阅读说明：本技术 用于递送核酸的新型羰基脂质和脂质纳米颗粒制剂 (Novel carbonyl lipid and lipid nanoparticle formulations for delivery of nucleic acids ) 是由 杜新曜 史蒂文·M·安塞尔 于 2018-04-27 设计创作，主要内容包括：提供了具有以下结构(I)或(II)中的一种的化合物：<Image he="304" wi="700" file="DDA0002335743290000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>或者其药学上可接受的盐、互变异构体或立体异构体,其中R3a、R3b、L1、L2、G1a、G1b、G2a、G2b和G3如本文所限定。还提供了所述化合物作为脂质纳米颗粒制剂的组分以用于递送治疗剂的用途、包含所述化合物的组合物及其使用和制备方法。(Provided are compounds having one of the following structures (I) or (II): or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein R3a, R3b, L1, L2, G1a, G1b, G2a, G2b, and G3 are as defined herein. Also provided are uses of the compounds as components of lipid nanoparticle formulations for the delivery of therapeutic agents, compositions comprising the compounds, and methods of use and preparation thereof.)

1. A compound having one of the following structures (I) or (II):

or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:

L¹is-O (C ═ O) R¹、-(C＝O)OR¹、-C(＝O)R¹、-OR¹、-S(O)_xR¹、-S-SR¹、-C(＝O)SR¹、-SC(＝O)R¹、-NR^aC(＝O)R¹、-C(＝O)NR^bR^c、-NR^aC(＝O)NR^bR^c、-OC(＝O)NR^bR^cor-NR^aC(＝O)OR¹；

L²is-O (C ═ O) R²、-(C＝O)OR²、-C(＝O)R²、-OR²、-S(O)_xR²、-S-SR²、-C(＝O)SR²、-SC(＝O)R²、-NR^dC(＝O)R²、-C(＝O)NR^eR^f、-NR^dC(＝O)NR^eR^f、-OC(＝O)NR^eR^f；-NR^dC(＝O)OR²Or to R²A direct bond of (a);

G^1aand G^2aEach independently is C₂-C₁₂Alkylene or C₂-C₁₂An alkenylene group;

G^1band G^2bEach independently is C₁-C₁₂Alkylene or C₂-C₁₂An alkenylene group;

G³is C₁-C₂₄Alkylene radical, C₂-C₂₄Alkenylene radical, C₃-C₈Cycloalkylene or C₃-C₈Cycloalkenylene;

R^a、R^b、R^dand R^eEach independently is H or C₁-C₁₂Alkyl or C₂-C₁₂An alkenyl group;

R^cand R^fEach independently is C₁-C₁₂Alkyl or C₂-C₁₂An alkenyl group;

R¹and R²Each independently of the other being branched C₆-C₂₄Alkyl or branched C₆-C₂₄An alkenyl group;

R^3ais-C (═ O) N (R)^4a)R^5aOR-C (═ O) OR⁶；

R^3bis-NR^4bC(＝O)R^5b；

R^4aIs C₁-C₁₂An alkyl group;

R^4bis H, C₁-C₁₂Alkyl or C₂-C₁₂An alkenyl group;

R^5ais H, C₁-C₈Alkyl or C₂-C₈An alkenyl group;

when R is^4bWhen is H, R^5bIs C₂-C₁₂Alkyl or C₂-C₁₂An alkenyl group; or when R is^4bIs C₁-C₁₂Alkyl or C₂-C₁₂When an alkenyl radical, R^5bIs C₁-C₁₂Alkyl or C₂-C₁₂An alkenyl group;

R⁶is H, aryl or aralkyl; and

x is 0, 1 or 2, and

wherein each alkyl, alkenyl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, aryl, and aralkyl is independently substituted or unsubstituted.

2. The compound of claim 1, wherein G³Is unsubstituted.

3. The compound of any one of claims 1 or 2, wherein G³Is C₁-C₁₂An alkylene group.

4. The compound of any one of claims 1-3, wherein G is³Is C₃-C₁₂An alkylene group.

5. The compound of any one of claims 1-4, having one of the following structures (IA) or (IIA):

wherein y1 and z1 are each independently integers in the range of 2 to 12, and y2 and z2 are each independently integers in the range of 1 to 12.

6. The compound of any one of claims 1-5, wherein L¹is-O (C ═ O) R¹、-(C＝O)OR¹or-C (═ O) NR^bR^cAnd L is²is-O (C ═ O) R²、-(C＝O)OR²or-C (═ O) NR^eR^f(ii) a Or wherein L¹And L²Each independently is-O (C ═ O) R¹OR- (C ═ O) OR¹。

7. The compound of claim 6, having the following structure (IB), (IC), (ID); (IE) (IIB) or (IIC):

8. the compound of claim 6, having the following structures (IF), (IG), (IH); (IJ), (IID) or (IIE):

9. the compound of any one of claims 5, 6, or 8, wherein y1, y2, z1, and z2 are each independently integers in the range of 2 to 12.

10. The compound of any one of claims 5, 6, or 8, wherein y1, y2, z1, and z2 are each independently integers in the range of 4 to 10.

11. The compound of any one of claims 1-10, wherein R¹And R²Each independently of the other being branched C₆-C₂₄An alkyl group.

12. The compound of claim 11, wherein R¹And R²Each independently having the structure:

wherein:

R^7aand R^7bIndependently at each occurrence is H or C₁-C₁₂An alkyl group; and

a is an integer of 2 to 12 and,

wherein each is selected from R^7a、R^7bAnd a, such that R¹And R²Each independently branched and independently containing from 6 to 20 carbon atoms.

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

14. The compound of any one of claims 12 or 13, wherein at least one occurrence of R^7aIs H.

15. The compound of any one of claims 12 or 13, wherein R^7aAt each occurrence is H.

16. The compound of any one of claims 12 or 15, wherein at least one occurrence of R^7bIs C₁-C₈An alkyl group.

17. The compound of claim 16, wherein C₁-C₈Alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl or n-octyl.

18. The compound of any one of claims 1-17, wherein R¹Or R²Or both independently have one of the following structures:

19. the compound of any one of claims 6-10, wherein R^b、R^c、R^eAnd R^fEach independently is C₃-C₁₂An alkyl group.

20. The compound of any one of claims 6-11, wherein R^b、R^c、R^eAnd R^fIs n-hexyl.

21. The compound of any one of claims 6-11, wherein R^b、R^c、R^eAnd R^fIs n-octyl.

22. The compound of any one of claims 1-21, wherein R^3ais-C (═ O) N (R)^4a)R^5a。

23. The compound of claim 22, wherein R^4aIs ethyl, propyl, n-butyl, n-hexyl, n-octyl or n-nonyl.

24. The compound of any one of claims 21-22, wherein R^5aIs H, methyl, ethyl, propyl, n-butyl, n-hexyl or n-octyl.

25. The compound of any one of claims 1-21, wherein R^3ais-C (═ O) OR⁶。

26. The compound of claim 25, wherein R⁶Is benzyl.

27. The compound of any one of claims 25 or 26, wherein R⁶Is H.

28. The compound of any one of claims 1-26, wherein R^4a、R^5aAnd R⁶Independently optionally substituted with one or more substituents selected from: -OR^g、-NR^gC(＝O)R^h、-C(＝O)NR^gR^h、-C(＝O)R^h、-OC(＝O)R^h、-C(＝O)OR^hand-ORⁱOH, wherein:

R^gindependently at each occurrence is H or C₁-C₆An alkyl group;

R^hindependently at each occurrence is C₁-C₆An alkyl group; and

Rⁱindependently at each occurrence is C₁-C₆An alkylene group.

29. The compound of any one of claims 1-28, wherein R^3aHas one of the following structures:

30. the compound of any one of claims 1-18, wherein R^4bIs H, methyl, ethyl, propyl or octyl.

31. The compound of claim 30, wherein R^4bAnd R^5bIndependently optionally substituted with one or more substituents selected from: -OR^g、-NR^gC(＝O)R^h、-C(＝O)NR^gR^h、-C(＝O)R^h、-OC(＝O)R^h、-C(＝O)OR^hand-OR^hOH, wherein:

R^gindependently at each occurrence is H or C₁-C₆An alkyl group;

R^hindependently at each occurrence is C₁-C₆An alkyl group; and

Rⁱindependently at each occurrence is C₁-C₆An alkylene group.

32. The compound of any one of claims 30 or 31, wherein R^5bIs methyl, ethyl, propyl, heptyl or octyl.

33. The compound of any one of claims 1-18, wherein R^3bHas one of the following structures:

34. a compound selected from the compounds in table 1.

35. A compound selected from the compounds in table 2.

36. A composition comprising a compound of any one of claims 1-35 and a therapeutic agent.

37. The composition of claim 36, further comprising one or more excipients selected from the group consisting of: neutral lipids, steroids, and polymer-conjugated lipids.

38. The composition of claim 37, wherein the composition comprises one or more neutral lipids selected from the group consisting of: DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.

39. The composition of claim 38, wherein the neutral lipid is DSPC.

40. The composition of any one of claims 36-39, wherein the molar ratio of the compound to the neutral lipid ranges from about 2:1 to about 8:1.

41. The composition of any one of claims 37-40, wherein the steroid is cholesterol.

42. The composition of claim 41, wherein the molar ratio of the compound to cholesterol ranges from 5:1 to 1: 1.

43. The composition of any one of claims 37-42, wherein the polymer-conjugated lipid is a pegylated lipid.

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

45. The composition of any one of claims 43 or 44, wherein the pegylated lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, or PEG dialkoxypropylcarbamate.

46. The composition of any one of claims 43 or 44, wherein the pegylated lipid has the following structure (III):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

R⁸and R⁹Each independently a straight or branched alkyl, alkenyl or alkynyl group of 10 to 30 carbon atoms, wherein the alkyl, alkenyl or alkynyl group is optionally interrupted by one or more ester linkages; and

the average value of w ranges from 30 to 60.

47. The composition of claim 46, wherein R⁸And R⁹Each independently a linear alkyl chain containing from 12 to 16 carbon atoms.

48. The composition of any one of claims 46 or 47, wherein the average value w is about 49.

49. The composition of any one of claims 36-48, wherein the therapeutic agent comprises a nucleic acid.

50. The composition of claim 49, wherein the nucleic acid is selected from the group consisting of antisense RNA and messenger RNA.

51. A method of administering a therapeutic agent to a patient in need thereof, the method comprising preparing or providing the composition of any one of claims 36-50, and administering the composition to the patient.

52. A lipid nanoparticle comprising the compound of any one of claims 1-35.

53. A pharmaceutical composition comprising the lipid nanoparticle of claim 52 and a pharmaceutically acceptable diluent or excipient.

Technical Field

Embodiments of the present invention generally relate to novel lipids that can be used in combination with other lipid components (such as neutral lipids, cholesterol, and polymer-conjugated lipids) to form lipid nanoparticles for the delivery of therapeutic agents, such as nucleic acids (e.g., oligonucleotides, messenger RNAs), in vitro and in vivo.

Description of the related Art

Many challenges associated with nucleic acid delivery affect the desired response in biological systems. Nucleic acid-based therapies have great potential, but there remains a need to more efficiently deliver nucleic acids to appropriate sites within cells or organisms to achieve this potential. Therapeutic nucleic acids include, for example, messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, immunostimulatory nucleic acids, antagomirs, animirs, mimetics, supermir, and aptamers. Some nucleic acids, such as mRNA or plasmids, can be used to achieve expression of a particular cell product, which would be useful in treating diseases associated with, for example, 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 or not that sequence is inherent to the system. The expression product of the nucleic acid may increase the existing level of the protein, replace a missing or non-functional form of the protein, or introduce a new protein and related functions in the cell or organism.

Some nucleic acids, such as miRNA inhibitors, can be used to achieve expression of specific cellular products that are modulated by mirnas, which would be useful in treating diseases associated with, for example, protein or enzyme deficiencies. Therapeutic applications of miRNA inhibition are extremely broad, as constructs can be synthesized to inhibit one or more mirnas, which in turn regulate expression of mRNA products. Inhibition of endogenous mirnas can increase the expression of its downstream target endogenous proteins and restore appropriate function in a cell or organism as a means of treating diseases associated with a particular miRNA or group of mirnas.

Other nucleic acids can down-regulate intracellular levels of a particular mRNA, and thus can down-regulate synthesis of the corresponding protein by processes such as RNA interference (RNAi) or complementary binding of antisense RNA. Antisense oligonucleotides and RNAi are also very widely used in therapy, since oligonucleotide constructs with any nucleotide sequence directed against a target mRNA can be synthesized. Targets may include mRNA from normal cells, mRNA associated with disease states (e.g., cancer), and mRNA from infectious agents (e.g., viruses). To date, antisense oligonucleotide constructs have shown the ability to specifically down-regulate target proteins by degrading homologous mrnas, both in vitro and in vivo models. In addition, antisense oligonucleotide constructs are currently being evaluated in clinical studies.

However, the current use of oligonucleotides in a therapeutic setting faces two problems. First, free RNA is susceptible to nuclease digestion in plasma. Secondly, the ability of free RNA to enter the intracellular compartment in which the relevant translation machinery is located is limited. Lipid nanoparticles formed from lipids and oligonucleotides formulated with other lipid components (e.g., neutral lipids, cholesterol, PEG, pegylated lipids) have been used to block the degradation of RNA in plasma and to facilitate the uptake of oligonucleotides by cells.

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

Brief description of the drawings

Briefly, embodiments of the present invention provide lipid compounds (including stereoisomers, pharmaceutically acceptable salts, prodrugs or tautomers thereof) that can be used alone or in combination with other lipid components (such as neutral lipids, charged lipids, steroids (including, for example, all sterols) and/or analogs thereof, and/or polymer-conjugated lipids to form lipid nanoparticles for the delivery of therapeutic agents.

In one embodiment, compounds having the following structure (I) are provided:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein R is^3a、L¹、L²、G^1a、G^2aAnd G³As defined herein.

In other embodiments, compounds having the following structure (II) are provided:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein R is^3b、L¹、L²、G^1b、G^2bAnd G³As defined herein.

Also provided are pharmaceutical compositions comprising one or more compounds of the above structures (I) or (II) and a therapeutic agent. Lipid Nanoparticles (LNPs) comprising one or more compounds of structure (I) or (II) are also provided. In some embodiments, the pharmaceutical composition and/or LNP further comprises one or more components selected from the group consisting of neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. The disclosed compositions can be used to form lipid nanoparticles for the delivery of therapeutic agents.

In other embodiments, the present invention provides a method for administering a therapeutic agent to a patient in need thereof, the method comprising preparing a composition comprising a compound of structure (I) or (II) and a lipid nanoparticle of the therapeutic agent, and delivering the composition to the patient. For example, the composition can comprise an LNP comprising one or more compounds of structure (I) or (II).

These and other aspects of the invention will become apparent upon reference to the following detailed description.

Detailed description of the invention

In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these details.

Embodiments of the present invention are based, in part, on the discovery of novel lipids: provides advantages when used in lipid nanoparticles for the in vivo delivery of active or therapeutic agents, such as nucleic acids, into mammalian cells. In particular, embodiments of the present invention provide nucleic acid-lipid nanoparticle compositions comprising one or more novel lipids described herein that provide increased nucleic acid activity and improved composition tolerance in vivo, as compared to previously described nucleic acid-lipid nanoparticle compositions, resulting in a significant increase in therapeutic index. For example, embodiments provide lipid nanoparticles comprising one or more compounds of structure (I) or (II).

In particular embodiments, the present invention provides novel lipids that enable the formulation of improved compositions for the delivery of mRNA and/or other oligonucleotides in vitro and in vivo. In some embodiments, these improved lipid nanoparticle compositions can be used to express proteins encoded by mRNA. In other embodiments, these improved lipid nanoparticle compositions can be used to upregulate endogenous protein expression by delivering miRNA inhibitors targeting a particular miRNA or a set of mirnas modulating a target mRNA or mrnas. In other embodiments, these improved lipid nanoparticle compositions can be used to down-regulate (e.g., silence) the protein level and/or mRNA level of a target gene. In some other embodiments, lipid nanoparticles can also be used to deliver mRNA and plasmids to express transgenes. In other embodiments, the lipid nanoparticle compositions can be used to induce pharmacological effects resulting from protein expression, such as increasing red blood cell production by delivering suitable erythropoietin mRNA, or protecting against infection by delivering mRNA encoding a suitable antigen or antibody.

Lipid nanoparticles and compositions of embodiments of the invention can be used for a variety of purposes, including the delivery of encapsulated or associated (e.g., complexed) therapeutic agents, such as nucleic acids, to cells in vitro and in vivo. Accordingly, embodiments of the present invention provide methods of treating or preventing a disease or disorder in a subject in need thereof by contacting the subject with a lipid nanoparticle encapsulating or associated with a suitable therapeutic agent, wherein the lipid nanoparticle comprises one or more novel lipids described herein.

As described herein, embodiments of the lipid nanoparticles of the present invention are particularly useful for the delivery of nucleic acids, including, e.g., mRNA, antisense oligonucleotides, plasmid DNA, microrna (miRNA), miRNA inhibitors (antagomir/antimir), complementary RNA that interferes with messenger RNA (microrna), DNA, multivalent RNA, Dicer substrate RNA, complementary DNA (cdna), and the like. Thus, the lipid nanoparticles and compositions of embodiments of the invention can be used to induce expression of a desired protein in vivo and in vitro by contacting a cell with a lipid nanoparticle comprising one or more of the novel lipids described herein, wherein the lipid nanoparticle encapsulates or associates with a nucleic acid that is expressed to produce the desired protein (e.g., a messenger RNA or plasmid encoding the desired protein) or to inhibit a process that terminates expression of an mRNA (e.g., a miRNA inhibitor). Alternatively, the lipid nanoparticles and compositions of embodiments of the invention can be used to reduce expression of target genes and proteins in vivo and in vitro by contacting a cell with a lipid nanoparticle comprising one or more novel lipids described herein (e.g., a compound of structure (I) or (II)), wherein the lipid nanoparticle encapsulates or is associated with a nucleic acid that reduces target gene expression (e.g., an antisense oligonucleotide or small interfering rna (sirna)). The lipid nanoparticles and compositions of embodiments of the invention can also be used to co-deliver different nucleic acids (e.g., mRNA and plasmid DNA), either separately or in combination, such as can be used to provide an effect that requires co-localization of different nucleic acids (e.g., mRNA encoding suitable gene modifying enzymes and DNA segments for incorporation into a host genome).

Nucleic acids for use with embodiments of the invention may be prepared according to any available technique. For mRNA, the main preparation method is, but not limited to, enzymatic synthesis (also known as in vitro transcription), which represents the currently most efficient method for producing long sequence-specific mRNA. In vitro transcription describes the process of template-directed synthesis of RNA molecules from an engineered DNA template comprising an upstream phage promoter sequence (e.g., including but not limited to promoter sequences from T7, T3, and SP6 escherichia coli phages) linked to a downstream sequence encoding a gene of interest. Template DNA for in vitro transcription can be prepared from a variety of sources using appropriate techniques well known in the art, including but not limited to plasmid DNA and polymerase chain reaction amplification (see Linpinsel, j.l and Conn, g.l., General protocols for preparation of plasmid DNA templates, and Bowman, j.c., Azizi, b., Lenz, t.k., Ray, p., and Williams, l.d.in RNA in vitro transcription and RNA purification by modification, pen g.l., eds, new york, n.y.humana Press 2012).

RNA transcription is performed in vitro using a linearized DNA template in the presence of the corresponding RNA polymerase and adenosine, guanosine, uridine and cytidine ribonucleoside triphosphates (rntps) under conditions that support polymerase activity while minimizing potential degradation of the resulting mRNA transcript. In vitro transcription can be performed using a variety of commercially available kits, including but not limited to the RiboMax Large Scale RNA production System (Promega), the MegaScript transcription kit (Life Technologies), as well as commercially available reagents, including RNA polymerase and rNTP. Methods for in vitro transcription of mRNA are well known in the art. (see, e.g., Lomock, R.,1972, In Vitro Transcription, Ann RevBiochem Vol. 41, 409-46; Kamakaka, R.T. and Kraus, W.L.2001.In Vitro Transcription. Current Protocols In Cell biology.2:11.6: 11.6.1-11.6.17; Beckert, B. and Masquida, B., (2010) Synthesis of RNA by In Vitro Transcription In RNA In Molecular Biology Vol. 703 (Neilson, H. editor), New York, N.Y.Humana Press, Bruss, unelle, J.L. and Green, R.,2013, Chapter Fiter-In Vitro Transcription In PCR, Vol. 530, all incorporated herein by reference DNA, PCR, Vol. 114).

The desired in vitro transcribed mRNA is then purified from the undesired components of the transcription or related reaction (including unincorporated rNTP, proteases, salts, short RNA oligomers, etc.). Techniques for isolating mRNA transcripts are well known in the art. Well known procedures include phenol/chloroform extraction or precipitation with alcohols (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride. Other non-limiting examples of purification procedures that can be used include size exclusion chromatography (Lukavsky, p.j. and Puglisi, j.d.,2004, Large-scale preparation and purification of polyacylamide-free RNA oligonucleotides, RNA v.10,889-893), silica-based affinity chromatography, and polyacrylamide gel electrophoresis (Bowman, j.c., azi, b., Lenz, t.k., Ray, p., and Williams, l.d.in RNA in vision transcription and RNA purification by condensation PAGE in communication and in vision RNA synthesis Methods v.941n g.l., editions, New York, n.y.manipulation, p.y.n. human expression and RNA synthesis Methods 2012). Purification can be performed using a variety of commercially available kits including, but not limited to, the SV Total Isolation System (Promega) and the In Vitro Transcription clean and Concentration Kit (In Vitro Transcription Cleanup and Concentration Kit) (Norgen Biotek).

Furthermore, although reverse transcription can produce large quantities of mRNA, the product can contain many abnormal RNA impurities associated with undesirable polymerase activity that may need to be removed from the full-length mRNA preparation. These include short RNAs produced by abortive transcription initiation, and double-stranded RNAs (dsrnas) produced by RNA-dependent RNA polymerase activity, RNA-initiated transcription from an RNA template, and self-complementary 3' extension. It has been demonstrated that these contaminants with dsRNA structures can lead to undesired immunostimulatory activity by interacting with various innate immunosensors in eukaryotic cells that function to recognize specific nucleic acid structures and induce effective immune responses. This in turn can significantly reduce mRNA translation because protein synthesis is reduced during the innate cellular immune response. Thus, additional techniques for removing these dsRNA contaminants have been developed and are known in the art, including but not limited to scalable HPLC Purification (see, e.g., Kariko, K., Muramatsu, H., Ludwig, J. and Weissman, D.,2011, Generation of the optimal mRNA for therapy: HPLCpurposion activities activation and promotion transformation of nucleic Acid-modified, protein-encoding mRNA, nucleic Acid Res, v.39142; Weisman, D., Pardi, N., Muramatsu, H., and Kariko, K., HPLC Purification of viral transformed RNA in Synthetic Messenger RNA and method, Molecular editor 969. Biodistribution H., 969. Biodistribution). HPLC purified mRNA is reported to be translated at much higher levels, particularly in primary cells and in vivo.

Various modifications have been described in the art for altering the specific properties and increasing the utility of in vitro transcribed mRNA. These include, but are not limited to, modifications to the 5 'and 3' ends of the mRNA. Endogenous eukaryotic mrnas typically contain a cap structure at the 5' -end of the mature molecule that plays an important role in mediating the binding of mRNA cap-binding proteins (CBPs), which in turn are responsible for enhancing the stability of the mRNA and the efficiency of mRNA translation in the cell. Thus, the highest level of protein expression is achieved with capped mRNA transcripts. The 5' -cap contains a 5' -5 ' -triphosphate linkage between the 5' -majority nucleotide (5 ' -mostnucleotide) and the guanine nucleotide. The conjugated guanine nucleotide is methylated at position N7. Additional modifications include methylation of the penultimate and penultimate majority of 5 '-nucleotides at the 2' -hydroxyl group.

A variety of different cap structures can be used to generate the 5' -cap of the in vitro transcribed synthetic mRNA. 5' -capping of synthetic mRNA can be performed with chemical cap analogs co-transcription (i.e., capping during in vitro transcription). For example, the anti-reverse cap analog (ARCA) cap contains a 5' -5 ' -triphosphate guanine-guanine linkage, wherein one guanine contains an N7 methyl group and a 3' -O-methyl group. However, during this co-transcription process, up to 20% of the transcripts remain uncapped, and the synthesized cap analogs differ from the 5' -cap structure of real cellular mRNA, potentially reducing translatability and cellular stability. Alternatively, the synthetic mRNA molecule may also be enzymatically capped after transcription. These can result in a more authentic 5 '-cap structure that more closely mimics, structurally or functionally, the endogenous 5' -cap, with enhanced binding of cap-binding proteins, extended half-life, reduced sensitivity to 5 'endonucleases, and/or reduced 5' decapping. Many Synthetic 5' -cap analogs have been developed and are known in the art to enhance mRNA stability and translatability (see, e.g., Grudzien-Nogalska, e., Kowalska, j., Su, w., Kuhn, a.n., sleprenkov, s.v., Darynkiewicz, e., Sahin, u., jemielite, j., and Rhoads, r.e., Synthetic mRNAs with a surfactant transformation and specificity in Synthetic Messenger RNA and Cell metabolism in Methods in Molecular Biology v.969 (rabinich, p.h. editors, 2013).

At the 3' -end, long-chain adenine nucleotides (poly-a tail) are typically added to mRNA molecules during RNA processing. Immediately after transcription, the 3 'end of the transcript is cleaved to release the 3' hydroxyl group where poly-a polymerase will add an adenine nucleotide chain to the RNA in a process known as polyadenylation. Poly-A tails have been shown extensively to enhance 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.14373-377; Guaniyogi, 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) taof mRNAs: Bodyguard inukaryotes, Bodyscher in bacterial, Cell, v.111, 611-613).

Poly (a) tailing of in vitro transcribed mRNA can be achieved using a variety of methods including, but not limited to, cloning of a poly (T) cluster (poly (T) track) into a DNA template, or post-transcriptional addition using a poly (a) polymerase. The first case allows in vitro transcription of mRNA with a defined length of poly (a) tail, depending on the size of the poly (T) cluster, but requires additional manipulation of the template. The latter case involves the enzymatic addition of a poly (a) tail to in vitro transcribed mRNA using a poly (a) polymerase that incorporates catalytic adenine residues into the 3' end of the RNA, which does not require additional manipulation of the DNA template, but results in mRNA with poly (a) tails of heterogeneous length. 5 '-capping and 3' -poly (A) Tailing may be performed using a variety of commercially available kits, including but not limited to poly (A) Polymerase Tailing kit (Poly (A) Polymerase Tailing kit) (EpiCenter), mMESSAGE mMACHINE T7 Ultra kit, and poly (A) Tailing kit (Poly (A) Tailing kit) (Life Technologies), as well as commercially available reagents, various ARCA caps, poly (A) Polymerase, and the like.

It has been reported that in addition to 5 'cap and 3' polyadenylation, other modifications of in vitro transcripts provide benefits related to translational efficiency and stability. It is well known in the art that pathogenic DNA and RNA can be recognized by a variety of sensors within eukaryotes and trigger potent innate immune responses. The ability to distinguish pathogenicity from self DNA and RNA has been shown to be based at least in part on structural and nucleoside modifications, since most nucleic acids of natural origin contain modified nucleosides. In contrast, RNA synthesized in vitro lacks these modifications, thus making it immunostimulatory, which in turn can inhibit efficient mRNA translation as outlined above. Introduction of modified nucleosides into In vitro transcribed mRNA can be used to prevent recognition and activation of RNA sensors, thereby alleviating this undesirable immunostimulatory activity and enhancing translation ability (see, e.g., Kariko, K. and Weissman, D.2007, Natural overcoat nucleic acid modifications, current optics diagnostic device, v.10523-532; Pardi, N., Muramatsu, H., Weissman, D.Kariko, K.in vitro transcription of RNA binding nucleic acids In synthetic RNA and modification of Cell biology, H.2019, biological coding of protein molecules, H.2013, H.D.; protein coding protein modification, expression; kariko, K., Muramatsu, H., Welsh, F.A., Ludwig, J., Kato, H., Akira, S., Weissman, D.,2008, Incorporation of pseudoudidine Into mRNAYIelds Superior nonlinear Vector With incorporated transformed regional capacity and Biological Stability, Mol Ther v.16, 1833-1840. Modified nucleosides and nucleotides used in modified RNA synthesis can be prepared, monitored, and utilized using general methods and procedures known in the art. A wide variety of nucleoside modifications are available which may be incorporated to some extent into in vitro transcribed mRNA, either alone or in combination with other modified nucleosides (see e.g. US 2012/0251618). It has been reported that in vitro synthesis of nucleoside-modified mrnas reduces the ability to activate immunosensors with concomitant enhanced translation.

Other components of an mRNA that can be modified to provide advantages in terms of translatability and stability include the 5 'and 3' untranslated regions (UTRs). Optimization of the UTR (which can result In advantageous 5 'and 3' UTRs from cellular or viral RNA), whether performed simultaneously or separately, has been shown to improve mRNA stability and translation efficiency of In vitro transcribed mRNA (see, e.g., Pardi, n., Muramatsu, h., Weissman, d., Kariko, k., In vitro transcription of long RNA contacting modified nucleotides In Synthetic Messenger RNA and cell metabolism modification In Methods In Molecular Biology v.969(Rabinovich, p.h. ed), 2013).

In addition to mRNA, other nucleic acid payloads may also be used in embodiments of the invention. For oligonucleotides, methods of preparation include, but are not limited to, chemical synthesis and enzymatic, chemical cleavage of longer precursors, in vitro transcription as described above, and the like. Methods for synthesizing DNA and RNA nucleotides are widely used and well known in the art (see, e.g., Gait, M.J. (eds.) Oligonucleotide synthesis: a reactive approach, Oxford [ Oxfordshire ], Washington, D.C.: IRL Press, 1984; and Herdewin, P. (eds.) Oligonucleotide synthesis: Methods and applications, Methods in Molecular Biology, v.288(Clifton, N.J.) Totowa, N.J.: Humana Press, 2005; both of which are incorporated herein by reference).

For plasmid DNA, preparations for use with embodiments of the present invention generally utilize, but are not limited to, amplification and isolation of plasmid DNA in vitro in liquid culture of bacteria containing the plasmid of interest. The presence of the gene encoding resistance to a particular antibiotic (penicillin, kanamycin, etc.) in the plasmid of interest allows those bacteria containing the plasmid of interest to be able to grow selectively in antibiotic-containing cultures. Methods for isolating plasmid DNA are widely used and well known in the art (see, e.g., Heilig, J., Elbin, K.L., 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., U.S.,

S.,

r. and Schmidt, S.R. (2008), Large-scale production of endoxin-free plasmids for transfer expression in mammariance culture, Biotechnol.Bioeng.,99: 557-; and US6197553B 1). Plasmid isolation can be performed using a variety of commercially available kits, including but not limited to the Plasmid plus (Qiagen), GenJET Plasmid maxiPrep (Thermo), and PureYield maxiPrep (Promega) kits, as well as commercially available reagents.

Various exemplary embodiments of the lipids, lipid nanoparticles, and compositions comprising the same of the present invention, and their use to deliver active agents (e.g., therapeutic agents) such as nucleic acids to modulate gene and protein expression are described in further detail below.

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

Throughout the specification and claims, the word "comprise" and variations such as "comprises" and "comprising" are to be interpreted in an open and inclusive sense, i.e., "including but not limited to," unless the context requires otherwise.

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

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

The phrase "inducing expression of a desired protein" refers to a nucleic acid that is capable of increasing expression of the desired protein. To examine the extent of protein expression, a test sample (e.g., a cultured cell sample expressing a desired protein) or a test mammal (e.g., a mammal, such as a human or animal model, such as a rodent (e.g., mouse) or non-human primate (e.g., monkey) model) can be contacted with a nucleic acid (e.g., a nucleic acid in combination with a lipid of the invention). The expression of the desired protein in the test sample or test animal is compared to the expression of the desired protein in a control sample (e.g., a cultured cell sample expressing the desired protein) or a control mammal (e.g., a mammal such as a human or animal model, such as a rodent (e.g., mouse) or non-human primate (e.g., monkey) model) that is not contacted with or administered the nucleic acid. When the desired protein is present in the control sample or control mammal, the expression of the desired protein in the control sample or control mammal can be assigned a value of 1.0. In particular embodiments, inducing expression of the desired protein can be achieved when the ratio of expression of the desired protein in the test sample or test mammal to the level of expression of the desired protein in the control sample or control mammal is greater than 1, e.g., about 1.1, 1.5, 2.0, 5.0, or 10.0. Inducing expression of the desired protein is achieved when any measurable level of the desired protein is detected in the test sample or the test mammal when the desired protein is not present in the control sample or the control mammal. One of ordinary skill in the art will appreciate appropriate assays for determining the level of protein expression in a sample, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function and phenotype assays, or assays based on reporter proteins that produce fluorescence or luminescence under appropriate conditions.

The phrase "inhibiting expression of a target gene" refers to a nucleic acid that is capable of silencing, reducing, or inhibiting expression of a target gene. To examine the extent of gene silencing, a test sample (e.g., a sample of cultured cells expressing a target gene) or a test mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g., mouse) or non-human primate (e.g., monkey) model) is contacted with a nucleic acid that silences, reduces, or inhibits expression of the target gene. The expression of the target gene in the test sample or test animal is compared to the expression of the target gene in a control sample (e.g., a cultured cell sample expressing the target gene) or a control mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g., mouse) or non-human primate (e.g., monkey) model) that has not been contacted with or administered the nucleic acid. The expression of the target gene in the control sample or control mammal can be assigned a value of 100%. In particular embodiments, silencing, inhibiting, or reducing expression of the target gene is achieved when the level of target gene expression in the test sample or the test mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% relative to the level of target gene expression in the control sample or the control mammal. In other words, the nucleic acid is capable of silencing, reducing or inhibiting expression of the target gene in the test sample or the test mammal by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to the level of expression of the target gene in a control sample or a control mammal that has not been contacted or administered the nucleic acid. Suitable assays for determining the expression level of a target gene include, but are not limited to, examining protein or mRNA levels using techniques known to those skilled in the art, such as, for example, dot blot, Northern blot, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those skilled in the art.

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

The term "nucleic acid" as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single-or double-stranded form, and includes DNA, RNA, and hybrids thereof. The DNA may be in the form of an antisense molecule, plasmid DNA, cDNA, PCR product, or vector. The RNA can be in the form of small hairpin RNA (shrna), messenger RNA (mrna), antisense RNA, miRNA, microrna, multivalent RNA, Dicer substrate RNA, or viral RNA (vrna), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring and non-naturally occurring and have similar binding properties as the reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral-methylphosphonate, 2' -O-methyl ribonucleotide, and peptide-nucleic acid (PNA). Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms and complementary sequences, as well as the sequence explicitly indicated. In particular, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res.,19:5081 (1991); Ohtsuka et al, J.biol.chem.,260: 2605-. "nucleotides" contain the following sugars: deoxyribose (DNA) or Ribose (RNA); a base and a phosphate group. Nucleotides are linked together 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, including but not limited to modifications to place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises a coding sequence of partial or entire length necessary for the production of a polypeptide or precursor polypeptide.

"Gene product" as used herein refers to a gene product such as an RNA transcript or polypeptide.

The term "lipid" refers to a group of organic compounds, which include, but are not limited to, esters of fatty acids, and are generally characterized as being poorly soluble in water but soluble in many organic solvents. They are generally divided into at least three categories: (1) "simple lipids" including fats and oils and waxes; (2) "complex lipids," including phospholipids and glycolipids; and (3) "derivatized lipids" such as steroids.

A "steroid" is a compound comprising the following carbon skeleton:

non-limiting examples of steroids include cholesterol and the like.

"cationic lipid" refers to a lipid capable of being positively charged. Exemplary cationic lipids include one or more amine groups bearing a positive charge. Preferred cationic lipids are ionizable such that they can exist in a positively charged or neutral form depending on pH. Under different pH conditions, ionization of cationic lipids affects the surface charge of lipid nanoparticles. This charge state can affect plasma protein uptake, blood clearance, and tissue distribution that are critical for intracellular delivery of nucleic acids (Semple, s.c., et al, adv.drug Deliv Rev 32:3-17(1998)) as well as the ability to form endosomolytic (endosomolytic) non-bilayer structures (Hafez, i.m., et al, Gene Ther 8:1188-1196 (2001)).

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

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

The term "charged lipid" refers to any of a variety of lipid species that exist in a positive or negative form that is independent of pH, within a useful physiological range (e.g., pH-3 to pH-9). The charged lipids may be of synthetic or natural origin. Examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyltrimethylammonium propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, dimethylaminoethanecarbamoyl sterol (e.g., DC-Chol).

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

In various embodiments, the lipid nanoparticle has an average diameter of about 30nm to about 150nm, about 40nm to about 150nm, about 50nm to about 150nm, about 60nm to about 130nm, about 70nm to about 110nm, about 70nm to about 100nm, about 80nm to about 100nm, about 90nm to about 100nm, about 70 to about 90nm, about 80nm to about 90nm, about 70nm to about 80nm, or about 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm, or 150nm, and is substantially non-toxic. In certain embodiments, the nucleic acid is resistant to degradation by nucleases in aqueous solution when present in the lipid nanoparticle. Lipid nanoparticles comprising nucleic acids and methods for their preparation are disclosed, for example, in U.S. patent publication nos. 2004/0142025, 2007/0042031 and PCT publication nos. WO2017/004143, WO 2015/199952, WO 2013/016058 and WO 2013/086373, the entire disclosures of which are incorporated herein by reference in their entirety for all purposes.

As used herein, "encapsulated lipid" refers to a lipid nanoparticle that provides complete encapsulation, partial encapsulation, or both, for an active or therapeutic agent, such as a nucleic acid (e.g., mRNA). In embodiments, the nucleic acid (e.g., mRNA) is completely encapsulated in the lipid nanoparticle.

As used herein, the term "aqueous solution" refers to a composition comprising water.

With respect to nucleic acid-lipid nanoparticles, "serum stable" means that nucleotides do not significantly degrade after exposure to serum or nuclease assays that would significantly degrade free DNA or RNA. Suitable assays include, for example, standard serum assays, dnase assays, or rnase assays.

As used herein, "systemic delivery" refers to the delivery of therapeutic products that can result in widespread exposure of active agents in the organism. Some administration techniques may result in systemic delivery of certain agents, but not others. Systemic delivery means that a useful, preferably therapeutic amount of the agent is exposed to the majority of the body. Systemic delivery of the 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 the lipid nanoparticle is by intravenous delivery.

As used herein, "local delivery" refers to the delivery of an active agent directly to a target site within an organism. For example, the agent may be delivered locally by direct injection into a disease site, such as a tumor, other target site, such as a site of inflammation, or a target organ (e.g., liver, heart, pancreas, kidney, etc.). Local delivery may also include local administration or local injection techniques, such as intramuscular, subcutaneous or intradermal injection. Local delivery does not exclude systemic pharmacological effects.

"alkyl" refers to a straight or branched hydrocarbon chain radical consisting only of carbon and hydrogen atoms, which is saturated, having, for example, one to twenty-four carbon atoms (C)₁-C₂₄Alkyl), four to twenty carbon atoms (C)₄-C₂₀Alkyl), six to sixteen carbon atoms (C)₆-C₁₆Alkyl), six to nine carbon atoms (C)₆-C₉Alkyl), one to fifteen carbon atoms (C)₁-C₁₅Alkyl), one to twelve carbon atoms (C)₁-C₁₂Alkyl), one to eight carbon atoms (C)₁-C₈Alkyl) or one to six carbon atoms (C)₁-C₆Alkyl) and is attached to the rest of the molecule by a single bond, such as methyl, ethyl, n-propyl, 1 methylethyl (isopropyl), n-butyl, n-pentyl, 1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, and the like. Unless otherwise specifically stated in the specification, an alkyl group is optionally substituted.

"alkenyl" means a straight or branched hydrocarbon chain radical consisting only of carbon and hydrogen atomsGroups containing one or more carbon-carbon double bonds and having, for example, two to twenty-four carbon atoms (C)₂-C₂₄Alkenyl), four to twenty carbon atoms (C)₄-C₂₀Alkenyl), six to sixteen carbon atoms (C)₆-C₁₆Alkenyl), six to nine carbon atoms (C)₆-C₉Alkenyl), from two to fifteen carbon atoms (C)₂-C₁₅Alkenyl), di-to twelve carbon atoms (C)₂-C₁₂Alkenyl), two to eight carbon atoms (C)₂-C₈Alkenyl) or two to six carbon atoms (C)₂-C₆Alkenyl) and is attached to the remainder of the molecule by a single bond, such as vinyl, prop-1-enyl, but-1-enyl, pent-1, 4-dienyl, and the like. Unless otherwise specifically stated in the specification, an alkenyl group is optionally substituted.

"alkynyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing one or more carbon-carbon triple bonds and having, for example, two to twenty-four carbon atoms (C)₂-C₂₄Alkynyl), four to twenty carbon atoms (C)₄-C₂₀Alkynyl), six to sixteen carbon atoms (C)₆-C₁₆Alkynyl), six to nine carbon atoms (C)₆-C₉Alkynyl), two to fifteen carbon atoms (C)₂-C₁₅Alkynyl), two to twelve carbon atoms (C)₂-C₁₂Alkynyl), two to eight carbon atoms (C)₂-C₈Alkynyl) or two to six carbon atoms (C)₂-C₆Alkynyl) and is attached to the remainder of the molecule by a single bond, such as ethynyl, propynyl, butynyl, pentynyl, and the like. Unless otherwise specifically stated in the specification, alkynyl groups are optionally substituted.

"alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain consisting only of carbon and hydrogen connecting the remainder of the molecule to a group, which is saturated and has, for example, one to twenty-four carbon atoms (C)₁-C₂₄Alkylene), one to fifteen carbon atoms (C)₁-C₁₅Alkylene), one to twelve carbon atoms (C)₁-C₁₂Alkylene) of one to eight carbon atoms(C₁-C₈Alkylene), one to six carbon atoms (C)₁-C₆Alkylene), two to four carbon atoms (C)₂-C₄Alkylene), one to two carbon atoms (C)₁-C₂Alkylene) such as methylene, ethylene, propylene, n-butylene, and the like. The alkylene chain is connected to the rest of the molecule by single bonds and to the group by single bonds. The point of attachment of the alkylene chain to the rest of the molecule and to the group may be through one or any two carbons in the chain. Unless otherwise specifically stated in the specification, the alkylene chain may be optionally substituted.

"alkenylene" or "alkenylene chain" refers to a straight or branched divalent hydrocarbon chain consisting only of carbon and hydrogen connecting the rest of the molecule to a group, containing one or more carbon-carbon double bonds and having, for example, two to twenty-four carbon atoms (C)₂-C₂₄Alkenylene), from two to fifteen carbon atoms (C)₂-C₁₅Alkenylene), di-to twelve carbon atoms (C)₂-C₁₂Alkenylene), two to eight carbon atoms (C)₂-C₈Alkenylene), two to six carbon atoms (C)₂-C₆Alkenylene) or two to four carbon atoms (C)₂-C₄Alkenylene), such as ethenylene, propenylene, n-butenyl, and the like. The alkenylene chain is connected to the rest of the molecule by single or double bonds and to the group by single or double bonds. The point of attachment of the alkenylene chain to the rest of the molecule and to the group may be through one or any two carbons in the chain. The alkenylene chain may be optionally substituted unless otherwise specifically indicated in the specification.

"aryl" refers to a carbocyclic ring system group comprising hydrogen, 6 to 18 carbon atoms, and at least one aromatic ring. For the purposes of the present invention, aryl groups are monocyclic, bicyclic, tricyclic or tetracyclic ring systems, which may include fused or bridged ring systems. Aryl groups include, but are not limited to, those derived from aceanthrylene, acenaphthene, acephenanthrylene, anthracene, azulene, benzene, phenanthrylene, aceanthrylene, azulene, and the like,

Fluoranthene, fluorene, paranAryl groups known as indacene, symindacene, indane, indene, naphthalene, phenalene, phenanthrene, obsidian, pyrene, and triphenylene. Unless otherwise specifically stated in the specification, the term "aryl" or the prefix "ar-" (such as in "aralkyl") is intended to include optionally substituted aryl groups.

"aralkyl" means a group of the formula-R_b-R_cWherein R is_bIs alkylene or alkenylene as defined above and R_cIs one or more aryl groups as defined above, such as benzyl, diphenylmethyl, and the like. Unless otherwise specifically stated in the specification, an aralkyl group is optionally substituted.

"cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon group consisting only of carbon and hydrogen atoms, which may include fused or bridged ring systems having 3 to 15 carbon atoms, 3 to 10 carbon atoms, or 3 to 8 carbon atoms, which are saturated and are connected to the rest of the molecule by single bonds. Monocyclic cycloalkyl groups include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic groups include, for example, adamantyl, norbornyl, decahydronaphthyl, 7-dimethyl-bicyclo [2.2.1] heptyl, and the like. Unless otherwise specifically stated in the specification, cycloalkyl groups may be optionally substituted.

"cycloalkylene" is a divalent cycloalkyl group. Unless otherwise specifically stated in the specification, cycloalkylene groups may be optionally substituted.

"cycloalkenyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems having 3 to 15 carbon atoms, 3 to 10 carbon atoms, or 3 to 8 carbon atoms, containing one or more carbon-carbon double bonds and being connected to the rest of the molecule by single bonds. Monocyclic cycloalkenyl groups include, for example, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl. Unless otherwise specifically stated in the specification, cycloalkenyl groups may be optionally substituted.

"cycloalkenylene" is a divalent cycloalkenyl group. Unless otherwise specifically stated in the specification, cycloalkenylene groups may be optionally substituted.

The term "substituted" as used herein means any of the above groups (e.g., alkyl, alkenyl, alkynyl, alkylene, alkenylene, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkylene, or cycloalkenylene), wherein at least one hydrogen atom is replaced by a bond to a non-hydrogen atom such as, but not limited to: halogen atoms such as F, Cl, Br or I; oxo (═ O); a hydroxyl group (-OH); c₁-C₁₂An alkyl group; a cycloalkyl group; - (C ═ O) OR'; -O (C ═ O) R'; -C (═ O) R'; -OR'; -S (O)_xR’；-S-SR’；-C(＝O)SR’；-SC(＝O)R’；-NR’R’；-NR’C(＝O)R’；-C(＝O)NR’R’；-NR’C(＝O)NR’R’；-OC(＝O)NR’R’；-NR’C(＝O)OR’；-NR’S(O)_xNR’R’；-NR’S(O)_xR'; and-S (O)_xNR ' R ', wherein R ' is independently at each occurrence H, C₁-C₁₅Alkyl or cycloalkyl, and x is 0, 1 or 2. In some embodiments, the substituent is C₁-C₁₂An alkyl group. In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group, such as fluoro. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group (-OR'). In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group (-NR 'R').

"optional" or "optionally" (e.g., optionally substituted) means that the subsequently described event can or can not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, "optionally substituted alkyl" means that the alkyl group may or may not be substituted, and the description includes both substituted alkyl groups and unsubstituted alkyl groups.

By "prodrug" is meant a compound that can be converted under physiological conditions or by solvolysis to a biologically active compound of structure (I) or (II). Thus, the term "prodrug" refers to a pharmaceutically acceptable metabolic precursor of a compound of structure (I) or (II). Prodrugs may be inactive when administered to a subject in need thereof, but convert in vivo to an active compound of structure (I) or (II). Prodrugs are generally rapidly converted in vivo, for example by hydrolysis in blood, to yield the parent compound of structure (I) or (II). Prodrug compounds generally provide the advantages of solubility, histocompatibility, or delayed release in mammalian organisms (see Bundgard, h., Design of produgs (1985), pages 7-9, pages 21-24 (Elsevier, Amsterdam)). A discussion of prodrugs is provided in Higuchi, t, et al, a.c. s.symposium Series, volume 14 and Bioreversible Carriers in Drug Design, editors b.roche, american pharmaceutical Association and Pergamon Press, 1987.

The term "prodrug" is also intended to include any covalently bonded carriers that release the active compound of structure (I) or (II) in vivo when such prodrug is administered to a mammalian subject. Prodrugs of compounds of structure (I) or (II) may be prepared by modifying functional groups present in the compounds of structure (I) or (II) in a manner that cleaves the modification to the parent compound of structure (I) or (II) in routine manipulation or in vivo. Prodrugs include compounds of structure (I) or (II) wherein a hydroxy, amino, or sulfhydryl group is bonded to any group that, when the prodrug of the compound of structure (I) or (II) is administered to a mammalian subject, cleaves to form a free hydroxy, free amino, or free sulfhydryl group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohol or amide derivatives of amine functional groups in compounds of structure (I) or (II), and the like.

The embodiments of the invention disclosed herein are also intended to encompass all pharmaceutically acceptable compounds of the compounds of structure (I) or (II) that are isotopically labeled by the replacement of one or more atoms with an atom having a different atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as²H、³H、¹¹C、¹³C、¹⁴C、¹³N、¹⁵N、¹⁵O、¹⁷O、¹⁸O、³¹P、³²P、³⁵S、¹⁸F、³⁶Cl、¹²³I and¹²⁵I. these radiolabeled compounds can be used to help determine or measure the effectiveness of a compound by characterizing, for example, the site or mode of action or binding affinity to a pharmacologically important site of action. Certain isotopically-labeled compounds of structure (I) or (II), for example those incorporating a radioisotope, are useful in drug and/or substrate tissue distribution studies. In view of the radioactive isotope tritium, i.e.³H and carbon-14 i.e¹⁴C incorporation is easy and detection means are readily available, and they can be used in particular for this purpose.

With heavier isotopes such as deuterium (i.e.²H) Substitution may provide certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and may therefore be preferred in certain circumstances.

With positron-emitting isotopes such as¹¹C、¹⁸F、¹⁵O and¹³n substitution can be used in Positron Emission Tomography (PET) studies to examine substrate receptor occupancy. Isotopically-labeled compounds of structure (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the preparations and examples set forth below using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously used.

The embodiments of the invention disclosed herein are also intended to encompass in vivo metabolites of the disclosed compounds. Such products may result primarily from enzymatic processes, e.g., from oxidation, reduction, hydrolysis, amidation, esterification, etc., of the administered compound. Accordingly, embodiments of the present invention include compounds produced by a method comprising administering a compound of the present invention to a mammal for a period of time sufficient to produce a metabolite thereof. Such products are typically identified by: administering a detectable dose of a radiolabeled compound of structure (I) or (II) to an animal such as rat, mouse, guinea pig, monkey, or a human, allowing sufficient time for metabolism to occur, and isolating its transformation products from urine, blood or other biological samples.

"stable compound" and "stable structure" are intended to refer to a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture and formulation into an effective therapeutic agent.

"mammal" includes humans as well as domestic animals such as laboratory animals and domestic pets (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits) and non-domestic animals (e.g., wild animals, etc.).

A "pharmaceutically acceptable carrier, diluent or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier that has been approved by the U.S. food and drug administration as being acceptable for use in humans or domestic animals.

"pharmaceutically acceptable salts" include acid addition salts and base addition salts.

"pharmaceutically acceptable acid addition salts" means salts which retain the biological potency and properties of the free base, are not biologically or otherwise undesirable, and are formed with inorganic acids (such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like) or with organic acids (such as, but not limited to, acetic acid, 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, dodecylsulfuric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1, 5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like).

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

Typically, crystallization yields a solvate of the compound of structure (I) or (II). As used herein, the term "solvate" refers to an aggregate of molecules comprising one or more compounds of structure (I) or (II) with one or more solvent molecules. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of the present invention may exist in hydrate forms (including monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate, and the like) as well as in corresponding solvated forms. In some embodiments, the compounds of structure (I) or (II) may exist as true solvates, while in other cases, the compounds of structure (I) or (II) may retain only extraneous water or be a mixture of water plus some extraneous solvent.

"pharmaceutical composition" refers to a formulation of a compound of structure (I) and a vehicle generally accepted in the art for delivering biologically active compounds to a mammal, such as a human. Such media include all pharmaceutically acceptable carriers, diluents or excipients therefor.

An "effective amount" or "therapeutically effective amount" refers to the amount of a compound of structure (I) or (II) or a lipid nanoparticle comprising a compound of structure (I) or (II) that is sufficient to effect treatment in a mammal, preferably a human, when administered to the mammal, preferably a human. The amount of lipid nanoparticles constituting a "therapeutically effective amount" of an embodiment of the present invention will vary depending on the compound, the condition and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one of ordinary skill in the art based on his own knowledge and the present disclosure.

As used herein, "treatment" or "treatment" encompasses treatment for a target disease or condition in a mammal, preferably a human, having the target disease or condition, and includes:

(i) preventing the disease or condition from occurring in a mammal, particularly when such mammal is susceptible to the condition but has not yet been diagnosed as having the condition;

(ii) inhibiting the disease or condition, i.e., arresting its development;

(iii) alleviating, i.e., causing regression of, the disease or condition; or

(iv) Relieving symptoms caused by the disease or condition, i.e., relieving pain, without addressing the underlying disease or condition. As used herein, the terms "disease" and "condition" may be used interchangeably, or may be different, in that a particular disease or condition may not have a known pathogen (such that the cause has not been ascertained), and thus has not been identified as a disease but merely as an undesirable condition or syndrome, with clinicians having identified more or less specific sets of symptoms.

A compound of structure (I) or (II), or a pharmaceutically acceptable salt thereof, may contain one or more asymmetric centers and may therefore give rise to enantiomers, diastereomers, and other stereoisomeric forms which may be defined in terms of absolute stereochemistry as (R) -or (S) -or as (D) -or (L) - (for amino acids). Embodiments of the present invention are intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R) -and (S) -, or (D) -and (L) -isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques such as chromatography and fractional crystallization. Conventional techniques for the preparation/separation of the individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral High Pressure Liquid Chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless otherwise indicated, the compounds are intended to include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

"stereoisomers" refers to compounds consisting of the same atoms bonded by the same bond but having different three-dimensional structures that are not interchangeable. The present invention contemplates various stereoisomers and mixtures thereof, and includes "enantiomers" which refers to two stereoisomers whose molecules are non-overlapping mirror images of each other.

"tautomer" refers to the transfer of a proton from one atom of a molecule to another atom of the same molecule. The invention includes tautomers of any of the compounds.

Compound (I)

In one aspect, the present invention provides novel lipid compounds that can be combined with other lipid components such as neutral lipids, charged lipids, steroids and/or polymer-conjugated lipids to form lipid nanoparticles with oligonucleotides. Without wishing to be bound by theory, it is believed that these lipid nanoparticles shield the oligonucleotides from degradation in serum and provide for efficient delivery of the oligonucleotides to cells in vitro and in vivo.

A compound of structure (I)

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

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

L¹is-O (C ═ O) R¹、-(C＝O)OR¹、-C(＝O)R¹、-OR¹、-S(O)_xR¹、-S-SR¹、

-C(＝O)SR¹、-SC(＝O)R¹、-NR^aC(＝O)R¹、-C(＝O)NR^bR^c、-NR^aC(＝O)NR^bR^c、-OC(＝O)NR^bR^cor-NR^aC(＝O)OR¹；

L²is-O (C ═ O) R²、-(C＝O)OR²、-C(＝O)R²、-OR²、-S(O)_xR²、-S-SR²、

-C(＝O)SR²、-SC(＝O)R²、-NR^dC(＝O)R²、-C(＝O)NR^eR^f、-NR^cC(＝O)NR^eR^f、-OC(＝O)NR^eR^f；-NR^dC(＝O)OR²Or to R²A direct bond of (a);

G^1aand G^2aEach independently is C₂-C₁₂Alkylene or C₂-C₁₂An alkenylene group;

G³is C₁-C₂₄Alkylene radical, C₂-C₂₄Alkenylene radical, C₃-C₈Cycloalkylene or C₃-C₈Cycloalkenylene;

R^a、R^b、R^dand R^eEach independently is H or C₁-C₁₂Alkyl or C₂-C₁₂An alkenyl group;

R^cand R^fEach independently is C₁-C₁₂Alkyl or C₂-C₁₂An alkenyl group;

R¹and R²Each independently of the other being branched C₆-C₂₄Alkyl or branched C₆-C₂₄An alkenyl group;

R^3ais-C (═ O) N (R)^4a)R^5aOR-C (═ O) OR⁶；

R^4aIs C₁-C₁₂An alkyl group;

R^5ais H or C₁-C₈Alkyl or C₂-C₈An alkenyl group;

R⁶is H, aryl or aralkyl; and

x is 0, 1 or 2, and

wherein each alkyl, alkenyl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, aryl, and aralkyl is independently substituted or unsubstituted.

In certain embodiments of structure (I), G³Is unsubstituted. In a more particular embodiment of structure (I), G³Is C₂-C₁₂Alkylene, e.g. in some embodiments of structure (I), G³Is C₃-C₇Alkylene, or in other embodiments of structure (I), G³Is C₃-C₁₂An alkylene group. In some embodiments of structure (I), G³Is C₂Or C₃An alkylene group.

In some of the foregoing embodiments of structure (I), the compounds have the following structure (IA):

wherein y1 and z1 are each independently an integer in the range of 2 to 12, such as an integer in the range of 2 to 6, for example 4.

In some of the foregoing embodiments of structure (I), L¹is-O (C ═ O) R¹、-(C＝O)OR¹or-C (═ O) NR^bR^cAnd L²is-O (C ═ O) R²、-(C＝O)OR²or-C (═ O) NR^eR^f. For example, in some embodiments of structure (I), L¹And L²Are each- (C ═ O) OR¹And- (C ═ O) OR². In other embodiments of structure (I), L¹Is- (C ═ O) OR¹And L is²is-C (═ O) NR^eR^f. In other embodiments of structure (I), L¹is-C (═ O) NR^bR^cAnd L is²is-C (═ O) NR^eR^f。

In other embodiments of the foregoing, the compound has one of the following structures (IB), (IC), (ID), or (IE):

in some of the foregoing embodiments, the compound has structure (IB), in other embodiments, the compound has structure (IC), and in other embodiments the compound has structure (ID). In other embodiments, the compound has the structure (IE).

In some of the various embodiments described above, the compound has one of the following structures (IF), (IG), (IH), or (IJ):

wherein y1 and z1 are each independently an integer in the range of 2 to 12, such as an integer in the range of 2 to 6, for example 4.

In some of the foregoing embodiments of structure (I), y1 and z1 are each independently integers in the range of 2 to 10,2 to 8, 4 to 10, or 4 to 7. For example, in some embodiments of structure (I), y1 is 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments of structure (I), z1 is 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments of structure (I), y1 and z1 are the same, while in other embodiments of structure (I), y1 and z1 are different.

In some of the foregoing embodiments of structure (I), R¹Or R²Or both are branched C₆-C₂₄An alkyl group. For example, in some embodiments of structure (I), R¹And R²Each independently having the structure:

wherein:

R^7aand R^7bIndependently at each occurrence is H or C₁-C₁₂An alkyl group; and

a is an integer of 2 to 12 and,

wherein each is selected from R^7a、R^7bAnd a, such that R¹And R²Each independently containing from 6 to 20 carbon atoms. For example, in some embodiments, a is an integer ranging from 5 to 9 or 8 to 12.

In some of the foregoing embodiments of structure (I), at least one occurrence of R^7aIs H. For example, in some embodiments of structure (I), R^7aAt each occurrence is H. In various other embodiments of the foregoing, at least one occurrence of R^7bIs C₁-C₈An alkyl group. For example, in some embodiments, C₁-C₈Alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl or n-octyl.

In various embodiments of structure (I), R¹Or R²Or both having one of the following structures:

in some of the foregoing embodiments of structure (I), R^b、R^c、R^eAnd R^fEach independently is C₃-C₁₂An alkyl group. For example, in some embodiments of structure (I), R^b、R^c、R^eAnd R^fIs n-hexyl and in other embodiments of structure (I), R^b、R^c、R^eAnd R^fIs n-octyl.

In some of the foregoing embodiments of structure (I), R^3ais-C (═ O) N (R)^4a)R^5a. In more particular embodiments of structure (I), R^4aIs ethyl, propyl, n-butyl, n-hexyl, n-octyl or n-nonyl. In certain embodiments of structure (I), R^5aIs H, methyl, ethyl, propyl, n-butyl, n-hexyl or n-octyl. In some of these embodiments of structure (I), R^4aAnd/or R^5aOptionally substituted with a substituent such as hydroxy.

In some embodiments of structure (I), R^3ais-C (═ O) OR⁶. In certain embodiments of structure (I), R⁶Is benzyl and in other embodiments, R⁶Is H.

In some of the foregoing embodiments of structure (I), R^4a、R^5aAnd R⁶Independently optionally substituted with one or more substituents selected from: -OR^g、-NR^gC(＝O)R^h、-C(＝O)NR^gR^h、-C(＝O)R^h、-OC(＝O)R^h、-C(＝O)OR^hand-ORⁱOH, wherein:

R^gindependently at each occurrence is H or C₁-C₆An alkyl group;

R^hindependently at each occurrence is C₁-C₆An alkyl group; and

Rⁱindependently at each occurrence is C₁-C₆An alkylene group.

In certain specific embodiments of structure (I), R^3aHas one of the following structures:

in various embodiments, the compound has one of the structures shown in table 1 below.

TABLE 1

Representative compounds of Structure (I)

A compound of structure (II)

In one embodiment, the compound has the following structure (II):

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

L¹is-O (C ═ O) R¹、-(C＝O)OR¹、-C(＝O)R¹、-OR¹、-S(O)_xR¹、-S-SR¹、

-C(＝O)SR¹、-SC(＝O)R¹、-NR^aC(＝O)R¹、-C(＝O)NR^bR^c、-NR^aC(＝O)NR^bR^c、-OC(＝O)NR^bR^cor-NR^aC(＝O)OR¹；

L²is-O (C ═ O) R²、-(C＝O)OR²、-C(＝O)R²、-OR²、-S(O)_xR²、-S-SR²、

-C(＝O)SR²、-SC(＝O)R²、-NR^dC(＝O)R²、-C(＝O)NR^eR^f、-NR^cC(＝O)NR^eR^f、-OC(＝O)NR^eR^f；-NR^dC(＝O)OR²Or a direct bond;

G^1band G^2bEach independently is C₁-C₁₂Alkylene or C₂-C₁₂An alkenylene group;

G³is C₁-C₂₄Alkylene radical, C₂-C₂₄Alkenylene radical, C₃-C₈Cycloalkylene radical, C₃-C₈Cycloalkenylene;

R^a、R^b、R^dand R^eEach independently is H or C₁-C₁₂Alkyl or C₂-C₁₂An alkenyl group;

R^cand R^fEach independently is C₁-C₁₂Alkyl or C₂-C₁₂An alkenyl group;

R¹and R²Each independently of the other being branched C₆-C₂₄Alkyl or branched C₆-C₂₄An alkenyl group;

R^3bis-NR^4bC(＝O)R^5b；

R^4bIs H, C₁-C₁₂Alkyl or C₂-C₁₂An alkenyl group;

when R is^4bWhen is H, R^5bIs C₂-C₁₂Alkyl or C₂-C₁₂An alkenyl group; or when R is^4bIs C₁-C₁₂Alkyl or C₂-C₁₂When an alkenyl radical, R⁵Is C₁-C₁₂Alkyl or C₂-C₁₂An alkenyl group; and

x is 0, 1 or 2, and

wherein each alkyl, alkenyl, alkylene, alkenylene, cycloalkylene, and cycloalkenylene is independently substituted or unsubstituted.

In certain embodiments of structure (II), G³Is unsubstituted. In a more particular embodiment of structure (II), G³Is C₁-C₁₂Alkylene radicals, e.g. G³Is C₃-C₅Alkylene or G³Is C₃-C₁₂An alkylene group.

In some of the foregoing embodiments, the compound has the following structure (IIA):

wherein y2 and z2 are each independently integers ranging from 1 to 12.

In some of the foregoing embodiments of structure (II), L¹And L²Each independently is-O (C ═ O) R¹OR- (C ═ O) OR¹。

In other embodiments of the foregoing, the compound has one of the following structures (IIB) or (IIC):

in some of the foregoing embodiments, the compound has structure (IIB), and in other embodiments, the compound has structure (IIC).

In some embodiments, the compound has one of the following structures (IID) or (IIE):

wherein y2 and z2 are each independently integers ranging from 1 to 12.

In some of the foregoing embodiments of structure (II), y2 and z2 are each independently integers ranging from 2 to 12, e.g., 2 to 10,2 to 8, 4 to 7, or 4 to 10. For example, in some embodiments of structure (II), y2 is 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments of structure (II), z2 is 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments of structure (II), y2 and z2 are the same, while in other embodiments of structure (II), y2 and z2 are different.

In some of the foregoing embodiments of structure (II), R¹Or R²Or both are branched C₆-C₂₄An alkyl group. For example, in some embodiments of structure (II), R¹And R²Each independently having the structure:

wherein:

R^7aand R^7bIndependently at each occurrence is H or C₁-C₁₂An alkyl group; and

a is an integer of 2 to 12 and,

wherein each is selected from R^7a、R^7bAnd a, such that R¹And R²Each independently containing from 6 to 20 carbon atoms. For example, in some embodiments, a is an integer ranging from 5 to 9 or 8 to 12.

In some of the foregoing embodiments of structure (II), at least one occurrence of R^7aIs H. For example, in some embodiments of structure (II), R^7aAt each occurrence is H. In various other embodiments of the foregoing, at least one occurrence of R^7bIs C₁-C₈An alkyl group. For example, in some embodiments of Structure (II), C₁-C₈Alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl or n-octyl.

In various embodiments of structure (II), R¹Or R²Or both having one of the following structures:

in some of the foregoing embodiments of structure (II), R^4bIs H, methyl, ethyl, propyl or octyl. In some embodiments of structure (II), R^5bIs methyl, ethyl, propyl, heptyl or octyl, for example n-heptyl or n-octyl.

In certain related embodiments of structure (II), R^4bAnd R^5bIndependently optionally substituted with one or more substituents each selected from: -OR^g、-NR^gC(＝O)R^h、-C(＝O)NR^gR^h、-C(＝O)R^h、-OC(＝O)R^h、-C(＝O)OR^hand-OR^hOH, wherein:

R^gindependently at each occurrence is H or C₁-C₆An alkyl group;

R^hindependently at each occurrence is C₁-C₆An alkyl group; and

Rⁱindependently at each occurrence is C₁-C₆An alkylene group.

In certain specific embodiments of structure (II), R^3bHas one of the following structures:

in various embodiments, the compound of structure (II) has one of the structures shown in table 2 below.

TABLE 2

Representative compounds of Structure II

It is to be understood that any embodiment of the compounds of structure (I) or (II) as shown above, as well as any particular substituents and/or variables of the compounds of structure (I) or (II) as shown above, may be independently combined with other embodiments and/or substituents and/or variables of the compounds of structure (I) or (II) to form embodiments of the present invention not specifically shown above. Additionally, if any particular R group, L group, G group, or substituent and/or list of variables a, x, y, or z is listed in a particular embodiment and/or claim, it is to be understood that each individual substituent and/or variable may be deleted from the particular embodiment and/or claim and the remaining list of substituents and/or variables is to be considered within the scope of the present invention.

It is understood that in this specification, combinations of substituents and/or variables of the depicted formulae are permissible only if such contribution results in a stable compound.

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

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

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

In various embodiments, the polymer-conjugated lipid is a pegylated lipid. For example, some embodiments include a pegylated diacylglycerol (PEG-DAG) such as 1- (monomethoxy-polyethylene glycol) -2, 3-dimyristoyl glycerol (PEG-DMG), a pegylated phosphatidylethanolamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O- (2 ', 3' -bis (tetradecanoyloxy) propyl-1-O- (ω -methoxy (polyethoxy) ethyl) succinate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as ω -methoxy (polyethoxy) ethyl-N- (2, 3-ditetradecyloxy) propyl) carbamate or 2, 3-ditetradecyloxy propyl-N- (. omega. -methoxy (polyethoxy) ethyl) carbamate. In various embodiments, the molar ratio of compound to pegylated lipid ranges from about 100:1 to about 20: 1.

In some embodiments, the composition comprises a pegylated lipid having the following structure (III):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

R⁸and R⁹Each independently is a linear or branched alkyl, alkenyl or alkynyl group containing 10 to 30 carbon atoms, wherein the alkyl, alkenyl or alkynyl group is optionally interrupted by one or more ester linkages; and

the average value of w ranges from 30 to 60.

In some embodiments, R⁸And R⁹Each independently a straight chain alkyl group containing 12 to 16 carbon atoms. In some embodiments, w has an average value in the range of 43 to 53. In other embodiments, the average value w is about 45. In various other embodiments, the average value w is about 49.

In some embodiments, Lipid Nanoparticles (LNPs) comprising any one or more compounds of structure (I) or (II) and a therapeutic agent are provided. For example, in some embodiments, the LNP comprises any one of the compounds of structure (I) or (II) and a therapeutic agent and one or more excipients selected from the group consisting of neutral lipids, steroids, and polymer-conjugated lipids.

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

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

In various embodiments of the LNP, the polymer-conjugated lipid is a pegylated lipid. For example, some embodiments include a pegylated diacylglycerol (PEG-DAG) such as 1- (monomethoxy-polyethylene glycol) -2, 3-dimyristoyl glycerol (PEG-DMG), a pegylated phosphatidylethanolamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O- (2 ', 3' -bis (tetradecanoyloxy) propyl-1-O- (ω -methoxy (polyethoxy) ethyl) succinate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as ω -methoxy (polyethoxy) ethyl-N- (2, 3-ditetradecyloxy) propyl) carbamate or 2, 3-ditetradecyloxy propyl-N- (. omega. -methoxy (polyethoxy) ethyl) carbamate. In various embodiments, the molar ratio of compound to pegylated lipid ranges from about 100:1 to about 20: 1.

In some embodiments, the LNP comprises a pegylated lipid having the following structure (III):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

R⁸and R⁹Each independently is a linear or branched alkyl, alkenyl or alkynyl group containing 10 to 30 carbon atoms, wherein the alkyl, alkenyl or alkynyl group is optionally interrupted by one or more ester linkages; and

the average value of w ranges from 30 to 60.

In some embodiments, R⁸And R⁹Each independently a straight chain alkyl group containing 12 to 16 carbon atoms. In some embodiments, w has an average value in the range of 43 to 53. In other embodiments, the average value w is about 45. In various other embodiments, the average value w is about 49.

Methods of making the above-described lipids, lipid nanoparticles, and compositions are described below and/or are known in the art, for example, in PCT publication nos. WO 2015/199952 and WO2017/004143, both of which are incorporated herein by reference in their entirety.

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

In other various embodiments, the present invention relates to a method for administering a therapeutic agent to a patient in need thereof, the method comprising preparing or providing any of the foregoing compositions and administering the composition to the patient.

For administration purposes, the compounds of structure (I) or (II), typically in the form of lipid nanoparticles combined with a therapeutic agent, may be administered as a chemical starting material or may be formulated into pharmaceutical compositions. Pharmaceutical compositions of embodiments of the invention comprise a compound of structure (I) or (II) (e.g., as a component in LNP) and one or more pharmaceutically acceptable carriers, diluents, or excipients. The compounds of structure (I) or (II) are present in the composition in an amount effective to form lipid nanoparticles and deliver a therapeutic agent, such as for treating a particular target disease or condition. Suitable concentrations and dosages can be readily determined by those skilled in the art.

Administration of the compositions and/or LNPs of embodiments of the invention can be via any acceptable mode of administration of the agents for similar utility. The pharmaceutical composition of the embodiment of the present invention may be formulated into preparations in the form of solid, semisolid, liquid or gas, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, peritoneal, sublingual, buccal, rectal, vaginal and intranasal. The term "intraperitoneal" as used herein includes subcutaneous injections, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques. The pharmaceutical compositions of the present invention are formulated such that the active ingredients contained therein are bioavailable upon administration of the composition to a patient. The composition to be administered to a subject or patient takes the form of one or more dosage units, where for example a tablet may be a single dosage unit and a container of a compound of structure (I) or (II) in aerosol form may contain a plurality of dosage units. The actual methods of making such dosage forms are known or will be apparent to those skilled in the art; see, for example, Remington, the science and Practice of Pharmacy, 20 th edition (Philadelphia College of Pharmacy and science, 2000). In any event, the compositions to be administered will contain a therapeutically effective amount of a compound of structure (I) or (II) (e.g., in the form of LNP comprising a therapeutic agent) or a pharmaceutically acceptable salt thereof for the treatment of the disease or condition of interest in accordance with the teachings of an embodiment of the present invention.

The pharmaceutical compositions of embodiments of the present invention may be in solid or liquid form. In one aspect, the carrier or carriers are particulate such that the composition is in the form of, for example, a tablet or powder. The carrier or carriers may be a liquid, wherein the composition is, for example, an oral syrup, an injectable liquid, or an aerosol, which may be used, for example, for administration by inhalation.

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

As a solid composition for oral administration, the pharmaceutical composition may be formulated into the form of powder, granules, compressed tablets, pills, capsules, chewing gum, wafer (wafer), and the like. Such solid compositions typically contain one or more inert diluents or edible carriers. Furthermore, one or more of the following may be present: a binder such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrin, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch, etc.; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; flavoring agents such as peppermint, methyl salicylate or orange flavoring; and a colorant.

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

The pharmaceutical compositions may be in the form of a liquid, such as an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred compositions comprise, in addition to a compound of the invention or LNP, one or more of a sweetener, preservative, dye/colorant and taste enhancer. In compositions intended for administration by injection, one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizing agents, and isotonic agents may be included.

Liquid pharmaceutical compositions of embodiments of the invention, whether they be in solution, suspension or other similar form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solutions preferably physiological saline, ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono-or diglycerides which may be employed as a solvent or suspending medium, polyethylene glycol, glycerol, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for adjusting tonicity such as sodium chloride or dextrose; agents used as cryoprotectants, such as sucrose or trehalose. The peritoneal preparation can be contained in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. The injectable pharmaceutical composition is preferably sterile.

The liquid pharmaceutical composition of the embodiments of the present invention intended for peritoneal or oral administration should contain an amount of the compound of structure (I) or (II) in order to obtain a suitable LNP.

The pharmaceutical compositions of embodiments of the invention may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The matrix may, for example, comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents (such as water and alcohol), and emulsifiers and stabilizers. The thickening agent may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or an iontophoresis device.

The pharmaceutical compositions of the embodiments of the invention may be intended for rectal administration, for example, in the form of suppositories that will melt in the rectum and release the drug. Compositions for rectal administration may contain an oily base as a suitable non-irritating excipient. Such bases include, but are not limited to, lanolin, cocoa butter, and polyethylene glycols.

The pharmaceutical compositions of embodiments of the present invention may comprise a variety of materials that modify the physical form of the solid or liquid dosage unit. For example, the composition may comprise a material that forms an envelope around the active ingredient. The material forming the coating is generally inert and may be selected from, for example, sugars, shellac, and other enteric coating agents. Alternatively, the active ingredient may be contained in a gelatin capsule.

Pharmaceutical compositions of embodiments of the invention in solid or liquid form may comprise an agent that binds to a compound of structure (I) or (II) and thereby facilitates delivery of the compound. Suitable agents that can function with this capability include monoclonal or polyclonal antibodies or proteins.

The pharmaceutical compositions of embodiments of the present invention may be comprised of dosage units that can be administered as an aerosol. The term "aerosol" is used to denote a variety of systems ranging from those of colloidal nature to those consisting of pressurized packaging. Delivery may be by liquefied or compressed gas or by a suitable pump system that dispenses the active ingredient. Aerosols comprising the compounds of structures (I) or (II) may be delivered in a single phase, two phase, or three phase system for delivery of one or more active ingredients. The delivery of the aerosol includes the necessary containers, activators, valves, sub-containers, etc., which together may form a kit. One skilled in the art can determine the preferred aerosol without undue experimentation.

The pharmaceutical compositions of the embodiments of the present invention may be prepared by methods well known in the pharmaceutical arts. For example, a pharmaceutical composition intended for administration by injection may be prepared by combining the lipid nanoparticles of the present invention with sterile distilled water or other carrier to form a solution. Surfactants may be added to promote the formation of a homogeneous solution or suspension. Surfactants are compounds that interact non-covalently with a compound of structure (I) or (II) to facilitate dissolution or uniform suspension of the compound in an aqueous delivery system.

The compositions of the embodiments of the present invention, or pharmaceutically acceptable salts thereof, are administered in therapeutically effective amounts, which will vary depending on a variety of factors including the activity of the particular therapeutic agent employed; metabolic stability and length of action of the therapeutic agent; the age, weight, general health, sex, and diet of the patient; mode and time of administration; the rate of excretion; a pharmaceutical composition; the severity of a particular disorder or condition; and the subject being treated.

The compositions of the embodiments of the present invention may also be administered simultaneously with, before, or after the administration of one or more other therapeutic agents. Such combination therapies include administration of a single pharmaceutical dosage formulation of a composition of an embodiment of the invention and one or more additional active agents, as well as administration of the composition of the invention and each active agent in its own separate pharmaceutical dosage formulation. For example, compositions of embodiments of the invention and other active agents may be administered to a patient in a single oral dosage composition (e.g., a tablet or capsule) or each agent administered in separate oral dosage formulations. When separate dosage formulations are used, the compound of structure (I) or (II) and the one or more additional active agents may be administered at substantially the same time, i.e., simultaneously, or at respectively staggered times, i.e., sequentially; combination therapy is understood to include all such regimens.

Methods for preparing the above compounds and compositions are described below and/or are known in the art.

One skilled in the art will appreciate that in the processes described herein, the functional groups of the intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxyl, amino, mercapto and carboxylic acid. Suitable hydroxyl protecting groups include trialkylsilyl or diarylalkylsilyl groups (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl, or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino and guanidino groups include t-butyloxycarbonyl, benzyloxycarbonyl and the like. Suitable thiol protecting groups include-C (O) -R '(where R' is alkyl, aryl or arylalkyl), p-methoxybenzyl, trityl and the like. Suitable carboxylic acid protecting groups include alkyl, aryl or arylalkyl esters. Protecting groups may be added or removed according to standard techniques known to those skilled in the art and as described herein. The use of protecting Groups is described in detail in Green, T.W. and P.G.M.Wutz, Protective Groups in Organic Synthesis (1999), 3 rd edition, Wiley. As will be appreciated by those skilled in the art, the protecting group may also be a polymer resin, such as Wang resin, Rink resin, or 2-chlorotrityl-chloride resin.

Furthermore, all compounds of structure (I) or (II) in free base or acid form can be converted into their pharmaceutically acceptable salts by methods known to those skilled in the art by treatment with suitable inorganic or organic bases or acids. Salts of the compounds of structure (I) or (II) may be converted to their free base or acid forms by standard techniques.

The following general reaction schemes illustrate methods for preparing compounds of structure (I) or (II), or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof,

wherein R is^3a、R^3b、L¹、L²、G^1a、G^1b、G^2a、G^2bAnd G³As defined herein. It is understood that one skilled in the art may be able to prepare these compounds by similar methods or by combining other methods known to one skilled in the art. It is also understood that one skilled in the art will be able to prepare other compounds of structure (I) or (II) not specifically illustrated below in a similar manner as described below by using the appropriate starting components and varying the synthesis parameters as necessary. In general, starting components are available from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybrid, Matrix Scientific, TCI, and Fluorochem USA, or synthesized according to sources known to those of skill in the art (see, e.g., Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5 th edition (Wiley, 12 months 2000)) or prepared as described in the present invention.

General reaction scheme 1

General reaction scheme 1 ("method a") provides an exemplary method for preparing a compound of structure (I). R in general reaction scheme 1¹、R²、R⁴、R⁵、R⁶Y and z are as defined herein. R', X, m and n refer to variables selected such that A-5, A-6, A-8 and A-10 are compounds having structure (I). For example, R' is R¹Or R²X is Br, m is y or z, and n is an integer ranging from 0 to 23. The compounds of structure a-1 are purchased or prepared according to methods known in the art. Amine/acid A-1 is protected with alcohol A-2 (e.g., benzyl alcohol) using suitable conditions and reagents (e.g., p-TSA) to obtain ester/amine A-3. Ester/amine A-3 is coupled with ester A-4 (e.g., using DIPEA) to give benzyl ester A-5. Compound A-5 optionally using appropriate conditions (e.g., Pd/C, H)₂) Deprotected to obtain acid A-6. Acid A-6 can be reacted with amine A-7 (e.g., using oxalyl chloride/DMF) to obtain amide A-8, or alternatively, with alcohol A-9 (e.g., using DCC/DMAP) to produce ester A-10.Each of A-5, A-6, A-8 and A-10 is a compound of structure (I).

General reaction scheme 2

General reaction scheme 2 ("method B") provides an exemplary method for preparing compounds of structure (I). R in general reaction scheme 2¹、R²、R⁴、R⁵、R⁶Y and z are as defined herein. R', X, m and n refer to variables selected such that B-6 is a compound having structure (I). For example, R' is R¹Or R²X is Br, m is y or z, and n is an integer ranging from 0 to 23. Compounds of structure B-1 are either purchased or prepared according to methods known in the art. The reaction of the protected amine/acid B-1 with the amine B-2 is carried out under suitable coupling conditions (e.g. NHS, DCC) to give the amide B-3. Following the deprotection step using acidic conditions (e.g., TFA), amine B-4 is coupled with ester B-5 under suitable conditions (e.g., DIPEA) to afford compound B-6 of structure (I).

General reaction scheme 3

General reaction scheme 3 ("method C") provides an exemplary method for preparing compounds of structure (I). R in general reaction scheme 3¹、R⁴、R⁵、R^e、R^fY and z are as defined herein. R', X, m and n refer to variables selected such that C-7 is a compound having structure (I). For example, R' is R¹Or R²X is Br, m is y or z, and n is an integer ranging from 0 to 23. Compounds of structures C-1, C-2, C-4 and C-5 are either purchased or prepared according to methods known in the art. The reaction of amine/acid C-1 with alcohol C-2 is carried out under suitable coupling conditions (e.g., p-TSA) to give amine/ester C-3. In parallel, amides are prepared by coupling the acid C-4 with the amine C-5 under suitable conditions (e.g., oxalyl chloride/DMF)And C-6. C-3 and C-6 are combined under basic conditions (e.g., DIPEA) to provide compound C-7 of structure (I).

General reaction scheme 4

General reaction scheme 4 ("method D") provides an exemplary method for preparing the compound of structure (II). R in general reaction scheme 1¹、R²、R⁴、R⁵Y and z are as defined herein. R', X, m and n refer to variables selected such that D-10 is a compound having structure (I). For example, R' is R¹Or R²X is Br, m is y or z, and n is an integer ranging from 0 to 23. Compounds of structure D-1 are either purchased or prepared according to methods known in the art. Reaction of D-1 with alcohol D-2 under appropriate conditions (e.g., KOH/heat) affords ester D-3, which ester D-3 can then be coupled with amine D-4 to yield protected amine D-5. Protected amine D-5 can be coupled with acid chloride D-6 using a basic reagent (e.g., triethylamine/DMAP) to provide D-7 and deprotected using an acid (e.g., TFA) to provide deprotected amine D-8. The final coupling between D-8 and D-9 can be carried out under basic conditions (e.g., DIPEA) to give compound D-10 of structure (II).

General reaction scheme 5

General reaction scheme 5 ("method E") provides an exemplary method for preparing a compound of structure (II). R in general reaction scheme 2¹、R²、R⁵Y and z are as defined herein. R', X, m and n refer to variables selected such that E-6 is a compound having structure (I). For example, R' is R¹Or R²X is Br, m is y or z, and n is an integer ranging from 0 to 23. Compounds of structure E-1 are either purchased or prepared according to methods known in the art. The reaction of E-1 with methanol to give the ester E-2 is carried out under suitable conditions, such as acetyl chloride. Can be through use of a caseSuch as methanol and diamine E-3 is added with heating to produce ester E-2. The resulting amide E-4 can be coupled with E-5 using basic conditions (e.g., DIPEA) to give compound E-6 of structure (II).

General reaction scheme 6

General reaction scheme 6 ("method F") provides an exemplary method for preparing the compound of structure (II). R in general reaction scheme 3¹、R²、R⁴、R⁵Y and z are as defined herein. R', X, m and n refer to variables selected such that F-8 is a compound having structure (I). For example, R' is R¹Or R²X is Br, m is y or z, and n is an integer ranging from 0 to 23. Compounds of structure F-1 are either purchased or prepared according to methods known in the art. Reaction of F-1 alcohol/amine F-2 under appropriate conditions (e.g. KCO)₃、Ce₂CO₃And NaI) to give the diester/alcohol F-3. Mesylate F-4 is obtained (e.g., using MsCl, triethylamine, and DMAP) and coupled with amine F-5 (e.g., using heat) to give diester F-6. The final coupling between F-6 and acid chloride F-7 is carried out using basic coupling conditions (e.g., triethylamine, DMAP) to give compound F-8 of structure (II).

General reaction scheme 7

General reaction scheme 7 ("method G") provides an exemplary method for preparing compounds of structure (II). R in general reaction scheme 4¹、R²、R⁴、R⁵Y and z are as defined herein. R', X, m and n refer to variables selected such that G-8 is a compound having structure (I). For example, R' is R¹Or R²X is Br, m is y or z, and n is an integer ranging from 0 to 23. Compounds of structure G-1 are either purchased or prepared according to methods known in the art. Reaction of mesylate G-1Coupling with amine G-2 (e.g., using heat to 75 ℃) to afford protected amine G-3, which is reacted with acid chloride G-4 (e.g., using triethylamine/DMAP) to afford amide G-5. The protecting group is removed under acidic conditions (e.g., TFA) and the amine G-6 is then coupled with G-7 under basic conditions (e.g., DIPEA) to provide compound G-8 of structure (II).

It is noted that various alternative strategies for preparing compounds of structure (I) or (II) are available to one of ordinary skill in the art. The use of protecting groups and other modifications to the above general reaction schemes 1-7 as desired will be apparent to those of ordinary skill in the art. The following examples are provided for purposes of illustration and not limitation.

Example 1

Luciferase mRNA in vivo evaluation using lipid nanoparticle compositions

The lipid of structure (I) or (II), DSPC, cholesterol and PEG-lipid were dissolved in ethanol in a molar ratio of 50:10:38.5:1.5 or 47.5:10:40.8: 1.7. Lipid Nanoparticles (LNPs) were prepared at a weight ratio of total lipid to mRNA of about 10:1 to 30: 1. Briefly, mRNA was diluted to 0.2mg/mL in 10 to 50mM citrate buffer (pH 4). The ethanolic lipid solution is mixed with the aqueous mRNA solution using a syringe pump in a ratio of about 1:5 to 1:3(vol/vol), with the total flow rate being higher than 15 mL/min. The ethanol was then removed and the external buffer was replaced by PBS by dialysis. Finally, the lipid nanoparticles were filtered through a 0.2 μm pore sterile filter.

Studies were performed in 6-8 week old female C57BL/6 mice (Charles River)8-10 week old CD-1(Harlan) mice (Charles River) according to guidelines established by the institutional Animal Care Committee (ACC) and the canadian animal care council (CCAC). Various doses of mRNA-lipid nanoparticles were administered systemically by tail vein injection, and animals were euthanized at specific time points (e.g., 4 hours) post-administration. The liver and spleen were collected in pre-weighed tubes, weighed, immediately snap frozen in liquid nitrogen and stored at-80 ℃ until processed for analysis.

For the liver, about 50mg was dissected in 2mL FastPrep tubes (MP Biomedicals, Solon OH) for analysis. 1/4 "ceramic spheres (MP Biomedicals) were added to each tube, and 500. mu.L of Glo Lysis Buffer (Glo Lysis Buffer) -GLB (Promega, Madison Wis.) equilibrated to room temperature was added to the liver tissue. Liver tissue was homogenized for 15 seconds at 2X 6.0m/s using a FastPrep24 instrument (MPBiomedials). The homogenate was incubated at room temperature for 5 minutes, then diluted 1:4 in GLB and evaluated using the SteadyGlo luciferase assay system (Promega). Specifically, 50 μ L of diluted homogenate was reacted with 50 μ L of SteadyGlo substrate, shaken for 10 seconds, followed by 5 minutes incubation, and then quantified using a CentroXS3LB960 luminometer (Berthold Technologies, Germany). The amount of protein assayed was determined by using BCA protein assay kit (Pierce, Rockford, IL). The Relative Luminescence Units (RLU) were then normalized to the total μ g protein determined. To convert RLU to ng luciferase, a standard curve was generated with QuantiLum recombinant luciferase (Promega).

Fluc mRNA from Trilink Biotechnologies (L-6107) will express luciferase protein originally isolated from the firefly, Photinus pyralis. FLuc is commonly used in mammalian cell culture to measure gene expression and cell viability. In the presence of the substrate fluorescein, it emits bioluminescence. The capped and polyadenylated mRNA was completely replaced by 5-methylcytidine and pseudouridine.