33-3.23(m, 4H), 3.23-3.15(m, 4H), 2.44-2.36(m, 5H), 2.27-2.26(m, 4H), 1.89-1.78(m, 4H), 1.70-1.13(m, 92H), 1.03-0.77(m, 14H). ESI-MS:C 63 H 125 Calculated m / z value for N3O3 = 972.0, measured value [M+H] + = 973.3.

[0230] Example 38 8,8'-(((1r,4r)-4-(hydroxymethyl)cyclohexyl)azandiyl)bis(N,N-didecyloctanamide)(compound(1r,3r)-I-24) [ka]

[0231] (1R,3R)-I-24 synthesis Compound (1R,3R)-I-24 was synthesized using ((1r,3r)-3-aminocyclobutyl)methanol according to general procedure A. The title compound was obtained by purification by automated flash chromatography (5-65% ethylethanol in hexane containing 1% Et3N) (198 mg, 77%). 1H NMR(400 MHz, CDCl3)δ 3.72-3.62(m, 2H), 3.32-3.23(m, 4H), 3.23-3.13(m, 4H), 2.40-2.32(m, 4H), 2.26(t, J= 7.6 Hz, 5H), 2.09-1.95(m, 2H), 1.93-1.84(m, 2H), 1.69-1.04(m, 88H), 0.92-0.81(m, 12H). ESI-MS:C 61 H 121 Calculated m / z value for N3O3 = 943.9, measured value [M+H] + =945.2.

[0232] Example 39 8,8'-(((1s,3s)-3-(hydroxymethyl)cyclobutyl)azandiyl)bis(N,N-didecyloctanamide)(compound(1s,3s)-I-24) [ka]

[0233] (1S,3S)-I-24 synthesis Compound (1S,3S)-I-24 was synthesized using ((1s,3s)-3-aminocyclobutyl)methanol according to general procedure A. The title compound was obtained by purification by automated flash chromatography (5-65% ethylethanol in hexane containing 1% Et3N) (195 mg, 76%). 1 H NMR(400 MHz, CDCl3)δ 3.58(d, J= 4.1 Hz, 2H), 3.34-3.23(m, 4H), 3.23-3.15(m, 4H), 2.99-2.89(m, 1H), 2.42-2.34(m, 4H), 2.31-2.16(m, 7H), 1.75-1.03(m, 88H), 0.92-0.82(m, 12H). ESI-MS:C 61 H 121 Calculated m / z value for N3O3 = 943.9, measured value [M+H] + =945.2.

[0234] Example 40 8,8'-(((1r,4r)-4-hydroxy-4-methylcyclohexyl)azandiyl)bis(N,N-didecyloctanamide)(compound (1r,4r)-I-25) [ka]

[0235] (1R,4R)-I-25 synthesis Compound (1R,4R)-I-25 was synthesized using (1r,4r)-4-amino-1-methylcyclohexane-1-ol according to general procedure A. The compound was purified by automated flash chromatography (5-65% ethylethanol in hexane containing 1% Et3N) to obtain the title compound (150 mg, 34%). 1 H NMR(400 MHz, CDCl3)δ 3.32-3.23(m, 4H), 3.23-3.15(m, 4H), 2.56-2.35(m, 5H), 2.31-2.22(m, 4H), 1.80-1.01(m, 89H), 0.93-0.79(m, 12H). ESI-MS:C 63 H 125 Calculated m / z value for N3O3 = 972.0, measured value [M+H] + =973.2.

[0236] Example 41 8,8'-((6-hydroxyspiro[3,3]heptan-2-yl)azandiyl)bis(N,N-didecyloctanamide)(Compound I-36) [ka]

[0237] I-36 synthesis Compound I-36 was synthesized using 6-aminospiro[3.3]heptan-2-ol according to general procedure A. The title compound was obtained by purification by automated flash chromatography (5-65% toluene in hexane containing 1% Et3N) (185 mg, 42%). 1H NMR(400 MHz, CDCl3)δ 4.22-4.12(m, 1H), 3.32-3.23(m, 4H), 3.23-3.14(m, 4H), 2.98-2.87(m, 1H), 2.48-237(m, 1H), 2.35-2.20(m, 9H), 2.13-1.97(m, 2H), 1.94-1.79(m, 4H), 1.69-1.02(m, 87H), 0.92-0.84(m, 12H). ESI-MS:C 63 H 123 Calculated m / z value for N3O3 = 970.0, measured value [M+H] + = 971.2.

[0238] Example 42 8,8'-(((1s,4s)-4-hydroxy-4-methylcyclohexyl)azandiyl)bis(N,N-didecyloctanamide)(compound (1s,4s)-I-25) [ka]

[0239] (1S,4S)-I-25 synthesis Compound (1S,4S)-I-25 was synthesized using (1s,4s)-4-amino-1-methylcyclohexane-1-ol according to general procedure A. The title compound was obtained by purification by automated flash chromatography (5-65% ethylethanol in hexane containing 1% Et3N) (145 mg, 33%). 1 H NMR(400 MHz, CDCl3)δ 3.32-3.23(m, 4H), 3.23-3.15(m, 4H), 2.45-2.37(m, 5H), 2.30-2.22(m, 4H), 1.74-1.04(m, 99H), 0.92-0.84(m, 12H). ESI-MS:C 63 H 125 Calculated m / z value for N3O3 = 972.0, measured value [M+H] + =973.2.

[0240] Example 43 8,8'-(((1r,3r,5s)-3,5-dihydroxycyclohexyl)azandiyl)bis(N,N-didecyloctanamide)(compound(1r,3R,5S)-I-34) [ka]

[0241] (1R,3R,5S)-I-34 synthesis Compound (1R,3R,5S)-I-34 was synthesized using (1R,3S,5r)-5-aminocyclohexane-1,3-diol hydrochloride according to general procedure A. It was purified by automated flash chromatography (5-65% ethylethanol in hexane containing 1% Et3N), followed by a second purification by automated flash chromatography (0-10% MeOH in chloroform) to obtain the title compound (46 mg, 12%). 1 H NMR(400 MHz, CDCl3)δ 3.80-3.60(m, 2H), 3.31-3.15(m, 8H), 3.05-2.36(m, 5H), 2.34-2.04(m, 8H), 2.01-1.06(m, 93H), 0.92-0.84(m, 12H). ESI-MS:C 62 H 123 Calculated m / z value for N3O4 = 974.0, measured value [M+H] + =975.2.

[0242] Example 44 8,8'-(((1s,3R,5S)-3,5-dihydroxycyclohexyl)azandiyl)bis(N,N-didecyloctanamide)(compound(1s,3R,5S)-I-34) [ka]

[0243] (1S,3R,5S)-I-34 synthesis Compound (1S,3R,5S)-I-34 was synthesized using (1R,3S,5s)-5-aminocyclohexane-1,3-diol hydrochloride according to general procedure A. The compound was purified by automated flash chromatography (5-65% ethylethanol in hexane containing 1% Et3N) to obtain the title compound (85 mg, 22%). 1 H NMR(400 MHz, CDCl3)δ 4.28(s, 2H), 3.74(s, 2H), 3.51-3.34(m, 1H), 3.31-3.15(m, 8H), 2.46-2.38(m, 4H), 2.27(t, J= 7.4 Hz, 4H), 2.09-1.97(m, 3H), 1.72-1.02(m, 92H), 0.92-0.83(m, J=6.9, 2.9 Hz, 12H). ESI-MS:C 62 H 123 Calculated m / z value for N3O4 = 974.0, measured value [M+H] + =975.2.

[0244] Example 45 8,8'-(((1S,2R)-2-(hydroxymethyl)cyclopropyl)azandiyl)bis(N,N-didecyloctanamide)(compound (1S,2R)-I-46) [ka]

[0245] (1S,2R)-I-46 synthesis Compound (1S,2R)-I-46 was synthesized using ((1R,2S)-2-aminocyclopropyl)methanol hydrochloride according to general procedure A. It was purified by automated flash chromatography (5-65% ethylethanol in hexane containing 1% Et3N), followed by a second purification by automated flash chromatography (0-10% MeOH in chloroform) to obtain the title compound (107 mg, 29%). 1H NMR(400 MHz, CDCl3)δ 4.14-4.07(m, 1H), 3.98-3.88(m, 1H), 3.32-3.22(m, 4H), 3.23-3.14(m, 4H), 2.60-2.47(m, 4H), 2.31-2.23(m, 4H), 2.05-1.98(m, 1H), 1.72-1.00(m, 90H), 0.96-0.84(m, 14H), 0.70-0.59(m, 1H). ESI-MS:C 60 H 119 Calculated m / z value for N3O3 = 929.9, measured value [M+H] + =931.1.

[0246] Example 46 8,8'-(((1S,2S)-2-(hydroxymethyl)cyclopropyl)azandiyl)bis(N,N-didecyloctanamide)(compound (1S,2S)-I-46) [ka]

[0247] (1s,2s)-I-46 synthesis Compound (1S,2S)-I-46 was synthesized using ((1S,2S)-2-aminocyclopropyl)methanol according to general procedure A. It was purified by automated flash chromatography (5-65% ethylethanol in hexane containing 1% Et3N), followed by a second purification by automated flash chromatography (0-10% MeOH in chloroform) to obtain the title compound (65 mg, 18%). 1 H NMR(400 MHz, CDCl3)δ 3.57-3.37(m, 2H), 3.32-3.23(m, 4H), 3.23-3.15(m, 4H), 2.56-2.51(m, 4H), 2.27(t, J= 7.5 Hz, 4H), 1.89-1.01(m, 91H), 0.92-0.83(m, 12H), 0.73-0.60(m, 1H), 0.51-0.38(m, 1H). ESI-MS:C 60 H 119Calculated m / z value for N3O3 = 929.9, measured value [M+H] + =931.1.

[0248] Example 47 8,8'-(((1-(hydroxymethyl)cyclobutyl)methyl)azandiyl)bis(N,N-didecyloctanamide)(Compound I-29) [ka]

[0249] I-29 synthesis Compound I-29 was synthesized using (1-(aminomethyl)cyclobutyl)methanol according to general procedure A. It was purified by automated flash chromatography (5-65% ethylethanol in hexane containing 1% Et3N) to obtain the title compound (168 mg, 44%). 1 H NMR(400 MHz, CDCl3)δ 6.17(broad s, 1H), 3.75(s, 2H), 3.32-3.23(m, 4H), 3.23-3.14(m, 4H), 2.55(s, 2H), 2.41-2.31(m, 4H), 2.31-2.20(m, 4H), 2.03-1.89(m, 1H), 1.87-1.74(m, 5H), 1.69-1.04(m, 90H), 0.92-0.83(m, 12H). ESI-MS:C 62 H 123 Calculated m / z value for N3O3 = 958.0, measured value [M+H] + = 959.2.

[0250] Example 48 8,8'-(((3-(hydroxymethyl)oxetan-3-yl)methyl)azandiyl)bis(N,N-didecyloctanamide)(Compound I-41) [ka]

[0251] I-41 synthesis Compound I-41 was synthesized using (3-(aminomethyl)oxetan-3-yl)methanol according to general procedure A. It was purified by automated flash chromatography (5-65% ethyl ethanol in hexane containing 1% Et3N) to obtain the title compound (150 mg, 39%). 1 H NMR(400 MHz, CDCl3)δ 5.69(broad s, 1H), 4.47(d, J= 6.0 Hz, 2H), 4.37(d, J= 6.0 Hz, 2H), 4.04(s, 2H), 3.32-3.24(m, 4H), 3.23-3.15(m, 4H), 2.84(s, 2H), 2.36-2.22(m, 8H), 1.72-0.99(m, 90H), 0.92-0.83(m, 12H). ESI-MS:C 61 H 121 Calculated m / z value for N3O4 = 959.9, measured value [M+H] + =961.2.

[0252] Example 49 8,8'-(((3R,4R)-4-hydroxytetrahydrofuran-3-yl)azandiyl)bis(N,N-didecyloctanamide) 8,8'-(((3S,4S)-4-hydroxytetrahydrofuran-3-yl)azandiyl)bis(N,N-didecyloctanamide) (Compound (rac)-(3RS,4RS)-I-40) [ka]

[0253] (rac)-(3RS,4RS)-I-40 synthesis A mixture of compound I-40 enantiomers was synthesized according to general procedure A using a mixture of (3R,4R)-4-aminotetrahydrofuran-3-ol and (3S,4S)-4-aminotetrahydrofuran-3-ol. The compound was purified by automated flash chromatography (5-65% ethyl phosphate in hexane containing 1% Et3N) to obtain the title compound (94 mg, 25%). 1H NMR(400 MHz, CDCl3)δ 4.14-4.07(m, 1H), 4.00-3.91(m, 2H), 3.89-3.81(m, 1H), 3.71(dd, J= 9.7, 7.9 Hz, 1H), 3.32-3.23(m, 4H), 3.21-3.17(m, 4H), 3.16-3.07(m, 1H), 2.58-2.42(m, 4H), 2.31-2.23(m, 4H), 1.73-1.05(m, 86H), 0.92-0.81(m, 12H). ESI-MS:C 60 H 119 Calculated m / z value for N3O4 = 945.9, measured value [M+H] + =947.2.

[0254] Example 50 8,8'-(((3R,4S)-4-hydroxytetrahydrofuran-3-yl)azandiyl)bis(N,N-didecyloctanamide) 8,8'-(((3S,4R)-4-hydroxytetrahydrofuran-3-yl)azandiyl)bis(N,N-didecyloctanamide) (Compound (rac)-(3RS,4SR)-I-40) [ka]

[0255] (rac)-(3RS,4SR)-I-40 synthesis A mixture of compound I-40 enantiomers was synthesized according to general procedure A using a mixture of (3S,4R)-4-aminotetrahydrofuran-3-ol hydrochloride and (3R,4S)-4-aminotetrahydrofuran-3-ol hydrochloride. The mixture was purified by automated flash chromatography (5-65% ethyl phosphate in hexane containing 1% Et3N), followed by a second purification by automated flash chromatography (50-100% ethyl phosphate in hexane) to obtain the title compound (90 mg, 24%). 1H NMR(400 MHz, CDCl3)δ 4.34-4.29(m, 1H), 4.05-3.19(m, 2H), 3.70-3.59(m, 2H), 3.32-3.24(m, 5H), 3.23-3.15(m, 4H), 2.55-2.42(m, 4H), 2.27(t, J= 7.4 Hz, 4H), 1.97-1.00(m, 87H), 0.92-0.81(m, 12H). ESI-MS:C 60 H 119 Calculated m / z value for N3O4 = 945.9, measured value [M+H] + =947.2.

[0256] Example 51 8,8'-((2-((1R,2R)-2-hydroxycyclohexyl)ethyl)azandiyl)bis(N,N-didecyloctanamide) 8,8'-((2-((1S,2S)-2-hydroxycyclohexyl)ethyl)azandiyl)bis(N,N-didecyloctanamide) (Compound (rac)-(1RS,2RS)-I-31) [ka]

[0257] (rac)-(1RS,2RS)-I-31 synthesis A mixture of enantiomers of compound I-31 was synthesized using rac-(1R,2S)-2-(2-aminoethyl)cyclohexane-1-ol according to general procedure B. The compound was purified by automated flash chromatography (5-65% ethyl hexane containing 1% Et3N) to obtain the title compound (86 mg, 29%). 1H NMR(400 MHz, CDCl3)δ 7.95(broad s, 1H), 3.32-3.23(m, 4H), 3.23-3.14(m, 4H), 3.10-3.00(m, 1H), 2.60-2.40(m, 4H), 2.36-2.22(m, 6H), 2.02-1.93(m, 1H), 1.77-0.98(m, 96H), 0.92-0.83(m, 12H). ESI-MS:C 64 H 127 Calculated m / z value for N3O3 = 986.0, measured value [M+H] + =987.2.

[0258] Example 52 8,8'-((2-((1R,2S)-2-hydroxycyclohexyl)ethyl)azandiyl)bis(N,N-didecyloctanamide) 8,8'-((2-((1S,2R)-2-hydroxycyclohexyl)ethyl)azandiyl)bis(N,N-didecyloctanamide)(compound(rac)-(1RS,2SR)-I-31) [ka]

[0259] (rac)-(1RS,2SR)-I-31 synthesis A mixture of enantiomers of compound I-31 was synthesized using rac-(1R,2R)-2-(2-aminoethyl)cyclohexane-1-ol according to general procedure B. The compound was purified by automated flash chromatography (5-65% ethyl hexane containing 1% Et3N) to obtain the title compound (92 mg, 25%). 1 H NMR(400 MHz, CDCl3)δ 7.95(broad s, 1H), 3.32-3.23(m, 4H), 3.23-3.14(m, 4H), 3.10-2.99(m, 1H), 2.60-2.38(m, 4H), 2.36-2.22(m, 6H), 2.01-1.93(m, 1H), 1.77-0.97(m, 97H), 0.92-0.83(m, 12H). ESI-MS:C 64 H127 Calculated m / z value for N3O3 = 986.0, measured value [M+H] + =987.2.

[0260] Example 53 8,8'-((((1R,2S)-2-(hydroxymethyl)cyclopropyl)methyl)azandiyl)bis(N,N-didecyloctanamide) 8,8'-((((1S,2R)-2-(hydroxymethyl)cyclopropyl)methyl)azandiyl)bis(N,N-didecyloctanamide)(compound(rac)-(1RS,2SR)-I-45) [ka]

[0261] (rac)-(1RS,2SR)-I-45 synthesis A mixture of enantiomers of compound I-45 was synthesized using rac-((1R,2S)-2-(aminomethyl)cyclopropyl)methanol according to general procedure B. The compound was purified by automated flash chromatography (5-65% ethylethanol in hexane containing 1% Et3N) to obtain the title compound (112 mg, 31%). 1 H NMR(400 MHz, CDCl3)δ 6.19(broad s, 1H), 4.03-3.91(m, 1H), 3.32-3.23(m, 4H), 3.23-3.12(m, 4H), 3.12-3.00(m, 1H), 2.75-2.58(m, 3H), 2.34-2.13(m, 7H), 1.73-0.96(m, 88H), 0.92-0.75(m, 13H), 0.26-0.13(m, 1H). ESI-MS:C 61 H 121 Calculated m / z value for N3O3 = 943.9, measured value [M+H] + =945.2.

[0262] Example 54 8,8'-((((1R,2R)-2-(hydroxymethyl)cyclopropyl)methyl)azandiyl)bis(N,N-didecyloctanamide) 8,8'-((((1S,2S)-2-(hydroxymethyl)cyclopropyl)methyl)azandiyl)bis(N,N-didecyloctanamide)(compound(rac)-(1RS,2RS)-I-45) [ka]

[0263] (rac)-(1RS,2RS)-I-45 synthesis A mixture of compound I-45 enantiomers was synthesized using rac-((1R,2R)-2-(aminomethyl)cyclopropyl)methanol according to general procedure B. The compound was purified by automated flash chromatography (5-65% ethylethanol in hexane containing 1% Et3N) to obtain the title compound (110 mg, 31%). 1 H NMR(400 MHz, CDCl3)δ 3.54-3.42(m, 2H), 3.32-3.23(m, 4H), 3.23-3.15(m, 4H), 2.51-2.39(m, 5H), 2.34-2.22(m, 5H), 2.00-1.02(m, 86H), 0.98-0.71(m, 14H), 0.50-0.41(m, 1H), 0.0.40-0.31(m, 1H). ESI-MS:C 61 H 121 Calculated m / z value for N3O3 = 943.9, measured value [M+H] + =945.2.

[0264] Example 55 8,8'-(((1R,2S)-2-hydroxycyclopentyl)azandiyl)bis(N,N-didecyloctanamide)(compound (1R,2S)-I-48) [ka]

[0265] (1R,2S)-I-48 synthesis Compound (1R,2S)-I-48 was synthesized using (1S,2R)-2-aminocyclopentane-1-ol hydrochloride according to general procedure B. It was purified by automated flash chromatography (5-65% ethylethanol in hexane containing 1% Et3N), followed by secondary flash chromatography (100% ethylethanol) to obtain the title compound (61 mg, 18%). 1 H NMR(400 MHz, CDCl3)δ 4.05-3.94(m, 1H), 3.32-3.23(m, 4H), 3.23-3.15(m, 4H), 2.75-2.66(m, 1H), 2.62-2.48(m, 4H), 2.31-2.22(m, 4H), 1.92-1.01(m, 100H), 0.92-0.83(m, 12H). ESI-MS:C 61 H 121 Calculated m / z value for N3O3 = 943.9, measured value [M+H] + =945.3.

[0266] Example 56 8,8'-(((1R,2R)-2-hydroxycyclopentyl)azandiyl)bis(N,N-didecyloctanamide)(compound (1R,2R)-I-48) [ka]

[0267] (1R,2R)-I-48 synthesis Compound (1R,2R)-I-48 was synthesized using (1R,2R)-2-aminocyclopentane-1-ol hydrochloride according to general procedure B. The title compound was obtained by purification by automated flash chromatography (5-65% ethylethanol in hexane containing 1% Et3N) (111 mg, 33%). 1H NMR(400 MHz, CDCl3)δ 3.95-3.83(m, 1H), 3.32-3.23(m, 4H), 3.23-3.15(m, 4H), 2.87-2.74(m, 1H), 2.55-2.20(m, 9H), 1.95-1.85(m, 1H), 1.81-1.02(m, 97H), 0.92-0.83(m, 12H). ESI-MS:C 61 H 121 Calculated m / z value for N3O3 = 943.9, measured value [M+H] + =945.3.

[0268] Example 57 8,8'-(((1R,2S)-2-hydroxycyclopentyl)azandiyl)bis(N,N-didecyloctanamide) 8,8'-(((1S,2R)-2-hydroxycyclopentyl)azandiyl)bis(N,N-didecyloctanamide) (Compound (rac)-(1RS,2SR)-I-48) [ka]

[0269] (rac)-(1RS,2SR)-I-48 synthesis A mixture of enantiomers of compound I-48 was synthesized using rac-(1R,2S)-2-aminocyclopentane-1-ol hydrochloride according to general procedure A. It was purified by automated flash chromatography (5-65% ethyl phosphate in hexane containing 1% Et3N), followed by secondary flash chromatography (50-100% ethyl phosphate in hexane) to obtain the title compound (68 mg, 20%). 1 H NMR(400 MHz, CDCl3)δ 4.04-3.95(m, 1H), 3.31-3.24(m, 4H), 3.23-3.15(m, 4H), 2.73-2.67(m, 1H), 2.62-2.49(m, 4H), 2.31-2.22(m, 4H), 1.91-1.00(m, 96H), 0.92-0.83(m, 12H). ESI-MS:C61 H 121 Calculated m / z value for N3O3 = 943.9, measured value [M+H] + =945.3.

[0270] Example 58 8,8'-(((1R,2R)-2-hydroxycyclopentyl)azandiyl)bis(N,N-didecyloctanamide) 8,8'-(((1S,2S)-2-hydroxycyclopentyl)azandiyl)bis(N,N-didecyloctanamide) (Compound (rac)-(1RS,2RS)-I-48) [ka]

[0271] (rac)-(1RS,2RS)-I-48 synthesis A mixture of enantiomers of compound I-48 was synthesized using rac-(1R,2R)-2-aminocyclopentan-1-ol according to general procedure A. The title compound was obtained by purification using automated flash chromatography with 5-65% ethylethanol in hexane containing 1% Et3N (130 mg, 39%). 1 H NMR(400 MHz, CDCl3)δ 3.94-3.81(m, 1H), 3.32-3.23(m, 4H), 3.23-3.15(m, 4H), 2.88-2.75(m, 1H), 2.55-2.34(m, 4H), 2.31-2.26(t, J= 7.6 Hz, 5H), 1.96-1.85(m, 1H), 1.79-1.02(m, 96H), 0.92-0.83(m, 12H). ESI-MS:C 61 H 121 Calculated m / z value for N3O3 = 943.9, measured value [M+H] + =945.2.

[0272] Example 59 8,8'-((4-hydroxycyclohexyl)azandiyl)bis(N,N-didecyloctanamide)(Compound I-9) [ka]

[0273] I-9 synthesis Compound I-9 was prepared by mixing compound (1S,4S)-I-9 (25 mg) and compound (1R,4R)-I-9 (25 mg) together to obtain the title compound (47 mg, 94%). 1 H NMR(400 MHz, CDCl3)δ 3.98(broad s, 0.5H), 3.54(broad s, 0.5H), 3.32-3.23(m, 4H), 3.23-3.15(m, 4H), 2.46-2.32(m, 5H), 2.30-2.22(m, 4H), 2.06-1.92(m, 1H), 1.89-1.73(m, 2H), 1.71-1.07(m, 105H), 0.94-0.83(m, 12H). ESI-MS:C 62 H 123 Calculated m / z value for N3O3 = 958.0, measured value [M+H] + = 959.2.

[0274] Example 60 8,8'-(((1s,4s)-4-hydroxycyclohexyl)azandiyl)bis(N,N-didecyl-2-fluorooctanamide)(compound (1s,4s)-I-49) [ka]

[0275] (1s,4s)-I-49 synthesis Compound (1s,4s)-I-49 was synthesized according to general procedure A using intermediate 6 (Example 32) and (1s,4s)-4-aminocyclohexane-1-ol. The title compound was obtained by purification by automated flash chromatography (5-100% ethylethanol in hexane containing 1% Et3N) (155 mg, 54%). 1H NMR(400 MHz, CDCl3)δ 5.12(dd, J= 8.4, 4.4 Hz, 1H), 4.99(dd, J= 8.5, 4.3 Hz, 1H), 4.01(s, 1H), 3.41-3.14(m, 8H), 2.52-2.40(m, 5H), 2.02-1.73(m, 6H), 1.72-1.09(m, 92H), 0.95-0.86(m, 12H). ESI-MS:C 62 H 121 Calculated m / z value for F2N3O3 = 993.9. Measured value [M+H] + = 995.3.

[0276] Example 61 8,8'-(((1r,4r)-4-hydroxycyclohexyl)azandiyl)bis(N,N-didecyl-2-fluorooctanamide)(compound (1r,4r)-I-49) [ka]

[0277] Synthesis of (1r,4r)-I-49 Compound (1R,4R)-I-49 was synthesized according to general procedure A using intermediate 6 (Example 32) and (1r,4r)-4-aminocyclohexane-1-ol. The title compound was obtained by purification by automated flash chromatography (5-100% ethyl phosphate in hexane containing 1% Et3N) (170 mg, 60%). 1 H NMR(400 MHz, CDCl3)δ 5.12(dd, J= 8.4, 4.4 Hz, 1H), 4.99(dd, J= 8.5, 4.3 Hz, 1H), 3.57(broad s, 1H), 3.41-3.14(m, 8H), 2.54-2.31(m, 5H), 2.09-1.72(m, 8H), 1.69-1.08(m, 90H), 0.95-0.86(m, 12H). ESI-MS:C 62 H 121 Calculated m / z value for F2N3O3 = 993.9. Measured value [M+H] + = 995.2.

[0278] Example 62 8,8'-(((1s,4s)-4-hydroxycyclohexyl)azandiyl)bis(N-(nonadecan-10-yl)octanamide)(compound (1s,4s)-I-50)

[0279] [ka] Synthesis of Intermediate 7 A mixture of (1s,4s)-4-aminocyclohexane-1-ol (4.34 mmol, 500 mg), ethyl 8-bromooctanoate (8.68 mmol, 2.18 g), DIEA (13.5 mmol, 2.34 mL), and potassium iodide (13.0 mmol, 2.16 g) in acetonitrile (8.7 mL) was stirred at 75°C for 19 hours. The reaction mixture was concentrated, the crude product was suspended in dichloromethane, and filtered. The filtrate was purified by automated flash chromatography (10% to 100% phosphate in hexane containing 1% Et3N) to obtain intermediate 7 (1.28 g, 65%).

[0280] [ka] Synthesis of Intermediate 8 A mixture of intermediate 7 (0.44 mmol, 200 mg) and potassium hydroxide (1.34 mmol, 75 mg) in methanol (0.88 mL) and water (0.88 mL) was stirred at room temperature for 20 hours. The reaction mixture was concentrated to obtain intermediate 8, which was used in subsequent steps without further purification.

[0281] [ka] (1s,4s)-I-50 synthesis A mixture of intermediate 8 (0.25 mmol, 100 mg), nonadecane-10-amine (0.5 mmol, 142 mg), DIEA (0.75 mmol, 0.13 mL), and HATU (0.5 mmol, 190 mg) in dichloromethane (1.25 mL) was stirred at room temperature for 20 hours. The reaction mixture was concentrated, and the crude product was partitioned between ethyl acetate and saturated NaHCO3. The organic layer was separated, dried over Na2SO4, and concentrated. It was purified by flash chromatography (5-100% ethyl acetate in hexane containing 1% Et3N) to obtain compound (1s,4s)-I-50 (75 mg, 32%). 1 H NMR(400 MHz, CDCl3)δ 5.25(d, J= 9.2 Hz, 2H), 4.05-3.88(m, 3H), 2.58-2.39(m, 5H), 2.18(t, J= 7.6 Hz, 4H), 1.92-1.83(m, 2H), 1.77-1.05(m, 105H), 0.95-0.84(m, 15H). ESI-MS:C 60 H 119 Calculated m / z value for N3O3 = 929.9. Measured value [M+H] + =931.2.

[0282] Example 63 8,8'-(((1s,4s)-4-hydroxycyclohexyl)azandiyl)bis(N-(deca-9-en-1-yl)-N-decyloctanamide)(compound(1s,4s)-I-51)

[0283] [ka] Synthesis of Intermediate 9 A mixture of decylamine (13.7 mmol, 2.15 g), 10-bromo-1-decene (4.6 mmol, 1.0 g), and DIEA (13.7 mmol, 2.38 mL) in acetonitrile (9.1 mL) was stirred at room temperature for 72 hours. The reaction mixture was concentrated, and the crude product was purified by flash chromatography (0% to 20% butyl in hexane containing 1% Et3N). Secondary purification was performed by flash chromatography (1% to 15% MeOH in DCM) to obtain intermediate 9 (538 mg, 40%).

[0284] [ka] (1s,4s)-I-51 synthesis A mixture of intermediate 8 (0.25 mmol, 100 mg), intermediate 9 (0.5 mmol, 148 mg), DIEA (1.50 mmol, 0.26 mL), and HATU (0.65 mmol, 247 mg) in dichloromethane (2.5 mL) was stirred at room temperature for 20 hours. The reaction mixture was concentrated, and the crude product was partitioned between butyl and saturated NaHCO3. The organic layer was separated, dried over Na2SO4, and concentrated. Compound (1s,4s)-I-51 was obtained (95 mg, 40%) by flash chromatography (5-100% butyl in hexane containing 1% Et3N). 1 H NMR(400 MHz, CDCl3)δ 5.88-5.73(m, 2H), 5.04-4.88(m, 4H), 4.01-3.92(m, 1H), 3.32-3.23(m, 4H), 3.23-3.15(m, 4H), 2.49-2.38(m, 5H), 2.30-2.22(m, 4H), 2.09-1.97(m, 4H), 1.92-1.75(m, 2H), 1.76-1.07(m, 89H), 0.93-0.81(m, 6H). ESI-MS:C 62 H 119 Calculated m / z value for N3O3 = 953.9. Measured value [M+H] + =955.2.

[0285] Example 64 8,8'-(((1s,4s)-4-hydroxycyclohexyl)azandiyl)bis(N-dodecyl-N-octyloctanamide)(compound (1s,4s)-I-62)

[0286] [ka] Synthesis of intermediate 10 A mixture of octylamine (24.1 mmol, 3.11 g), 1-bromododecane (8.0 mmol, 2.0 g), and DIEA (24.1 mmol, 4.2 mL) in acetonitrile (16 mL) was stirred at 50°C for 20 hours. The reaction mixture was concentrated, and the crude product was purified by flash chromatography (1% to 15% MeOH in DCM) to obtain intermediate 10 (1.75 g, 73%).

[0287] [ka] (1s,4s)-I-62 synthesis Compound (1S,4S)-I-62 was prepared from intermediates 8 and 10 according to the procedure for compound (1s,4s)-I-51 (Example 63). Yield (67 mg, 28%). 1 H NMR(400 MHz, CDCl3)δ 4.02-3.92(m, 1H), 3.32-3.23(m, 4H), 3.23-3.15(m, 4H), 2.50-2.38(m, 5H), 2.31-2.22(m, 4H), 1.86-1.79(m, 2H), 1.72-1.10(m, 96H), 0.92-0.83(m, 12H). ESI-MS:C 62 H 123 Calculated m / z value for N3O3 = 958.0. Measured value [M+H] + =959.3.

[0288] Example 65 8,8'-(((1s,4s)-4-hydroxycyclohexyl)azandiyl)bis(N-decyl-N-hexyloctanamide)(compound (1s,4s)-I-52)

[0289] [ka] Synthesis of intermediate 11 A mixture of hexylamine (24.1 mmol, 2.44 g), 1-bromododecane (8.0 mmol, 2.0 g), and DIEA (24.1 mmol, 4.2 mL) in acetonitrile (16 mL) was stirred at 50°C for 20 hours. The reaction mixture was concentrated, and the crude product was purified by flash chromatography (1% to 15% MeOH in DCM) to obtain intermediate 11 (1.7 g, 79%).

[0290] [ka] (1S,4S)-I-52 synthesis Compound (1S,4S)-I-52 was prepared from intermediates 8 and 11 according to the procedure for compound (1s,4s)-I-51 (Example 63). Yield (33 mg, 15%). 1 H NMR(400 MHz, CDCl3)δ 4.02-3.91(m, 1H), 3.32-3.24(m, 4H), 3.23-3.15(m, 4H), 2.51-2.38(m, 5H), 2.31-2.22(m, 4H), 1.89-1.79(m, 2H), 1.76-1.05(m, 88H), 0.94-0.83(m, 12H). ESI-MS:C 58 H 115 Calculated m / z value for N3O3 = 901.9. Measured value [M+H] + =903.2.

[0291] Example 66 8,8'-(((1s,4s)-4-hydroxycyclohexyl)azandiyl)bis(N,N-di(deca-9-en-1-yl)octanamide)(compound (1s,4s)-I-53)

[0292] [ka] Synthesis of intermediate 12 A mixture of deca-9-en-1-amine (6.84 mmol, 1.06 g), 10-bromodeca-1-ene (2.28 mmol, 500 mg), and DIEA (6.84 mmol, 1.19 mL) in acetonitrile (4.6 mL) was stirred at room temperature for 20 hours. The reaction mixture was concentrated, and the crude product was purified by flash chromatography (1% to 15% MeOH in DCM). Secondary purification was performed by flash chromatography (1% to 10% MeOH in CHCl3) to obtain intermediate 12 (0.36 g, 54%).

[0293] [ka] (1S,4S)-I-53 synthesis Compound (1S,4S)-I-53 was prepared from intermediates 8 and 12 according to the procedure for compound (1S,4S)-I-51 (Example 63). Yield (150 mg, 48%). 1 H NMR(400 MHz, CDCl3)δ 5.83(m, 4H), 5.07-4.90(m, 8H), 4.05-3.96(m, 1H), 3.34-3.26(m, 4H), 3.25-3.17(m, 4H), 2.51-2.40(m, 5H), 2.33-2.25(m, 4H), 2.12-2.00(m, 8H), 1.90-1.81(m, 2H), 1.74-1.06(m, 84H). ESI-MS:C 62 H 115 Calculated m / z value for N3O3 = 949.9. Measured value [M+H] + =951.2.

[0294] Example 67 8,8'-(((1s,4s)-4-hydroxycyclohexyl)azandiyl)bis(N-butyl-N-(heptadecan-9-yl)octanamide)(compound (1s,4s)-I-54)

[0295] [ka] Synthesis of intermediate 13 A mixture of heptadecane-9-one (5.89 mmol, 1.5 g), butylamine (8.84 mmol, 0.87 mL), acetic acid (8.84 mmol, 0.51 mL), and sodium triacetoxyborohydride (23.6 mmol, 5.0 g) in dichloroethane (29.5 mL) was stirred at room temperature for 20 hours. Further sodium triacetoxyborohydride (9.4 mmol, 2.0 g) was added, and the reaction mixture was stirred at room temperature for 20 hours. The reaction mixture was concentrated, and the crude product was partitioned between toluene and saturated NaHCO3. The organic layer was separated, dried over Na2SO4, and concentrated. It was purified by flash chromatography (1%-10% MeOH in CHCl3) to obtain intermediate 13 (1.29 g, 70%).

[0296] [ka] (1S,4S)-I-54 synthesis Compound (1S,4S)-I-54 was prepared from intermediates 8 and 13 according to the procedure for compound (1S,4S)-I-51 (Example 63). Yield (130 mg, 35%). 1 H NMR(400 MHz, CDCl3)δ 4.45(broad s, 1H), 4.05-3.96(m, 1H), 3.70-3.58(m, 1H), 3.12-3.00(m, 4H), 2.58-2.38(m, 5H), 2.35-2.26(m, 4H), 1.89-1.81(m, 2H), 1.77-1.06(m, 97H), 1.02-0.85(m, 18H). ESI-MS:C 64 H 127 Calculated m / z value for N3O3 = 986.0. Measured value [M+H] + =987.2.

[0297] Example 68 N,N-Didecyl-8-((8-(Dodecyl(octyl)amino)-8-Oxooctyl)((1s,4s)-4-Hydroxycyclohexyl)amino)Octanaamide(Compound (1s,4s)-I-55)

[0298] [ka] Synthesis of intermediate 14 A mixture of (1s,4s)-4-aminocyclohexane-1-ol (18.3 mmol, 2.11 g), intermediate 1 (4.58 mmol, 2.3 g), DIEA (14.2 mmol, 2.47 mL), and potassium iodide (13.7 mmol, 2.28 g) in acetonitrile (9.2 mL) was heated at 75°C for 20 hours. The reaction mixture was concentrated, and the resulting crude product was suspended in 95:5:1 (hexane:SiO:Et3N) and filtered. The filtrate was purified by flash chromatography (100% SiO), followed by a second flash chromatography (1%-10% MeOH in CHCl3) to obtain intermediate 14 (1.0 g, 41%).

[0299] [ka] Synthesis of intermediate 15 A mixture of intermediate 14 (1.6 mmol, 850 mg), ethyl 8-bromooctanoate (1.6 mmol, 400 mg), DIEA (4.9 mmol, 0.86 mL), and potassium iodide (4.8 mmol, 790 mg) was heated at 75°C for 20 hours. The reaction mixture was concentrated, and the resulting crude product was suspended in 95:5:1 (hexane:SiO:Et3N) and filtered. The filtrate was purified by flash chromatography (5-100% SiO in hexane containing 1% Et3N) to obtain intermediate 15 (700 mg, 63%).

[0300] [ka] Synthesis of intermediate 16 Intermediate 15 (0.99 mmol, 0.70 g) and a methanol:water (4 mL:0.4 mL) mixture in sodium hydroxide (25 mmol, 1 g) were heated at 60°C for 2 hours. The reaction mixture was diluted with water and extracted with SiO2. The organic layer was separated, dried over Na2SO4, and concentrated to obtain intermediate 16 (600 mg, 89%).

[0301] [ka] (1S,4S)-I-55 synthesis A mixture of intermediate 16 (0.12 mmol, 80 mg), intermediate 10 (0.12 mmol, 35 mg), DIEA (0.47 mmol, 0.082 mL), and HATU (0.15 mmol, 58 mg) in dichloromethane (1.2 mL) was stirred at room temperature for 20 hours. The reaction mixture was concentrated, and the crude product was partitioned between butyl and saturated NaHCO3. The organic layer was separated, dried over Na2SO4, and concentrated. The product was purified by flash chromatography (5-100% butyl in hexane containing 1% Et3N) to obtain the title compound (33 mg, 29%). 1 H NMR(400 MHz, CDCl3)δ 4.05-3.98(m, 1H), 3.35-3.27(m, 4H), 3.26-3.18(m, 4H), 2.57-2.40(m, 5H), 2.34-2.26(m, 4H), 1.92-1.82(m, 2H), 1.78-1.09(m, 95H), 0.96-0.87(m, 12H). ESI-MS:C 62 H 123 Calculated m / z value for N3O3 = 958.0. Measured value [M+H] + = 959.2.

[0302] Example 69 N,N-Didecyl-8-((8-(Dodecyl(hexyl)amino)-8-oxooctyl)((1s,4s)-4-hydroxycyclohexyl)amino)octanamide(compound(1s,4s)-I-56) [ka]

[0303] (1s,4s)-I-56 synthesis Compound (1S,4S)-I-56 was prepared from intermediates 16 and 11 according to the procedure of compound (1s,4s)-I-55 (Example 68). Yield (26 mg, 24%).1 H NMR(400 MHz, CDCl3)δ 4.05-3.98(m, 1H), 3.35-3.27(m, 4H), 3.27-3.18(m, 4H), 2.57-2.40(m, 5H), 2.34-2.26(m, 4H), 1.92-1.82(m, 2H), 1.77-1.09(m, 91H), 0.91(td, J= 6.9, 2.8 Hz, 12H). ESI-MS:C 60 H 119 Calculated m / z value for N3O3 = 929.9. Measured value [M+H] + =931.2.

[0304] Example 70 N,N-Di(deca-9-en-1-yl)-8-((8-(didecylamino)-8-oxooctyl)((1s,4s)-4-hydroxycyclohexyl)amino)octanamide(compound(1s,4s)-I-57) [ka]

[0305] (1S,4S)-I-57 synthesis Compound (1S,4S)-I-57 was prepared from intermediates 16 and 12 according to the procedure of compound (1s,4s)-I-55 (Example 68). Yield (30 mg, 21%). 1 H NMR(400 MHz, CDCl3)δ 5.92-5.77(m, 2H), 5.08-4.92(m, 4H), 4.01(s, 1H), 3.35-3.27(m, 4H), 3.27-3.18(m, 4H), 2.59-2.39(m, 4H), 2.34-2.26(m, 4H), 2.13-2.01(m, 4H), 1.93-1.80(m, 2H), 1.79-1.07(m, 85H), 0.91(td, J= 6.8, 3.1 Hz, 6H). ESI-MS:C 62 H 119 Calculated m / z value for N3O3 = 953.9. Measured value [M+H] + =955.2.

[0306] Example 71 N,N-Didecyl-8-(((1s,4s)-4-Hydroxycyclohexyl)(8-(Nonadecane-10-ylamino)-8-oxooctyl)amino)octanamide(Compound (1s,4s)-I-58) [ka]

[0307] (1S,4S)-I-58 synthesis Compound (1S,4S)-I-58 was prepared from intermediate 16 and nonadecane-10-amine according to the procedure for compound (1S,4S)-I-55 (Example 68). Yield (36 mg, 26%). 1 H NMR(400 MHz, CDCl3)δ 5.22(d, J= 9.1 Hz, 1H), 4.03-3.84(m, 2H), 3.28(t, J= 7.7 Hz, 2H), 3.20(t, J= 7.8 Hz, 2H), 2.55-2.37(m, 5H), 2.27(t, J= 7.6 Hz, 2H), 2.16(t, J= 7.6 Hz, 2H), 1.88-1.80(m, 2H), 1.75-1.03(m, 93H), 0.93-0.84(m, 12H). ESI-MS:C 61 H 121 Calculated m / z value for N3O3 = 943.9. Measured value [M+H] + =945.3.

[0308] Example 72 N-butyl-8-((8-(didecylamino)-8-oxooctyl)((1s,4s)-4-hydroxycyclohexyl)amino)-N-(heptadecan-9-yl)octanamide(compound(1s,4s)-I-59) [ka]

[0309] (1S,4S)-I-59 synthesis Compound (1S,4S)-I-59 was prepared from intermediates 16 and 13 according to the procedure for compound (1S,4S)-I-55 (Example 68). Yield (39 mg, 27%). 1 H NMR(400 MHz, CDCl3)δ 4.02-3.96(m, 1H), 3.67-3.57(m, 1H), 3.28(t, J= 7.7 Hz, 2H), 3.20(t, J= 7.8 Hz, 2H), 3.12-2.97(m, 2H), 2.55-2.36(m, 5H), 2.33-2.23(m, 4H), 1.90-1.78(m, 2H), 1.76-1.05(m, 97H), 1.00-0.84(m, 15H). ESI-MS:C 63 H 125 Calculated m / z value for N3O3 = 972.0. Measured value [M+H] + = 973.3.

[0310] Example 73 10,10'-(((1s,4s)-4-hydroxycyclohexyl)azandiyl)bis(N,N-didecyldecanamide)(compound (1s,4s)-I-60)

[0311] [ka] Synthesis of intermediate 17 Intermediate 17 was prepared from 10-bromodecanoic acid according to the procedure for intermediate 1 (Example 5). Yield (5.07 g, 40%).

[0312] [ka] (1s,4s)-I-60 synthesis Compound (1S,4S)-I-60 was synthesized according to general procedure A using a mixture of intermediate 17 and (1s,4s)-4-aminocyclohexane-1-ol. The title compound was obtained by purification by automated flash chromatography (5-100% SiO in hexane containing 1% Et3N) (yield 97 mg, 33%). 1H NMR(400 MHz, CDCl3)δ 4.04-3.98(m, 1H), 3.34-3.26(m, 4H), 3.25-3.17(m, 4H), 2.55-2.38(m, 5H), 2.33-2.25(m, 4H), 1.92-1.80(m, 2H), 1.77-1.05(m, 106H), 0.95-0.86(m, 12H). ESI-MS:C 66 H 131 Calculated m / z value for N3O3 = 1014.0. Measured value [M+H] + = 1015.3.

[0313] Example 74 N,N-Didecyl-10-((6-(Didecylamino)-6-oxohexyl)((1s,4s)-4-hydroxycyclohexyl)amino)decanamide (compound (1s,4s)-I-61)

[0314] [ka] Synthesis of intermediate 18 A mixture of (1s,4s)-4-aminocyclohexane-1-ol (3.77 mmol, 434 mg), intermediate 17 (0.94 mmol, 0.50 g), DIEA (2.92 mmol, 0.510 mL), and potassium iodide (2.83 mmol, 469 mg) in acetonitrile (1.9 mL) was heated at 140 °C by microwave irradiation for 1 hour. The reaction mixture was concentrated, and the resulting crude product was suspended in 95:5:1 (hexane:SiO:Et3N) and filtered. The filtrate was purified by flash chromatography (25% to 100% SiO in hexane), and then by second flash chromatography (1% to 10% MeOH in CHCl3) to obtain intermediate 18 (440 mg, 83%).

[0315] Synthesis of intermediate 19 Intermediate 19 was prepared from 6-bromohexanoic acid according to the procedure for intermediate 1 (Example 5). Yield (4.6 g, 76%).

[0316] [ka]

[0317] (1s,4s)-I-61 synthesis Compound (1S,4S)-I-61 was synthesized from intermediates 18 and 19 according to the procedure of intermediate 15 (Example 68). Yield (90 mg, 53%). 1 H NMR(400 MHz, CDCl3)δ 4.02-3.94(m, 1H), 3.32-3.23(m, 4H), 3.23-3.15(m, 4H), 2.52-2.37(m, 5H), 2.31-2.22(m, 4H), 1.90-1.77(m, 2H), 1.76-1.04(m, 97H), 0.92-0.83(m, 12H). ESI-MS:C 62 H 123 Calculated m / z value for N3O3 = 958.0. Measured value [M+H] + =959.3.

[0318] Example 75 N,N'-((((1s,4s)-4-hydroxycyclohexyl)azandiyl)bis(octane-8,1-diyl))bis(N-decyldecanamide)(compound(1s,4s)-I-63)

[0319] [ka] (1s,4s)-I-63 synthesis Compound (1S,4S)-I-63 was prepared from Int4-1 (Example 33) and (1s,4s)-4-aminocyclohexane-1-ol according to general procedure B. The compound was purified by automated flash chromatography (5-100% ethylethanol in hexane containing 1% Et3N) to obtain the title compound (122 mg, 59%). 1H NMR(400 MHz, CDCl3)δ 4.02-3.95(m, 1H), 3.32-3.23(m, 4H), 3.23-3.15(m, 4H), 2.54-2.34(m, 5H), 2.31-2.22(m, 4H), 1.89-1.77(m, 2H), 1.74-1.04(m, 94H), 0.92-0.84(m, 12H). ESI-MS:C 62 H 123 Calculated m / z value for N3O3 = 958.0, measured value [M+H] + =959.3.

[0320] Example 76 N-decyl-N-(8-((8-(didecylamino)-8-oxooctyl)((1s,4s)-4-hydroxycyclohexyl)amino)octyl)decanamide (compound (1s,4s)-I-47)

[0321] [ka] (1s,4s)-I-47 synthesis A mixture of intermediate 14 (0.21 mmol, 112 mg), Int4-1 (0.14 mmol, 70 mg), DIEA (0.43 mmol, 0.075 mL), and potassium iodide (0.42 mmol, 69 mg) was heated by microwave irradiation at 160°C for 3 hours. The reaction mixture was concentrated, and the resulting crude product was suspended in 95:5:1 (hexane:HCl:Et3N) and filtered. The filtrate was purified by flash chromatography (5-100% HCl in hexane containing 1% Et3N) to obtain the title compound (45 mg, 34%). 1 H NMR(400 MHz, CDCl3)δ 4.03-3.94(m, 1H), 3.32-3.23(m, 4H), 3.23-3.15(m, 4H), 2.52-2.35(m, 5H), 2.31-2.22(m, 4H), 1.89-1.78(m, 2H), 1.73-1.02(m, 97H), 0.92-0.84(m, 12H). ESI-MS:C 62 H 123Calculated m / z value for N3O3 = 958.0, measured value [M+H] + = 959.2.

[0322] Example 77 N-decyl-N-(8-((8-(didecylamino)-8-oxooctyl)((1r,4r)-4-hydroxycyclohexyl)amino)octyl)decanamide (compound (1r,4r)-I-47)

[0323] [ka] Synthesis of intermediate 20 A mixture of (1r,4r)-4-aminocyclohexane-1-ol (3.99 mmol, 458 mg), intermediate 1 (1.0 mmol, 500 mg), DIEA (3.1 mmol, 0.54 mL), and potassium iodide (3.0 mmol, 495 mg) in acetonitrile (2.0 mL) was heated by microwave irradiation at 140 °C for 40 minutes. The reaction mixture was concentrated, and the resulting crude product was suspended in 95:5:1 (hexane:HCl:Et3N) and filtered. The filtrate was purified by flash chromatography (10% to 100% HCl in hexane), and then by second flash chromatography (1% to 12% MeOH in dichloromethane) to obtain intermediate 20 (363 mg, 68%).

[0324] Synthesis of (1r,4r)-I-47 A mixture of intermediate 20 (0.24 mmol, 130 mg), Int4-1 (0.29 mmol, 146 mg), DIEA (0.75 mmol, 0.13 mL), and potassium iodide (0.73 mmol, 121 mg) was heated by microwave irradiation at 140°C for 40 minutes. The reaction mixture was concentrated, and the resulting crude product was suspended in 95:5:1 (hexane:siRNA:Et3N) and filtered. The filtrate was purified by flash chromatography (5-100% siRNA in hexane containing 1% Et3N) to obtain the title compound (160 mg, 69%). 1H NMR(400 MHz, CDCl3)δ 3.60-3.49(m, 1H), 3.32-3.23(m, 4H), 3.23-3.15(m, 4H), 2.51-2.31(m, 5H), 2.31-2.21(m, 4H), 2.06-1.93(m, 2H), 1.84-1.02(m, 97H), 0.92-0.81(m, 12H). ESI-MS:C 62 H 123 Calculated m / z value for N3O3 = 958.0, measured value [M+H] + = 959.2.

[0325] The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referenced herein, including U.S. Provisional Patent Application No. 63 / 500,511 filed May 5, 2023, are incorporated herein by full attribution. Aspects of the embodiments may be modified to provide further embodiments by adopting concepts from various patents, applications, and publications as needed.

[0326] These and other modifications may be made to the embodiments in light of the detailed description above. In general, the terms used in the following claims should not be interpreted as limiting the claims to the specific embodiments disclosed in the specification and claims, but rather as encompassing all possible embodiments, along with the entire scope of equivalents to which rights are granted. Thus, the claims are not limited by this disclosure.

Claims

1. Structure (I): 【Chemistry 1】 (I) [In the formula, A is one or more fluoro, hydroxyl, C 1 -C 6 Alkyl or C 1 -C 6 A 3- to 10-membered carbocyclic or oxygen-containing heterocyclic ring, which may be substituted with alkylhydroxyl substituents; R 1 is -NR a C(=O)R 3 or -C(=O)NR b R c wherein; R 2 -NR d C(=O)R 4 Or -C(=O)NR e R f And; R 3 and R 4 Each is independent of C 6 -C 24 Alkyl or C 6 -C 24 It is an alkenil; R a , R b , R d and R e H and C are independent of each other. 1 -C 20 Alkyl or C 2 -C 20 It is an alkenil; R c and R f Each is independent of C 1 -C 20 Alkyl or C 2 -C 20 It is an alkenil; L 1 and L 2 Each can be directly joined or C 1 -C 6 It is alkylene; and L 2a and L 2b Each is independent of C 4 -C 12 It is alkylene; Each alkyl, alkylene, and alkenyl may be substituted with one or more fluoropolymers. or compounds having pharmaceutically acceptable salts or stereoisomers thereof.

2. R 1 However, -C(=O)NR b R c And R 2 However, -C(=O)NR e R f The compound according to claim 1.

3. R 1 However, -NR a C(=O)R 3 And R 2 However, -NR d C(=O)R 4 The compound according to claim 1.

4. R 1 However, -NR a C(=O)R 3 And R 2 However, -C(=O)NR e R f The compound according to claim 1.

5. R a , R b , R c , R d , R e and R f Each of them operates independently, C 1 -C 20 A compound according to any one of claims 1 to 4, wherein the compound is alkyl.

6. R a , R b , R c , R d , R e and R f Each of them operates independently, C 8 -C 19 The compound according to claim 5, wherein it is alkyl.

7. R a , R b , R c , R d , R e and R f Each of them operates independently, C 8 , C 9 , C 10 or C 19 The compound according to claim 6, wherein it is alkyl.

8. R 3 and R 4 each independently is C 6 -C 19 alkyl, the compound according to any one of claims 1 or 3 to 7.

9. R 3 and R 4 Each of them operates independently, C 8 -C 10 The compound according to claim 8, wherein it is alkyl.

10. R 3 and R 4 are each independently C 8 , C 9 or C 10 alkyl, the compound according to claim 9.

11. L 1 The compound according to any one of claims 1 to 10, wherein the bond is direct.

12. L 1 However, C 1 -C 6 A compound according to any one of claims 1 to 10, which is an alkylene.

13. L 1 However, C 1 or C 2 A compound according to any one of claims 1 to 10, which is an alkylene.

14. L 2 The compound according to any one of claims 1 to 13, wherein the bond is direct.

15. L 2 However, C 1 -C 6 The compound according to any one of claims 1 to 13, which is an alkylene.

16. L 2 However, C 1 or C 2 The compound according to any one of claims 1 to 13, which is an alkylene.

17. L 2a and L 2b Each of them operates independently, C 6 -C 10 The compound according to any one of claims 1 to 16, which is an alkylene.

18. L 2a and L 2b Each of them operates independently, C 6 , C 7 , C 8 , C 9 or C 10 The compound according to any one of claims 1 to 16, which is an alkylene.

19. A contains one or more hydroxyls or C 1 -C 6 C may be substituted with alkylhydroxyl substituents. 3 -C 10 A compound according to any one of claims 1 to 18, wherein the compound is a carbocyclic ring.

20. The compound according to claim 19, wherein A is a monocyclic compound.

21. The compound according to claim 19, wherein A is a bicyclic compound.

22. The compound according to claim 19, wherein A is a spirocyclic compound.

23. A contains one or more hydroxyls or C 1 -C 6 C may be substituted with alkylhydroxyl substituents. 3 -C 8 The compound according to claim 19, wherein the compound is a carbocyclic ring.

24. The compound according to claim 19, wherein A is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl.

25. A contains one or more hydroxyls or C 1 -C 6 The compound according to any one of claims 1 to 18, which is a 3 to 10-membered oxygen-containing heterocyclic ring that may be substituted with alkylhydroxyl substituents.

26. A contains one or more hydroxyls or C 1 -C 6 The compound according to claim 24, which is a 3- to 6-membered oxygen-containing heterocyclic ring that may be substituted with an alkylhydroxyl substituent.

27. The compound according to claim 24, wherein A is oxyranil, oxetanil, tetrahydrofuranil, or tetrahydropyranil.

28. A compound according to any one of claims 1 to 27, wherein A is unsubstituted.

29. The compound according to any one of claims 1 to 27, wherein A is substituted with hydroxyl. 【Request Item 30】 【Chemistry 2】 However, the structure is as follows: 【Transformation 3】 A compound according to any one of claims 1 to 29, having one of the above or a stereoisomer thereof. 【Request Item 31】 【Chemistry 4】 However, the structure is as follows: 【Transformation 5】 【Transformation 6】 A compound according to any one of claims 1 to 30, having one of the above.

32. R a , R b , R c , R d , R e , R f , L 1 , L 2 , L 2a and L 2b The compound according to any one of claims 1 to 31, wherein one or more of the compounds are substituted with one or more fluorine atoms.

33. The compound is an enantiomer pair or combination of the following structures (I): 【Transformation 7】 【Transformation 8】 The compound according to claim 1, which is a racemic mixture of one of the following.

34. Structure below: 【Chemistry 9】 【Chemistry 10】 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】 【Chemistry 16】 【Chemistry 17】 [Chemistry 18] 【Chemistry 19】 【Chemistry 20】 The compound according to claim 1, having one of the following.

35. Lipid nanoparticles comprising the compound and therapeutic agent according to any one of claims 1 to 34.

36. A composition comprising a compound according to any one of claims 1 to 34 or nanoparticles according to claim 35, and a therapeutic agent.

37. Lipid nanoparticles or compositions according to claim 35 or 36, further comprising one or more additives selected from neutral lipids, steroids, and polymer-conjugated lipids.

38. Lipid nanoparticles or composition according to claim 37, comprising one or more neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

39. The lipid nanoparticle or composition according to claim 38, wherein the neutral lipid is DSPC.

40. Lipid nanoparticles or composition according to any one of claims 37 to 39, wherein the molar ratio of the compound to the neutral lipid is in the range of about 2:1 to about 8:

1.

41. Lipid nanoparticles or composition according to any one of claims 37 to 40, wherein the steroid is cholesterol.

42. The lipid nanoparticles or composition according to claim 41, wherein the molar ratio of the compound to cholesterol is in the range of 5:1 to 1:1 or 2:1 to 1:

1.

43. Lipid nanoparticles or composition according to any one of claims 37 to 42, wherein the polymer conjugate lipid is a pegylated lipid.

44. The lipid nanoparticles or composition according to claim 43, wherein the molar ratio of the compound to the pegylated lipid is in the range of about 100:1 to about 20:1 or about 100:1 to about 10:

1.

45. The lipid nanoparticles or composition according to claim 43 or 44, wherein the pegylated lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, or PEG-dialkoxypropyl carbamate.

46. The pegylated lipid has the following structure (II): 【Chemistry 21】 (II) [In the formula, R 10 and R 11 Each of these is independently a linear or branched alkyl, alkenyl, or alkynyl of 10 to 30 carbon atoms, and the alkyl, alkenyl, or alkynyl may be cleaved by one or more ester bonds; and [w has a value between 30 and 60] The lipid nanoparticles or composition according to claim 43 or 44, having a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

47. R 10 and R 11 The lipid nanoparticle or composition according to claim 46, wherein each is independently a linear alkyl chain containing 12 to 16 carbon atoms.

48. The lipid nanoparticles or composition according to claim 46 or 47, wherein the lipid nanoparticles or composition comprises a plurality of compounds of structure (II), and the average value of w for the plurality is about 49.

49. A lipid nanoparticle or composition according to any one of claims 35 to 48, wherein the therapeutic agent comprises nucleic acid.

50. Lipid nanoparticles or composition according to claim 49, wherein the nucleic acid is selected from antisense and messenger RNA.

51. A method for administering a therapeutic agent to a patient in need thereof, the method comprising preparing and providing lipid nanoparticles or a composition according to any one of claims 35 to 50, and administering the composition to a patient.

52. A pharmaceutical composition comprising lipid nanoparticles according to claim 35 and a pharmaceutically acceptable diluent or additive.

53. A method for inducing protein expression in a patient in need thereof, the method comprising administering to the patient the pharmaceutical composition according to claim 52, wherein the lipid nanoparticles contain mRNA encoding the protein.

54. The method according to claim 53, wherein the protein is an antigen and the method is for inducing an immune response in a patient.

55. The method according to claim 53, wherein the protein is an antigen and the method is for vaccinating a patient against a pathogen.

56. The method according to claim 53, wherein the protein is for gene editing.

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