Lipid compounds and uses thereof

Abstract

Compounds are provided having the following structure (I): or a pharmaceutically acceptable salt, N-oxide, tautomer, or stereoisomer thereof, wherein R 1 , G 1 , W and m, n, o and p are as defined herein. Use of the compounds as components of lipid nanoparticle formulations for delivering nucleic acids, compositions comprising the compounds and methods for their use and preparation are also provided. [Formula 1] TIFF2025538939000136.tif2993

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 590,756, filed October 16, 2023, and U.S. Provisional Patent Application No. 63 / 382,389, filed November 4, 2022. The entire contents of each of the above-mentioned applications are incorporated herein by reference.

[0002] Sequence Listing Reference This application has been filed electronically via EFS-Web and contains a sequence listing that has been submitted electronically in .xml format. The .xml file contains a sequence listing entitled "PC072882A Sequence Listing.xml," which was created on October 17, 2023, and is 25 KB in size. The sequence listing contained in this .xml file is a part of the present specification and is incorporated by reference in its entirety.

[0003] Background of the Invention The present invention relates to novel ionizable lipid compounds. The present invention also relates to the preparation of ionizable lipid compounds and intermediates used in the preparation, compositions containing ionizable lipid compounds, and the use of ionizable lipid compounds, including in combination with other lipid components such as neutral lipids, cholesterol, and polymer-conjugated lipids, to form lipid nanoparticles with oligonucleotides to facilitate intracellular delivery of therapeutic nucleic acids both in vitro and in vivo. [Background technology]

[0004] Numerous challenges exist with the delivery of nucleic acids to affect desired responses in biological systems. Nucleic acid-based therapeutics have enormous potential, but realizing this potential still requires more effective delivery of nucleic acids to appropriate sites within cells or organisms. Therapeutic nucleic acids include, for example, messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, immunostimulatory nucleic acids, antagomirs, antimirs, mimetics, supermirs, and aptamers. Some nucleic acids, such as mRNA or plasmids, can be used to induce the expression of specific cellular products that are useful, for example, in the treatment of diseases associated with protein or enzyme deficiency or as vaccines. The therapeutic applications of translatable nucleotide delivery are extremely broad, as constructs can be synthesized to produce any selected protein sequence, whether resident in the system or not. The expression product of a nucleic acid can increase existing levels of a protein, replace a missing or non-functional version of a protein, or introduce a new protein and associated functionality within a cell or organism.

[0005] However, two problems currently confront the use of oligonucleotides in therapeutic settings. First, free RNA is susceptible to nuclease digestion in plasma. Second, free RNA has limited access to intracellular compartments where the relevant translation machinery resides. Lipid nanoparticles formed from oligonucleotides and ionizable lipids containing other lipid components, such as neutral lipids, cholesterol, PEG, and PEGylated lipids, have been used to block RNA degradation in plasma and promote cellular uptake of oligonucleotides. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, there is still a need for improved lipid compounds and lipid nanoparticles for delivering oligonucleotides.Preferably, these lipid nanoparticles will provide an optimal drug:lipid ratio, protect nucleic acid from degradation and clearance in serum, be suitable for systemic or local delivery, and provide intracellular delivery of nucleic acid.In addition, these lipid-nucleic acid particles should be well tolerated and provide a sufficient therapeutic index so that treatment of patients with an effective dose of nucleic acid does not involve unacceptable toxicity and / or risk to patients.In addition, there is a need to identify ionizable lipid-containing nanoparticle compositions with improved colloidal stability and tissue or cell specificity for oligonucleotide delivery. [Means for solving the problem]

[0007] The present invention provides, in part, lipid compounds of formula (I) and their pharmaceutically acceptable salts, N-oxides, tautomers, or stereoisomers. Such lipid compounds, including their pharmaceutically acceptable salts, N-oxides, tautomers, or stereoisomers, can be used alone or in combination with other lipid components, such as neutral lipids, charged lipids, steroids (including, for example, cholesterol) and / or their analogs, and / or polymer-conjugated lipids, to form lipid nanoparticles for delivering therapeutic agents. In some examples, lipid nanoparticles are used to deliver nucleic acids, such as antisense and / or messenger RNA. Pharmaceutical compositions containing the lipid compounds of the present invention, their pharmaceutically acceptable salts, N-oxides, tautomers, or stereoisomers, alone or in combination with additional therapeutic agents, are also provided. The present invention also provides, in part, methods for preparing such lipid compounds, or their pharmaceutically acceptable salts, N-oxides, tautomers, or stereoisomers, and compositions of the present invention, as well as methods of using the above to treat various diseases or conditions, such as those caused by infectious entities and / or protein dysfunction. This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used solely as an aid in determining the scope of the claimed subject matter.

[0008] In one embodiment of the present invention, a compound of formula (I)

[0009] [ka] or a pharmaceutically acceptable salt, N-oxide, tautomer, or stereoisomer thereof. [In the formula, m, n, o, and p each independently represent 1 to 3; G 1 is C 1~12 Alkylene or C 2~12is alkenylene, R 1 is -N(R 2 )R 3 , -OR 4 , CN, -N(R 4 ) (heteroaryl), -O(CH2) q OH, -(OCH2CH2) r OH, -OC(=O)R 5 , -N(R 4 )C(=O)R 5 , -N(R 4 )S(O)2R 5 , -N(R 4 )C(=O)N(R 2 )R 3 , -OC(=O)N(R 2 )R 3 , -N(R 4 )C(=O)OR 5 , -N(R 4 )C(=S)N(R 2 )R 3 , -N(R 4 )C(=NR 6 )N(R 2 )R 3 ,or

[0010] [ka] and R 2 and R 3 are independently H, C 1~6 Alkyl, C 3~8 cycloalkyl, or aryl, or R 2 and R 3 form a heterocyclic ring together with the nitrogen atom to which they are attached, R 4 is H, C 1~6 Alkyl or C 3~8 is cycloalkyl, R 5 is C 1~6 Alkyl or C 1~6 C optionally substituted with alkyl 3~8 is cycloalkyl, R6 are H, CN, NO2, C 1~6 Alkyl, OR 5 , S(O)2R 5 , or S(O)N(R 2 )R 3 and q is 2 to 6; r is 1 to 6; W is

[0011] [ka] and X is N or CH; G 2 and G 3 are each independently, C 1~12 Alkylene or C 2~12 is alkenylene, L 1 and L 2 are each independently -C(=O)OR 7 , -OC(=O)R 7 , -OC(=O)(CH2) r C(=O)OR 7 , -OC(=O)(CH2) r OC(=O)R 7 , -OC(=O)N(R 4 )R 7 , -N(R 4 )C(=O)OR 7 , -N(R 4 )C(=O)N(R 4 )R 7 , -OC(=O)OR 7 , or -S-SR 7 and R 7 is C 6~24 Alkyl, C 6~24 Alkenyl, or C 6~24 alkynyl, F, C 1~6 Alkoxy, C 3~8 Cycloalkyl, or C 3~8 optionally substituted by cycloalkenyl; L 1 and L 2 R7 may be the same or different].

[0012] In one aspect, the present disclosure provides a compound of formula (Ia):

[0013] [ka] or a pharmaceutically acceptable salt, N-oxide, tautomer or stereoisomer thereof wherein m, n, o, and p are each independently 1 or 2.

[0014] In another aspect, the present disclosure provides a compound of formula (Ib):

[0015] [ka] or a pharmaceutically acceptable salt, N-oxide, tautomer or stereoisomer thereof wherein m, n, o, and p are each independently 1 or 2.

[0016] In another aspect, the present disclosure provides a compound of formula (Ic):

[0017] [ka] or a pharmaceutically acceptable salt, N-oxide, tautomer or stereoisomer thereof wherein m and n each independently represent 1 or 2; o and p are each 1].

[0018] In a further aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (3-(4-hydroxybutyl)-3-azaspiro[5.5]undecane-9,9-diyl)bis(methylene)bis(2-heptylnonanoate); 2-(3-(4-hydroxybutyl)-3-azaspiro[5.5]undecan-9-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-cyclobutyldecanoate); 2-(9-(5-hydroxypentyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(2-(4-hydroxybutyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-hexyldecanoate); 3-((2-hexyldecanoyl)oxy)-2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl palmitate; 2-(9-(3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(3-(1-methylcyclopropane-1-carboxamido)propyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(9-(3-hydroxypropyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(3-(ethylsulfonamido)propyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(7-(4-hydroxybutyl)-2,7-diazaspiro[4.4]nonan-2-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(7-(4-hydroxybutyl)-2,7-diazaspiro[3.5]nonan-2-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(8-(4-hydroxybutyl)-2,8-diazaspiro[4.5]decan-2-yl)propane-1,3-diylbis(2-heptylnonanoate); 3-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)pentane-1,5-diylbis(2-hexyldecanoate); 2-(7-(5-hydroxypentyl)-2,7-diazaspiro[4.4]nonan-2-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(2-(5-hydroxypentyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(7-(5-hydroxypentyl)-2,7-diazaspiro[3.5]nonan-2-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)pentane-1,5-diylbis(2-hexyldecanoate); 2-(2-(4-hydroxybutyl)-2,7-diazaspiro[3.5]nonan-7-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(5-hydroxypentan-2-yl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(2-(4-hydroxybutyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-octyldecanoate); 2-(9-(5-hydroxypentyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptyldecanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-butyldecanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(3-hexylundecanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-pentyldecanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-(cyclobutylmethyl)decanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptyltetradecanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-(cyclopentylmethyl)decanoate); 2-(7-(4-hydroxybutyl)-2,7-diazaspiro[4.4]nonan-2-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(7-(4-hydroxybutyl)-2,7-diazaspiro[3.5]nonan-2-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-(cyclopent-3-en-1-ylmethyl)decanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-(2-cyclobutylethyl)decanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-(cyclohexylmethyl)decanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(4,5-dibutylnonanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(3,3-dibutylnonanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(4-heptylundecanoate); 2-(7-(5-hydroxypentyl)-2,7-diazaspiro[4.4]nonan-2-yl)propane-1,3-diylbis(2-heptylnonanoate); 3-(Decanoyloxy)-2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl 2-hexyldecanoate; 3-((2-hexyldecanoyl)oxy)-2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl (Z)-dodec-5-enoate; 3-((2-(cyclobutylmethyl)decanoyl)oxy)-2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl 2-hexyldecanoate; 3-((2-hexyldecanoyl)oxy)-2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl 2-butylundecanoate; 3-((4,5-dibutylnonanoyl)oxy)-2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl palmitate; 3-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)pentane-1,5-diylbis(2-heptylnonanoate); 2-(2-(5-hydroxypentyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(7-(5-hydroxypentyl)-2,7-diazaspiro[3.5]nonan-2-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(2-(4-hydroxybutyl)-2,7-diazaspiro[3.5]nonan-7-yl)propane-1,3-diylbis(2-hexyldecanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate); rac-(2R,3R)-8-(2-hydroxyethyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate); rac-(7R,8R)-2-(3-hydroxypropyl)-2-azaspiro[4.4]nonane-7,8-diylbis(2-heptylnonanoate); rac-(2R,3R)-8-(3-hydroxypropyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate); rac-(2R,3R)-8-(3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate); rac-(2R,3R)-8-(5-hydroxypentyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate); (2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate); (2S,3S)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-octyldecanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptyltetradecanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-hexyldecanoate); rac-O,O'-((2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diyl)di(pentadecan-8-yl)disuccinate; rac-O' 1 ,O 1 -((2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diyl)7,7'-di(pentadecan-8-yl)di(heptanedioate); rac-(((2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diyl)bis(oxy))bis(6-oxohexane-6,1-diyl)bis(2-heptylnonanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-pentyldecanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptyldecanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-hexylundecanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-hexylnonanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-butyldecanoate); rac-(2R,3R)-3-((2-ethylnonanoyl)oxy)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decan-2-yl 2-hexyldecanoate; and Bis(3-pentyloctyl) 3-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)pentanedioate; or a pharmaceutically acceptable salt thereof.

[0019] In another embodiment of the present invention, there is provided a pharmaceutical composition comprising a nucleic acid, at least one pharmaceutically acceptable excipient, and a compound described herein, or a pharmaceutically acceptable salt thereof. In a further embodiment of the present invention, there is provided a method for administering a nucleic acid to a subject in need thereof, comprising preparing or providing a pharmaceutical composition described herein and administering the pharmaceutical composition to the subject.

[0020] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. [Brief explanation of the drawings]

[0021] [Figure 1]1 is a graph showing the 50% neutralization titer, geometric mean titer (GMT) ratio relative to the ALC-0315 control, two weeks after two doses of LNP 0.2 μg (modRNA Flu HA / California) in Balb / c mice. Study #1 and Study #2 were performed using the same protocol (see Example 74). DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention may be more readily understood by reference to the following detailed description of embodiments of the invention and the examples contained herein. It should be understood that the present invention is not limited to specific synthetic methods of preparation, which may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting.

[0023] Embodiments of the present invention are described below, where, for convenience, embodiment 1 (E1) is the same as the embodiment of formula (I) presented above. Exemplary embodiments (E) of the present invention provided herein include:

[0024] E1 Formula (I)

[0025] [ka] or a pharmaceutically acceptable salt, N-oxide, tautomer, or stereoisomer thereof [In the formula, m, n, o, and p each independently represent 1 to 3; G 1 is C 1~12 Alkylene or C 2~12 is alkenylene, R 1 is -N(R 2 )R 3 , -OR 4 , CN, -N(R 4) (heteroaryl), -O(CH2) q OH, -(OCH2CH2) r OH, -OC(=O)R 5 , -N(R 4 )C(=O)R 5 , -N(R 4 )S(O)2R 5 , -N(R 4 )C(=O)N(R 2 )R 3 , -OC(=O)N(R 2 )R 3 , -N(R 4 )C(=O)OR 5 , -N(R 4 )C(=S)N(R 2 )R 3 , -N(R 4 )C(=NR 6 )N(R 2 )R 3 ,or

[0026] [ka] and R 2 and R 3 are independently H, C 1~6 Alkyl, C 3~8 cycloalkyl, or aryl, or R 2 and R 3 form a heterocyclic ring together with the nitrogen atom to which they are attached, R 4 is H, C 1~6 Alkyl or C 3~8 is cycloalkyl, R 5 is C 1~6 Alkyl or C 1~6 C optionally substituted with alkyl 3~8 is cycloalkyl, R 6 are H, CN, NO2, C 1~6 Alkyl, OR 5 , S(O)2R 5 , or S(O)N(R 2 )R3 and q is between 2 and 6 r is 1 to 6; W is

[0027] [ka] and X is N or CH; G 2 and G 3 are each independently, C 1~12 Alkylene or C 2~12 is alkenylene, L 1 and L 2 are each independently -C(=O)OR 7 , -OC(=O)R 7 , -OC(=O)(CH2) r C(=O)OR 7 , -OC(=O)(CH2) r OC(=O)R 7 , -OC(=O)N(R 4 )R 7 , -N(R 4 )C(=O)OR 7 , -N(R 4 )C(=O)N(R 4 )R 7 , -OC(=O)OR 7 , or -S-SR 7 and R 7 is C 6~24 Alkyl, C 6~24 Alkenyl, or C 6~24 alkynyl, F, C 1~6 Alkoxy, C 3~8 Cycloalkyl, or C 3~8 optionally substituted by cycloalkenyl; L 1 and L 2 R 7 may be the same or different].

[0028] E2 Formula (Ia)

[0029] [ka] or a pharmaceutically acceptable salt, N-oxide, tautomer, or stereoisomer thereof. wherein m, n, o, and p are each independently 1 or 2.

[0030] E3 Formula (Ib)

[0031] [ka] or a pharmaceutically acceptable salt, N-oxide, tautomer, or stereoisomer thereof. wherein m, n, o, and p are each independently 1 or 2.

[0032] E4 Formula (Ic)

[0033] [ka] or a pharmaceutically acceptable salt, N-oxide, tautomer, or stereoisomer thereof. wherein m and n each independently represent 1 or 2; o and p are each 1].

[0034] E5 G 1 But C 1~12 is alkylene, R 1 But -OH or

[0035] [ka] and R 2 and R 3 However, independently, H, C 1~6 Alkyl or C3~8 cycloalkyl or R 2 and R 3 form a heterocyclic ring together with the nitrogen atom to which they are attached, R 4 But H, C 1~6 Alkyl or C 3~8 is cycloalkyl, G 2 and G 3 However, each independently, C 1~12 is alkylene, L 1 and L 2 are respectively -OC(=O)R 7 and R 7 But C 6~24 Alkyl, C 6~24 Alkenyl, or C 6~24 alkynyl, F, C 1~6 Alkoxy, C 3~8 Cycloalkyl, or C 3~8 optionally substituted by cycloalkenyl; L 1 and L 2 R 7 The compound of any one of embodiments E1 to E4, wherein may be the same or different.

[0036] E6 R 7 But the following structure:

[0037] [ka] The compound of any one of embodiments E1 to E5, having

[0038] E7 R 1 The compound of any one of embodiments E1-E6, wherein is OH.

[0039] E8 R 1 but,

[0040] [ka] The compound of any one of embodiments E1 to E6, wherein

[0041] E9 G 1 The compound of any one of Embodiments E1 through E8, wherein is C2-C5 alkylene.

[0042] E10 G 1 The compound of any one of Embodiments E1 through E8, wherein is C3-C5 alkylene.

[0043] E11 (3-(4-hydroxybutyl)-3-azaspiro[5.5]undecane-9,9-diyl)bis(methylene)bis(2-heptylnonanoate); 2-(3-(4-hydroxybutyl)-3-azaspiro[5.5]undecan-9-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-cyclobutyldecanoate); 2-(9-(5-hydroxypentyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(2-(4-hydroxybutyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-hexyldecanoate); 3-((2-hexyldecanoyl)oxy)-2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl palmitate; 2-(9-(3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(3-(1-methylcyclopropane-1-carboxamido)propyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(9-(3-hydroxypropyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(3-(ethylsulfonamido)propyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(7-(4-hydroxybutyl)-2,7-diazaspiro[4.4]nonan-2-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(7-(4-hydroxybutyl)-2,7-diazaspiro[3.5]nonan-2-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(8-(4-hydroxybutyl)-2,8-diazaspiro[4.5]decan-2-yl)propane-1,3-diylbis(2-heptylnonanoate); 3-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)pentane-1,5-diylbis(2-hexyldecanoate); 2-(7-(5-hydroxypentyl)-2,7-diazaspiro[4.4]nonan-2-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(2-(5-hydroxypentyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(7-(5-hydroxypentyl)-2,7-diazaspiro[3.5]nonan-2-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)pentane-1,5-diylbis(2-hexyldecanoate); 2-(2-(4-hydroxybutyl)-2,7-diazaspiro[3.5]nonan-7-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(5-hydroxypentan-2-yl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(2-(4-hydroxybutyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-octyldecanoate); 2-(9-(5-hydroxypentyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptyldecanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-butyldecanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(3-hexylundecanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-pentyldecanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-(cyclobutylmethyl)decanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptyltetradecanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-(cyclopentylmethyl)decanoate); 2-(7-(4-hydroxybutyl)-2,7-diazaspiro[4.4]nonan-2-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(7-(4-hydroxybutyl)-2,7-diazaspiro[3.5]nonan-2-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-(cyclopent-3-en-1-ylmethyl)decanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-(2-cyclobutylethyl)decanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-(cyclohexylmethyl)decanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(4,5-dibutylnonanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(3,3-dibutylnonanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(4-heptylundecanoate); 2-(7-(5-hydroxypentyl)-2,7-diazaspiro[4.4]nonan-2-yl)propane-1,3-diylbis(2-heptylnonanoate); 3-(Decanoyloxy)-2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl 2-hexyldecanoate; 3-((2-hexyldecanoyl)oxy)-2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl (Z)-dodec-5-enoate; 3-((2-(cyclobutylmethyl)decanoyl)oxy)-2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl 2-hexyldecanoate; 3-((2-hexyldecanoyl)oxy)-2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl 2-butylundecanoate; 3-((4,5-dibutylnonanoyl)oxy)-2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl palmitate; 3-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)pentane-1,5-diylbis(2-heptylnonanoate); 2-(2-(5-hydroxypentyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(7-(5-hydroxypentyl)-2,7-diazaspiro[3.5]nonan-2-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(2-(4-hydroxybutyl)-2,7-diazaspiro[3.5]nonan-7-yl)propane-1,3-diylbis(2-hexyldecanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate); rac-(2R,3R)-8-(2-hydroxyethyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate); rac-(7R,8R)-2-(3-hydroxypropyl)-2-azaspiro[4.4]nonane-7,8-diylbis(2-heptylnonanoate); rac-(2R,3R)-8-(3-hydroxypropyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate); rac-(2R,3R)-8-(3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate); rac-(2R,3R)-8-(5-hydroxypentyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate); (2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate); (2S,3S)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-octyldecanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptyltetradecanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-hexyldecanoate); rac-O,O'-((2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diyl)di(pentadecan-8-yl)disuccinate; rac-O' 1 ,O 1-((2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diyl)7,7'-di(pentadecan-8-yl)di(heptanedioate); rac-(((2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diyl)bis(oxy))bis(6-oxohexane-6,1-diyl)bis(2-heptylnonanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-pentyldecanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptyldecanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-hexylundecanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-hexylnonanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-butyldecanoate); rac-(2R,3R)-3-((2-ethylnonanoyl)oxy)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decan-2-yl 2-hexyldecanoate; and Bis(3-pentyloctyl) 3-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)pentanedioate; or a pharmaceutically acceptable salt thereof.

[0044] E12 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(7-(4-hydroxybutyl)-2,7-diazaspiro[3.5]nonan-2-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(2-(4-hydroxybutyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(9-(5-hydroxypentyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-hexyldecanoate); 2-(2-(4-hydroxybutyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(9-(3-hydroxypropyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(3-hexylundecanoate); 2-(7-(4-hydroxybutyl)-2,7-diazaspiro[4.4]nonan-2-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(7-(5-hydroxypentyl)-2,7-diazaspiro[3.5]nonan-2-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(2-(4-hydroxybutyl)-2,7-diazaspiro[3.5]nonan-7-yl)propane-1,3-diylbis(2-hexyldecanoate); and rac-(2R,3R)-8-(5-hydroxypentyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate); or a pharmaceutically acceptable salt thereof.

[0045] E13 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate), or a pharmaceutically acceptable salt thereof.

[0046] E14 2-(7-(4-hydroxybutyl)-2,7-diazaspiro[3.5]nonan-2-yl)propane-1,3-diylbis(2-heptylnonanoate), or a pharmaceutically acceptable salt thereof.

[0047] E15 2-(2-(4-hydroxybutyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-heptylnonanoate), or a pharmaceutically acceptable salt thereof.

[0048] E16 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-hexyldecanoate), or a pharmaceutically acceptable salt thereof.

[0049] E17 2-(9-(5-hydroxypentyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate), or a pharmaceutically acceptable salt thereof.

[0050] E18 rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate), or a pharmaceutically acceptable salt thereof.

[0051] E19 rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-hexyldecanoate), or a pharmaceutically acceptable salt thereof.

[0052] E20 2-(2-(4-hydroxybutyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-hexyldecanoate), or a pharmaceutically acceptable salt thereof.

[0053] E21 2-(9-(3-hydroxypropyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate), or a pharmaceutically acceptable salt thereof.

[0054] E22 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(3-hexylundecanoate), or a pharmaceutically acceptable salt thereof.

[0055] E23 2-(7-(4-hydroxybutyl)-2,7-diazaspiro[4.4]nonan-2-yl)propane-1,3-diylbis(2-hexyldecanoate), or a pharmaceutically acceptable salt thereof.

[0056] E24 2-(7-(5-hydroxypentyl)-2,7-diazaspiro[3.5]nonan-2-yl)propane-1,3-diylbis(2-hexyldecanoate), or a pharmaceutically acceptable salt thereof.

[0057] E25 2-(2-(4-hydroxybutyl)-2,7-diazaspiro[3.5]nonan-7-yl)propane-1,3-diylbis(2-hexyldecanoate), or a pharmaceutically acceptable salt thereof.

[0058] E26 rac-(2R,3R)-8-(5-hydroxypentyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate), or a pharmaceutically acceptable salt thereof.

[0059] E27 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate).

[0060] E28 2-(7-(4-hydroxybutyl)-2,7-diazaspiro[3.5]nonan-2-yl)propane-1,3-diylbis(2-heptylnonanoate).

[0061] E29 2-(2-(4-hydroxybutyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-heptylnonanoate).

[0062] E30 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-hexyldecanoate).

[0063] E31 2-(9-(5-hydroxypentyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate).

[0064] E32 rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate).

[0065] E33 rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-hexyldecanoate).

[0066] E34 2-(2-(4-hydroxybutyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-hexyldecanoate).

[0067] E35 2-(9-(3-hydroxypropyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate).

[0068] E36 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(3-hexylundecanoate).

[0069] E37 2-(7-(4-hydroxybutyl)-2,7-diazaspiro[4.4]nonan-2-yl)propane-1,3-diylbis(2-hexyldecanoate).

[0070] E38 2-(7-(5-hydroxypentyl)-2,7-diazaspiro[3.5]nonan-2-yl)propane-1,3-diylbis(2-hexyldecanoate).

[0071] E39 2-(2-(4-hydroxybutyl)-2,7-diazaspiro[3.5]nonan-7-yl)propane-1,3-diylbis(2-hexyldecanoate).

[0072] E40 rac-(2R,3R)-8-(5-hydroxypentyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate).

[0073] E41 A pharmaceutical composition comprising a nucleic acid, at least one pharmaceutically acceptable excipient, and a compound according to any one of embodiments E1 to E40, or a pharmaceutically acceptable salt thereof.

[0074] E42 The pharmaceutical composition of embodiment E41, wherein the pharmaceutically acceptable excipient is selected from the group consisting of neutral lipids, steroids, and polymer-conjugated lipids.

[0075] E43 The pharmaceutical composition of any one of embodiments E41-E42, comprising 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), phophatidylethanolamines, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM), or a combination thereof.

[0076] E44 The pharmaceutical composition of any one of embodiments E42-E43, wherein the steroid is cholesterol.

[0077] E45 The pharmaceutical composition of any one of embodiments E42 to E44, wherein the polymer-conjugated lipid is a PEGylated lipid.

[0078] E46 The pharmaceutical composition of embodiment E45, wherein the PEGylated lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, PEG-DMG, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), or PEG dialkyoxypropylcarbamate.

[0079] E47 The pharmaceutical composition of any one of embodiments E41 to E46, wherein the nucleic acid is RNA.

[0080] E48 The pharmaceutical composition of embodiment E47, wherein the RNA is messenger RNA.

[0081] E49 The pharmaceutical composition of any one of embodiments E47-E48, wherein the RNA is a modRNA or a saRNA.

[0082] E50 A method for administering a nucleic acid to a subject in need thereof, comprising preparing or providing a pharmaceutical composition of any one of embodiments E41 to E49 and administering the pharmaceutical composition to the subject.

[0083] E51 A method of making a compound of any one of embodiments E1 to E40, comprising any of the methods described in the Examples provided herein.

[0084] E52 A method of making the pharmaceutical composition of any one of embodiments E41 to E49, comprising combining a nucleic acid, at least one pharmaceutically acceptable excipient, and a compound according to any one of embodiments E1 to E40.

[0085] E53 A compound according to any one of embodiments E1 to E40 for use as a component of a medicament.

[0086] E54 The compound of embodiment E53, wherein the medicament is a vaccine.

[0087] E55 Use of a compound according to any one of embodiments E1 to E40 for the manufacture of a medicament.

[0088] E56 The use of embodiment E55, wherein the medicament is a vaccine.

[0089] Each embodiment described herein can be combined with any other embodiment described herein that is not inconsistent with the combined embodiment. In addition, any of the compounds described in the Examples, or a pharmaceutically acceptable salt thereof, may be claimed individually, or together with one or more other compounds of the Examples, or a pharmaceutically acceptable salt thereof, as a group, for any of the embodiments described herein.

[0090] Furthermore, each of the embodiments described herein includes within its scope pharmaceutically acceptable salts of the compounds described herein.

[0091] The section headings used herein are for general information purposes only and are not to be construed as limiting the subject matter described.

[0092] All references cited herein, including patent applications, patent publications, and UniProtKB accession numbers, are hereby incorporated by reference to the same extent as if each individual reference were specifically and individually indicated to be incorporated by reference in its entirety.

[0093] definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have meanings commonly understood by those of ordinary skill in the art.

[0094] The invention described herein may be suitably practiced in the absence of any element not specifically disclosed herein.

[0095] "Compounds of the invention" include compounds of Formula I, I(a), I(b), and / or I(c), their pharmaceutically acceptable salts, N-oxides, tautomers, or stereoisomers, as well as novel intermediates used in their preparation. One of ordinary skill in the art will recognize that compounds of the invention include conformational isomers (e.g., cis and trans isomers) and all optical isomers (e.g., enantiomers and diastereoisomers), where they may exist, as well as racemates, diastereoisomers, and other mixtures of such isomers, and tautomers thereof. One of ordinary skill in the art will also recognize that compounds of the invention include solvates, hydrates, isomorphs, polymorphs, esters, salt forms, prodrugs, and isotopically labeled versions (including deuterium substitutions) thereof, where they may be formed.

[0096] As used herein, the singular forms "a," "an," and "the" include plural references unless otherwise indicated. For example, "a" substituent includes one or more substituents.

[0097] As used herein, the term "about," when used to modify a parameter defined by a numerical value (e.g., a dose of XXX), means that the parameter can vary 10% above or below the numerical value stated for that parameter. For example, a dose of about 5 mg means 5%±10%, and can be, for example, 4.5 mg and 5.5 mg, or any value therebetween.

[0098] As used herein, the term "aqueous solution" refers to a composition that includes water.

[0099] If substituents are described as being "independently selected" from a group, then each substituent is selected independently of the others. Thus, each substituent can be the same or different from the other substituents.

[0100] "Optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances in which the event or circumstance occurs and instances in which it does not occur.

[0101] The terms "optionally substituted" and "substituted or unsubstituted" are used interchangeably to indicate that a particular group being described may have no non-hydrogen substituents (e.g., unsubstituted) or that the group may have one or more non-hydrogen substituents (e.g., substituted). Unless otherwise specified, the total number of substituents that may be present is equal to the number of H atoms present on the unsubstituted form of the group being described. When an optional substituent is attached through a double bond, such as an oxo (=O) substituent, the group occupies two available valencies and the total number of other substituents included is reduced by two. When optional substituents are independently selected from a list of alternatives, the selected groups may be the same or different. It will be understood that throughout this disclosure, the number and nature of optional substituents will be limited to the extent that such substitution makes chemical sense to one of ordinary skill in the art.

[0102] "Halogen" or "halo" refers to fluoro, chloro, bromo, and iodo (F, Cl, Br, I).

[0103] "Cyano" refers to a substituent having a carbon atom joined to a nitrogen atom by a triple bond, eg, --C.ident.N.

[0104] "Hydroxy" refers to the group --OH.

[0105] "Oxo" refers to a double-bonded oxygen (=O).

[0106] "Alkyl" refers to a saturated monovalent aliphatic hydrocarbon radical having the specified number of carbon atoms, including straight-chain or branched-chain groups. Alkyl groups include, but are not limited to, groups having 1 to 12 carbon atoms ("C1-C 12The alkyl group may contain 1 to 8 carbon atoms ("C1-C8 alkyl"), 1 to 6 carbon atoms ("C1-C6 alkyl"), 1 to 5 carbon atoms ("C1-C5 alkyl"), 1 to 4 carbon atoms ("C1-C4 alkyl"), 1 to 3 carbon atoms ("C1-C3 alkyl"), or 1 to 2 carbon atoms ("C1-C2 alkyl"). Examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, and the like. Alkyl groups may be optionally substituted, unsubstituted, or substituted, as further defined herein.

[0107] An "alkylene" or "alkylene chain" is a group consisting of only carbon and hydrogen, saturated, e.g., 1 to 24 carbon atoms (C1 to C 24 Alkylene, 1 to 15 carbon atoms (C1 to C 15 Alkylene, 1 to 12 carbon atoms (C1 to C 12 "Ci-C alkylene" refers to a straight or branched divalent hydrocarbon chain connecting the rest of the molecule to a radical group, e.g., methylene, ethylene, propylene, n-butylene, and the like, having 1 to 8 carbon atoms (Ci-C alkylene), 1 to 6 carbon atoms (Ci-C alkylene), 2 to 4 carbon atoms (C2-C4 alkylene), or 1 to 2 carbon atoms (Ci-C2 alkylene). The alkylene chain is attached to the rest of the molecule through a single or double bond and to the radical group through a single or double bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, the alkylene chain may be substituted.

[0108] "Fluoroalkyl" refers to an alkyl group, as defined herein, in which one to all of the alkyl group's hydrogen atoms have been replaced with fluoro atoms. Examples include, but are not limited to, fluoromethyl, difluoromethyl, 2-fluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, and 1,2,2,2-tetrafluoroethyl. Examples of fully substituted fluoroalkyl groups (also referred to as perfluoroalkyl groups) include trifluoromethyl (-CF3) and pentafluoroethyl (-CF5).

[0109] "Alkoxy" refers to an alkyl group, as defined herein, single-bonded to an oxygen atom. The point of attachment of the alkoxy radical to the molecule is through the oxygen atom. The alkoxy radical is sometimes depicted as alkyl-O-. Alkoxy groups may contain, but are not limited to, 1 to 8 carbon atoms ("C1-C8 alkoxy"), 1 to 6 carbon atoms ("C1-C6 alkoxy"), 1 to 4 carbon atoms ("C1-C4 alkoxy"), or 1 to 3 carbon atoms ("C1-C3 alkoxy"). Alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isobutoxy, and the like.

[0110] "Alkoxyalkyl" refers to an alkyl group, as defined herein, substituted with an alkoxy group, as defined herein. Examples include, but are not limited to, CH3OCH2- and CH3CH2OCH2-.

[0111] "Alkenyl" refers to a monovalent aliphatic hydrocarbon radical, including straight- or branched-chain groups, consisting of at least two carbon atoms and at least one carbon-carbon double bond. For example, as used herein, the term "C2-C6 alkenyl" refers to a straight- or branched-chain unsaturated radical of 2 to 6 carbon atoms, including, but not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-, 2-, or 3-butenyl, and the like.

[0112] An "alkenylene" or "alkenylene chain" consists solely of carbon and hydrogen, and consists of at least two carbon atoms and at least one carbon-carbon double bond, and can have, for example, 1 to 24 carbon atoms (C1 to C 24 Alkenylene, 1 to 15 carbon atoms (C1 to C 15 Alkenylene, 1 to 12 carbon atoms (C1 to C 12 Alkylene refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, e.g., ethenylene, propenylene, n-butenylene, and the like, having 1 to 8 carbon atoms (C1-C8 alkenylene), 1 to 6 carbon atoms (C1-C6 alkenylene), 2 to 4 carbon atoms (C2-C4 alkenylene), or 1 to 2 carbon atoms (C1-C2 alkenylene). The alkenylene chain is attached to the rest of the molecule through a single or double bond and to the radical group through a single or double bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, an alkylene chain may be substituted.

[0113] "Alkynyl" refers to a monovalent aliphatic hydrocarbon radical, including straight- or branched-chain groups, consisting of at least two carbon atoms and at least one carbon-carbon triple bond. Examples include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-, 2-, or 3-butynyl, and the like.

[0114] "Cycloalkyl" or "carbocyclic ring" refers to a fully saturated hydrocarbon ring system having the specified number of carbon atoms, which may be a monocyclic, bridged, spirocyclic, or fused bicyclic or polycyclic ring system, linked to the base molecule through a carbon atom of the cycloalkyl ring. Cycloalkyl groups include, but are not limited to, cycloalkyl groups containing 3 to 12 carbon atoms ("C3-C6"). 12The cycloalkyl group may contain 3 to 8 carbon atoms ("C-C cycloalkyl"), 3 to 6 carbon atoms ("C-C cycloalkyl"), 3 to 5 carbon atoms ("C-C cycloalkyl"), or 3 to 4 carbon atoms ("C-C cycloalkyl"). Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantanyl, and the like. Cycloalkyl groups may be optionally substituted, unsubstituted, or substituted, as further defined herein.

[0115] Illustrative examples of cycloalkyl rings include, but are not limited to, the following:

[0116] [ka]

[0117] "Cycloalkenyl" refers to a hydrocarbon ring system having a specified number of carbon atoms containing at least one carbon-carbon double bond, which may be a monocyclic, bridged, spirocyclic, or fused bicyclic or polycyclic ring system linked to the base molecule through a carbon atom of the cycloalkenyl ring. Cycloalkenyl groups include, but are not limited to, cycloalkenyl groups containing 3 to 12 carbon atoms ("C3-C6"). 12 The cycloalkenyl group may contain 3 to 8 carbon atoms ("C-C cycloalkenyl"), 3 to 6 carbon atoms ("C-C cycloalkenyl"), 3 to 5 carbon atoms ("C-C cycloalkenyl"), or 3 to 4 carbon atoms ("C-C cycloalkenyl"). Examples include, but are not limited to, cyclopentenyl, cyclohexenyl, cycloheptenyl, and the like. Cycloalkenyl groups can be optionally substituted, unsubstituted, or substituted, as further defined herein.

[0118] "Cycloalkoxy" refers to a cycloalkyl group, as defined herein, that is single-bonded to an oxygen atom. The point of attachment of the cycloalkoxy radical to the molecule is through the oxygen atom. The cycloalkoxy radical may also be designated as cycloalkyl-O-. Cycloalkoxy groups may contain, but are not limited to, 3 to 8 carbon atoms ("C3-C8 cycloalkoxy"), 3 to 6 carbon atoms ("C3-C6 cycloalkoxy"), and 3 to 4 carbon atoms ("C3-C4 cycloalkoxy"). Cycloalkoxy groups include, but are not limited to, cyclopropoxy, cyclobutoxy, cyclopentoxy, and the like.

[0119] "Heterocycloalkyl" refers to a group in which the ring S atom is optionally substituted with one or two oxo groups (e.g., S(O) q , where q is 0, 1, or 2), a heterocycloalkyl ring refers to a fully saturated ring system having the specified number of ring atoms connected to the base molecule through a ring atom which may be C or N, and containing at least one heteroatom selected from N, O, and S as a ring member. Heterocycloalkyl rings include rings that are spirocyclic, bridged, or fused to one or more other heterocycloalkyl or carbocyclic rings, and such spirocyclic, bridged, or fused rings may themselves be saturated, partially unsaturated to the extent that unsaturation or aromaticity makes chemical sense, or aromatic, provided that the point of attachment to the base molecule is an atom of the heterocycloalkyl portion of the ring system. Heterocycloalkyl rings include rings that are N, O, and S(O) qHeterocycloalkyl rings may contain 1 to 4 heteroatoms selected from as ring members, or 1 to 2 ring heteroatoms, provided that such heterocycloalkyl rings do not contain two adjacent oxygen or sulfur atoms. Heterocycloalkyl rings may be optionally substituted, unsubstituted, or substituted, as further defined herein. Such substituents may be present on the heterocyclic ring attached to the base molecule, or on spirocyclic, bridged, or fused rings attached thereto. Heterocycloalkyl rings may include, but are not limited to, 3- to 8-membered heterocyclyl groups, e.g., 4- to 7- or 4- to 6-membered heterocycloalkyl groups, as defined herein.

[0120] Illustrative examples of heterocycloalkyl rings include, but are not limited to,

[0121] [ka] It includes a monovalent radical of the formula:

[0122] Illustrative examples of bridged and fused heterocycloalkyl groups include, but are not limited to,

[0123] [ka] It includes a monovalent radical of the formula:

[0124] As used herein, "spirocyclic" refers to a bicyclic moiety in which each ring is independently a cycloalkyl, cycloalkenyl, or heterocycloalkyl ring as defined herein, and both rings share only one common carbon atom. The spirocyclic ring may be attached to the molecule by a carbon or nitrogen atom.

[0125] "Aryl" or "aromatic" means that all carbon atoms in the ring are sp 2Aryl groups refer to hybridized, pi-conjugated, monocyclic, bicyclic (e.g., biaryl, fused), or polycyclic ring systems containing the specified number of ring atoms. Aryl groups include, but are not limited to, groups containing 6 to 20 carbon atoms ("C6-C6"). 20 aryl), 6 to 14 carbon atoms (C6-C 14 aryl), 6 to 12 carbon atoms (C6 to C 12 aryl"), or 6 to 10 carbon atoms ("C6-C 10 A fused aryl group may contain an aryl ring (e.g., a phenyl ring) fused to another aryl ring. Examples include, but are not limited to, phenyl, biphenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, and indenyl. Aryl groups may be optionally substituted, unsubstituted, or substituted, as further defined herein.

[0126] Similarly, a "heteroaryl" or "heteroaromatic" is one in which all carbon atoms in the ring are sp 2"Heteroaryl" refers to a hybridized, pi-electron conjugated, monocyclic, bicyclic (e.g., heterobiaryl, fused), or polycyclic ring system containing a specified number of ring atoms and including at least one heteroatom selected from N, O, and S as a ring member in the ring. Heteroaryl groups may contain, but are not limited to, 5 to 20 ring atoms ("5-20-membered heteroaryl"), 5 to 14 ring atoms ("5-14-membered heteroaryl"), 5 to 12 ring atoms ("5-12-membered heteroaryl"), 5 to 10 ring atoms ("5-10-membered heteroaryl"), 5 to 9 ring atoms ("5-9-membered heteroaryl"), or 5 to 6 ring atoms ("5-6-membered heteroaryl"). The heteroaryl ring is attached to the base molecule through a ring atom of the heteroaromatic ring. Thus, either a 5- or 6-membered heteroaryl ring, alone or in a fused configuration, may be attached to the base molecule through a ring C or N atom. Examples of heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thiophenyl, pyrazolyl, imidazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridinyl, pyridizinyl, pyrimidinyl, pyrazinyl, benzofuranyl, benzothiophenyl, indolyl, benzamidazolyl, indazolyl, quinolinyl, isoquinolinyl, purinyl, triazinyl, naphthyridinyl, cinnolinyl, quinazolinyl, quinoxalinyl, and carbazolyl. Examples of 5- or 6-membered heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thiophenyl, pyrazolyl, imidazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, triazolyl, pyridinyl, pyrimidinyl, pyrazinyl, and pyridazinyl rings. Heteroaryl groups may be optionally substituted, unsubstituted or substituted as further defined herein.

[0127] Examples of monocyclic heteroaryl groups include, but are not limited to,

[0128] [ka] Contains a monovalent radical of

[0129] Illustrative examples of fused ring heteroaryl groups include, but are not limited to:

[0130] [ka]

[0131] [ka] Includes:

[0132] "Amino" refers to the group -NH2, which is unsubstituted. Where amino is described as substituted or optionally substituted, the term includes groups of the form -NR'R" where each of R' and R" is defined as further described herein. For example, "alkylamino" refers to the group -NR'R" where one of R' and R" is an alkyl moiety and the other is H, and "dialkylamino" refers to -NR'R" where both R' and R" are alkyl moieties having the specified number of carbon atoms (e.g., -NH(C1-C4 alkyl) or -N(C1-C4 alkyl)2).

[0133] "Aminoalkyl" refers to an alkyl group, as defined above, that is substituted with one, two, or three amino groups, as defined herein.

[0134] The term "pharmaceutically acceptable" means a substance (e.g., a compound described herein) and any salts thereof, or a composition containing a substance or salt of the invention, that is suitable for administration to a subject or patient.

[0135] "Deuterium enrichment factor" as used herein means the ratio between the deuterium abundance and the natural abundance of deuterium, respectively, relative to the hydrogen abundance. Atomic locations designated as having deuterium typically have an atomic ratio of at least 1000 (15% deuterium incorporation), at least 2000 (30% deuterium incorporation), at least 3000 (45% deuterium incorporation), at least 3500 (52.5% deuterium incorporation), at least 3500 (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 ( and a deuterium enrichment factor of at least 6000 (90% deuterium incorporation rate), at least 6333.3 (95% deuterium incorporation rate), at least 6466.7 (97% deuterium incorporation rate), at least 6600 (99% deuterium incorporation rate), or at least 6633.3 (99.5% deuterium incorporation rate).

[0136] salt Salts encompassed within the term "pharmaceutically acceptable salt" generally refer to compounds prepared by reacting a free base or free acid with a suitable organic or inorganic acid, or a suitable organic or inorganic base, respectively, to obtain a salt of a compound of the present invention suitable for administration to a subject or patient.

[0137] In addition, compounds of formula I may include other salts of such compounds, which are not necessarily pharmaceutically acceptable salts, but which may be useful as intermediates for one or more of the following: 1) preparing compounds of formula I; 2) purifying compounds of formula I; 3) separating enantiomers of compounds of formula I; or 4) separating diastereoisomers of compounds of formula I.

[0138] Suitable acid addition salts for pharmaceutically acceptable salts may be formed from acids which form non-toxic salts. Examples include, but are not limited to, acetate, adipate, aspartate, benzoate, besylate, bicarbonate / carbonate, hydrogensulfate / sulfate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hybenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, phosphate ... These include gallate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate, 1,5-naphthalenedisulfonate, and xinofoate.

[0139] Hemisalts of acids and bases, such as hemisulfates and hemicalcium salts, may also be formed.

[0140] For a review of suitable salts, see Paulekun, GS et al., Trends in Active Pharmaceutical Ingredient Salt Selection Based on Analysis of the Orange Book Database, J. Med. Chem. 2007;50(26), 6665-6672.

[0141] Pharmaceutically acceptable salts of the compounds of the present invention can be prepared by, but are not limited to, the following procedure: (i) reacting a compound of the present invention with a desired acid; (ii) removing an acid- or base-labile protecting group from a suitable precursor of a compound of the invention, or ring-opening a suitable cyclic precursor, such as a lactone or lactam, using a desired acid; or (iii) converting one salt of a compound of the invention into another salt (which can be accomplished by reaction with an appropriate acid or by a suitable ion exchange procedure); It can be prepared by methods well known to those skilled in the art, including

[0142] These procedures are typically carried out in solution, and the resulting salt may precipitate and be collected by filtration or may be recovered by evaporation of the solvent.

[0143] solvate The compound of the present invention and its pharmaceutically acceptable salt can exist in non-solvated and solvated form.The term " solvate " is used herein to describe the molecular complex that comprises compound and one or more solvent molecules, for example, ethanol.The term " hydrate " can be used when solvent is water.

[0144] Compounds of Formula I may include pharmaceutically acceptable solvates of such compounds. In addition, compounds of Formula I may also include other solvates of such compounds that are not necessarily pharmaceutically acceptable solvates, but that may be useful as intermediates for one or more of: 1) preparing compounds of Formula I; 2) purifying compounds of Formula I; 3) separating enantiomers of compounds of Formula I; or 4) separating diastereoisomers of compounds of Formula I.

[0145] The currently accepted classification system for organic hydrates is one that defines isolated site, channel, or metal ion coordinated hydrates. See Polymorphism in Pharmaceutical Solids by KR Morris (HGBrittain, ed., Marcel Dekker, 1995). Isolated site hydrates are hydrates in which the water molecules are isolated from direct contact with each other by intervening organic molecules. In channel hydrates, the water molecules reside in lattice channels where they are adjacent to other water molecules. In metal ion coordinated hydrates, the water molecules are bound to the metal ion.

[0146] When the solvent or water is tightly bound, the complex may have a well-defined stoichiometry independent of humidity. However, when the solvent or water is weakly bound, as in channel solvates and hygroscopic compounds, the water / solvent content may be dependent on humidity and drying conditions. In such cases, non-stoichiometry becomes the norm.

[0147] solid form The compounds of the present invention can exist in a continuum of solid states ranging from completely amorphous to completely crystalline. The term "amorphous" refers to a state in which the material lacks long-range order at the molecular level and can exhibit the physical properties of a solid or a liquid, depending on temperature. Typically, such materials do not exhibit a distinctive X-ray diffraction pattern and are more formally described as liquids, while exhibiting the properties of a solid. Upon heating, a change from solid to liquid properties occurs, which is typically characterized by a second-order change of state ("glass transition"). The term "crystalline" refers to a solid phase in which the material has an internal structure that is regularly ordered at the molecular level and exhibits a distinctive X-ray diffraction pattern with defined peaks. When such materials are heated sufficiently, they also exhibit the properties of a liquid, but the change from solid to liquid is typically characterized by a first-order phase change ("melting point").

[0148] The compounds of the present invention may also exist in a mesomorphic state (mesophase or liquid crystal) when subjected to suitable conditions. The mesomorphic state is intermediate between the true crystalline state and the true liquid state (either melt or solution) and consists of two-dimensional order at the molecular level. Mesomorphic states that arise as a result of a change in temperature are described as "thermotropic," while those that arise upon the addition of a second component, such as water or another solvent, are described as "lyotropic." Compounds that have the potential to form lyotropic mesophases are described as "amphiphilic," and may be ionic (-COO - Na + , -COO - K + , or -SO3 - Na + ) or non-ionic (-N- N + They consist of molecules with polar head groups (e.g., (CH3)3). For more information, see Crystals and the Polarizing Microscope, N.H. Hartshorne and A. Stuart, 4th ed. (Edward Arnold, 1970).

[0149] stereoisomer Some compounds of the present invention may exist as two or more stereoisomers. Stereoisomers of a compound may include cis and trans isomers (geometric isomers), optical isomers such as R and S enantiomers, diastereoisomers, rotamers, atropisomers, and conformational isomers. For example, compounds of the present invention containing one or more asymmetric carbon atoms may exist as two or more stereoisomers. When a compound of the present invention contains an alkenyl or alkenylene group, geometric cis / trans (or Z / E) isomers may occur. Cis / trans isomers may also exist in saturated rings.

[0150] Salts of compounds of the invention can also contain counterions that are optically active (eg, d-lactate or l-lysine) or racemic (eg, dl-tartrate or dl-arginine).

[0151] Cis / trans isomers can be separated by conventional techniques well known to those skilled in the art, such as chromatography and fractional crystallization.

[0152] Conventional techniques for preparing / isolating individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of the racemate (or racemate of a salt or derivative) using, for example, chiral high-pressure liquid chromatography (HPLC). Alternatively, the racemate (or racemic precursor) can be reacted with a suitable optically active compound, for example, an alcohol, or, if the compound of the present invention contains an acidic or basic moiety, with a base or acid such as 1-phenylethylamine or tartaric acid. The resulting mixture of diastereoisomers can be separated by chromatography, fractional crystallization, or both techniques, and one or both of the diastereoisomers can be converted to the corresponding pure enantiomer by means well known to those skilled in the art. Chromatography can be used to obtain the chiral compounds of the present invention (and their chiral precursors) in enantiomerically enriched form, typically by HPLC concentration of the eluent to yield the enriched mixture. Chiral chromatography using subcritical and supercritical fluids can be used. Methods for chiral chromatography useful in some embodiments of the present invention are known in the art (see, e.g., Smith, Roger M., Loughborough University, Loughborough, UK; Chromatographic Science Series (1998), 75 (Supercritical Fluid Chromatography with Packed Columns), 223-249 and references cited therein).

[0153] When any racemate crystallizes, two different types of crystals can occur. The first type is the racemate (true racemate) mentioned above, which produces a single uniform crystal form containing both enantiomers in equimolar amounts. The second type is a racemic mixture or conglomerate, which produces two crystalline forms in equimolar amounts, each containing a single enantiomer. Both crystalline forms present in a racemic mixture have the same physical properties, but they may have different physical properties compared to a true racemate. Racemic mixtures can be separated by conventional techniques known to those skilled in the art. For example, see Stereochemistry of Organic Compounds by EL Eliel and SH Wilen (Wiley, 1994).

[0154] Tautomerism Tautomeric isomerism ("tautomerism") can occur where structural isomers are interconvertible via a low energy barrier. This can take the form of proton tautomerism in compounds of the invention that contain, for example, imino / amino, keto / enol, or oxime / nitroso groups, lactam / lactim, or so-called valence tautomerism in compounds that contain aromatic moieties. It follows that a single compound may exhibit more than one type of isomerism. In particular, the bis(amino)cyclobut-3-ene-1,2-dione moiety contained within the compounds of the invention can tautomerize as shown below and is included within the scope of the invention.

[0155] [ka]

[0156] For simplicity, the compounds of the invention are depicted herein in a single tautomeric form, however, it should be emphasized that all possible tautomeric forms are included within the scope of the invention.

[0157] Isotopes The present invention includes all isotopically labeled compounds of the present invention in which one or more atoms are replaced by an atom having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number predominant in nature.

[0158] Examples of isotopes suitable for inclusion in the compounds of the invention include: 2 H (D, deuterium) and 3 Hydrogen such as H (T, tritium), 11 C. 13 C and 14 Carbon, such as C 36 chlorine such as Cl, 18 Fluorine such as F, 123 I and 125 Iodine, such as I 13 N and 15 Nitrogen such as N 15 O. 17 O and 18 Oxygen, such as O 32 Phosphorus, such as P, 35 Isotopes of sulfur such as S may be included.

[0159] Certain isotopically labeled compounds of the present invention, for example, those incorporating a radioactive isotope, are useful in either or both drug and / or substrate tissue distribution studies. 3 H, and carbon-14, i.e., 14 C are particularly useful for this purpose given their ease of incorporation and rapid means of detection.

[0160] Deuterium, e.g., 2 Substitution with H may confer certain therapeutic advantages resulting from greater metabolic stability.

[0161] Positron-emitting isotopes, e.g. 11 C. 18 F, 15 O and 13Substitution with N may be useful in positron emission tomography (PET) studies to examine substrate receptor occupancy. Substitution with deuterium may confer certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life, reduced dosage requirements, decreased CYP450 inhibition (competitive or time-dependent), or improved therapeutic index or tolerability.

[0162] In some embodiments, the present disclosure provides deuterium-labeled (or deuterated) compounds and salts, wherein the formulas and variables of such compounds and salts are each independently as described herein. "Deuterated" means that at least one of the atoms in the compound is deuterium at an abundance greater than the natural abundance of deuterium (typically approximately 0.015%). Those skilled in the art recognize that in chemical compounds containing hydrogen atoms, the hydrogen atom is actually a mixture of H and D, with approximately 0.015% being D. The concentration of deuterium incorporated in the deuterium-labeled compounds and salts of the present invention can be defined by the deuterium enrichment factor. It is understood that one or more deuterium atoms can be exchanged for hydrogen under physiological conditions.

[0163] In some embodiments, the deuterium compound is selected from any one of the compounds described in Tables 6A-6G shown in the Examples section.

[0164] In some embodiments, one or more hydrogen atoms at certain metabolic sites in the compounds of the invention are deuterated.

[0165] Isotopically labeled compounds of the present invention can generally be prepared by conventional techniques known to those skilled in the art, or by processes analogous to those described in the accompanying Examples and Preparations, substituting an appropriate isotopically labeled reagent for the previously used unlabeled reagent.

[0166] Solvates in accordance with the present invention include those wherein the solvent of crystallization may be isotopically substituted, eg, D2O, d6-acetone, d6-DMSO.

[0167] Metabolites Active metabolites of the compounds of the invention, i.e., compounds formed in vivo upon drug administration, often by oxidation or dealkylation, are also included within the scope of the invention. Some examples of metabolites according to the invention include, but are not limited to: (i) When the compound of the present invention contains an alkyl group, its hydroxyalkyl derivative (-CH > -COH): (ii) if the compound of the invention contains an alkoxy group, its hydroxy derivative (-OR → -OH); (iii) if the compound of the invention contains a tertiary amino group, its secondary amino derivative (-NRR' → -NHR or -NHR'); (iv) When the compound of the present invention contains a tertiary amino group, its N-oxide derivative (-NRR ’ → -N(O)RR'); (v) If the compound of the invention contains a secondary amino group, its primary derivative (-NHR → -NH2); (vi) When the compound of the invention contains a phenyl moiety, its phenol derivative (-Ph → -PhOH); (vii) When the compound of the invention contains an amide group, its carboxylic acid derivative (-CONH2 → COOH); Contains, (viii) If a compound contains a hydroxy or carboxylic acid group, the compound may be metabolized, for example, by conjugation with glucuronic acid to form a glucuronide. Other pathways of conjugation metabolism exist. These pathways are often known as phase 2 metabolism and include, for example, sulfation or acetylation. Other functional groups, such as NH groups, may also undergo conjugation.

[0168] lipid nanoparticles Disclosed herein are novel ionizable lipids that provide advantages when used in lipid nanoparticles for delivering active or therapeutic agents, such as nucleic acids, to mammalian cells. In particular, embodiments of the present invention provide nucleic acid-lipid nanoparticle compositions comprising one or more of the novel ionizable lipids described herein, which provide increased nucleic acid activity and improved tolerability of the compositions in vivo, resulting in an increased therapeutic index, when compared to previously described nucleic acid-lipid nanoparticle compositions.

[0169] In certain embodiments, the present invention provides novel ionizable lipids that enable the formulation of improved compositions for in vitro and in vivo delivery of mRNA and / or other oligonucleotides. In some embodiments, these improved lipid nanoparticle compositions are useful for expressing proteins encoded by mRNA. In other embodiments, these improved lipid nanoparticle compositions are useful for upregulating endogenous protein expression by delivering miRNA inhibitors that target a specific miRNA or group of miRNAs that regulate a target mRNA or several mRNAs. In other embodiments, these improved lipid nanoparticle compositions are useful for downregulating (e.g., silencing) the protein and / or mRNA levels of target genes. In some other embodiments, lipid nanoparticles are also useful for delivering mRNA and plasmids for transgene expression. In still other embodiments, lipid nanoparticle compositions are useful for eliciting pharmacological effects resulting from protein expression, such as increased red blood cell production through delivery of a suitable erythropoietin mRNA, or protection from infection through delivery of a suitable antigen- or antibody-encoding mRNA. The present disclosure also provides for RNA molecules that are messenger RNA (mRNA), which can be either nucleoside-modified RNA (modRNA) or self-replicating RNA (saRNA). In some embodiments, the RNA is mRNA. In some embodiments, the RNA is modRNA. In other embodiments, the RNA is saRNA.

[0170] The lipid nanoparticles and compositions of the present invention can be used for a variety of purposes, including delivering encapsulated or associated (e.g., complexed) therapeutic agents, such as nucleic acids, to cells, both in vitro and in vivo. Accordingly, embodiments of the present invention provide methods of treating or preventing a disease or disorder in a subject in need thereof by contacting the subject with lipid nanoparticles that encapsulate or are associated with a suitable therapeutic agent, wherein the lipid nanoparticles comprise one or more of the novel ionizable lipids described herein.

[0171] As described herein, lipid nanoparticles are particularly useful for delivering nucleic acids, including, for example, mRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomir / antimir), messenger-RNA-interfering complementary RNA (micRNA), DNA, polyvalent RNA, Dicer substrate RNA, complementary DNA (cDNA), circular DNA (ceDNA), small interfering RNA (siRNA), etc. Thus, the lipid nanoparticles and compositions of the present invention can be used to induce expression of a desired protein both in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more of the novel ionizable lipids described herein, wherein the lipid nanoparticles encapsulate or are associated with a nucleic acid to be expressed to produce the desired protein (e.g., messenger RNA or a plasmid encoding the desired protein) or to inhibit a process that silences mRNA expression (e.g., an miRNA inhibitor). Alternatively, lipid nanoparticles and compositions can be used to reduce target gene and protein expression both in vitro and in vivo by contacting cells with lipid nanoparticles containing one or more of the novel ionizable lipids described herein, where the lipid nanoparticles encapsulate or are associated with a nucleic acid (e.g., an antisense oligonucleotide or small interfering RNA (siRNA)) that reduces target gene expression. The lipid nanoparticles and compositions of the present invention can also be used to co-deliver various nucleic acids (e.g., mRNA and plasmid DNA), either separately or in combination, such as to provide effects that require the co-localization of various nucleic acids (e.g., mRNA encoding a suitable gene-modifying enzyme and a DNA segment for integration into the host genome).

[0172] Nucleic acids for use in the present invention can be prepared according to any available technique. For mRNA, the primary method of preparation is, but is not limited to, enzymatic synthesis (also known as in vitro transcription), which currently represents the most efficient method for generating long, sequence-specific mRNA. In vitro transcription describes the process of template-directed synthesis of RNA molecules from an engineered DNA template composed of an upstream bacteriophage promoter sequence (e.g., including but not limited to, those from T7, T3, and SP6 coliphages) linked to a downstream sequence encoding a gene of interest. Template DNA can be prepared for in vitro transcription from several sources using suitable techniques well known in the art, including, but not limited to, plasmid DNA and polymerase chain reaction amplification (see Linpinsel, JL and Conn, GL, General protocols for preparation of plasmid DNA template, and Bowman, JC, Azizi, B., Lenz, TK, Ray, P. and Williams, LD, RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods, v.941, Conn GL (ed.), New York, NY Humana Press, 2012).

[0173] RNA transcription occurs in vitro using a linearized DNA template in the presence of the corresponding RNA polymerase and adenosine, guanosine, uridine, and cytidine ribonucleoside triphosphates (rNTPs) under conditions that support polymerase activity while minimizing the potential degradation of the resulting mRNA transcripts.In vitro transcription can be carried out using a variety of commercially available kits, including but not limited to, RiboMax Large Scale RNA Production System (Promega), MegaScript Transcription Kit (Life Technologies), and commercially available reagents containing RNA polymerase and rNTPs.Methods for in vitro transcription of mRNA are well known in the art. (See, e.g., Losick, R., 1972, In vitro transcription, Ann Rev Biochem, v. 41, 409-46; Kamaka, RT and Kraus, WL, 2001, In Vitro Transcription. Current Protocols in Cell Biology. 2:11.6:11.6.1-11.6.17; Beckert, B. and Masquida, B., (2010) Synthesis of RNA by In Vitro Transcription in RNA in Methods in Molecular Biology, v. 703 (Neilson, H., ed.), New York, NY Humana Press, 2010; Brunelle, JL and Green, R., 2013, Chapter Five—In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology, v. 530, 101-114, all of which are incorporated herein by reference.)

[0174] The desired, in vitro transcribed mRNA is then purified from undesired components of the transcription or related reaction, including unincorporated rNTPs, protein enzymes, salts, short RNA oligos, etc. Techniques for isolating mRNA transcripts are well known in the art. Well-known procedures include phenol / chloroform extraction or precipitation with alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride. Additional, non-limiting examples of purification procedures that can be used include size exclusion chromatography (Lukavsky, PJ and Puglisi, JD, 2004, Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides, RNA, v.10, 889-893), silica-based affinity chromatography, and polyacrylamide gel electrophoresis (Bowman, JC, Azizi, B., Lenz, TK, Ray, P. and Williams, LD, RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA synthesis Methods, v.941, Conn GL (ed.), New York, NY Humana Press, 2012). Purification can be performed using various commercially available kits, including, but not limited to, the SV Total Isolation System (Promega) and the In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).

[0175] Furthermore, while reverse transcription can produce large amounts of mRNA, the product may contain several aberrant RNA impurities associated with undesired polymerase activity that may need to be removed from full-length mRNA preparations. These include short RNAs resulting from incomplete transcription initiation and RNA-dependent RNA polymerase activity, RNA-primed transcription from RNA templates, and double-stranded RNA (dsRNA) generated by self-complementary 3' extension. It has been demonstrated that these contaminants with dsRNA structures can result in undesirable immunostimulatory activity through interactions with various innate immune sensors in eukaryotic cells that recognize specific nucleic acid structures and function to elicit potent immune responses. This, in turn, can dramatically reduce mRNA translation due to reduced protein synthesis during the cell's innate immune response. Therefore, additional techniques for removing these dsRNA contaminants have been developed and are known in the art, including, but not limited to, scalable HPLC purification (see, for example, Kariko, K., Muramatsu, H., Ludwig, J., and Weissman, D., 2011, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA, Nucl Acid Res, v.39, el42; Weissman, D., Pardi, N., Muramatsu, H., and Kariko, K., HPLC Purification of in vitro transcribed long RNA in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology, v.969 (ed. Rabinovich, PH), 2013). HPLC-purified mRNA has been reported to be translated at significantly higher levels, especially in primary cells and in vivo.

[0176] A wide variety of modifications have been described in the art that can be used to alter specific properties of in vitro transcribed mRNA and improve its utility. These include, but are not limited to, modifications to the 5' and 3' ends of mRNA. Endogenous eukaryotic mRNAs typically contain a cap structure at the 5' end of the mature molecule, which plays an important role in mediating the binding of mRNA cap-binding protein (CBP), thereby enhancing intracellular mRNA stability and the efficiency of mRNA translation. Therefore, the highest level of protein expression is achieved with capped mRNA transcripts. The 5'-cap contains a 5'-5'-triphosphate bond between the 5'-most nucleotide and a guanine nucleotide. The conjugated guanine nucleotide is methylated at the N7 position. Additional modifications include methylation of the 5'-most and penultimate 5'-most nucleotides on the 2'-hydroxyl group.

[0177] Multiple distinct cap structures can be used to generate the 5'-cap of in vitro transcribed synthetic mRNA. 5'-capping of synthetic mRNA can be performed by co-transcription with a chemical cap analog (e.g., capping during in vitro transcription). For example, the anti-reverse cap analog (ARCA) cap contains a 5'-5'-triphosphate guanine-guanine linkage in which one guanine contains an N7 methyl group as well as a 3'-O-methyl group. However, up to 20% of transcripts remain uncapped during this co-transcription process, rendering the synthetic cap analog non-identical to the 5'-cap structure of authentic cellular mRNA, potentially reducing translatability and cellular stability. Alternatively, synthetic mRNA molecules can be enzymatically capped post-transcriptionally. These can generate more authentic 5'-cap structures that structurally or functionally mimic endogenous 5'-caps with enhanced cap-binding protein binding, increased half-life, reduced susceptibility to 5' endonucleases, and / or reduced 5' decapping. A number of synthetic 5'-cap analogs have been developed to enhance mRNA stability and translatability and are known in the art (see, e.g., Grudzien-Nogalska, E., Kowalska, J., Su, W., Kuhn, AN, Slepenkov, SV, Darynkiewicz, E., Sahin, U., Jemielity, J., and Rhoads, RE, Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology, v. 969 (ed. Rabinovich, PH), 2013).

[0178] At the 3' end, a long chain of adenine nucleotides (a poly-A tail) is usually added to mRNA molecules during RNA processing. Immediately after transcription, the 3' end of the transcript is cleaved, freeing a 3' hydroxyl, in response to which poly-A polymerase adds a chain of adenine nucleotides to the RNA in a process called polyadenylation. The poly(A) tail has been widely shown to enhance both the translation efficiency and stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly(A), poly(A) binding protein and the regulation of mRNA stability, Trends Bio Sci, v. 14, 373-377; Guhaniyogi, J. and Brewer, G., 2001, Regulation of mRNA stability in mammalian cells, Gene, v. 265, 11-23; Dreyfus, M. and Regnier, P., 2002, The poly(A) tail of mRNAs: Bodyguard in eukaryotes, scavenger in bacteria, Cell, v. 111, 611-613).

[0179] Poly(A) tailing of in vitro transcribed mRNA can be achieved using various techniques, including, but not limited to, cloning a poly(T) tract into a DNA template or post-transcriptional addition using poly(A) polymerase. The first case allows for in vitro transcription of mRNAs with poly(A) tails of defined lengths, depending on the size of the poly(T) tract, but requires additional template manipulation. The latter case involves enzymatically adding a poly(A) tail to in vitro transcribed mRNA using poly(A) polymerase, which catalyzes the incorporation of adenine residues into the 3' end of the RNA, and does not require additional DNA template manipulation, but results in mRNAs with poly(A) tails of heterogeneous lengths. 5'-capping and 3'-poly(A) tailing can be performed using various commercially available kits, including, but not limited to, Poly(A) Polymerase Tailing Kit (EpiCenter), mMESSAGE mMACHINE T7 Ultra Kit, and Poly(A) Tailing Kit (Life Technologies), as well as using commercially available reagents, various ARCA caps, poly(A) polymerases, etc.

[0180] In addition to 5' cap and 3' polyadenylation, other modifications of in vitro transcripts have been reported to provide benefits such as translation efficiency and stability.It is well known in the art that pathogen DNA and RNA can be recognized by various sensors in eukaryotic cells and trigger a strong innate immune response.Since most nucleic acids from natural sources contain modified nucleosides, it has been shown that the ability to distinguish between pathogen DNA and RNA and self-DNA and RNA is at least partially based on structure and nucleoside modification.In contrast, in vitro synthesized RNA lacks these modifications and therefore becomes immunostimulatory, which can in turn inhibit effective 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, thus alleviating this undesired immunostimulatory activity and enhancing translational capacity (e.g., Kariko, K. and Weissman, D., 2007, Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: implications for therapeutic RNA development, Curr Opin Drug Discov Devel, v.10, 523-532; Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology, v.969 (ed. Rabinovich, PH), 2013; Kariko, K., Muramatsu, H., Welsh, FA, Ludwig, J., Kato, H., Akira, S., Weissman, D., 2008, Incorporation of Pseudouridine Into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability, Mol Ther, v.16, 1833-1840). Modified nucleosides and nucleotides used in the synthesis of modified RNA can be prepared, monitored, and utilized using general methods and procedures known in the art. A wide variety of nucleoside modifications are available that can be incorporated into in vitro transcribed mRNA, either alone or in combination with other modified nucleosides (see, for example, US2012 / 0251618).In vitro synthesis of nucleoside-modified mRNA has been reported to decrease its ability to activate immune sensors while simultaneously enhancing its translational capacity.

[0181] Other components of mRNA (modRNA) that can be modified to provide benefits in terms of translatability and stability include the 5' and 3' untranslated regions (UTRs). Optimizing both or independently the UTRs (preferred 5' and 3' UTRs can be obtained from cellular or viral RNA) has been shown to increase mRNA stability and translation efficiency of in vitro transcribed mRNA (see, e.g., Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology, v.969 (ed. Rabinovich, PH), 2013).

[0182] "Modified RNA" or "modRNA" refers to an RNA molecule having at least one addition, deletion, substitution, and / or modification of one or more nucleotides to form a non-naturally occurring nucleotide structure (e.g., other than A, C, T, G, or U). Such modifications may refer to the addition of non-nucleotide material to an endogenous RNA nucleotide or to the 5' and / or 3' end of the RNA. In one embodiment, such a modRNA contains at least one modified nucleotide, e.g., a modification to the base of a nucleotide. For example, modified nucleotides may replace one or more uridine and / or cytidine nucleotides. For example, these substitutions may occur at any instance of uridine and / or cytidine in the RNA sequence, or may occur only for selected uridine and / or cytidine nucleotides. Such modifications to standard nucleotides in the RNA may include non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For example, at least one uridine nucleotide can be replaced with N1-methylpseudouridine in the RNA sequence. Other such modified nucleotides are known to those skilled in the art. Such modified RNA molecules are considered analogs of naturally occurring RNA. In some embodiments, RNA is produced by in vitro transcription using a DNA template, where DNA refers to a nucleic acid containing deoxyribonucleotides.

[0183] In some embodiments, the RNA molecule may be saRNA. "Self-replicating RNA," "self-replicating RNA," and "replicon" refer to RNA capable of self-replication. Self-replicating RNA molecules can be generated, for example, by using replication elements derived from alphaviruses and replacing structural viral polypeptides with nucleotide sequences encoding a polypeptide of interest. Self-replicating RNA molecules are typically positive-strand molecules that can be directly translated after delivery to cells; this translation results in an RNA-dependent RNA polymerase, which then produces both antisense and sense transcripts from the delivered RNA. The delivered RNA results in the production of multiple daughter RNA molecules. These daughter RNA molecules, as well as collinear subgenomic transcripts, can themselves be translated to result in the in situ expression of the encoded protein of interest, e.g., a viral antigen, or can be transcribed to provide additional transcripts in the same orientation as the delivered RNA, which can be translated to result in the in situ expression of the antigen. The overall result of this series of transcriptions is an amplification of the number of introduced saRNA molecules, so that the encoded gene of interest, e.g., a viral antigen, becomes the major polypeptide product of the cell.

[0184] In some embodiments, the self-replicating RNA contains at least one or more genes, including any one or combination of viral replicase, viral protease, viral helicase, and other nonstructural viral proteins. In some embodiments, the self-replicating RNA may contain 5' and 3' tractive replication sequences and, optionally, a heterologous sequence encoding a desired amino acid sequence (e.g., an antigen of interest). A subgenomic promoter directing expression of the heterologous sequence may be included in the self-replicating RNA. Optionally, the heterologous sequence (e.g., an antigen of interest) may be fused in-frame to other coding regions within the self-replicating RNA and / or may be under the control of an internal ribosome entry site (IRES).

[0185] In addition to mRNA, other nucleic acid payloads can be used in the present invention. For oligonucleotides, preparation methods include, but are not limited to, chemical synthesis of long precursors, enzymatic or chemical cleavage, in vitro transcription, etc., as described above. Methods for synthesizing DNA and RNA nucleotides are widely used and well known in the art (see, for example, Gait, MJ (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, DC: IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v.288 (Clifton, NJ) Totowa, NJ: Humana Press, 2005, both of which are incorporated herein by reference).

[0186] For plasmid DNA, preparation for use in the present invention typically involves, but is not limited to, in vitro propagation and isolation of the plasmid DNA in liquid cultures of bacteria containing the plasmid of interest. The presence of a gene on the plasmid of interest that encodes resistance to a particular antibiotic (penicillin, kanamycin, etc.) allows bacteria containing the plasmid of interest to be selectively grown in cultures containing the antibiotic. Methods for isolating plasmid DNA are widely used and well known in the art (see, e.g., Heilig, J., Elbing, KL, and Brent, R (2001) Large-Scale Preparation of Plasmid DNA. Current Protocols in Molecular Biology., 41:11:1.7:1.7.1-1.7.16; Rozkov, A., Larsson, B., Gillstrom, S., Bjornestedt, R., and Schmidt, SR (2008) Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture. Biotechnol. Bioeng., 99:557-566; and US6197553B1). Plasmid isolation can be performed using a variety of commercially available kits, including, but not limited to, Plasmid Plus (Qiagen), GenJET plasmid MaxiPrep (Thermo), and Pure Yield MaxiPrep (Promega) kits, as well as using commercially available reagents.

[0187] As used herein, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form, including DNA, RNA, and hybrids thereof. DNA may be in the form of an antisense molecule, plasmid DNA, cDNA, PCR product, or vector. RNA may be in the form of small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, polyvalent RNA, Dicer substrate RNA, or viral RNA (vRNA), and combinations thereof. Nucleic acids include synthetic, natural, and non-natural nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that have similar binding properties to the reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conventionally modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences, as well as the explicitly indicated sequence. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together via phosphate groups."Base" includes purines and pyrimidines, which further include the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0188] The term "gene" refers to a nucleic acid (eg, DNA or RNA) sequence that comprises partial or full-length coding sequences necessary for the production of a polypeptide or precursor polypeptide.

[0189] "Gene product," as used herein, refers to the product of a gene, such as an RNA transcript or a polypeptide.

[0190] The term "lipid" refers to a group of organic compounds, including, but not limited to, esters of fatty acids, generally characterized by poor solubility in water but solubility in many organic solvents. Lipids are typically divided into at least three classes: (1) "simple lipids," which include fats and oils and waxes; (2) "complex lipids," which include phospholipids and glycolipids; and (3) "derived lipids," such as steroids.

[0191] "Steroids" are compounds with the following carbon skeleton:

[0192] [ka] Non-limiting examples of steroids include cholesterol, and the like.

[0193] "Ionizable lipid" refers to a lipid capable of carrying a positive charge. Exemplary ionizable lipids contain one or more amine groups that carry a positive charge. Preferred ionizable lipids are ionizable so that they can exist in a positively charged or neutral form, depending on the pH. The ionization of ionizable lipids affects the surface charge of lipid nanoparticles under different pH conditions. This charge state can affect plasma protein absorption, blood clearance, and tissue distribution (Semple, SC et al., Adv. Drug Deliv Rev, 32:3-17 (1998)), as well as the ability to form endosomolytic non-bilayer structures important for intracellular delivery of nucleic acids (Hafez, IM et al., Gene Ther 8:1188-1196 (2001)). As used herein, "ionizable lipid" can also include, but is not limited to, "cationic lipids."

[0194] The term "polymer-conjugated lipid" refers to a molecule that contains both a lipid portion and a polymer portion. An example of a polymer-conjugated lipid is a PEGylated lipid. The term "PEGylated lipid" refers to a molecule that contains both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG) and the like.

[0195] The term "neutral lipid" refers to any of several lipid species that exist in either an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, but are not limited to, phosphotidylcholines 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), phophatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM), ceramide, steroids such as sterols, and derivatives thereof. Neutral lipids may be synthetic or naturally derived.

[0196] The term "charged lipid" refers to any of several lipid species that exist in positively or negatively charged form at any pH within a useful physiological range, e.g., from about pH 3 to about pH 9. Charged lipids can be synthetic or naturally occurring. Examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyltrimethylammonium-propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, and dimethylaminoethanecarbamoylsterol (e.g., DC-Chol).

[0197] The term "lipid nanoparticle" refers to a particle having a dimension on the order of at least one nanometer (e.g., 1-1,000 nm) comprising one or more compounds of structure (I) or other specified ionizable lipids. In some embodiments, the lipid nanoparticles are included in a formulation that can be used to deliver an active drug or therapeutic agent, such as a nucleic acid (e.g., mRNA), to a desired target site (e.g., a cell, tissue, organ, tumor, or the like). In some embodiments, the lipid nanoparticles of the present invention comprise a nucleic acid. Such lipid nanoparticles typically comprise a compound of structure (I) and one or more excipients selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. In some embodiments, the active drug or therapeutic agent, e.g., a nucleic acid, is encapsulated in the lipid portion of the lipid nanoparticle or in the aqueous space enclosed by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects elicited by the host organism's or cell's machinery, such as a harmful immune response.

[0198] In various embodiments, the lipid nanoparticles are about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, 35 nm, The lipid nanoparticles have an average diameter of 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, or any size or range therebetween. In various embodiments, the lipid nanoparticles are substantially non-toxic. In certain embodiments, nucleic acids, when present in the lipid nanoparticles, are resistant to degradation by nucleases in aqueous solution. Lipid nanoparticles containing nucleic acids and methods for their preparation are disclosed, for example, in U.S. Patent Application Publication Nos. 2004 / 0142025, 2007 / 0042031 and PCT Publication Nos. WO2013 / 016058 and WO2013 / 086373, the entire disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0199] As used herein, "polydispersity index," or "PDI," is a ratio that describes the uniformity of the particle size distribution of a system. A value of less than 0.3 indicates a relatively narrow particle size distribution.

[0200] As used herein, "lipid encapsulating" refers to a lipid nanoparticle that provides an active or therapeutic agent, such as a nucleic acid (e.g., mRNA), by complete encapsulation, partial encapsulation, or both. In one embodiment, the nucleic acid (e.g., mRNA) is completely encapsulated within the lipid nanoparticle.

[0201] As used herein, "encapsulation efficiency" refers to the percentage of therapeutic agent that becomes part of the nanoparticle composition relative to the total initial amount of therapeutic agent used in the preparation. Encapsulation efficiency (EE%) is calculated by dividing (total therapeutic agent added - free unentrapped therapeutic agent) by total therapeutic agent added.

[0202] As used herein, "size" or "average size" in the context of lipid nanoparticles refers to the average diameter of the nanoparticle composition.

[0203] "Serum stable" in relation to nucleic acid-lipid nanoparticles means that the nucleotides are not significantly degraded after exposure to serum or nuclease assays that significantly degrade free DNA or RNA. Suitable assays include, for example, standard serum assays, DNAse assays, or RNAse assays.

[0204] "Stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.

[0205] In one embodiment, the composition disclosed herein comprises lipid.For example, the composition may comprise lipid and mRNA (for example, modRNA or saRNA), and lipid and mRNA (for example, modRNA or saRNA) can form nanoparticles together, thereby producing mRNA-containing nanoparticles that comprise lipid.Lipid can encapsulate or associate with mRNA in the form of lipid nanoparticle (LNP), and can help the stability, cell entry and intracellular release of RNA / lipid nanoparticles.

[0206] In some examples, the LNP comprises a micelle, a solid lipid nanoparticle, a nanoemulsion, a liposome, or the like, or a combination thereof.

[0207] The lipid components of the LNP can include, for example, ionizable lipids, neutral lipids, such as phospholipids (unsaturated lipids, such as DOPE or DSPC), polymer-lipid conjugates (e.g., PEGylated lipids), structured lipids, or any combination thereof. The elements of the lipid components can be provided in designated fractions. Further disclosed herein are ionizable lipids, polymer-lipid conjugates, structured lipids, and neutral lipids suitable for the methods of the present disclosure.

[0208] In certain embodiments, the lipid component of the lipid nanoparticles comprises from about 0 mol% to about 60 mol% ionizable lipids (e.g., at least about, at most about, between any two thereof, or exactly 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, , 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol% ionizable lipids; about 0 mol% to about 60 mol% phospholipids (e.g., at least about, at most about, between any two thereof, or exactly 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol% ionizable lipids); , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol% phospholipids; about 0 mol% to about 60 mol% structural lipids (e.g., at least about, at most about, or between any two thereof). , or exactly 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 mol% structural lipids);about 0 mol% to about 60 mol% polymer-lipid conjugates (e.g., at least about, at most about, between any two thereof, or exactly 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol% polymer-lipid conjugate; about 0 mol% to about 60 mol% ionizable lipid (e.g., at least about, at most about, between any two thereof, or exactly 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol% polymer-lipid conjugate); 3, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol% ionizable lipids); and / or about 0 mol% to about 60 mol% neutral lipids (e.g., at least about, at most about, between any two thereof, or , 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol% neutral lipids). LNPs can have any amount of the above lipid components, provided that the total mol% does not exceed 100%. As used herein, "mol percent" or "mol%" refers to the molar percentage of a component relative to the total moles of all lipid components in an LNP (e.g., the total moles of ionizable lipids, neutral lipids, steroids, and polymer-conjugated lipids);

[0209] In some embodiments, the lipid component of the lipid nanoparticles comprises about 35 mol% to about 55 mol% of ionizable lipid compounds, about 5 mol% to about 25 mol% of phospholipids, about 30 mol% to about 50 mol% of structural lipids, and about 0 mol% to about 10 mol% of polymer-lipid conjugates. In certain embodiments, the lipid component comprises about 50 mol% of ionizable lipids, about 10 mol% of phospholipids, about 40 mol% of structural lipids, and about 1.5 mol% of polymer-lipid conjugates. In another specific embodiment, the lipid component comprises about 40 mol% of ionizable lipids, about 20 mol% of phospholipids, about 40 mol% of structural lipids, and about 1.5 mol% of polymer-lipid conjugates. In certain embodiments, the lipid component of the lipid nanoparticles comprises the ionizable lipids, phospholipids, structural lipids, and polymer-lipid conjugates in a molar ratio of about 47.5:10:40.7:1.8.

[0210] In some embodiments, the lipid component of the lipid nanoparticles comprises about 0 mol% to about 10 mol% of ionizable lipid compounds, about 40 mol% to about 60 mol% of phospholipids, and about 40 mol% to about 60 mol% of structural lipids. In certain embodiments, the lipid component comprises about 2 mol% of ionizable lipids, about 49 mol% of phospholipids, and about 49 mol% of structural lipids. In certain embodiments, the lipid component of the lipid nanoparticles comprises the ionizable lipids, phospholipids, and structural lipids in a molar ratio of about 1.8:49.1:49.1.

[0211] In some embodiments, phospholipid can be DOPE or DSPC.In other embodiments, polymer-lipid conjugate can be PEG-DMG, and / or structured lipid can be cholesterol.In other embodiments, polymer-lipid conjugate can be PEG-2000DMG, and / or structured lipid can be cholesterol.

[0212] In some aspects, the lipid nanoparticles are i) ionizable lipids between 40 and 50 mol percent; ii) phospholipids and / or neutral lipids; iii) structured lipids; iv) polymer-conjugated lipids; and v) Therapeutic agents (i.e., RNA) encapsulated within or associated with lipid nanoparticles Includes:

[0213] In some aspects, the lipid nanoparticles are i) between 0 and 10 mol% ionizable lipids; ii) phospholipids and / or neutral lipids; and iii) Steroids Includes:

[0214] In some embodiments, the lipid nanoparticles have 41-50 mol percent, 42-50 mol percent, 43-50 mol percent, 44-50 mol percent, 45-50 mol percent, 46-50 mol percent, or 47-50 mol percent ionizable lipid, or any mol percent or range therebetween. In certain specific embodiments, the lipid nanoparticles have a molecular weight of at least about, at most about, between any two of, or exactly 41.0, 41.1, 41.2, 41.3, 41.4, 41.5, 41.6, 41.7, 41.8, 41.9, 42.0, 42.1, 42.2, 42.3, 42.4, 42.5, 42.6, 42.7, 42.8, 42.9, 43.0, 43.1, 43.2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44.0, 44.1, 44.2, 44.3, 44.4, 44.5, 44.6, 44.7, 44.8, 44.9, 45.0, 45.1, 45.2, 45.3, 45.4, 45.5, 45.6, 45.7, 45.8, 45.9, 46.0, 46.1, 46.2, 46.3, 46.4, 46.5, 46.6, 46.7, 46.8, 46.9, 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, 48.1, 48.2, 48.4, 48.5, 48.6, 48.7, 48.8, 48.9, 49.0, 49.1, 5.2, 45.3, 45.4, 45.5, 45.6, 45.7, 45.8, 45.9, 46.0, 46.1, 46.2, 46.3, 46.4, 46.5, 46.6, 46.7, 46.8, 46.9, 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8 , 47.9, 48.0, 48.1, 48.2, 48.3, 48.4, 48.5, 48.6, 48.7, 48.8, 48.9, 49.0, 49.1, 49.2, 49.3, 49.4, 49.5, 49.6, 49.7, 49.8, 49.9, or 50 mol percent ionizable lipids.

[0215] In other embodiments, the lipid nanoparticles comprise 0-10 mol percent ionizable lipids. In certain specific embodiments, the lipid nanoparticles comprise at least about, at most about, between any two thereof, or exactly 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mol percent ionizable lipids.

[0216] In some embodiments, the phospholipids and / or neutral lipids are present at a concentration of 5-15 mol percent, 7-13 mol percent, or 9-11 mol percent, or any mol percent or range therebetween. In certain embodiments, the phospholipids and / or neutral lipids are at least about, at most about, between any two thereof, or exactly 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10. Present in concentrations of 1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, or 15 mol percent. In certain specific embodiments, the phospholipids and / or neutral lipids are present at a concentration of about 9.5, 10, or 10.5 mol percent.

[0217] In other embodiments, the phospholipids and / or neutral lipids are present at a concentration of 40-60 mol%, or any molar percent or range therebetween. In certain embodiments, the phospholipids and / or neutral lipids are present at a concentration of at least about, at most about, between any two thereof, or exactly 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol percent. In certain specific embodiments, the phospholipids and / or neutral lipids are present at a concentration of about 48, 49, or 50 mol percent.

[0218] In some embodiments, the molar ratio of ionizable lipid to phospholipid and / or neutral lipid is about 4.1:1.0 to about 4.9:1.0, about 4.5:1.0 to about 4.8:1.0, or about 4.7:1.0 to 4.8:1.0, or any molar ratio or range therebetween. In other embodiments, the molar ratio of phospholipid and / or neutral lipid to ionizable lipid is 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, or 1:4.9.

[0219] In some embodiments, the structured lipid is a steroid. In some embodiments, the steroid is cholesterol. In some embodiments, the structured lipid is present at a concentration of 39-49 mol percent, 40-46 mol percent, 40-44 mol percent, 40-42 mol percent, 42-44 mol percent, or 44-46 mol percent, or any molar percentage or range therebetween. In certain specific embodiments, the structured lipid is at least about, at most about, between any two thereof, or exactly 39, 39.1, 39.2, 39.3, 39.4, 39.5, 39.6, 39.7, 39.8, 39.9, 40, 40.1, 40.2, 40.3, 40.4, 40.5, 40.6, 40.7, 40.8, 40.9, 41, 41.1, 41.2, 41.3, 41.4, 41.5, 41.6, 41.7, 41.8, 41.9, 42, 42.1, 42.2, 42.3, 42.4, 42.5, 42.6, 42.7, 42.8, 42.9, 43, 43.1, 43.2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44, 44.1, 44.2, 44.3, 44.4, 44.5, 44.6, 44.7, 44.8, 44.9, 45, 45.1, 45.2, 45.3, 45.4, 45.5, 45.6, 45.7, 45.8, 45.9, 46, 46.1, 46.2, 46.3, 46.4, 46.5 , 46.6, 46.7, 46.8, 46.9, 47, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, 48, 48.1, 48.2, 48.3, 48.4, 48.5, 48.6, 48.7, 48.8, 48.9, or 49 mol percent. In certain specific embodiments, the structured lipid is present at a concentration of 40, 41, 42, 43, 44, 45, or 46 mol percent.

[0220] In other embodiments, the structured lipids are present at a concentration ranging from 40 to 60 mol%. In certain embodiments, the structured lipids are present at a concentration of at least about, at most about, between any two thereof, or exactly 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol percent. In certain specific embodiments, the structured lipids are present at a concentration of about 48, 49, or 50 mol percent.

[0221] In certain embodiments, the molar ratio of ionizable lipid to structural lipid ranges from 1.0:0.9 to 1.0:1.2, or from 1.0:1.0 to 1.0:1.2, such as 1:0.9, 1:1, 1:1.1, or 1:1.2.

[0222] In a preferred embodiment, the ionizable lipid has the following structure (I):

[0223] [ka] or a pharmaceutically acceptable salt, N-oxide, tautomer, or stereoisomer thereof. [In the formula, m, n, o, and p each independently represent 1 to 3; G 1 is C 1~12 Alkylene or C 2~12 is alkenylene, R 1 is -N(R 2 )R 3 , -OR 4 , CN, -N(R 4 ) (heteroaryl), -O(CH2) q OH, -(OCH2CH2) r OH, -OC(=O)R 5 , -N(R 4 )C(=O)R 5 , -N(R 4 )S(O)2R 5 , -N(R 4 )C(=O)N(R 2 )R3 , -OC(=O)N(R 2 )R 3 , -N(R 4 )C(=O)OR 5 , -N(R 4 )C(=S)N(R 2 )R 3 , -N(R 4 )C(=NR 6 )N(R 2 )R 3 ,or

[0224] [ka] and R 2 and R 3 are independently H, C 1~6 Alkyl, C 3~8 cycloalkyl, or aryl, or R 2 and R 3 form a heterocyclic ring together with the nitrogen atom to which they are attached, R 4 is H, C 1~6 Alkyl or C 3~8 is cycloalkyl, R 5 is C 1~6 Alkyl or C 1~6 C optionally substituted with alkyl 3~8 is cycloalkyl, R 6 are H, CN, NO2, C 1~6 Alkyl, OR 5 , S(O)2R 5 , or S(O)N(R 2 )R 3 and q is 2 to 6; r is 1 to 6; W is

[0225] [ka] and X is N or CH; G 2 and G 3 are each independently, C 1~12 Alkylene or C 2~12 is alkenylene, L 1 and L 2 are each independently -C(=O)OR 7 , -OC(=O)R 7 , -OC(=O)(CH2) r C(=O)OR 7 , -OC(=O)(CH2) r OC(=O)R 7 , -OC(=O)N(R 4 )R 7 , -N(R 4 )C(=O)OR 7 , -N(R 4 )C(=O)N(R 4 )R 7 , -OC(=O)OR 7 , or -S-SR 7 and R 7 is C 6~24 Alkyl, C 6~24 Alkenyl, or C 6~24 alkynyl, F, C 1~6 Alkoxy, C 3~8 Cycloalkyl, or C 3~8 optionally substituted by cycloalkenyl; L 1 and L 2 R 7 may be the same or different].

[0226] The lipid component of the lipid nanoparticle composition may include one or more molecules containing a polymer such as polyethylene glycol, e.g., PEG, or a PEG-modified lipid. Such species may alternatively be referred to as PEGylated lipids. A PEG lipid is a lipid modified with polyethylene glycol. The PEG lipid may be selected from the non-limiting group including PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the PEG lipid may be a PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid. As used herein, the term "PEG lipid" refers to a polyethylene glycol (PEG)-modified lipid. Non-limiting examples of PEG-lipid include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugate (for example, PEG-CerCl4 or PEG-CerC20), PEG-modified dialkylamine and PEG-modified 1,2-diacyloxypropan-3-amine.Such lipids are also referred to as PEGylated lipids.In some embodiments, the PEG-lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.In some embodiments, the PEG-modified lipid is a modified form of PEG-DMG.

[0227] In some embodiments, the PEG-modified lipid has formula (IV):

[0228] [ka] is a PEG lipid having [In the formula, R8 and R9 are each independently a linear or branched saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, the alkyl chain optionally being interrupted by one or more ester bonds; and w has an average value in the range of 30 to 60.

[0229] In some embodiments, the polymer-conjugated lipid has formula (IV):

[0230] [ka] POZ is a polyoxazoline (POZ) lipid comprising: POZ is known in the art and is described in WO / 2020 / 264505, filed June 29, 2020, and PCT / US2020 / 040140.

[0231] In some embodiments, the PEGylated lipid has the following structure (II):

[0232] [ka] or a pharmaceutically acceptable salt, N-oxide, tautomer or stereoisomer thereof, wherein R 10 and R 11 are each independently a linear or branched saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, the alkyl chain optionally being interrupted by one or more ester bonds, and z has an average value ranging from 30 to 60, with the proviso that when z is 42, R 10 and R 11 provided that R together are not n-octadecyl. 10 and R 11 are each independently a linear saturated alkyl chain containing 12 to 16 carbon atoms. In some embodiments of the PEGylated lipid, z is about 45.

[0233] In some embodiments, the PEGylated lipid has the following structure:

[0234] [ka] wherein n has an average value in the range of 40 to 50. In a preferred embodiment, the composition comprises an ionizable lipid as described herein and a compound having the following structure:

[0235] [ka] The PEGylated lipid comprises one of:

[0236] In some embodiments of the PEGylated lipids described above, R 10 and R 11 are each independently a linear or branched saturated or unsaturated alkyl chain containing 12 carbon atoms. In some embodiments of the PEGylated lipids described above, R 10 and R 11 are each independently a linear or branched saturated or unsaturated alkyl chain containing 14 carbon atoms. In some embodiments of the PEGylated lipids described above, R 10 and R 11 Each of the alkyl groups independently represents a linear or branched saturated or unsaturated alkyl chain containing 16 carbon atoms. Further exemplary lipids and related formulations are disclosed in, for example, U.S. Patent No. 9,737,619, filed February 14, 2017, U.S. Patent No. 10,166,298, filed October 28, 2016, and International Patent Application PCT / US2017 / 058619, filed October 26, 2017, the disclosures of which are incorporated herein by reference in their entirety.

[0237] In preferred embodiments, the composition further comprises nucleic acid. In preferred embodiments, the nucleic acid comprises messenger RNA. In some embodiments, the composition further comprises one or more excipients selected from neutral lipids and steroids. In some embodiments, the composition comprises one or more neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. Preferably, in some embodiments, the neutral lipid is DSPC. Preferably, in some embodiments, the steroid is cholesterol.

[0238] LNPs may contain one or more components described herein. In some embodiments, the LNP formulations of the present disclosure contain at least one lipid nanoparticle component. The lipid nanoparticles may contain a lipid component and one or more additional components, such as therapeutic and / or prophylactic agents, e.g., nucleic acids. LNPs may be designed for one or more specific applications or targets. The components of the LNP may be selected based on a particular application or target and / or based on the efficacy, toxicity, cost, ease of use, availability, or other characteristics of one or more components. Similarly, a particular formulation of LNP may be selected for a particular application or target, for example, according to the efficacy and toxicity of a particular combination of components. The efficacy and tolerability of an LNP formulation may be affected by the stability of the formulation.

[0239] Lipid nanoparticles can also be designed for one or more specific uses or targets. For example, LNPs can be designed to deliver therapeutic and / or prophylactic agents, such as RNA, to specific cells, tissues, organs, or systems or groups within a mammalian body. The physiochemical properties of lipid nanoparticles can also be modified to increase selectivity for specific bodily targets. For example, particle size can be adjusted based on the fenestration sizes of various organs. The therapeutic and / or prophylactic agents included in LNPs can also be selected based on one or more desired delivery targets. For example, therapeutic and / or prophylactic agents can be selected for a particular indication, condition, disease, or disorder and / or for delivery (e.g., localized or specific delivery) to specific cells, tissues, organs, or systems or groups thereof. In certain embodiments, LNPs can contain mRNA encoding a polypeptide of interest that can be translated within cells to produce the polypeptide of interest. Such compositions can be designed to be specifically delivered to a specific organ. In some embodiments, the composition can be designed to be specifically delivered to the mammalian liver. In some embodiments, the composition can be designed to be specifically delivered to lymph nodes. In some aspects, the composition can be designed for specific delivery to the spleen of a mammal.

[0240] In some embodiments, a polymer may be included and / or used to encapsulate or partially encapsulate the LNP. The polymer may be biodegradable and / or biocompatible. The polymer may be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and / or polyarylates. For example, polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly(L-lactic-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), and poly(D,L-lactide-co-PPO-co-D,L-lactide).L-lactide), polyalkyl cyanoacrylates, polyurethanes, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohol (PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone (PVP), polysiloxanes, polystyrene, polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocellulose, hydroxypropyl cellulose, carboxymethyl cellulose cellulose, polymers of acrylic acid, such as poly(methyl (meth)acrylate) (PMMA), poly(ethyl (meth)acrylate), poly(butyl (meth)acrylate), poly(isobutyl (meth)acrylate), poly(hexyl (meth)acrylate), poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), and and copolymers and blends thereof, polydioxolane and its copolymers, polyhydroxyalkanoates, polypropylene fumarates, polyoxymethylenes, poloxamers, poloxamines, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, poly(N-acryloylmorpholine) (PAcM), poly(2-methyl-2-oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), and polyglycerol.

[0241] In some embodiments, a surface-modifying agent may be included and / or used to encapsulate or partially encapsulate the LNP. Surface-modifying agents may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., ionizable surfactants such as dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrins), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., acetylcysteine, Artemisia grandiflora, bromelain, papain, Cleodendrum, bromhexine, carbocysteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4, dornase alfa, neltenexin, and erdosteine), and / or DNases (e.g., rhDNase). The surface-modifying agent can be disposed within the nanoparticle and / or on the surface of the LNP (eg, by coating, adsorption, covalent bonding, or other process).

[0242] LNP may contain one or more functionalized lipids.For example, lipids can be functionalized with alkyne groups that can undergo cycloaddition when exposed to azide under suitable reaction conditions.In particular, lipid bilayers can be functionalized in this manner with one or more groups that are useful for membrane permeation, cell recognition, or facilitating imaging.The surface of LNP can also be conjugated with one or more useful antibodies.Functional groups and conjugates that are useful for targeted cell delivery, imaging, and membrane permeation are well known in the art.

[0243] In addition to these components, lipid nanoparticles may contain any substance useful in pharmaceutical compositions.For example, lipid nanoparticles may contain one or more pharmaceutically acceptable excipients or subcomponents, such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, surfactants, buffers, preservatives, and other species.

[0244] Surfactants and / or emulsifiers include, but are not limited to, natural emulsifiers (e.g., gum arabic, alginic acid, sodium alginate, cholesterol, and lecithin), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [TWEEN® 20], polyoxyethylene sorbitan [TWEEN® 60], polyoxyethylene sorbitan monooleate [TWEEN® 80], sorbitan monopalmitate [SPAN® 40], sorbitan monostearate [SPAN® 60], sorbitan tristearate [SPAN® 65], glyceryl monooleate, sorbitan monooleate [SPAN® 80]), polyoxyethylene esters (e.g., polyoxyethylene monostearate [MYRJ® 45], sorbitan monooleate [MYRJ® 45]), and the like. ], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether [BRIJ® 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLURONIC® F68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or combinations thereof.

[0245] Examples of preservatives may include, but are not limited to, antioxidants, chelating agents, free radical scavengers, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Examples of antioxidants include, but are not limited to, alpha-tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfite. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate. Examples of antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Examples of antifungal preservatives include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Examples of alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoates, and / or phenylethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and / or phytic acid.Other preservatives include, but are not limited to, tocopherol, tocopheryl acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN® II, NEOLONE™, KATHON™, and / or EUXYL®. Exemplary free radical scavengers include butylated hydroxytoluene (BHT or butylhydroxytoluene) and / or deferoxamine. In some preferred embodiments, the composition is preservative-free.

[0246] Examples of buffering agents include, but are not limited to, citrate buffer solution, acetate buffer solution, phosphate buffer solution, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate mixture, tromethamine, aminosulfonic acid buffer (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and / or combinations thereof. In some embodiments, the concentration of the buffer in the composition is about 10 mM. In some embodiments, the buffer concentration can be equal to, at least equal to, at most equal to, or at any two of the following: 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM, or any range or value derivable therein. In specific embodiments, the buffer concentration is 10 mM. The buffer can be neutral pH, pH 6.5-8.5, pH 7.0-8.0, or pH 7.2-7.6. In some embodiments, the buffering agent can be at pH 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, or 8.5, or any range or value derivable therein. In a specific embodiment, the buffering agent is at pH 7.4.

[0247] In some embodiments, formulations comprising LNPs may further comprise a salt, e.g., a chloride salt. In some embodiments, formulations comprising LNPs may further comprise a sugar, such as a disaccharide. In some embodiments, the formulation further comprises a sugar rather than a salt, e.g., a chloride salt. In some embodiments, the LNPs may further comprise one or more hydrophobic small molecules, such as vitamins (e.g., vitamin A or vitamin E) or sterols. Carbohydrates may include simple sugars (e.g., glucose) and / or polysaccharides (e.g., glycogen and its derivatives and analogs).

[0248] The characteristics of LNPs may depend on their constituents. For example, LNPs containing cholesterol as a structural lipid may have different characteristics from LNPs containing different structural lipids. As used herein, the term "structured lipid" refers to sterols and also to lipids containing sterol moieties. As defined herein, "sterol" is a subgroup of steroids consisting of steroid alcohols. In some embodiments, the structured lipid is a steroid. In some embodiments, the structured lipid is cholesterol. In some embodiments, the structured lipid is a cholesterol analog. In some embodiments, the structured lipid is alpha-tocopherol.

[0249] In some embodiments, the characteristics of LNP can depend on the absolute or relative amount of its constituents.For example, the LNP that contains a higher molar fraction of phospholipids can have different characteristics from the LNP that contains a lower molar fraction of phospholipids.The characteristics can also vary depending on the method and conditions of preparing lipid nanoparticles.Generally, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.

[0250] The phospholipid moiety can be selected from the non-limiting group consisting of, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and / or sphingomyelin. The fatty acid moiety can be selected from the non-limiting group consisting of, for example, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and / or docosahexaenoic acid. Certain phospholipids can promote membrane fusion. In some embodiments, ionizable phospholipids can interact with one or more negatively charged phospholipids in membranes (e.g., cellular or intracellular membranes). The fusion of phospholipids to the membrane allows one or more components (e.g., therapeutic agents) of the lipid-containing composition (e.g., LNP) to pass through the membrane, enabling, for example, delivery of one or more components to a target tissue. Non-natural phospholipid species, including natural species with modifications and substitutions, including branching, oxidation, cyclization, and alkynes, are also contemplated. In some embodiments, the phospholipid may be functionalized with and / or crosslinked to one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced with triple bonds). Under appropriate reaction conditions, the alkyne group can undergo copper-catalyzed cycloaddition upon exposure to azide. Such reactions can be useful in functionalizing the lipid bilayer of nanoparticle compositions to facilitate membrane penetration or cellular recognition, or in conjugating nanoparticle compositions to useful components such as targeting or imaging moieties (e.g., dyes). Phospholipids include, but are not limited to, glycerophospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and / or phosphatidic acid.Phospholipids also include phosphosphingolipids, such as sphingomyelin.In some embodiments, the phospholipids that are useful or potentially useful in the present invention are analogs and / or variants of DSPC.

[0251] Formulations containing amphiphilic polymers and / or lipid nanoparticles can be formulated, in whole or in part, as pharmaceutical compositions. Pharmaceutical compositions may contain one or more amphiphilic polymers and one or more lipid nanoparticles. For example, pharmaceutical compositions may contain one or more amphiphilic polymers and one or more lipid nanoparticles, and may contain one or more different therapeutic and / or prophylactic agents. Pharmaceutical compositions may further contain one or more pharmaceutically acceptable excipients and / or accessory ingredients, such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and medicaments are available, for example, in Remington's The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro; Lippincott, Williams & Wilkins, Balti, MD, 2006. Conventional excipients and / or accessory ingredients can be used in any pharmaceutical composition, except that any conventional excipient or accessory ingredient may be incompatible with one or more components of the LNP or one or more amphiphilic polymers in the formulations of the present disclosure. An excipient or accessory ingredient may be incompatible with the LNP component or amphiphilic polymer of the formulation if its combination with the component or amphiphilic polymer could result in any undesired biological or otherwise adverse effect.

[0252] In some embodiments, the composition may include a pharmaceutically acceptable carrier and / or vehicle. In some embodiments, the composition may further include pyrogen-free water, isotonic saline and / or a buffer solution, such as a phosphate or citrate buffer solution. In some embodiments, the composition may include water and / or a buffer containing sodium salts, e.g., at least 50 mM sodium salts, calcium salts, in some embodiments at least 0.01 mM calcium salts, and optionally potassium salts, in some embodiments at least 3 mM potassium salts. In some embodiments, the sodium, calcium, and / or, optionally, potassium salts may be present in the form of their halides, e.g., chlorides, iodides, or bromides, or in the form of their hydroxides, carbonates, bicarbonates, and / or sulfates, etc. Examples of sodium salts include, for example, NaCl, NaI, NaBr, Na2CO3, NaHCO3, and Na2SO4; examples of potassium salts include, for example, KCl, KI, KBr, K2CO3, KHCO3, and K2SO4; and examples of calcium salts include, for example, CaCl2, CaI2, CaBr2, CaCO3, CaSO4, and Ca(OH)2. In some embodiments, organic anions of the above-mentioned cations may be contained in the buffer solution. In some embodiments, the composition may include a salt selected from sodium chloride (NaCl), calcium chloride (CaCl2), and / or potassium chloride (KCl), and additional anions may be present in addition to chloride. CaCl2 may also be replaced with another salt, for example, KCl. In some embodiments, the injection buffer can be hypertonic, isotonic, or hypotonic relative to a specific reference medium. For example, the buffer can have a higher, the same, or lower salt content than a specific reference medium. Such concentrations of salts as described above can be used to minimize cell damage due to osmotic or other concentration effects. The concentration of salt in the composition can be from about 70 mM to about 140 mM.For example, the salt concentration can be equal to, at least equal to, at most equal to, or between any two of 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM, or any range or value derivable therein. The salt can be at neutral pH, pH 6.5-8.5, pH 7.0-8.0, or pH 7.2-7.6. For example, the salt can be at a pH equal to, at least any one of, at most any one of, or between any two of 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, or 8.5, or any range or value derivable therein.

[0253] In some embodiments, one or more excipients or accessory ingredients may comprise more than 50% of the total mass or volume of a pharmaceutical composition comprising an LNP. For example, one or more excipients or accessory ingredients may comprise 50%, 60%, 70%, 80%, 90%, or more by pharmaceutical convention. In some embodiments, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. Examples of excipients, which refer to ingredients in a composition that are not the active ingredient, include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, disintegrants, coatings, plasticizers, compression agents, wet granulation agents, and / or colorants. Preservatives for use in the compositions disclosed herein include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens and / or thimerosal.As used herein, "pharmaceutically acceptable carrier" includes any and all aqueous solvents (e.g., water, alcohol / aqueous solutions, saline, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), tonicity agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, fluid and nutrient supplements, and similar materials known to those skilled in the art, as well as combinations thereof. Diluents, or thinning or thinning agents, include, but are not limited to, ethanol, glycerol, water, sugars such as lactose, sucrose, mannitol, and sorbitol, as well as starches derived from wheat, corn, rice, and potato, and / or celluloses such as microcrystalline cellulose. The amount of diluent in the composition can range from about 10% to about 90% by weight, from about 25% to about 75% by weight, from about 30% to about 60%, or from about 12% to about 60% by weight of the total composition.

[0254] In some embodiments, the excipients are approved for human and veterinary use. In some embodiments, the excipients are approved by the U.S. Food and Drug Administration. In some embodiments, the excipients are pharmaceutical grade. In some embodiments, the excipients meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia. The relative amounts of one or more amphiphilic polymers, one or more lipid nanoparticles, one or more pharmaceutically acceptable excipients, and / or any additional components in a pharmaceutical composition according to the present disclosure will vary depending on the identity, size, and / or condition of the subject being treated, as well as the route by which the composition is to be administered. By way of example, the pharmaceutical composition may contain 0.1% to 100% (wt / wt) of one or more lipid nanoparticles. As another example, the pharmaceutical composition may contain 0.1% to 15% (wt / vol) of one or more amphiphilic polymers (e.g., 0.5%, 1%, 2.5%, 5%, 10%, or 12.5% ​​w / v).

[0255] The pH and precise concentration of various components in pharmaceutical compositions are adjusted according to well-known parameters.The use of such media and agents for pharmaceutical active substances is well known in the art.Except where any conventional media or agent is incompatible with active ingredient, its use in immunogenic therapeutic compositions is contemplated.

[0256] In certain embodiments, the lipid nanoparticles and / or pharmaceutical compositions of the present disclosure are refrigerated or frozen for storage or shipping (e.g., at a temperature of 10°C or lower, e.g., at a temperature of about 4°C, between about -150°C and about 10°C (e.g., about 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, 4°C, 3°C, 2°C, 1°C, 0°C, -1°C, -2°C, -3°C, -4°C, -5°C, -6°C). , -7°C, -8°C, -9°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C, or -150°C), or at a temperature between about -80°C and about -20°C (e.g., about -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, or -80°C). For example, a pharmaceutical composition comprising one or more amphiphilic polymers and one or more lipid nanoparticles may be in solution or solid form (e.g., via lyophilization) that is refrigerated for storage and / or shipping, e.g., at about -20°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, or 90°C.

[0257] In certain embodiments, the present disclosure provides a method for preparing lipid nanoparticles and / or pharmaceutical compositions thereof by adding an effective amount of an amphiphilic polymer, and maintaining the lipid nanoparticles and / or pharmaceutical compositions thereof at a temperature of 10°C or less, e.g., at a temperature of about 4°C, between about -150°C and about 10°C (e.g., about 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, 4°C, 3°C, 2°C, 1°C, 0°C, -1°C, -2°C, -3°C, -4°C, -5°C, -6°C, -7°C, -8°C, -9°C). The present invention also relates to methods of increasing the stability of lipid nanoparticles by storing them at temperatures between about -80°C and about -20°C (e.g., about -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C, or -150°C), or at temperatures between about -80°C and about -20°C (e.g., about -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, or -80°C).

[0258] The chemical properties of the LNPs, LNP suspensions, lyophilized LNP compositions, or LNP formulations of the present disclosure can be characterized by various methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of the LNPs. Dynamic light scattering or potentiometry (e.g., potentiometric titration) can be used to measure the zeta potential. Dynamic light scattering can also be used to determine particle size. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure multiple characteristics of the LNPs, such as particle size, polydispersity index, and / or zeta potential.

[0259] The average size of the LNPs can be between tens of nanometers and hundreds of nanometers, as measured, for example, by dynamic light scattering (DLS). For example, the average size can be about 40 nm to about 150 nm, e.g., about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average size of the LNPs may be about 50 nm to about 100 nm, about 50 nm to about 90 nm, about 50 nm to about 80 nm, about 50 nm to about 70 nm, about 50 nm to about 60 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 60 nm to about 80 nm, about 60 nm to about 70 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, about 70 nm to about 80 nm, about 80 nm to about 100 nm, about 80 nm to about 90 nm, or about 90 nm to about 100 nm. In certain embodiments, the average size of the LNPs may be about 70 nm to about 100 nm. In certain embodiments, the average size may be about 80 nm. In other embodiments, the average size may be about 100 nm.

[0260] LNPs can be relatively uniform. The polydispersity index can be used to indicate the uniformity of LNPs, e.g., the particle size distribution of lipid nanoparticles. A small polydispersity index (e.g., less than 0.3) generally indicates a narrow particle size distribution. LNPs can have a polydispersity index of about 0 to about 0.25, e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of LNPs can be about 0.10 to about 0.20.

[0261] The zeta potential of LNP can be used to indicate the kinetic potential of composition.For example, zeta potential can describe the surface charge of LNP.The lipid nanoparticles with relatively low positive or negative charge are generally desired.Because the species with higher charge can interact undesirably with cells, tissues and other elements in the body. In some embodiments, the zeta potential of the LNP can be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV.

[0262] The efficiency of encapsulation of a therapeutic agent and / or prophylactic agent describes the amount of therapeutic agent and / or prophylactic agent encapsulated or otherwise associated with the LNP after preparation relative to the initial amount provided. A high encapsulation efficiency (e.g., approaching 100%) is desirable. The encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic agent and / or prophylactic agent in a solution containing lipid nanoparticles before and after disruption of the lipid nanoparticles with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic agent and / or prophylactic agent (e.g., RNA) in the solution. For the lipid nanoparticles described herein, the encapsulation efficiency of a therapeutic agent and / or prophylactic agent can be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%. In some embodiments, the LNP encapsulation efficiency of an LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the present disclosure produced in the presence of empty LNPs (e.g., lipid nanoparticles comprising a lipid listed herein but not encapsulating any nucleic acid) is about 50% or more, about 55% or more, about 60% or more, or more than the LNP encapsulation efficiency of an LNP, LNP suspension, lyophilized LNP composition, or LNP formulation produced by a comparable method in the presence of a lower concentration of empty LNPs or in the absence of empty LNPs. or higher, about 65% or higher, about 70% or higher, about 75% or higher, about 80% or higher, about 8% or higher, about 90% or higher, about 91% or higher, about 92% or higher, about 93% or higher, about 94% or higher, about 95% or higher, about 96% or higher, about 97% or higher, about 98% or higher, or about 99% or higher.

[0263] In some embodiments, electrophoresis (eg, capillary electrophoresis) and / or chromatography (eg, reverse-phase liquid chromatography) can be used to examine mRNA integrity.

[0264] In some embodiments, the LNP integrity of an LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the present disclosure produced in the presence of empty LNPs (e.g., lipid nanoparticles that include a lipid listed herein but do not encapsulate any nucleic acid) is about 20% or more, about 25% or more, about 30% or more, about 35% or more, or about 40% or more higher than the LNP integrity of an LNP, LNP suspension, lyophilized LNP composition, or LNP formulation produced by a comparable method in the presence of a lower concentration of empty LNPs or in the absence of empty LNPs. or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more.

[0265] In some embodiments, the LNP integrity of LNPs, LNP suspensions, lyophilized LNP compositions, and / or LNP formulations of the present disclosure produced in the presence of empty LNPs (e.g., lipid nanoparticles that include a lipid enumerated herein but do not encapsulate any nucleic acid) is about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more than the LNP integrity of LNPs, LNP suspensions, lyophilized LNP compositions, or LNP formulations produced by a comparable method in the presence of a lower concentration of empty LNPs or in the absence of empty LNPs. , about 80% or more, about 90% or more, about 1 fold or more, about 2 fold or more, about 3 fold or more, about 4 fold or more, about 5 fold or more, about 10 fold or more, about 20 fold or more, about 30 fold or more, about 40 fold or more, about 50 fold or more, about 100 fold or more, about 200 fold or more, about 300 fold or more, about 400 fold or more, about 500 fold or more, about 1000 fold or more, about 2000 fold or more, about 3000 fold or more, about 4000 fold or more, about 5000 fold or more, or about 10000 fold or more higher.

[0266] In some embodiments, the Txo% of LNPs, LNP suspensions, lyophilized LNP compositions, and / or LNP formulations of the present disclosure produced in the presence of empty LNPs (e.g., lipid nanoparticles comprising a lipid enumerated herein but not encapsulating any nucleic acid) is about 12 months or more, about 15 months or more, about 18 months or more, about 21 months or more, about 24 months or more, about 27 months or more, about 30 months or more, about 33 months or more, about 36 months or more, about 48 months or more, about 60 months or more, about 72 months or more, about 84 months or more, about 96 months or more, about 108 months or more, or about 120 months or more than the Txo% of LNPs, LNP suspensions, lyophilized LNP compositions, and / or LNP formulations produced by a comparable method in the presence of a lower concentration of empty LNPs or in the absence of empty LNPs. In some embodiments, the Txo% of LNPs, LNP suspensions, lyophilized LNP compositions, and / or LNP formulations of the present disclosure produced in the presence of empty LNPs (e.g., lipid nanoparticles comprising a lipid enumerated herein but not encapsulating any nucleic acid) is about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 1-fold or more, about 2-fold or more, about 3-fold or more, about 4-fold or more, or about 5-fold or more longer than the Txo% of LNPs, LNP suspensions, lyophilized LNP compositions, and / or LNP formulations produced by a comparable method in the presence of a lower concentration of empty LNPs or in the absence of empty LNPs.

[0267] In some embodiments, the T of an LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the present disclosure produced in the presence of empty LNPs (e.g., lipid nanoparticles comprising a lipid enumerated herein but not encapsulating any nucleic acid) is about 12 months or more, about 15 months or more, about 18 months or more, about 21 months or more, about 24 months or more, about 27 months or more, about 30 months or more, about 33 months or more, about 36 months or more, about 48 months or more, about 60 months or more, about 72 months or more, about 84 months or more, about 96 months or more, about 108 months or more, or about 120 months or more longer than the T of an LNP, LNP suspension, lyophilized LNP composition, or LNP formulation produced by a comparable method in the presence of a lower concentration of empty LNPs or in the absence of empty LNPs.

[0268] In some embodiments, the T of an LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the present disclosure produced in the presence of empty LNPs (e.g., lipid nanoparticles comprising a lipid enumerated herein but not encapsulating any nucleic acid) is about 5% or more higher, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 1-fold or more, about 2-fold or more, about 3-fold or more, about 4-fold or more, or about 5-fold or more longer than the T of an LNP, LNP suspension, lyophilized LNP composition, or LNP formulation produced by a comparable method in the presence of a lower concentration of empty LNPs or in the absence of empty LNPs.

[0269] As used herein, "Tx" refers to the amount of time it takes for the nucleic acid integrity (e.g., mRNA integrity) of an LNP, LNP suspension, lyophilized LNP composition, or LNP formulation to deteriorate to about X of the initial integrity of the nucleic acid (e.g., mRNA) used to prepare the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation. For example, "T80" refers to the amount of time it takes for the nucleic acid integrity (e.g., mRNA integrity) of an LNP, LNP suspension, lyophilized LNP composition, or LNP formulation to deteriorate to about 80% of the initial integrity of the nucleic acid (e.g., mRNA) used to prepare the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation. In another example, "T1 / 2" refers to the amount of time it takes for the nucleic acid integrity (e.g., mRNA integrity) of an LNP, LNP suspension, lyophilized LNP composition, or LNP formulation to decrease to about half the initial integrity of the nucleic acid (e.g., mRNA) used to prepare the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation.

[0270] The amount of therapeutic and / or prophylactic agent in an LNP can depend on the size, composition, desired target and / or use, or other characteristics of the lipid nanoparticle and the properties of the therapeutic and / or prophylactic agent. For example, the amount of RNA useful in an LNP can depend on the size, sequence, and other characteristics of the RNA. The relative amounts of therapeutic and / or prophylactic agent (e.g., pharmaceutical agent) and other components (e.g., lipids) in an LNP can also vary. In some embodiments, the wt / wt ratio of lipid component to therapeutic and / or prophylactic agent in an LNP can be about 5:1 to about 60:1, e.g., 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and 60:1. For example, the wt / wt ratio of lipid component to therapeutic and / or prophylactic agent can be from about 10:1 to about 40:1. In certain embodiments, the wt / wt ratio is about 20:1. The amount of therapeutic and / or prophylactic agent in the LNP can be measured, for example, using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

[0271] In some embodiments, the ratio of mRNA to lipid (e.g., N:P, where N represents the moles of ionizable lipid and P represents the moles of phosphate present as part of the nucleic acid backbone) in the LNP ranges from 2:1 to 30:1, e.g., 3:1 to 22:1. In other embodiments, N:P ranges from 6:1 to 20:1 or 2:1 to 12:1. Exemplary N:P ranges include about 3:1, about 6:1, about 12:1, and about 22:1.

[0272] Various exemplary embodiments of the ionizable lipids of the present invention, lipid nanoparticles and compositions comprising same, 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.

[0273] RNA encapsulation The RNA in the RNA product solution can be encapsulated, and the RNA solution can further comprise at least one encapsulating agent.In one embodiment, the encapsulating agent comprises lipid, lipid nanoparticle (LNP), lipoplex, polymer particle, polyplex, monolithic delivery system, or a combination thereof.In some embodiments, one, two, three, four, five, or more of the above-mentioned elements can be excluded as the encapsulating agent.

[0274] In one embodiment, the encapsulating agent is a lipid, resulting in lipid nanoparticle (LNP)-encapsulated RNA. Without intending to be bound by any theory, it is believed that cationic or cationic ionizable lipids or lipid-like substances and / or cationic polymers combine with nucleic acids to form aggregates that result in colloidally stable particles.

[0275] Lipids can be naturally occurring or synthetic lipids.However, lipids are usually biological substances.Biological lipids are well known in the art, and include, for example, neutral lipids, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulfatides, lipids including ether- and ester-linked fatty acids and polymerizable lipids, and combinations thereof.Lipids are substances that are insoluble in water and can be extracted with organic solvents.Compounds other than those specifically described herein are also understood by those skilled in the art as lipids, and are included in the compositions and methods of the present disclosure.Lipid components and non-lipids can be bound to each other by either covalent or non-covalent bonds.

[0276] In some embodiments, LNPs can be designed to protect RNA molecules (e.g., saRNA, mRNA) from extracellular Rnase and / or can be engineered to deliver RNA to target cells systemically. In some embodiments, when RNA molecules are administered intravenously to a subject in need thereof, such LNPs can be particularly useful for delivering RNA molecules (e.g., saRNA, mRNA). In some embodiments, when RNA molecules are administered intramuscularly to a subject in need thereof, such LNPs can be particularly useful for delivering RNA molecules (e.g., saRNA, mRNA). In some embodiments, when RNA molecules are administered intradermally to a subject in need thereof, such LNPs can be particularly useful for delivering RNA molecules (e.g., saRNA, mRNA). In some embodiments, when RNA molecules are administered intranasally to a subject in need thereof, such LNPs can be particularly useful for delivering RNA molecules (e.g., saRNA, mRNA).

[0277] In one embodiment, the RNA in the RNA product solution is at a concentration of <1 mg / mL. In another embodiment, the RNA is at a concentration of at least 0.05 mg or at least about 0.05 mg / mL. In another embodiment, the RNA is at a concentration of at least 0.5 mg / mL or at least about 0.5 mg / mL. In another embodiment, the RNA is at a concentration of at least 1 mg or at least about 1 mg / mL. In another embodiment, the RNA concentration is 0.05 mg / mL or about 0.05 mg / mL to about 0.5 mg / mL. In another embodiment, the RNA is at a concentration of at least 10 mg / mL. In another embodiment, the RNA is at a concentration of at least 50 mg / mL. In some embodiments, the RNA is at a concentration of at least, at most, exactly between any two thereof (inclusively or exclusively), or about 0.05 mg / mL, 0.5 mg / mL, 1 mg / mL, 10 mg / mL, 50 mg / mL, 75 mg / mL, 100 mg / mL, 150 mg / mL, 200 mg / mL, 250 mg / mL, 300 mg / mL, 400 mg / mL, or more.

[0278] The present disclosure provides RNA product solutions and lipid preparation mixtures or compositions thereof, comprising at least one RNA encoding, for example, an antigen, complexed with, encapsulated in, and / or formulated with one or more lipids to form lipid nanoparticles (LNPs), liposomes, lipoplexes, and / or nanoliposomes. In some embodiments, the compositions comprise lipid nanoparticles.

[0279] Lipid nanoparticles or LNPs refer to any form of particle that is produced when cationic lipids and optionally one or more additional lipids are combined, for example, in an aqueous environment and / or in the presence of RNA. In some embodiments, lipid nanoparticles are included in a formulation that can be used to deliver active drugs or therapeutic agents, such as nucleic acids (e.g., mRNA), to target sites of interest (e.g., cells, tissues, organs, tumors, and the like). In some embodiments, the lipid nanoparticles of the present disclosure contain nucleic acids (e.g., mRNA). Such lipid nanoparticles typically contain cationic lipids and one or more excipients, such as one or more neutral lipids, charged lipids, steroids, polymer-conjugated lipids, or combinations thereof. In some embodiments, LNPs contain at least one cationic (e.g., ionizable) lipid, at least one neutral (e.g., non-cationic) lipid, at least one structural lipid (e.g., steroid), and / or at least one polymer-conjugated lipid (e.g., polyethylene glycol (PEG)-modified lipid). In some embodiments, one, two, three, or more of the above-mentioned excipients may be excluded from the LNP.

[0280] In some embodiments, LNPs comprise 20-60 mol% cationic (e.g., ionizable) lipids. For example, LNPs may comprise 20-50 mol%, 20-40 mol%, 20-30 mol%, 30-60 mol%, 30-50 mol%, 30-40 mol%, 40-60 mol%, 40-50 mol%, or 50-60 mol% cationic (e.g., ionizable) lipids. In some embodiments, LNPs comprise at least, at most, exactly, or between any two of these (inclusively or exclusively) 20 mol%, 30 mol%, 40 mol%, 50, or 60 mol% cationic (e.g., ionizable) lipids. In some embodiments, LNPs comprise 45-55 mole percent (mol%) cationic (e.g., ionizable) lipids. For example, the LNP may or may not include at least, at most, exactly, or between any two thereof (inclusively or exclusively) 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mol% cationic (e.g., ionizable) lipids.

[0281] In some embodiments, LNPs comprise 5-25 mol% neutral (e.g., non-cationic) lipids. For example, LNPs may comprise 5-20 mol%, 5-15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, or 20-25 mol% neutral (e.g., non-cationic) lipids. In some embodiments, LNPs are at least, at most, exactly, or between any two of these (inclusively or exclusively) 5 mol%, 10 mol%, 15 mol%, 20 mol%, or 25 mol% neutral (e.g., non-cationic) lipids. In some embodiments, LNPs comprise 5-15 mol% neutral (e.g., non-cationic) lipids. For example, the LNP may comprise at least, at most, exactly, or between any two thereof (inclusively or exclusively) 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mol % neutral (e.g., non-cationic) lipids.

[0282] In some embodiments, LNPs comprise 25-55 mol% structural lipids (e.g., steroids). For example, LNPs may comprise 25-50 mol%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25-30 mol%, 30-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 45-55 mol%, 45-50 mol%, or 50-55 mol% structural lipids (e.g., steroids). In some embodiments, the LNPs are at least, at most, exactly, or between any two of these (inclusive or exclusive) 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, or 55 mol% structural lipids (e.g., steroids). In some embodiments, the LNPs contain 35-40 mol% structural lipids (e.g., steroids). For example, the LNPs may contain at least, at most, exactly, or between any two of these (inclusive or exclusive) 35, 36, 37, 38, 39, or 40 mol% structural lipids (e.g., steroids).

[0283] In some embodiments, the LNP comprises 0.5-15 mol% of a polymer-conjugated lipid (e.g., a polyethylene glycol (PEG)-modified lipid). For example, the lipid nanoparticle may comprise 0.5-10 mol%, 0.5-5 mol%, 1-15 mol%, 1-10 mol%, 1-5 mol%, 2-15 mol%, 2-10 mol%, 2-5 mol%, 5-15 mol%, 5-10 mol%, or 10-15 mol% of a polymer-conjugated lipid (e.g., a polyethylene glycol (PEG)-modified lipid). In some embodiments, lipid LNPs may contain at least, at most, exactly, or between any two of these (inclusively or exclusively) 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol% polymer-conjugated lipids (e.g., polyethylene glycol (PEG)-modified lipids). In some embodiments, LNPs contain 1-2 mol% polymer-conjugated lipids (e.g., polyethylene glycol (PEG)-modified lipids). For example, LNPs may contain at least, at most, exactly, or between any two of these (inclusively or exclusively) 1, 1.5, or 2 mol% polymer-conjugated lipids (e.g., polyethylene glycol (PEG)-modified lipids).

[0284] In some embodiments, the LNPs comprise 20-75 mol% cationic (e.g., ionizable) lipids (e.g., at least, at most, exactly, or between any two of them (inclusively or exclusively) 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, and 75%), and 0.5-25 mol% neutral (e.g., non-cationic) lipids (e.g., at least, at most, exactly, or between any two of them (inclusively or exclusively) 0.5%, 2.25%, 4%, 5.75%, 7.5%, 9.25%, 11%, 12.75%, 14.5%, 16.25%, 18%, 19.75%, 21.5%, 23.25%, and 25%). , 5-55 mol% structural lipids (e.g., sterols), e.g., non-cationic lipids (e.g., at least, at most, exactly, or between any two thereof (inclusively or exclusively) 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, and 55%), and 0.5-20 mol% polymer-conjugated lipids (e.g., polyethylene glycol (PEG)-modified lipids) (e.g., at least, at most, exactly, or between any two thereof (inclusively or exclusively) 0.5%, 2%, 3.5%, 5%, 6.5%, 8%, 9.5%, 11%, 12.5%, 14%, 15.5%, 17%, 18.5%, and 20%). In some embodiments, one, two, three, or more types of lipids may be excluded from the LNP.

[0285] In some non-limiting embodiments, the lipid molar ratios are 50 / 10 / 38.5 / 1.5 (cationic lipid / neutral lipid / structural lipid / polymer-conjugated lipid mol%), 60 / 7.5 / 31 / 1.5 (cationic lipid / neutral lipid / structural lipid / polymer-conjugated lipid mol%), 57.5 / 7.5 / 31.5 / 3.5 (cationic lipid / neutral lipid / structural lipid / polymer-conjugated lipid mol%), 57.2 / 7.1 / 34.3 / 1.4 (cationic lipid / neutral lipid / structural lipid / polymer-conjugated lipid mol%), 40 / 15 / 40 / 5 (cationic lipid / neutral lipid / structural lipid / polymer-conjugated lipid mol%), ...50 / 10 / 38.5 / 1.5 (cationic lipid / neutral lipid / structural lipid / polymer-conjugated lipid mol%), 50 / 10 / 38.5 / 1. The lipid mixtures were: 50 / 10 / 35 / 4.5 / 0.5 (cationic lipid / neutral lipid / structural lipid / polymer-conjugated lipid mol%), 50 / 10 / 35 / 5 (cationic lipid / neutral lipid / structural lipid / polymer-conjugated lipid mol%), 40 / 10 / 40 / 10 (cationic lipid / neutral lipid / structural lipid / polymer-conjugated lipid mol%), 35 / 15 / 40 / 10 (cationic lipid / neutral lipid / structural lipid / polymer-conjugated lipid mol%), or 52 / 13 / 30 / 5 (cationic lipid / neutral lipid / structural lipid / polymer-conjugated lipid mol%).

[0286] In some embodiments, an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), can be encapsulated in the lipid portion of the lipid nanoparticle and / or the aqueous space enclosed 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 host organism or cellular machinery, for example, a harmful immune response. The nucleic acid (e.g., mRNA) or a portion thereof can be associated and complexed with the lipid nanoparticle. The lipid nanoparticle can comprise any lipid capable of binding nucleic acid and / or forming particles in which one or more nucleic acids are encapsulated.

[0287] In some embodiments, provided RNA molecules (e.g., saRNA, mRNA) can be formulated using LNPs. In some embodiments, lipid nanoparticles can be 1 nm or about 1-500 nm (e.g., at least, at most, exactly, or between (inclusively or exclusively) 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 90 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 nm). In some embodiments, the lipid nanoparticles are at least, at most, exactly or approximately 30 nm or about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or at least, at most, exactly or approximately or between (inclusively or exclusively) 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. The term "mean diameter" refers to the average hydrodynamic diameter of particles measured by dynamic laser light scattering (DLS) with data analysis using the so-called cumulant algorithm, which results in the so-called Z-average, which has a dimension of length, and the polydispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp. 4814-4820, ISO 13321). Herein, the terms "average diameter," "diameter," or "size" in reference to particles are used synonymously with the Z-average value.

[0288] The LNPs described herein may exhibit a polydispersity index of less than 0.5, or less than about 0.5, 0.4, 0.3, or 0.2 or less. By way of example, the LNPs may or may not exhibit a polydispersity index of at least, at most, exactly, or therebetween (inclusively or exclusively) 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5. The polydispersity index is calculated in some embodiments based on dynamic light scattering measurements by so-called cumulant analysis, as mentioned in the definition of "mean diameter." Under certain conditions, this can be understood as a measurement of the size distribution of a population of nanoparticles.

[0289] In some embodiments, the LNPs of the disclosure comprise or do not comprise an N:P ratio of 2:1 or about 2:1 to about 30:1, e.g., at least, at most, exactly, or therebetween (inclusively or exclusively) 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, or 30:1. In some embodiments, the LNPs of the disclosure comprise an N:P ratio of 6:1 or about 6:1. In some embodiments, the LNPs of the present disclosure comprise an N:P ratio of 3:1 or about 3:1.

[0290] In some embodiments, the LNPs of the disclosure have a ratio of 5:1 or about 5:1 to about 100:1, e.g., at least, at most, exactly, or between (inclusively or exclusively) 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1, 80:1, 81: 0:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1 , 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79 1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1, 90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, or 100:1. In some embodiments, the LNPs of the present disclosure comprise a wt / wt ratio of ionizable cationic lipid component to RNA of at or about 20:1. In some embodiments, the LNPs of the present disclosure comprise a wt / wt ratio of ionizable cationic lipid component to RNA of at or about 10:1.

[0291] In certain embodiments, nucleic acids (e.g., RNA molecules) are resistant to degradation by nucleases in aqueous solution when present in the provided LNPs. In some embodiments, the LNPs are liver-targeting lipid nanoparticles. In some embodiments, the LNPs are cationic lipid nanoparticles comprising one or more cationic lipids (e.g., those described herein). In some embodiments, the cationic LNPs may comprise at least one cationic lipid, at least one polymer-conjugated lipid, and at least one helper lipid (e.g., at least one neutral lipid).

[0292] In certain embodiments, the RNA solution and its lipid preparation mixture or composition have a concentration of at least, at most, exactly between (inclusively or exclusively) or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57% %, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of particular lipids, lipid species, or non-lipid components disclosed herein, such as lipid-like substances and / or cationic polymers and / or adjuvants, antigens, peptides, polypeptides, sugars, nucleic acids or other substances or as would be known to one of skill in the art.

[0293] The LNPs described herein can be produced using components, compositions, and methods generally known in the art, such as those described in PCT / US2016 / 052352; PCT / US2016 / 068300; PCT / US2017 / 037551; PCT / US2015 / 027400; PCT / US2016 / 047406; PCT / US201600, all of which are incorporated by reference in their entirety.

[0129] PCT / US2016 / 014280; PCT / US2016 / 014280; PCT / US2017 / 038426; PCT / US2014 / 027077; PCT / US2014 / 055394; PCT / US2016 / 52117; PCT / US2012 / 069610; PCT / US2017 / 027492; PCT / US2016 / 059575 and PCT / US2016 / 069491. Other non-limiting examples of methods for preparing LNPs can be found, for example, in WO2022 / 032154, the disclosure of which is incorporated herein by reference in its entirety.

[0294] For example, a method for preparing LNPs may include obtaining a colloid from at least one cationic or cationically ionizable lipid or lipid-like substance and / or at least one cationic polymer, and mixing the colloid with nucleic acid to obtain nucleic acid particles. As used herein, the term "colloid" refers to a type of homogeneous mixture in which the dispersed particles do not settle. The insoluble particles in the mixture are microscopic and have a particle size between 1 and 1000 nanometers. The mixture may also be referred to as a colloid or colloidal suspension. Sometimes, the term "colloid" refers only to the particles in the mixture, rather than the entire suspension.

[0295] For the preparation of colloids comprising at least one cationic or cationically ionizable lipid or lipid-like substance and / or at least one cationic polymer, suitably adapted methods conventionally used to prepare liposomal vesicles are applicable here. The most commonly used methods for preparing liposomal vesicles share the following basic steps: (i) dissolving lipids in an organic solvent, (ii) drying the resulting solution, and (iii) hydrating the dried lipids (using various aqueous media).

[0296] In the film hydration method, lipids are first dissolved in a suitable organic solvent and dried to obtain a thin film at the bottom of a flask. The resulting lipid film is hydrated using an appropriate aqueous medium to produce a liposome dispersion. An additional downsizing step may also be included.

[0297] Reverse phase evaporation is an alternative method to film hydration for preparing liposomal vesicles, involving the formation of a water-in-oil emulsion between an aqueous phase and a lipid-containing organic phase. Brief sonication of this mixture is necessary to homogenize the system. Removal of the organic phase under reduced pressure yields a milky gel that subsequently transforms into a liposomal suspension.

[0298] The term "ethanol injection technique" refers to the process of rapidly injecting an ethanol solution containing lipids into an aqueous solution through a needle. This action disperses the lipids throughout the solution and promotes the formation of lipid structures, e.g., lipid vesicles, such as liposomes. Generally, the RNA lipoplex particles described herein can be obtained by adding RNA to a colloidal liposome dispersion. Using the ethanol injection technique, such a colloidal liposome dispersion is formed in some embodiments as follows: an ethanol solution containing lipids, e.g., cationic lipids and additional lipids, is injected into an aqueous solution under stirring. In some embodiments, the RNA lipoplex particles described herein can be obtained without an extrusion step. The term "extrusion" or "extrusion" refers to the creation of particles with a fixed cross-sectional profile. In particular, this refers to particle downsizing, whereby the particles pass through a filter with defined pores.

[0299] Other methods for preparing colloids with organic solvent-free properties can also be used in accordance with the present disclosure.

[0300] In some embodiments, LNP-encapsulated RNA can be produced by rapidly mixing an RNA solution (e.g., an RNA product solution) and a lipid preparation described herein (e.g., comprising at least one cationic lipid and optionally one or more other lipid components in an organic solvent) under conditions that cause a sudden change in the solubility of the lipid components, thereby driving the lipids to self-assemble into the form of LNPs. In some embodiments, suitable buffering agents include Tris, histidine, citrate, acetate, phosphate, and / or succinate. In some embodiments, one, two, three, or more of the above-mentioned buffering agents are excluded. The pH of the liquid formulation is related to the pKa of the encapsulating agent (e.g., cationic lipid). The pH of the acidifying buffer can be at least half a pH scale lower than the pKa of the encapsulating agent (e.g., cationic lipid), and the pH of the final buffer can be at least half a pH scale higher than the pKa of the encapsulating agent (e.g., cationic lipid). In some embodiments, the properties of cationic lipid are selected so that the initial formation of particles occurs by association with the backbone of oppositely charged nucleic acid (for example, RNA).Thus, particles are formed around nucleic acid, which can, for example, in some embodiments, achieve significantly higher encapsulation efficiency than that achieved under the absence of the interaction between nucleic acid and at least one of lipid components.In certain embodiments, when nucleic acid is present in lipid nanoparticles, it is resistant to degradation by nuclease in aqueous solution.

[0301] Lipid nanoparticles containing 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. WO 2013 / 016058 and WO 2013 / 086373, the entire disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0302] Some embodiments described herein relate to compositions, methods, and uses comprising more than one nucleic acid species, e.g., two, three, four, five, six, or even more species, such as RNA species. In an LNP formulation, each nucleic acid species can be formulated separately as an individual LNP formulation. In this case, each individual LNP formulation will contain one nucleic acid species. The individual LNP formulations may exist as separate entities, e.g., in separate containers. Such formulations can be obtained by providing each nucleic acid species separately (typically in the form of a nucleic acid-containing solution) together with suitable cationic or cationically ionizable lipids or lipid-like substances and cationic polymers that enable the formation of LNPs. Each particle will exclusively contain the specific nucleic acid species provided when the particle is formed (individual microparticle formulation).

[0303] In some embodiments, a composition, e.g., a pharmaceutical composition, comprises more than one individual LNP formulation. Each pharmaceutical composition is referred to as a mixed LNP formulation. A mixed LNP formulation according to the present invention can be obtained by separately forming individual LNP formulations as described above, and then mixing the individual LNP formulations. The mixing step can produce a formulation comprising a mixed population of nucleic acid-containing LNPs. The individual LNP populations can be contained together in one container containing a mixed population of individual LNP formulations.

[0304] Alternatively, different nucleic acid species can be formulated together as a combined LNP preparation.This preparation can be obtained by providing a combination preparation (typically a combined solution) of various RNA species together with suitable cationic or cationic ionizable lipid or lipid-like substance and cationic polymer, which allows LNP formation.In contrast to mixed LNP preparations, combined LNP preparations will typically comprise LNPs that contain more than one RNA species.In combined LNP compositions, various RNA species are typically present together in a single particle.

[0305] A. Cationic polymeric substances Polymeric materials are commonly used for nanoparticle-based delivery, given their high chemical flexibility. Typically, cationic polymers are used to electrostatically condense negatively charged nucleic acids into nanoparticles. These positively charged groups often consist of amines that change protonation state in the pH range between 5.5 and 7.5, which is thought to result in an ionic imbalance that leads to endosomal rupture. Polymers such as poly-L-lysine, polyamidoamine, protamine, and polyethyleneimine, as well as naturally occurring polymers such as chitosan, have all been applied to nucleic acid delivery and are suitable as cationic materials useful in some embodiments herein. In addition, some researchers have synthesized polymeric materials specifically for nucleic acid delivery. Poly(P-amino esters), in particular, have been widely used in nucleic acid delivery due to their ease of synthesis and biodegradability. In some embodiments, such synthetic materials may be suitable for use as cationic materials herein.

[0306] As used herein, "polymeric material" is given its ordinary meaning, e.g., a molecular structure comprising one or more repeating units (monomers) linked by covalent bonds. In some embodiments, such repeating units may all be identical; alternatively, in some cases, more than one type of repeating unit may be present in the polymeric material. In some cases, the polymeric material is biologically derived, e.g., a biopolymer such as a protein. In some cases, additional moieties, e.g., targeting moieties such as those described herein, may also be present in the polymeric material.

[0307] Those skilled in the art will recognize that when more than one type of repeat unit is present in a polymer (or polymer portion), the polymer (or polymer portion) is said to be a "copolymer." In some embodiments, a polymer (or polymer portion) utilized in accordance with the present disclosure may be a copolymer. The repeat units forming the copolymer may be arranged in any manner. For example, in some embodiments, the repeat units may be arranged in a random order; alternatively, or in addition, in some embodiments, the repeat units may be arranged in an alternating order or as a "block" copolymer, e.g., a copolymer including one or more regions each including a first repeat unit (e.g., a first block) and one or more regions each including a second repeat unit (e.g., a second block). A block copolymer may have two (diblock copolymer), three (triblock copolymer), or more distinct blocks.

[0308] In certain embodiments, the polymeric material for use according to the present disclosure is biocompatible. A biocompatible material is typically a material that does not cause significant cell death at moderate concentrations. In certain embodiments, the biocompatible material is biodegradable, e.g., capable of chemically and / or biologically degrading in a physiological environment, such as within the body. In certain embodiments, the polymeric material may be or include protamine or polyalkyleneimine, particularly protamine.

[0309] As those skilled in the art will recognize, the term "protamine" is often used to refer to any of a variety of relatively low molecular weight, strongly basic proteins that are rich in arginine and are found in the sperm cells of various animals (such as fish) in place of somatic histones, particularly in association with DNA. In particular, the term "protamine" is often used to refer to a protein found in fish sperm that is strongly basic, soluble in water, does not coagulate with heat, and yields mainly arginine upon hydrolysis. In purified form, they are used in long-acting insulin preparations to neutralize the anticoagulant effect of heparin.

[0310] In some aspects, the term "protamine," as used herein, refers to any protamine amino acid sequence, including fragments thereof, obtained or derived from natural or biological sources, and / or multimeric forms of said amino acid sequence or fragments thereof, as well as polypeptides that are man-made, specifically designed for a particular purpose, and cannot be isolated from natural or biological sources (synthetic).

[0311] In some embodiments, the polyalkyleneimine comprises polyethyleneimine and / or polypropyleneimine. In some embodiments, the polyalkyleneimine is polyethyleneimine (PEI). In some embodiments, the polyalkyleneimine is a linear polyalkyleneimine, for example, linear polyethyleneimine (PEI).

[0312] Cationic materials (e.g., polymeric materials, including polycationic polymers) contemplated for use herein include those that can electrostatically bind to nucleic acids. In some aspects, cationic polymeric materials contemplated for use herein include any cationic polymeric material with which nucleic acids can associate, for example, by forming a complex with the nucleic acid or by forming a vesicle in which the nucleic acid is entrapped or encapsulated.

[0313] In some embodiments, the particles described herein may include polymers other than cationic polymers, such as non-cationic and / or anionic polymeric materials. Collectively, anionic and neutral polymeric materials are referred to herein as non-cationic polymeric materials.

[0314] B. Lipids and Lipid-like Substances The terms "lipid" and "lipid-like substance" are used herein to refer to molecules that contain one or more hydrophobic moieties or groups and, optionally, one or more hydrophilic moieties or groups. In this disclosure, lipids and lipid-like substances can be cationic, anionic, or neutral. Neutral lipids or lipid-like substances exist in an uncharged or neutral zwitterionic form at a selected pH.

[0315] The term "lipid" refers to a group of organic compounds characterized by being insoluble in water but soluble in many organic solvents. Generally, lipids can be divided into eight categories: fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides, sterol lipids, and even sterol-containing metabolites such as cholesterol, and prenol lipids. Examples of fatty acids include, but are not limited to, fatty esters and fatty acid amides. Examples of glycerolipids include, but are not limited to, glycosylglycerols and glycerophospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine). Examples of sphingolipids include, but are not limited to, ceramides, phosphosphingolipids (e.g., sphingomyelin, phosphocholine), and glycosphingolipids (e.g., cerebrosides, gangliosides). Examples of sterol lipids include, but are not limited to, cholesterol and its derivatives and tocopherol and its derivatives. In some embodiments, one, two, three, four, five, or more types of lipids may be excluded from the LNPs of the present disclosure.

[0316] The terms "lipid-like material," "lipid-like compound," or "lipid-like molecule" refer to substances that are structurally and / or functionally related to lipids but cannot be considered lipids in the strict sense. For example, this term includes compounds that can form amphiphilic layers such as those present in vesicles, multilamellar / unilamellar liposomes, or membranes in aqueous environments, and includes surfactants or synthetic compounds that have both hydrophilic and hydrophobic portions. Generally speaking, this term refers to molecules that contain hydrophilic and hydrophobic portions with different structural organizations that may or may not resemble the structure of lipids.

[0317] In some embodiments, the RNA solution and its lipid preparation mixture or composition may contain cationic lipids, neutral lipids, cholesterol, and / or polymer (e.g., polyethylene glycol)-conjugated lipids that form lipid nanoparticles that contain RNA molecules. Thus, in some embodiments, LNPs may contain cationic lipids and one or more excipients, such as one or more neutral lipids, charged lipids, steroids or steroid analogs (e.g., cholesterol), polymer-conjugated lipids (e.g., PEG-lipids), or combinations thereof. In some embodiments, one, two, three, or more of the above-mentioned excipients may be excluded from the LNPs of the present disclosure. In some embodiments, the lipids are present in the composition in an amount that is effective to form lipid nanoparticles and deliver therapeutic agents, such as RNA molecules, for treating a particular disease or condition of interest. In some embodiments, the LNPs contain or encapsulate nucleic acid molecules.

[0318] i. Cationic lipids Cationic or cationically ionizable lipids or lipid-like substances refer to lipids or lipid-like substances that can have a positive charge and can electrostatically bind to nucleic acids. As used herein, "cationic lipid" or "cationic lipid-like substance" refers to lipids or lipid-like substances that have a net positive charge. Cationic lipids or lipid-like substances bind to negatively charged nucleic acids through electrostatic interactions. Generally, cationic lipids have a lipophilic moiety such as a sterol, an acyl chain, a diacyl or more acyl chains, and the lipid head group typically carries a positive charge. Exemplary cationic lipids contain one or more amine groups that are positively charged. Cationic lipids can encapsulate negatively charged RNA.

[0319] In some embodiments, cationic lipid can be ionized, so that it can have a positive charge or exist in a neutral form depending on pH.The ionization of cationic lipid affects the surface charge of lipid nanoparticles under various pH conditions.Without wishing to be bound by theory, this ionizable behavior is believed to enhance efficacy by helping endosomal escape and reducing toxicity compared with particles that remain cationic at physiological pH.For the purpose of the present disclosure, such " cationically ionizable " lipid or lipid-like substance is included in the term " cationic lipid " or " cationic lipid-like substance ", unless the context is inconsistent.

[0320] In some embodiments, the cationic lipids may comprise 10 or about 10 mol% to about 100 mol%, about 20 mol% to about 100 mol%, about 30 mol% to about 100 mol%, about 40 mol% to about 100 mol%, or about 50 mol% to about 100 mol% of the total lipids present in the particle. In some embodiments, the cationic lipids may or may not comprise at least, at most, exactly, or therebetween (inclusively or exclusively) 10 mol%, 20 mol%, 30 mol%, 40 mol%, 50 mol%, 60 mol%, 70 mol%, 80 mol%, 90 mol%, or 100 mol%, or any range or value derivable therein, of the total lipids present in the particle.

[0321] In a preferred embodiment, the ionizable lipid has the following structure (I):

[0322] [ka] or a pharmaceutically acceptable salt, N-oxide, tautomer, or stereoisomer thereof. [In the formula, m, n, o, and p each independently represent 1 to 3; G 1 is C 1~12 Alkylene or C 2~12 is alkenylene, R 1 is -N(R 2 )R 3 , -OR 4 , CN, -N(R 4 ) (heteroaryl), -O(CH2) q OH, -(OCH2CH2) r OH, -OC(=O)R 5 , -N(R 4 )C(=O)R 5 , -N(R 4 )S(O)2R 5 , -N(R 4 )C(=O)N(R 2 )R 3 , -OC(=O)N(R 2 )R 3 , -N(R 4 )C(=O)OR 5 , -N(R 4 )C(=S)N(R 2 )R 3 , -N(R 4 )C(=NR 6 )N(R 2 )R 3 ,or

[0323] [ka] and R 2 and R 3 are independently H, C1~6 Alkyl, C 3~8 cycloalkyl, or aryl, or R 2 and R 3 form a heterocyclic ring together with the nitrogen atom to which they are attached, R 4 is H, C 1~6 Alkyl or C 3~8 is cycloalkyl, R 5 is C 1~6 Alkyl or C 1~6 C optionally substituted with alkyl 3~8 is cycloalkyl, R 6 are H, CN, NO2, C 1~6 Alkyl, OR 5 , S(O)2R 5 , or S(O)N(R 2 )R 3 and q is 2 to 6; r is 1 to 6; W is

[0324] [ka] and X is N or CH; G 2 and G 3 are each independently, C 1~12 Alkylene or C 2~12 is alkenylene, L 1 and L 2 are each independently -C(=O)OR 7 , -OC(=O)R 7 , -OC(=O)(CH2) r C(=O)OR 7 , -OC(=O)(CH2) r OC(=O)R 7 , -OC(=O)N(R 4 )R 7 , -N(R 4 )C(=O)OR 7 , -N(R4 )C(=O)N(R 4 )R 7 , -OC(=O)OR 7 , or -S-SR 7 and R 7 is C 6~24 Alkyl, C 6~24 Alkenyl, or C 6~24 alkynyl, F, C 1~6 Alkoxy, C 3~8 Cycloalkyl, or C 3~8 optionally substituted by cycloalkenyl; L 1 and L 2 R 7 may be the same or different].

[0325] In some embodiments, RNA-LNPs comprise one or more of the cationic / ionizable lipids described herein, RNA molecules, and neutral lipids, steroids, PEGylated lipids, or combinations thereof. When more than one cationic lipid is incorporated into LNPs, these percentages apply to the combination of cationic lipids. In one embodiment, the cationic lipid is present in LNPs in an amount of at least, at most, exactly, or between (inclusively or exclusively), or about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mole percent (mol%), etc. In some embodiments, two or more cationic lipids are incorporated into LNPs. When more than one cationic lipid is incorporated into LNPs, these percentages apply to the combined cationic lipids.

[0326] ii. Polymer-conjugated lipids In some embodiments, the LNP comprises a polymer-conjugated lipid. The term "polymer-conjugated lipid" refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer-conjugated lipid is a PEGylated lipid (e.g., polyethylene glycol-lipid, PEG-lipid). In certain embodiments, the LNP comprises an additional, stabilizing lipid that is a PEGylated lipid. The term "PEGylated lipid" refers to a molecule comprising both a lipid portion and a polyethylene glycol portion.

[0327] PEGylated lipids are known in the art and include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, and mixtures thereof. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, PEG-DSG, PEG-DPG, and PEG-s-DMG (1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol). In one embodiment, the polyethylene glycol-lipid is N-[(methoxypolyethylene glycol)2000)carbamoyl]-1,2-dimyristyloxylpropyl-3-amine (PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is PEG-2000-DMG. In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG. In other embodiments, the LNP is a PEGylated diacylglycerol (PEG-DAG), e.g., 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG), e.g., 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-((O-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), PEGylated ceramide (PEG-cer), or include PEG dialkoxypropyl carbamates, such as co-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecaneoxy)propyl)carbamate or 2,3-di(tetradecaneoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. PEG lipids are disclosed, for example, in U.S. Pat. No. 9,737,619, the entire disclosure of which is incorporated herein by reference in its entirety for all purposes. In some embodiments, one, two, three, four, five, or more of the above-mentioned PEGylated lipids may be excluded from the LNPs of the present disclosure.

[0328] In some embodiments, the composition has the following structure:

[0329] [ka] or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. [In the formula, R 8 and R 9 are each independently a linear or branched saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, the alkyl chain optionally being interrupted by one or more ester bonds, and w has an average value in the range of 30 to 60. 8 and R 9 are each independently a linear saturated alkyl chain containing 12 to 16 carbon atoms. In some aspects, w has an average value ranging from 43 to 53. In other aspects, the average w is 45 or about 45. In other different embodiments, the average w is 49 or about 49.

[0330] In some embodiments, the lipid nanoparticles comprise a polymer-conjugated lipid. In one embodiment, the lipid nanoparticles have the formula:

[0331] [ka] The compound includes 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), having the formula:

[0332] In various embodiments, the molar ratio of cationic lipid to PEGylated lipid ranges from 100 or about 100:1 to about 20:1, e.g., 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1, or any range or value derivable therein.

[0333] In certain embodiments, PEG-lipids are present or absent in the LNPs, e.g., in an amount of 1 or about 1 to about 10 mole percent (mol%) (at least, at most, exactly, or therebetween (inclusively or exclusively) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mol%) relative to the total lipid content of the nanoparticle.

[0334] In some embodiments, the ratio of PEG in a lipid nanoparticle formulation may be increased or decreased and / or the carbon chain length of the PEG lipid may be modified to alter the pharmacokinetics and / or biodistribution of the lipid nanoparticle formulation.

[0335] iii. Additional fats In certain embodiments, LNP comprises one or more additional lipids or lipid-like substances that stabilize particles during their formation.Suitable stabilizing or structural lipids include non-cationic lipids, such as neutral lipids and anionic lipids.Without being bound by any theory, LNP formulation can be optimized by adding other hydrophobic moieties, such as cholesterol and lipids, in addition to ionizable / cationic lipids or lipid-like substances, to improve particle stability and the effectiveness of nucleic acid delivery.

[0336] As used herein, "anionic lipid" refers to any lipid that is negatively charged at a selected pH. The term "neutral lipid" refers to any one of several lipid species that exist at physiological pH in either an uncharged or neutral zwitterionic form. In some embodiments, the additional lipid comprises one of the following neutral lipid components: (1) a phospholipid, (2) cholesterol or a derivative thereof, or (3) a mixture of a phospholipid and cholesterol or a derivative thereof.

[0337] Representative neutral lipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, ceramide, sphingomyelin, dihydro-sphingomyelin, cephalin, and cerebroside. Exemplary phospholipids include, for example, phosphatidylcholine, for example, diacylphosphatidylcholine, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), diarachidoylphosphatidylcholine (DPPG), dioleo ...choline (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPG), dioleoylphosphatidylglycerol (DPPG), di phosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), and 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC);and phosphatidylethanolamines, such as diacylphosphatidylethanolamines, such as dioleoyl-phosphatidylethanolamine (DOPE), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), disulfide phosphatidylethanolamine (DSPE), diisopropyl methyl esters of phosphoethanolamine (MPE ... Stearoyl-phosphatidylethanolamine (DSPE), 1-phytanoyl-phosphatidylethanolamine (DpyPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidiethanolamine (SOPE), 1,2-dielideyl-sn-glycero-3-phosphoethanolamine (transDOPE), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME) 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof. In some embodiments, one, two, three, four, five, or more of the above-mentioned neutral lipids may be excluded from the LNPs of the present disclosure.

[0338] In one aspect, the neutral lipid has the formula:

[0339] [ka] and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) having the formula:

[0340] In some embodiments, the LNPs comprise a neutral lipid, wherein the neutral lipid comprises one or more of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and / or SM. In some embodiments, one, two, three, four, five, or more of the above-mentioned neutral lipids may be excluded from the LNPs of the present disclosure.

[0341] In various embodiments, the LNP further comprises a steroid or steroid analog. A "steroid" is a compound having the following carbon skeleton:

[0342] [ka] It is a compound comprising:

[0343] In certain embodiments, the steroid or steroid analog is cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof. In some embodiments, one, two, three, four, five, or more of the above-mentioned steroids or steroid analogs may be excluded from the LNPs of the present disclosure. In certain embodiments, the steroid or steroid analog is cholesterol. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and its derivatives, and mixtures thereof. In some embodiments, one, two, three, four, five, or more of the above-mentioned cholesterol derivatives may be excluded from the LNPs of the present disclosure. In one embodiment, cholesterol is represented by the formula:

[0344] [ka] It has.

[0345] Without being bound by any theory, the amount of at least one cationic lipid relative to the amount of at least one additional lipid can affect important nucleic acid particle properties, such as charge, particle size, stability, tissue selectivity, and nucleic acid bioactivity. Thus, in some embodiments, the molar ratio of cationic lipid to neutral lipid ranges from about 2:1 to about 8:1, or from about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1.

[0346] In some embodiments, non-cationic lipids, e.g., neutral lipids (e.g., one or more phospholipids and / or cholesterol), may comprise 0 or about 0 mol% to about 90 mol%, 0 or about 0 mol% to about 80 mol%, 0 or about 0 mol% to about 70 mol%, 0 or about 0 mol% to about 60 mol%, or 0 or about 0 mol% to about 50 mol% of the total lipids present in the particle. In some embodiments, non-cationic lipids, e.g., neutral lipids (e.g., one or more phospholipids and / or cholesterol), may or may not comprise at least, at most, exactly, or between (inclusively or exclusively) 0 mol%, 10 mol%, 20 mol%, 30 mol%, 40 mol%, 50 mol%, 60 mol%, 70 mol%, 80 mol%, or 90 mol% of the total lipids present in the particle.

[0347] Pharmaceutical Composition In another embodiment, the invention comprises a pharmaceutical composition, for the purposes of which the compound per se or a pharmaceutically acceptable salt thereof will simply be referred to as the compound of the invention.

[0348] A "pharmaceutical composition" refers to a mixture of one or more compounds of the invention, or pharmaceutically acceptable salts, solvates, hydrates or prodrugs thereof, as the active ingredient, and at least one pharmaceutically acceptable excipient.

[0349] The term "excipient" is used herein to describe any ingredient other than the compound(s) of the invention. The choice of excipient will to a large extent depend on factors such as the mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

[0350] As used herein, "excipient" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity and absorption delaying agents, carriers, diluents, and the like. Examples of excipients include one or more of water, saline, phosphate buffer solution, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. Isotonicity adjusting agents, such as sugars, sodium chloride, or polyalcohols such as mannitol or sorbitol, may also be included in the composition. Examples of excipients also include various organic solvents (such as hydrates and solvates). Pharmaceutical compositions may contain additional excipients, such as flavoring agents, binders / binding agents, lubricants, disintegrants, sweeteners or flavoring agents, coloring agents, and the like, if desired. For example, for oral administration, tablets containing various excipients such as citric acid can be used with various disintegrating agents such as starch, alginic acid, and certain complex silicates, and binders such as sucrose, gelatin, and gum arabic. Non-limiting examples of excipients include calcium carbonate, calcium phosphate, various sugars and starch types, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols. Additionally, lubricants such as magnesium stearate, sodium lauryl sulfate, and talc are often useful for tableting. Similar types of solid compositions can also be used in filled soft and hard gelatin capsules. Non-limiting examples of excipients include lactose or milk sugar and high molecular weight polyethylene glycols. When aqueous suspensions or elixirs are desired for oral administration, the active compound therein can be combined with various sweeteners or flavoring agents, colorings or pigments, and, if desired, emulsifying or suspending agents, along with additional excipients such as water, ethanol, propylene glycol, glycerin, or combinations thereof.

[0351] Examples of excipients also include pharmaceutically acceptable substances that enhance the shelf life or effectiveness of the compound, such as wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffers.

[0352] The compositions of the present invention may be in a variety of forms, including, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, capsules, pills, powders, liposomes, and suppositories. The form depends on the intended mode of administration and therapeutic application.

[0353] Typical compositions are in the form of injectable or infusible solutions, such as compositions similar to those generally used for passive immunization of humans with antibodies.One mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular).In another embodiment, the compound is administered by intravenous infusion or injection.In yet another embodiment, the compound is administered by intramuscular or subcutaneous injection.

[0354] Oral administration of solid dosage forms can be provided in separate units, such as hard or soft capsules, pills, cachets, lozenges, or tablets, each containing a predetermined amount of at least one compound of the present invention.In another embodiment, oral administration can be in the form of powder or granules.In another embodiment, oral dosage forms are sublingual, such as lozenges.In such solid dosage forms, the compound of the present invention is usually combined with one or more adjuvants.Such capsules or tablets can contain controlled-release formulations.In the case of capsules, tablets, and pills, dosage forms can contain buffering agents or be prepared with enteric coatings.

[0355] In another embodiment, oral administration may be in the form of a liquid dosage form.Liquid dosage forms for oral administration include, for example, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art (e.g., water).Such compositions may also contain one or more auxiliary agents, such as wetting agents, emulsifying agents, suspending agents, flavoring agents (e.g., sweeteners), and / or aromatic agents.

[0356] In another embodiment, the present invention includes parenteral dosage forms. "Parenteral administration" includes, for example, subcutaneous injection, intravenous injection, intraperitoneal injection, intramuscular injection, intrasternal injection, and infusion. Injectable preparations (for example, sterile injectable aqueous or oily suspensions) can be formulated according to known techniques using one or more suitable dispersants, wetting agents, or suspending agents.

[0357] In another embodiment, the present invention encompasses topical dosage forms. "Topical administration" includes, for example, dermal and transdermal administration, such as via transdermal patches or iontophoretic devices, intraocular administration, or intranasal or inhalation administration. Compositions for topical administration also include, for example, topical gels, sprays, ointments, and creams. Topical formulations may include compounds that enhance absorption or penetration of the active ingredient through the skin or other affected areas. When administering the compounds of the present invention via a transdermal device, administration can be achieved using a patch, either of the reservoir and porous membrane type or of the solid matrix variety. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibers, adhesive plasters, and microemulsions. Liposomes can also be used. Typical excipients include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol, and propylene glycol. Permeation enhancers can be incorporated. See, e.g., B.C. Finnin and T.M. Morgan, J. Pharm. Sci., vol. 88, pp. 955-958, 1999.

[0358] Formulations suitable for topical administration to the eye include, for example, eye drops in which the compounds of the present invention are dissolved or suspended in a suitable vehicle. Typical formulations suitable for ocular or aural administration may be in the form of drops of micronized suspensions or solutions in isotonic, pH-adjusted, sterile saline. Other formulations suitable for ocular and aural administration include ointments, biodegradable (i.e., absorbent gel sponges, collagen) and non-biodegradable (i.e., silicone) implants, wafers, lenses, and microparticle or vesicle systems such as niosomes or liposomes. Polymers such as cross-linked polyacrylic acid, polyvinyl alcohol, hyaluronic acid, cellulose-based polymers such as hydroxypropylmethylcellulose, hydroxyethylcellulose, or methylcellulose, or heteropolysaccharide polymers such as gellan gum, together with preservatives such as benzalkonium chloride, can also be incorporated. Such formulations can also be delivered by iontophoresis.

[0359] For intranasal administration, the compounds of the present invention are conveniently delivered in the form of a solution or suspension from a pump-type spray container that the patient squeezes or pumps, or as an aerosol spray delivery from a pressurized container or nebulizer using a suitable propellant. Formulations suitable for intranasal administration are typically administered in the form of a dry powder from a dry powder inhaler (either alone or as a mixture, for example, in a dry blend with lactose, or as mixed component particles, for example, mixed with a phospholipid such as phosphatidylcholine), or as an aerosol spray from a pressurized container, pump, spray, atomizer (preferably an atomizer that uses electrohydrodynamics to produce a fine mist), or nebulizer with or without a suitable propellant, such as 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane. For intranasal use, the powder may contain a bioadhesive, for example, chitosan or cyclodextrin.

[0360] In another embodiment, the present invention includes a rectal dosage form. Such a rectal dosage form may be, for example, in the form of a suppository. Cocoa butter is a traditional suppository base, although various alternatives can be used where appropriate.

[0361] Other excipients and modes of administration known in the pharmaceutical art can also be used.The pharmaceutical compositions of the present invention can be prepared by any of the well-known techniques of pharmacy, such as effective formulation and administration procedures.The above discussions on effective formulation and administration procedures are well known in the art and are described in standard textbooks.Drug formulations are discussed, for example, in Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania, 1975; Liberman et al., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Kibbe et al., eds., Handbook of Pharmaceutical Excipients (3rd Edition), American Pharmaceutical Association, Washington, 1999.

[0362] Acceptable excipients are non-toxic to subjects at the dosages and concentrations employed and may include one or more of the following: 1) buffers such as phosphates, citrates, and other organic acids; 2) salts such as sodium chloride; 3) antioxidants such as ascorbic acid and methionine; 4) preservatives such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol; 5) alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, or m-cresol; 6) low molecular weight (less than about 10 residues) polypeptides; 7) serum albumin, 9) amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; 10) monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; 11) chelating agents such as EDTA; 12) sugars such as sucrose, mannitol, trehalose, or sorbitol; 13) salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), or 14) non-ionic surfactants such as polysorbates (e.g., polysorbate 20 or polysorbate 80), poloxamers, or polyethylene glycol (PEG).

[0363] For oral administration, the compositions can be provided in the form of tablets or capsules containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 75.0, 100, 125, 150, 175, 200, 250, or 500 milligrams of the active ingredient for symptomatic adjustment of dosage to the patient. Medicaments typically contain from about 0.01 mg to about 500 mg of the active ingredient, or in another embodiment, from about 1 mg to about 100 mg of the active ingredient. Intravenously, doses can range from about 0.01 to about 10 mg / kg / minute during a constant rate infusion.

[0364] Liposomes containing the compounds of the present invention can be prepared by methods known in the art (see, for example, Chang, HI; Yeh, MK; Clinical development of liposome-based drugs: formulation, characterization, and therapeutic efficacy; Int J Nanomedicine 2012;7;49-60). Particularly useful liposomes can be generated by the reverse-phase evaporation method using a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.

[0365] The compounds of the present invention can also be entrapped in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, prepared by coacervation technology or interfacial polymerization, respectively, colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or macroemulsions.Such techniques are disclosed in Remington, The Science and Practice of Pharmacy, 20th Edition, Mack Publishing (2000).

[0366] Sustained-release preparations can be used.Suitable examples of sustained-release preparations include the semipermeable matrix of solid hydrophobic polymer that contains the compound of the present invention, and this matrix is ​​in the form of shaped article, for example, film or microcapsule.Examples of sustained-release matrix include polyester, hydrogel (for example, poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactide, L-glutamic acid and 7-ethyl-L-glutamate copolymer, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymer (such as that used in leuprolide acetate for depot suspensions (injectable microspheres that consist of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0367] Preparations used for intravenous administration must be sterile. This can be easily achieved, for example, by filtration through a sterile filtration membrane. The compound of the present invention is generally placed into a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.

[0368] Suitable emulsions can be prepared using commercially available lipid emulsions, such as lipid emulsions containing soybean oil, lipid emulsions for intravenous administration (e.g., containing safflower oil, soybean oil, egg phospholipids, and glycerin in water), emulsions containing soybean oil and medium-chain triglycerides, and lipid emulsions of cottonseed oil. The active ingredient can be dissolved in a premixed emulsion composition, or alternatively, can be dissolved in an emulsion formed by mixing oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and phospholipids (e.g., egg phospholipids, soybean phospholipids, or soybean lecithin) with water. It will be appreciated that other ingredients, such as glycerol or glucose, can be added to adjust the tonicity of the emulsion. Suitable emulsions typically contain up to 20% oil, for example, between 5 and 20%. The lipid emulsion may contain lipid droplets between 0.1 and 1.0 μm, in particular between 0.1 and 0.5 μm, and have a pH in the range of 5.5 to 8.0.

[0369] For example, an emulsion composition may be prepared by mixing a compound of the present invention with a lipid emulsion comprising soybean oil or its components (soybean oil, egg phospholipids, glycerol, and water).

[0370] Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as set forth above. In some embodiments, the compositions are administered by oral or nasal respiratory route for local or systemic effect. Compositions, preferably in sterile pharmaceutically acceptable solvents, can be nebulized by using gas. The nebulized solution can be breathed directly from the nebulizing device, or the nebulizing device can be attached to a face mask, tent, or intermittent positive pressure respirator. Solution, suspension, or powder compositions can be administered, preferably orally or nasally, from a device that delivers the formulation in an appropriate manner.

[0371] A drug formulation intermediate (DPI) is a partially processed material that requires further processing steps before becoming a bulk formulation. The compounds of the present invention can be formulated into a drug formulation intermediate DPI that contains the active ingredient in a form with a higher free energy than the crystalline form. One reason for using a DPI is to improve oral absorption characteristics due to low solubility, slow dissolution, improved transport through the mucin layer adjacent to epithelial cells, and in some cases, limitations due to biological barriers such as metabolism and transporters. Other reasons may include improved solid-state stability and downstream manufacturability. In one embodiment, the drug formulation intermediate contains a compound of the present invention isolated and stabilized in an amorphous state (e.g., an amorphous solid dispersion (ASD)). Many techniques are known in the art for producing ASDs that produce materials suitable for incorporation into bulk formulations, such as spray-dried dispersions (SDDs), melt extrudates (often referred to as HMEs), co-precipitates, amorphous drug nanoparticles, and nanoadsorbates. In one embodiment, the amorphous solid dispersion contains a compound of the present invention and a polymeric excipient. Other excipients, as well as concentrations of excipients and compounds of the invention, are well known in the art and are described in standard textbooks, see, for example, "Amorphous Solid Dispersions Theory and Practice" by Navnit Shah et al.

[0372] "Systemic delivery," as used herein, refers to the delivery of a therapeutic product that can result in widespread exposure of the active agent in the body. Some administration techniques can 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 most of the body. Systemic delivery of lipid nanoparticles can be by any means known in the art, including, for example, intravenous, intraarterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery.

[0373] "Local delivery" as used herein refers to the delivery of an active agent directly to a target site in a living body.For example, the agent can be delivered locally by directly injecting into a disease site, such as a tumor, other target site, such as an inflammatory site, or into a target organ, such as the liver, heart, pancreas, kidney, and the like.Local delivery can also include local application or local injection techniques, such as intramuscular, subcutaneous, or intradermal injection.Local delivery does not interfere with systemic pharmacological effects.

[0374] Administration and Dosage The terms "treating," "treat," or "treatment" as used herein include both preventative, e.g., protective, and palliative treatment, e.g., reducing, alleviating, or slowing the progression of a patient's disease (or condition) or any tissue damage associated with a disease.

[0375] As used herein, the terms "subject," "individual," or "patient," used interchangeably, refer to any animal, including mammals. Mammals according to the present invention include dogs, cats, cows, goats, horses, sheep, pigs, rodents, rabbits, primates, humans, and the like, including mammals in utero. In one embodiment, humans are the preferred subject. Human subjects may be of any gender and at any stage of development.

[0376] As used herein, the phrase "therapeutically effective amount" or "effective amount" refers to an amount of an active compound or pharmaceutical agent, such as a nucleic acid, that elicits the biological or medical response in a tissue, system, animal, individual, or human that is being sought by a researcher, veterinarian, physician, or other clinician, which may include one or more of the following: (1) Preventing disease; e.g., preventing a disease, condition, or disorder in an individual who is susceptible to the disease, condition, or disorder but who has not yet experienced or exhibited the pathology or symptomology of the disease; (2) inhibiting a disease; e.g., inhibiting a disease, condition, or disorder (e.g., arresting (or slowing) further development of the pathology or symptomology, or both) in an individual experiencing or exhibiting the pathology or symptomology of the disease, condition, or disorder; and (3) Ameliorating a disease; e.g., ameliorating a disease, condition, or disorder (e.g., reversing the pathology or symptomology, or both) in an individual experiencing or exhibiting the pathology or symptomology of the disease, condition, or disorder.

[0377] An "effective amount" or "therapeutically effective amount" of a nucleic acid is an amount sufficient to produce a desired effect, e.g., increased or inhibited expression of a target sequence compared to the normal expression level detected in the absence of the nucleic acid. Increased expression of a target sequence is achieved when any measurable level of expression product not present in the absence of the nucleic acid is detected. When the expression product is present at a certain level before contact with the nucleic acid, increased expression is achieved when the fold increase over the value obtained with the nucleic acid, such as mRNA, is about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10,000, or more times greater than the control. Inhibition of expression of a target gene or target sequence is achieved when the value obtained using a nucleic acid, such as an antisense oligonucleotide, is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% relative to the 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 of skill in the art, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of a suitable reporter protein, and phenotypic assays known to those of skill in the art.

[0378] The phrase "induce the expression of a desired protein" refers to the ability of a nucleic acid to increase the expression of a desired protein.To investigate the level of protein expression, a test sample (e.g., a cell sample in culture that expresses a desired protein) or a test mammal (e.g., a mammal, such as a human, or an animal model, such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model) is contacted with a nucleic acid (e.g., the nucleic acid combined with the lipid of the present invention).The expression of the desired protein in the test sample or test animal is compared with the expression of the desired protein in a control sample (e.g., a cell sample in culture that expresses a desired protein) or a control mammal (e.g., a mammal, such as a human, or an animal model, such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model) that has not been contacted with or administered with a nucleic acid.If 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 certain embodiments, induction of expression of a desired protein is achieved when the ratio of the expression of the desired protein in a test sample or test mammal to the level of expression of the desired protein in a control sample or control mammal is greater than 1, for example, about 1.1, 1.5, 2.0, 5.0, or 10.0. If the desired protein is not present in the control sample or control mammal, induction of expression of a desired protein is achieved when any measurable level of the desired protein is detected in the test sample or test mammal. Those skilled in the art will recognize suitable assays for determining protein expression levels in a sample, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays, or assays based on reporter proteins that can produce fluorescence or luminescence under appropriate conditions.

[0379] The phrase "inhibit the expression of target gene" refers to the ability of nucleic acid to silence, reduce or inhibit the expression of target gene.To investigate the degree of gene silencing, a test sample (e.g., a cell sample in culture that expresses target gene) or a test mammal (e.g., a mammal, such as a human, or an animal model, such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model) is contacted with the nucleic acid that silences, reduces or inhibits the expression of target gene.The expression of the target gene in the test sample or test animal is compared with the expression of the target gene in a control sample (e.g., a cell sample in culture that expresses target gene) or a control mammal (e.g., a mammal, such as a human, or an animal model, such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model) that is not contacted with nucleic acid or administered with nucleic acid.The expression of the target gene in the control sample or the control mammal can be assigned a value of 100%. In certain embodiments, silencing, inhibition, or reduction of expression of a target gene is achieved when the level of expression of the target gene in a test sample or test mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% of the level of expression of the target gene in a control sample or control mammal. In other words, nucleic acid can silence, reduce or inhibit the expression of target gene in test sample or test mammal by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to the expression level of target gene in control sample or control mammal that is not contacted with nucleic acid or not administered nucleic acid.Suitable assays for determining the expression level of target gene include, but are not limited to, protein or mRNA level examination using techniques known to those skilled in the art, such as dot blot, Northern blot, in situ hybridization, ELISA, immunoprecipitation, enzyme function and phenotypic assay known to those skilled in the art.

[0380] Typically, the compound of the present invention is administered in an amount effective for treating the conditions described herein or for delivering the active agent for treating the conditions described herein.The compound of the present invention can be administered as compound itself or alternatively as pharmaceutically acceptable salt.For the purpose of administration and dosage, the compound itself or its pharmaceutically acceptable salt will simply be referred to as the compound of the present invention.

[0381] The compounds of the present invention are administered by any suitable route, in the form of a pharmaceutical composition adapted to such a route, and in a dose effective for the intended treatment. The compounds of the present invention can be administered orally, rectally, vaginally, parenterally, topically, intranasally, or by inhalation.

[0382] The compounds of the present invention can be administered orally, either by swallowing, so that the compound enters the gastrointestinal tract, or by using buccal or sublingual administration, by which the compound enters the blood stream directly from the mouth.

[0383] In another embodiment, the compound of the present invention can be administered parenterally, for example, directly into the bloodstream, muscle, or internal organs.Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, and subcutaneous.Suitable devices for parenteral administration include needle (including fine needle) injectors, needle-free injectors, and infusion techniques.

[0384] In another embodiment, the compounds of the present invention can be administered topically to the skin or mucosa, i.e., dermally or transdermally. In another embodiment, the compounds of the present invention can be administered intranasally or by inhalation. In another embodiment, the compounds of the present invention can be administered rectally or vaginally. In another embodiment, the compounds of the present invention can be administered directly to the eye or ear.

[0385] Dosing regimens for compounds of the invention or compositions containing compounds are based on a variety of factors, including the patient's species, age, weight, sex, and medical condition; the severity of the condition; the route of administration; and the activity of the particular compound used. Thus, dosing regimens can vary widely. In one embodiment, the total daily dose of a compound of the invention is typically about 0.01 to about 100 mg / kg (i.e., mg of compound of the invention per kg of body weight) for the treatment of the indications discussed herein or for delivery of active agents by use of a compound of the invention. In another embodiment, the total daily dose of a compound of the invention is about 0.00001 to about 50 mg / kg, or in another embodiment, about 0.0001 to about 30 mg / kg. It is not uncommon to repeat administration of a compound of the invention multiple times per day (typically up to four times). Multiple doses per day can typically be used to increase the total daily dose, if desired.

[0386] Treatment Methods and Uses The compounds of the present invention can be useful for treating or preventing diseases, disorders, or conditions, or for delivering active drugs for treating or preventing diseases, disorders, or conditions.In particular, such compositions can be useful in treating or preventing diseases, disorders, or conditions characterized by missing or abnormal protein or polypeptide activity.Diseases, disorders, and / or conditions characterized by dysfunctional or abnormal protein or polypeptide activity to which the compositions can be administered include, but are not limited to, rare diseases, infectious diseases (both as vaccines and therapeutic agents), cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, heart and renal vascular diseases, and metabolic diseases.

[0387] Simultaneous administration The compounds of the invention can be administered alone or in combination with one or more additional therapeutic agents. The invention provides any of the uses, methods, or compositions defined herein, employing a compound of the invention, or a pharmaceutically acceptable salt thereof, in combination with one or more other therapeutic agents discussed herein.

[0388] Administration of two or more compounds "in combination" means that all of the compounds are administered close enough in time to affect the treatment of the subject.Two or more compounds can be administered synchronously or sequentially, via the same or different administration routes, with the same or different administration schedules, with or without specific time limits, depending on the treatment regimen.In addition, synchronous administration can be achieved by mixing the compounds before administration, or by administering the compounds in separate dosage forms at the same time but at the same or different administration sites.Examples of "in combination" include, but are not limited to, "simultaneous administration," "co-administration," "synchronous administration," "sequential administration," and "administered synchronously."

[0389] The compound of the present invention and one or more other therapeutic agents can be administered as a fixed or non-fixed combination of active ingredients. The term "fixed combination" means that the compound of the present invention, or a pharmaceutically acceptable salt thereof, and one or more other therapeutic agents are both administered to a subject synchronously in a single composition or dosage. The term "non-fixed combination" means that the compound of the present invention, or a pharmaceutically acceptable salt thereof, and one or more other therapeutic agents are formulated as separate compositions or dosages so that they can be administered to a subject in need thereof synchronously or at different times with varying intervening time periods, such that such administration results in effective levels of the two or more compounds in the subject's body.

[0390] These agents and compounds of the present invention can be combined with a pharmaceutically acceptable vehicle such as saline, Ringer's solution, dextrose solution, and the like. The particular administration regimen, e.g., dosage, timing, and repetition, will depend on the particular individual and their medical history.

[0391] kit Another aspect of the present invention provides a kit comprising a compound of the present invention or a pharmaceutical composition comprising a compound of the present invention. The kit may include a diagnostic or therapeutic agent in addition to the compound of the present invention or a pharmaceutical composition thereof. The kit may also include instructions for use in a diagnostic or therapeutic method. In some embodiments, the kit includes a compound or a pharmaceutical composition thereof and a diagnostic agent.

[0392] In another embodiment, the present invention includes a kit suitable for use in carrying out the treatment methods described herein. In one embodiment, the kit contains a first dosage form containing one or more compounds of the present invention in an amount sufficient to carry out the method of the present invention. In another embodiment, the kit includes one or more compounds of the present invention in an amount sufficient to carry out the method of the present invention and a container for the dosage amount.

[0393] Synthesis method The compounds of the present invention can be synthesized by synthetic routes, including processes similar to those well known in the chemical arts, particularly in light of the description contained herein.Starting materials are generally available from commercial sources or can be prepared using methods well known to those skilled in the art.Many of the compounds used herein are related to or can be derived from compounds that have one or more scientific importance and commercial needs.Therefore, such compounds can be one or more of: 1) commercially available; 2) reported in the literature; or 3) prepared by those skilled in the art using materials reported in the literature from other commercially available materials.

[0394] For illustrative purposes, the reaction schemes shown below provide potential routes for synthesizing the compounds of the present invention, as well as key intermediates. For more detailed descriptions of the individual reaction steps, see the Examples section below. Those skilled in the art will recognize that other synthetic routes can be used to synthesize the compounds of the present invention. While specific starting materials and reagents are discussed below, other starting materials and reagents can be substituted to obtain one or more of the various derivatives and / or reaction conditions. Additionally, many of the compounds prepared by the methods described below can be further modified in light of this disclosure using conventional chemistry well known to those skilled in the art.

[0395] Those skilled in the art will recognize that the experimental conditions depicted in the following schemes are illustrative of conditions suitable for carrying out the transformations shown, and that it may be necessary or desirable to vary the exact conditions used to prepare the compounds of the invention. Furthermore, it will be recognized that it may be necessary or desirable to carry out the transformations in a different order than that depicted in the schemes, or to modify one or more of the transformations to obtain the desired compounds of the invention.

[0396] In preparing the compounds of the present invention, it is noted that some of the preparative methods useful for preparing the compounds described herein may require protection of remote functional groups (e.g., primary amines, secondary amines, carboxyls, etc. in precursors of the compounds of the present invention). The need for such protection will vary depending on the nature of the remote functional group and the conditions of the preparation method. One skilled in the art will readily determine the need for such protection. The use of such protection / deprotection methods is also within the skill of the art. For a review of protecting groups and their uses, see March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 8th Edition; or Green's Protective Groups in Organic Synthesis, 5th Edition, edited by Wuts, PGM.

[0397] For example, if a compound contains an amine or carboxylic acid functional group, such a functional group, if left unprotected, may interfere with reactions at other sites of the molecule.Therefore, such functional groups can be protected with a suitable protecting group (PG) that can be removed in a subsequent step.Suitable protecting groups for amine and carboxylic acid protection include protecting groups commonly used in peptide synthesis (such as N-tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), and 9-fluorenylmethyleneoxycarbonyl (Fmoc) for amines and lower alkyl or benzyl esters for carboxylic acids), which are generally not chemically reactive under the reaction conditions described and can typically be removed without chemically changing other functional groups in the compounds of the present invention.

[0398] General Experimental Details In the non-limiting examples and preparations presented in the description which illustrate the present invention, and in the following schemes, reference may be made to the following abbreviations, definitions, and analytical procedures:

[0399] 1 H NMR spectra were recorded on a Bruker 400 MHz spectrometer. Chemical shifts are reported in parts per million (ppm), and all spectra are referenced to their residual non-deuterated solvent peaks as follows: CHCl (7.26 ppm), CDOD (3.31 ppm), DMSO-d (2.50 ppm), and DO (4.75 ppm). Coupling constants (J) are reported to the nearest 0.1 Hz. Multiplicities are reported as follows: singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad singlet (br s). Exchangeable protons are not always observed.

[0400] LCMS data were acquired using an Agilent Prime-6125B instrument, an Agilent Poroshell 120 EC-C18 3.0 × 30 mm column, 2.7 μm, and an acetonitrile / water gradient with TFA modifier, using an evaporative light scattering detector (ELSD) (see Methods A and B). Preparative chiral supercritical fluid chromatography (prep-SFC) was performed using a DAICEL ChiralPAK-AD, -AS, -IC, DAICEL ChiralCEL-OJ, or -OD column; fraction collection was driven using a gradient eluting with a CO mixture containing 0.1% NH3H2O ​​in EtOH and UV detection. LCMS-ELSD purity was verified by the following analytical method and expressed as area %: Instrument = Agilent 1260 Infinity with 6150MSD; Column = Waters XBridge C8 100 × 2.1 mm, 3.5 μm; Mobile phase A, 0.05% DFA in water; Mobile phase B, 0.05% DFA in acetonitrile; Gradient = 50% to 100% (solvent B) over 5 min, hold at 100% for 2 min, flow rate 1.0 mL / min, total time 7.0 min, 40 °C. LC-CAD purity was verified by the following analytical method and expressed as area %: Instrument: Thermo Vanquish F; Column: Waters XBridge C8 4.6 × 150 mm, 3.5 μm; Mobile phase A: 1 L water + 0.05% TFA; Mobile phase B: 1 L acetonitrile + 0.05% TFA; Gradient: 50% to 100% (solvent B) over 10 min, hold at 100% for 5 min, flow rate: 1.0 mL / min, total time: 15.0 min, 40 °C. Charged aerosol detection (CAD) data collection rate: 10 Hz; Vaporizer temperature: 35 °C.

[0401] LCMS-ELSD method used to monitor the reaction: Method A: Analytical LCMS data collected on an Agilent Prime-6125B instrument; Column: Agilent Poroshell 120 EC-C18 3.0 x 30 mm, 2.7 μm; Mobile phase A: water (4 L) + TFA (1.5 mL); Mobile phase B: acetonitrile (4 L) + TFA (0.75 mL); Gradient: 5% to 95% (solvent B) over 0.4 min, hold at 100% for 0.3 min, flow rate 2.0 mL / min, total time 1.0 min, 50 °C.

[0402] Method B: Analytical LCMS data collected on an Agilent Prime-6125B instrument; Column: Agilent Poroshell 120 EC-C18 3.0 x 30 mm, 2.7 μm; Mobile phase A: water (4 L) + TFA (1.5 mL); Mobile phase B: acetonitrile (4 L) + TFA (0.75 mL); Gradient: 95% to 100% (solvent B) over 0.4 min, hold at 100% for 0.3 min, flow rate 2.0 mL / min, total time 1.0 min, 50 °C.

[0403] Abbreviation °2θ is degrees two theta; AcCl is acetyl chloride; AcOH is acetic acid; APCI is atmospheric pressure chemical ionization; aq is aqueous; Bn is benzyl; Boc is tert-butoxycarbonyl; BocO is di-tert-butyl dicarbonate; br is broad; tBu is tert-butyl; tBuOH is tert-butanol; tBuOK is potassium tert-butoxide; °C is degrees Celsius; CDCl3 is deuterochloroform; CD3OD or MeOD_d4 is deuterated methanol; CDI is 1,1′-carbonyldiimidazole; δ is the chemical shift; d is a double line; dd is double doublet; ddd is a double double double line; dt is a triple doublet; DCE is 1,2-dichloroethane; DCM is dichloromethane; methylene chloride; DIAD is diisopropyl azodicarboxylate; DIPEA is N-ethyldiisopropylamine, also known as N,N-diisopropylethylamine; DMA is N,N-dimethylacetamide; DME is 1,2-dimethoxyethane; DMAP is 4-dimethylaminopyridine; DMF is N,N-dimethylformamide; DMSO is dimethyl sulfoxide; DMSO-d6 is deuterodimethyl sulfoxide; EDC is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide; EDC.HCl is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride; ESI is electrospray ionization; Et2O is diethyl ether; EtOAc is ethyl acetate; EtOH is ethanol; Et3N is triethylamine; g is grams; HATU is 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; HPLC is high pressure liquid chromatography; HOBt is 1-hydroxybenzotriazole hydrate; hr(s) is the time; IPA is isopropyl alcohol; iPrOAc is isopropyl acetate; L is liters; LCMS is liquid chromatography mass spectrometry; m is a multiplet; M is moles; m-CPBA is 3-chloroperbenzoic acid; MeCN is acetonitrile; MeMgBr is methylmagnesium bromide; MeNHOMe HCl is N,O-dimethylhydroxylamine hydrochloride; MeOH is methanol; 2-MeTHF is 2-methyltetrahydrofuran; mg is milligrams; MHz is megahertz; min(s) is minutes; mL is milliliters; mmol is millimole; mol is moles; MS(m / z) is the mass spectrum peak; MsCl is mesyl chloride; MTBE is tert-butyl methyl ether; NMR is nuclear magnetic resonance; Pd / C is palladium on carbon; Pd2(dba)3 is palladium tris(dibenzylideneacetone)dipalladium(0); Pd(dppf)Cl2 is [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II); Pd(PPh3)4 is tetrakis(triphenylphosphine)palladium(0); Pet. ether is a petroleum fraction consisting of aliphatic hydrocarbons and boiling in the range of 35-60°C; PMB is para-methoxybenzyl; PMB-NH2 is para-methoxybenzylamine; PPh3 is triphenylphosphine; pH is the hydrogen ion potential; ppm is parts per million; PSD is a position sensitive device; psi is pounds per square inch; PXRD is powder X-ray diffraction; q is a quartet; rt is room temperature; RT is retention time; s is a singlet; SEM-Cl is 2-(trimethylsilyl)ethoxymethyl chloride; SFC is supercritical fluid chromatography; t is the triple line; TBAF is tert-butylammonium fluoride; TBDMSCl is tert-butyldimethylsilyl chloride; TFA is trifluoroacetic acid; THF is tetrahydrofuran; TLC is thin layer chromatography; TMEDA is N,N,N'N'-tetramethylethylenediamine; TMSCl is trimethylsilyl chloride; TMSCN is trimethylsilyl cyanide; TMSCHN2 is (diazomethyl)trimethylsilane; TsCl is p-toluenesulfonyl chloride; Ts2O is p-toluenesulfonic anhydride; μL is microliter; μmol is micromole.

[0404] The schemes described below are intended to provide an overview of the methods used in the preparation of compounds of the present invention. Some of the compounds of the present invention contain one or more chiral centers. In the following schemes, general methods for preparing compounds are shown in either racemic or enantiomerically enriched form. It will be apparent to those skilled in the art that all synthetic transformations can be carried out in exactly the same manner, regardless of whether the material is enantiomerically enriched or racemic. Furthermore, resolution into the desired optically active material can be carried out at any desired point in the sequence using well-known methods, such as those described herein and in the chemical literature.

[0405] Common methods: Unless otherwise stated, the variables in Schemes AK have the same meanings as defined herein.

[0406] In some cases, compounds described in general Schemes A-J or having Formula I, I(a), I(b), or I(c) may contain protecting groups, which can be added or removed at additional steps in the synthetic sequence using conditions known in the art (March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 8th Edition, or Green's Protective Groups in Organic Synthesis, 5th Edition, edited by Wuts, PGM). Compounds at each step can be purified by standard techniques, such as column chromatography, crystallization, or reverse-phase SFC or HPLC. Variables m, n, o, p, s, or t are as defined in the embodiments, schemes, examples, and claims herein.

[0407] [ka]

[0408] Compounds of formula A-4 can be prepared from spirocyclic aminodiols of general structure A-1, as depicted in Scheme A (where X can be either carbon or nitrogen). Alkylation of the amine with an appropriate alkyl halide (or equivalent alkylating agent, e.g., alkyl sulfonate ester) bearing a side-chain protected hydroxyl group can lead to compounds of formula A-2 (s=1-11). Acylation of the free hydroxyl group with an acid chloride or carboxylic acid can introduce additional molecular framework, and cleavage of the alcohol protecting group leads to compounds of formula A-4.

[0409] [ka]

[0410] Compounds of formula A-1 (X=N) used in Scheme A can be prepared from readily available spirocyclic diamines having the general formula B-1, as described in Scheme B. Reductive amination with commercially available protected dihydroxyacetone (B-2), followed by acid hydrolysis, leads to the diol. If an acid-labile nitrogen protecting group is not used, an additional deprotection step can lead to compounds of general formula A-1 (where X=N).

[0411] [ka]

[0412] Alternatively, compounds of formula A-1 used in Scheme A (where X = CH) can be prepared from spirocyclic aminoketones of general formula C-1 as described in Scheme C. Homologation to derive the vinyl diester (C-2), followed by reduction of the ester and olefin, can lead to compounds of formula C-3. Removal of the nitrogen protecting group affords compounds of formula A-1 (where X = CH).

[0413] [ka]

[0414] Compounds of formula D-6 can be prepared from spirocyclic aminodiesters of general formula D-1, as shown in Scheme D. Reduction of the ester generates diols of formula D-2, followed immediately by deprotection to afford aminodiols of general formula D-3. Alkylation of the amine with an appropriate alkyl halide (or equivalent alkylating agent, e.g., alkylsulfonate ester) bearing a side-chain protected hydroxyl group can lead to compounds of formula D-4 (s=1-11). Acylation of the free hydroxyl group with an acid chloride or carboxylic acid can introduce additional molecular framework, and cleavage of the alcohol protecting group leads to compounds of formula D-6 (s=1-11).

[0415] [ka]

[0416] Compounds of general formula E-7 can be prepared from readily available dienes of formula E-1 (t = 0-1) as shown in Scheme E. Compounds of general formula E-1 can be prepared by bis-allylation of 1,3-diones as described in Tetrahedron 2015, 71, 129; Tetrahedron Lett. 2011, 52, 4204. Spirocyclic aminoolefins of formula E-2 can be readily prepared by ring-closing metathesis of dienes. Subsequent reduction of the dione to an alkane can provide aminospirocycles of general formula E-4. Epoxidation of the cyclic olefin followed by hydrolytic ring-opening can provide spirocyclic diols with general structure E-6. Final cleavage of the nitrogen protecting group liberates spirocyclic aminodiols (t = 0-1) with general formula E-7.

[0417] [ka]

[0418] Compounds with the general structure F-3 can be readily prepared from spirocyclic aminodiols of formula E-7 (t=0-1) by the method shown in Scheme F. Alkylation of the amine with an appropriate alkyl halide (or equivalent alkylating agent, e.g., alkylsulfonate ester) bearing a side-chain protected hydroxyl group can lead to compounds of formula F-1 (s=1-11). Acylation of the free hydroxyl group with an acid chloride or carboxylic acid can introduce additional molecular framework, and cleavage of the alcohol protecting group leads to compounds of formula F-3 (s=1-11, t=0-1).

[0419] [ka]

[0420] Compounds with the general structure G-3 can be readily prepared from spirocyclic aminodiols of formula A-1 by the method shown in Scheme G. Alkylation of the amine with an appropriate alkyl halide (or equivalent alkylating agent, e.g., alkyl sulfonate ester) bearing a side-chain protected nitrogen can lead to compounds of formula G-1 (s=1-11). Acylation of the free hydroxyl groups of the diol with acid chlorides or carboxylic acids can introduce additional molecular framework, and cleavage of the nitrogen protecting group leads to compounds of formula G-3 (s=1-11).

[0421] [ka]

[0422] Additional compounds of general formula (I) can be prepared according to Scheme H (where s = 1-11). Compounds of formula G-3 can be reacted with carboxylic acids or acid chlorides to give compounds of formula H-1. Compounds of formula H-2 can be prepared by a substitution reaction between compounds of formula G-3 and commercially available 3-methoxy-4-(methylamino)cyclobut-3-ene-1,2-dione. Compounds of formula G-3 can also be reacted with sulfonyl chlorides to give compounds of general formula H-3.

[0423] [ka]

[0424] Compounds with the general structure I-3 can be readily prepared from spirocyclic aminodiols of formula E-7 (t=0-1) by the method shown in Scheme I. Alkylation of the amine with an appropriate alkyl halide (or equivalent alkylating agent, e.g., alkyl sulfonate ester) bearing a side-chain protected nitrogen can lead to compounds of formula I-1 (s=1-11). Acylation of the free hydroxyl groups of the diol with acid chlorides or carboxylic acids can introduce additional molecular frameworks, and subsequent cleavage of the nitrogen protecting group leads to compounds of formula I-3 (s=1-11, t=0-1).

[0425] [ka]

[0426] Additional compounds of general formula (I) can be prepared according to Scheme J (where s = 1-11, t = 0-1). Compounds of general formula J-1 can be prepared by a substitution reaction between I-3 and commercially available 3-methoxy-4-(methylamino)cyclobut-3-ene-1,2-dione. Compounds of formula I-3 can be reacted with a carboxylic acid or acid chloride to give compounds of formula J-2. Compounds of formula I-3 can also be reacted with a sulfonyl chloride to give compounds of general formula J-3.

[0427] [ka]

[0428] General structure R 7 Carboxylic acids of COH may be obtained from commercial sources (e.g., 2-octyldecanoic acid, 2-heptylnonanoic acid, and 2-hexyldecanoic acid), prepared by procedures known in the literature, or prepared as described in Scheme K (where n=1-20). Carboxylic acids of formula K-1, which may be obtained from commercial sources or prepared by procedures known in the literature, are reacted sequentially with one equivalent of NaH, followed by additional strong base, such as LDA. The resulting dianion is then converted to an alkyl halide RX (where X=I or Br; R=C). 1~18 Alkyl, C 3~8 Cycloalkyl, and C 3~8 C substituted by cycloalkyl 1~6 alkyl) to give carboxylic acids of formula K-2. The requisite alkyl halides RX may be obtained from commercial sources or may be prepared by procedures known in the literature. [Example]

[0429] In order that this invention may be better understood, the following examples are set forth. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

[0430] The compounds and intermediates described below were named using the naming conventions provided with ChemDraw, Version 20.1.1.125 (Perkin Elmer). The naming conventions provided with ChemDraw, Version 20.1.1.125 are well known to those skilled in the art, and are generally considered to be compatible with the IUPAC (International Union of Pure and Applied Chemistry) recommendations for organic chemistry nomenclature or the CAS index rules. Unless otherwise indicated, all reactants were commercially available without further purification or prepared using methods known in the literature.

[0431] Synthesis of carboxylic acid tails A1 7-Oxo-7-(pentadecan-8-yloxy)heptanoic acid

[0432] [ka] Pentadecan-8-ol (4.28 g, 18.7 mmol) and EDCI (3.95 g, 20.6 mmol) were added to a solution of heptanedioic acid (3.0 g, 18.7 mmol) in DCM (208 mL). DMAP (343 mg, 2.81 mmol) was added, and the reaction mixture was stirred at 45 °C. After 30 h, the mixture was partitioned between saturated NaHCO and DCM. The organic layer was dried over NaSO and concentrated in vacuo. The residue was purified by silica gel chromatography to give 7-oxo-7-(pentadecan-8-yloxy)heptanoic acid (2.07 g, 30% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 4.88-4.85 (m, 1H), 2.37 (t, J = 7.5 Hz, 2H), 2.30 (t, J = 7.5 Hz, 2H), 1.66 (h, J = 7.4 Hz, 4H), 1.55-1.51 (m, 4H), 1.43-1.37 (m, 2H), 1.26 (m, 20H), 0.87 (t, J = 6.7 Hz, 6H).

[0433] A2 4-oxo-4-(pentadecan-8-yloxy)butanoic acid

[0434] [ka] DMAP (244 mg, 2.0 mmol) and pentadecan-8-ol (2.74 g, 12.0 mmol) were added to a solution of dihydrofuran-2,5-dione (1.0 g, 10.0 mmol) in toluene (20.0 mL). Triethylamine (303 mg, 3.0 mmol) was added, and the mixture was stirred at 110 °C for 16 hours. The reaction mixture was cooled to room temperature, filtered, and concentrated in vacuo. The residue was extracted with DCM. The combined organic layers were dried over NaSO and filtered. The filtrate was concentrated in vacuo. The crude product was purified by silica gel chromatography to give 4-oxo-4-(pentadecan-8-yloxy)butanoic acid (1.20 g, 73% yield) as a white solid. 1 H NMR (400 MHz, CDCl3) δ 4.89 (p, J = 6.3 Hz, 1H), 2.73-2.57 (m, 4H), 1.52-1.49 (m, 4H), 1.27 (m, 21H), 0.88 (t, J = 6.8 Hz, 6H).

[0435] A3 6-((2-heptylnonanoyl)oxy)hexanoic acid

[0436] [ka] Step 1: 6-(tert-butoxy)-6-oxohexyl 2-heptylnonanoate DCC (9.13 g, 44.3 mmol) was added to a solution of tert-butyl 6-hydroxyhexanoate (5.0 g, 26.6 mmol), 2-heptylnonanoic acid (A12) (5.68 g, 22.1 mmol), and DMAP (1.35 g, 11.1 mmol) in dichloroethane (100 mL). The reaction mixture was stirred at 85 °C for 16 h. After that time, the mixture was filtered to remove solids (with the aid of PET ether). The filtrate was dried over NaSO and concentrated in vacuo. The residue was purified by silica gel chromatography to give 6-(tert-butoxy)-6-oxohexyl 2-heptylnonanoate (3.6 g, 32%) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 4.06 (t, J = 6.6 Hz, 2H), 2.37-2.25 (m, 1H), 2.22 (t, J = 7.4 Hz, 2H), 1.69-1.51 (m, 6H), 1.44 (s, 9H), 1.40-1.35 (m, 4H), 1.29-1.20 (m, 20H), 0.87 (t, J = 6.8 Hz, 6H).

[0437] Step 2: 6-((2-heptylnonanoyl)oxy)hexanoic acid Trifluoroacetic acid (967 mg, 8.48 mmol) was added to a solution of 6-(tert-butoxy)-6-oxohexyl 2-heptylnonanoate (3.62 g, 8.48 mmol) in DMC (25 mL), and the reaction mixture was stirred at room temperature for 2 hours. The mixture was concentrated, and the residue was purified by silica gel chromatography to give 6-((2-heptylnonanoyl)oxy)hexanoic acid as a pale yellow oil. LCMS (ESI): C 22 H 43 O4 [M+H] + Calculated value 371.3; measured value 371.4.

[0438] Representative Procedure for the Synthesis of Carboxylic Acid Tail by Alkylation of Carboxylic Acid Using Alkyl Halides

[0439] A4 2-(Cyclobutylmethyl)decanoic acid

[0440] [ka] At 0°C, a solution of decanoic acid (A18) (30.0 g, 174 mmol) in THF (500.0 mL) was added dropwise to a solution of NaH (11.5 g, 287 mmol) in THF (500.0 mL). LDA (37.3 g, 348 mmol) was then added dropwise to the mixture. The mixture was stirred at room temperature for 2.5 hours to give a light yellow suspension. To the suspension, (bromomethyl)cyclobutene (38.9 g, 261 mmol) was added dropwise. The yellow mixture immediately turned to a white suspension. The reaction mixture was stirred at 75°C for an additional 14 hours. After stirring for the specified time, the reaction mixture was quenched with HCl (2 N, 500 mL) to give a clear yellow solution. The organic layer was separated, and the aqueous layer was extracted with EtOAc (300 mL x 5). The combined organic layers were dried over Na2SO4 and filtered. The filtrate was concentrated in vacuo, and the residue was purified by silica gel column chromatography to give 2-(cyclobutylmethyl)decanoic acid (26 g, 63% yield) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 2.40-2.20 (m, 2H), 2.12-1.99 (m, 2H), 1.94-1.72 (m, 3H), 1.69-1.53 (m, 5H), 1.51-1.40 (m, 1H), 1.36-1.26 (m, 12H), 0.90 (t, J= 6.7 Hz, 3H).

[0441] The following carboxylic acids A5-A11 were prepared following the representative procedure described for A4.

[0442] [Table 1-1]

[0443] [Table 1-2]

[0444] The following carboxylic acids A12-A26 are either commercially available or readily prepared by one skilled in the art.

[0445] [Table 2-1]

[0446] [Table 2-2]

[0447] Example 1 (3-(4-hydroxybutyl)-3-azaspiro[5.5]undecane-9,9-diyl)bis(methylene)bis(2-heptylnonanoate)(1)

[0448] [ka] Step 1: tert-Butyl 9,9-bis(hydroxymethyl)-3-azaspiro[5.5]undecane-3-carboxylate To a solution of 3-(tert-butyl) 9,9-diethyl 3-azaspiro[5.5]undecane-3,9,9-tricarboxylate (6.0 g, 20 mmol) in EtOH (60 mL) at 0 °C, NaBH (3.43 g, 90.6 mmol) was added, and the mixture was stirred at this temperature for 1 h. Then, it was heated to 60 °C and stirred for 8 h. The solution was cooled to room temperature and slowly quenched with HO (10 mL) and acetone (20 mL). The mixture was then stirred at room temperature for 1 h to give a yellow precipitate. The solid was collected by filtration. The filter cake was washed with HO (3 mL × 5) and dried to give tert-butyl 9,9-bis(hydroxymethyl)-3-azaspiro[5.5]undecane-3-carboxylate (4.50 g, 90% yield) as a yellow solid. The crude product was used directly in the next step without further purification. 1H NMR (400 MHz, CD3OD) δ 3.48-3.45 (m, 4H), 3.38-3.34 (m, 4H), 1.45 (s, 9H), 1.42-1.36 (m, 12H). LCMS (ESI): C 13 H 24 NO4[M+H-tBu] + Calculated value: 258.16, measured value: 258.0.

[0449] Step 2: (3-Azaspiro[5.5]undecane-9,9-diyl)dimethanol To a solution of tert-butyl 9,9-bis(hydroxymethyl)-3-azaspiro[5.5]undecane-3-carboxylate (4.50 g, 14.36 mmol) in DCM (20 mL) was added HCl (4 M in dioxane, 20 mL, 80 mmol) at room temperature. The solution was stirred at room temperature for 1 h, the solvent was removed in vacuo, and the resulting solid was triturated with MTBE to give (3-azaspiro[5.5]undecane-9,9-diyl)dimethanol (3.0 g, 98% yield) as a yellow solid. 1 H NMR (400 MHz, CD3OD) δ 3.46 (m, 4H), 3.16-3.14 (m, 4H), 1.69 (m, 4H), 1.50-1.36 (m, 8H). LCMS (ESI): C 12 H 24 NO2[M+H] + Calculated value: 214.17, measured value: 214.1.

[0450] Step 3: (3-(4-((tert-butyldimethylsilyl)oxy)butyl)-3-azaspiro[5.5]undecane-9,9-diyl)dimethanol General Alkylation Procedure To a solution of (3-azaspiro[5.5]undecane-9,9-diyl)dimethanol (3.0 g, 10 mmol) in DMF (30 mL) and MeOH (5 mL) at room temperature, 4-bromobutoxy-tert-butyl-dimethylsilane (4.51 g, 16.9 mmol) was added, followed by K2CO3 (11.7 g, 84.4 mmol). The mixture was stirred at 70 °C for 6 h, quenched with HO (25 mL), and extracted with EtOAc (40 mL × 3). The organic layers were combined, washed with brine (20 mL × 3) to remove DMF, and concentrated in vacuo. The residual material was purified by column chromatography to give (3-(4-((tert-butyldimethylsilyl)oxy)butyl)-3-azaspiro[5.5]undecane-9,9-diyl)dimethanol (2.20 g, 40% yield) as a white solid. 1 H NMR (400 MHz, CDCl3) δ 3.59-3.56 (m, 6H), 2.67 (br s, 2H), 2.40 (br s, 5H), 1.66-1.40 (m, 8H), 1.31 (s, 8H), 0.84 (s, 9H), 0.00 (s, 6H). LCMS (ESI): C 22 H 46 NO3Si [M+H] + Calculated value: 400.32, measured value: 400.2.

[0451] Step 4: (3-(4-((tert-butyldimethylsilyl)oxy)butyl)-3-azaspiro[5.5]undecane-9,9-diyl)bis(methylene)bis(2-heptylnonanoate) General acylation procedure To a solution of (3-(4-((tert-butyldimethylsilyl)oxy)butyl)-3-azaspiro[5.5]undecane-9,9-diyl)dimethanol (2.20 g, 5.50 mmol) in DCM (30 mL) was added 2-heptylnonanoic acid (A12) (2.89 g, 11.3 mmol), DCC (2.84 g, 13.8 mmol), followed by DMAP (202 mg, 1.65 mmol). The reaction mixture was heated to reflux and stirred for 40 h. After this time, the reaction mixture was filtered over Celite and concentrated in vacuo. The residue was purified by silica gel column chromatography to give (3-(4-((tert-butyldimethylsilyl)oxy)butyl)-3-azaspiro[5.5]undecane-9,9-diyl)bis(methylene)bis(2-heptylnonanoate) (3.0 g, yield 62.2%) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 3.97 (s, 4H), 3.65-3.55 (m, 2H), 2.38-2.28 (m, 8H), 1.65-1.32 (m, 24H), 1.24 (s, 40H), 0.91-0.84 (m, 21H), 0.03 (s, 6H). LCMS (ESI): C 54 H 106 O5Si [M+H] + Calculated value: 876.78, measured value: 876.6.

[0452] Step 5: (3-(4-hydroxybutyl)-3-azaspiro[5.5]undecane-9,9-diyl)bis(methylene)bis(2-heptylnonanoate) General HCl-mediated tert-butyldimethylsilyl deprotection To a solution of (3-(4-((tert-butyldimethylsilyl)oxy)butyl)-3-azaspiro[5.5]undecane-9,9-diyl)bis(methylene)bis(2-heptylnonanoate) (3.0 g, 3.42 mmol) in DCM (15 mL) was added HCl (4 M dioxane, 25 mL, 50 mmol), and the resulting solution was stirred at room temperature for 1 h. After this time, the mixture was concentrated in vacuo, and the residue was neutralized with saturated NaHCO (60 mL). The mixture was extracted with DCM (35 mL × 4), and the combined organic layers were washed with brine (70 mL), dried over NaSO, filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography followed by additional supercritical fluid chromatography to give (3-(4-hydroxybutyl)-3-azaspiro[5.5]undecane-9,9-diyl)bis(methylene)bis(2-heptylnonanoate) (1.21 g, 46.3% yield) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 3.96 (s, 4H), 3.57-3.54 (m, 2H), 2.49-2.43 (m, 4H), 2.41-2.36 (m, 3H), 2.35-2.27 (m, 2H), 1.70-1.64 (m, 4H), 1.63-1.48 (m, 8H), 1.46-1.32 (m, 12H), 1.32-1.12 (m, 40H), 0.86 (t, J = 6.8 Hz, 12H). LCMS (ESI): C 48 H 92 O5 [M+H] + Calculated value: 762.69, measured value: 762.5.

[0453] Example 2 2-(3-(4-hydroxybutyl)-3-azaspiro[5.5]undecan-9-yl)propane-1,3-diylbis(2-heptylnonanoate)(2)

[0454] [ka] Step 1: Diethyl 2-(3-(tert-butoxycarbonyl)-3-azaspiro[5.5]undecane-9-ylidene)malonate To a flask containing THF (50.0 mL) at 0 °C, TiCl (6.26 g, 33.0 mmol) was slowly added, followed by the dropwise addition of a mixture of tert-butyl 9-oxo-3-azaspiro[5.5]undecane-3-carboxylate (4.2 g, 16 mmol), diethyl malonate (2.64 g, 16.5 mmol), and pyridine (4.97 g, 62.8 mmol) in THF (40.0 mL). The red suspension was slowly warmed to room temperature and stirred for 16 h. The mixture was then quenched with saturated NaHCO (120 mL) until a clear orange liquid was obtained. The aqueous phase was extracted with ethyl acetate (60 mL × 5), and the combined organic phases were dried over NaSO and concentrated in vacuo. Purification by column chromatography gave diethyl 2-(3-(tert-butoxycarbonyl)-3-azaspiro[5.5]undecan-9-ylidene)malonate (4.16 g, 65% yield) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 4.22 (q, J = 7.1 Hz, 4H), 3.41-3.34 (m, 4H), 2.58-2.50 (m, 4H), 1.62-1.54 (m, 4H), 1.44 (br s, 13H), 1.28 (t, J = 7.1 Hz, 6H). LCMS (ESI): C 18 H 28 No. 6 [M-tBu+H] + Calculated value: 354.2; measured value: 354.1.

[0455] Step 2: tert-Butyl 9-(1,3-dihydroxypropan-2-yl)-3-azaspiro[5.5]undecane-3-carboxylate To a solution of diethyl 2-(3-(tert-butoxycarbonyl)-3-azaspiro[5.5]undecan-9-ylidene)malonate (6.40 g, 15.63 mmol) in EtOH (100.0 mL) was added NaBH4 (5.0 g, 132.2 mmol), and the mixture was stirred for 4 h at room temperature, then heated to 65 °C and stirred for an additional 12 h. At this point, the mixture was slowly quenched with saturated NH4Cl (100 mL) and then concentrated in vacuo to remove EtOH. The resulting aqueous layer was extracted with DCM (50 mL × 5). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. Purification using silica gel column chromatography gave tert-butyl 9-(1,3-dihydroxypropan-2-yl)-3-azaspiro[5.5]undecane-3-carboxylate (3.10 g, 60.7% yield) as a colorless solid. 1 H NMR (400 MHz, CDCl3) δ 4.06 (dd, J = 10.8, 4.5 Hz, 1H), 3.89-3.66 (m, 3H), 3.36-3.29 (m, 4H), 1.82-1.48 (m, 5H), 1.44 (s, 11H), 1.31-0.99 (m, 7H). LCMS (ESI): C 18 H 34 No. 4 [M+H] + Calculated value: 328.2; measured value: 328.1.

[0456] Step 3: 2-(3-azaspiro[5.5]undecan-9-yl)propane-1,3-diol To a solution of tert-butyl 9-(1,3-dihydroxypropan-2-yl)-3-azaspiro[5.5]undecane-3-carboxylate (3.10 g, 9.48 mmol) in MeOH (25.0 mL) at room temperature, 2 M HCl (25.0 mL, 50.0 mmol) was added, and the mixture was stirred at room temperature for 14 hours. The mixture was concentrated in vacuo to give 2-(3-azaspiro[5.5]undecan-9-yl)propane-1,3-diol (2.5 g, 99.9%, crude) as a white solid. The material was used directly in subsequent transformations. LCMS (ESI): C 13 H 26 NO2[M+H] + Calculated value: 228.2; measured value: 228.1.

[0457] Step 4: 2-(3-(4-((tert-butyldimethylsilyl)oxy)butyl)-3-azaspiro[5.5]undecan-9-yl)propane-1,3-diol This compound was prepared according to the general alkylation procedure as described in Example 1 (Step 3). 2-(3-Azaspiro[5.5]undecan-9-yl)propane-1,3-diol (2.50 g, 9.48 mmol) gave 2-(3-(4-((tert-butyldimethylsilyl)oxy)butyl)-3-azaspiro[5.5]undecan-9-yl)propane-1,3-diol (3.02 g, 77% yield) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 3.90-3.69 (m, 4H), 3.61-3.58 (t, J = 5.9 Hz, 2H), 2.39-2.31 (m, 6H), 1.68-1.63 (m, 2H), 1.60-1.42 (m, 9H), 1.40-1.32 (m, 3H), 1.25-0.96 (m, 4H), 0.87 (s, 9H), 0.02 (s, 6H). LCMS (ESI): C 23 H 48 NO3Si [M+H] + Calculated value: 414.3; Measured value: 414.3.

[0458] Step 5: 2-(3-(4-((tert-butyldimethylsilyl)oxy)butyl)-3-azaspiro[5.5]undecan-9-yl)propane-1,3-diylbis(2-heptylnonanoate) This compound was prepared according to the general acylation procedure as described in Example 1 (Step 4). 2-(3-(4-((tert-butyldimethylsilyl)oxy)butyl)-3-azaspiro[5.5]undecan-9-yl)propane-1,3-diol (3.02 g, 7.30 mmol) gave 2-(3-(4-((tert-butyldimethylsilyl)oxy)butyl)-3-azaspiro[5.5]undecan-9-yl)propane-1,3-diylbis(2-heptylnonanoate) (4.97 g, 76.4% yield) as a pale yellow oil.

[0459] Step 6: 2-(3-(4-hydroxybutyl)-3-azaspiro[5.5]undecan-9-yl)propane-1,3-diylbis(2-heptylnonanoate) This compound was prepared according to the general tert-butyldimethylsilyl deprotection procedure as described in Example 1 (Step 5). 2-(3-(4-((tert-butyldimethylsilyl)oxy)butyl)-3-azaspiro[5.5]undecan-9-yl)propane-1,3-diylbis(2-heptylnonanoate) (4.97 g, 5.58 mmol) gave 2-(3-(4-hydroxybutyl)-3-azaspiro[5.5]undecan-9-yl)propane-1,3-diylbis(2-heptylnonanoate) (1.70 g, 39.3% yield) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 4.18 (dd, J = 11.2, 4.9 Hz, 2H), 4.04 (dd, J = 11.2, 6.3 Hz, 2H), 3.56 (m, 2H), 2.57-2.37 (m, 6H), 2.34-2.25 (m, 2H), 1.83-1.79 (m, 1H), 1.74-1.63 (m, 6H), 1.61-1.52 (m, 7H), 1.46-1.36 (m, 7H), 1.24 (br s, 43H), 1.06-0.99 (m, 2H), 0.86 (t, J = 6.7 Hz, 12H). LCMS (ESI): C 49 H 94 No. 5 [M+H] + Calculated value: 776.7; measured value: 776.9.

[0460] Example 3 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate)(3)

[0461] [ka] Step 1: tert-butyl 9-(2,2-dimethyl-1,3-dioxan-5-yl)-3,9-diazaspiro[5.5]undecane-3-carboxylate General reductive amination procedure To a solution of tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate hydrochloride (9.13 g, 31.39 mmol) in DCM (150 mL) was added EtN (3.18 g, 31.4 mmol). After stirring at room temperature for 10 minutes, 2,2-dimethyl-1,3-dioxan-5-one (4.09 g, 31.4 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. The mixture was cooled to 0 °C, and Na(OAc)BH (6.65 g, 31.4 mmol) was slowly added. The mixture was warmed to room temperature and stirred for an additional 26 hours. At this point, LCMS showed the desired product as the major peak, and the reaction was quenched with saturated NaHCO until no more gas was produced. The mixture was extracted with DCM (50 mL × 5), and the combined organic layers were dried over NaSO, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography to give tert-butyl 9-(2,2-dimethyl-1,3-dioxan-5-yl)-3,9-diazaspiro[5.5]undecane-3-carboxylate (8.28 g, yield 71.6%) as a pale yellow solid. 1 H NMR (400 MHz, CDCl3) δ 3.96 (dd, J = 11.7, 5.2 Hz, 2H), 3.81 (dd, J = 11.6, 8.4 Hz, 2H), 3.42-3.31 (m, 5H), 2.65-2.48 (m, 5H), 1.57-1.26 (m, 24H). LCMS (ESI): C 20 H 37 N2O4 [M+H] + Calculated value: 369.27, measured value: 370.3.

[0462] Step 2: 2-(3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diol General acetal hydrolysis / boc deprotection procedure To a solution of tert-butyl 9-(2,2-dimethyl-1,3-dioxan-5-yl)-3,9-diazaspiro[5.5]undecane-3-carboxylate (8.28 g, 22.46 mmol) in MeOH (30 mL) was added aqueous HCl (30 mL, 30 mmol), and the mixture was stirred at room temperature for 16 hours. The mixture was dried under lyophilization to give 2-(3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diol (5.13 g, 100%, crude) as a yellow solid. The material was used as is in the subsequent transformation. LCMS (ESI): C 12 H 25 N2O2 [M+H] + The calculated value was 229.18 and the measured value was 229.2.

[0463] Step 3: 2-(9-(4-((tert-butyldimethylsilyl)oxy)butyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diol This compound was prepared according to the general alkylation procedure as described in Example 1 (Step 3). 2-(3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diol (5.03 g, 22.03 mmol) gave 2-(9-(4-((tert-butyldimethylsilyl)oxy)butyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diol (2.25 g, 25% yield) as a yellow solid. 1 H NMR (400 MHz, CDCl3) δ 3.63-3.59 (m, 6H), 2.76 (p, J = 6.5 Hz, 1H), 2.66 (t, J = 5.5 Hz, 4H), 2.44-2.32 (m, 6H), 1.55-1.48 (m, 13H), 0.88 (s, 9H), 0.04 (s, 6H). LCMS (ESI): C 22 H 47 N2O3Si [M+H] + Calculated value: 415.33, measured value: 415.3.

[0464] Step 4: 2-(9-(4-((tert-butyldimethylsilyl)oxy)butyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate) This compound was prepared according to the general acylation procedure as described in Example 1 (Step 4). 2-(9-(4-((tert-butyldimethylsilyl)oxy)butyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diol (1.88 g, 4.53 mmol) and 2-heptylnonanoic acid (A12) (2.56 g, 9.97 mmol) gave 2-(9-(4-((tert-butyldimethylsilyl)oxy)butyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate) (3.55 g, 87.9% yield) as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ 4.29 (dd, J = 11.5, 6.3 Hz, 2H), 4.07 (dd, J = 11.5, 5.5 Hz, 2H), 3.60 (t, J = 6.2 Hz, 2H), 2.98 (p, J = 5.8 Hz, 1H), 2.60 (t, J = 5.3 Hz, 4H), 2.55-2.40 (m, 6H), 2.36-2.28 (m, 2H), 1.69-1.35 (m, 20H), 1.26 (d, J = 8.6 Hz, 40H), 0.91-0.83 (m, 21H), 0.03 (s, 6H).

[0465] Step 5: 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate) This compound was prepared according to the general tert-butyldimethylsilyl deprotection procedure as described in Example 1 (Step 5). 2-(9-(4-((tert-butyldimethylsilyl)oxy)butyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate) (3.55 g, 3.98 mmol) gave 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptyl-nonanoate) (1.64 g, 53% yield) as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ 4.29 (dd, J = 11.5, 6.3 Hz, 2H), 4.06 (dd, J = 11.5, 5.5 Hz, 2H), 3.56-3.35 (m, 2H), 2.98-2.95 (m, 1H), 2.60-2.57 (m, 4H), 2.52-2.37 (m, 7H), 2.35-2.28 (m, 2H), 1.68-1.64 (m, 5H), 1.63-1.49 (m, 7H), 1.47-1.37 (m, 8H), 1.27-1.23 (m, 40H), 0.87 (t, J = 6.7 Hz, 12H). LCMS (ESI): C 48 H 93 N2O5 [M+H] + Calculated value: 777.70, measured value: 777.7.

[0466] Example 4 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-cyclobutyldecanoate)(4)

[0467] [ka] Step 1: 2-(9-(4-((tert-butyldimethylsilyl)oxy)butyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-cyclobutyldecanoate) This compound was prepared according to the general acylation procedure as described in Example 1 (Step 4). 2-(9-(4-((tert-butyldimethylsilyl)oxy)butyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diol ((Example 3, Step 3)) (0.500 g, 1.21 mmol) and 2-cyclobutyldecanoic acid (A5) (0.764 g, 3.38 mmol) gave 2-(9-(4-((tert-butyldimethylsilyl)oxy)butyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-cyclobutyldecanoate) (0.95 g, 95% yield) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 4.29 (dd, J= 11.6, 6.0 Hz, 2H), 4.08 (dd, J=11.5, 5.5 Hz, 2H), 3.62 (t, J= 6.1 Hz, 2H), 2.99-2.93 (m, 1H), 2.61 (t, J= 5.5 Hz, 4H), 2.53-2.40 (m, 8H), 2.37-2.34 (m, 3H), 2.11-1.92 (m, 5H),1.90-1.64(m, 10H), 1.62-1.36 (m, 16H), 1.33-1.20 (m, 20H), 0.92-0.86 (m, 15H), 0.06 (s, 6H).

[0468] Step 2: 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-cyclobutyldecanoate) General TBAF-mediated tert-butyldimethylsilyl deprotection procedure TBAF (1.81 g, 6.92 mmol) was added to a solution of 2-(9-(4-((tert-butyldimethylsilyl)-oxy)butyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-cyclobutyldecanoate) (1.15 g, 1.38 mmol) in THF (12.0 mL). The mixture was stirred at room temperature for 3 hours. The reaction mixture was diluted with EtOAc (10 mL) and washed with HO (15 mL × 3) and brine (15 mL × 2). The combined organic layers were dried over NaSO and filtered. The filtrate was concentrated in vacuo, and the residue was purified by silica gel column chromatography to give 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-cyclobutyl-decanoate) (0.35 g, 35% yield) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 4.29 (dd, J= 11.6, 6.1 Hz, 2H), 4.07 (dd, J=11.5, 5.5 Hz, 2H), 3.64-3.58 (m, 2H), 2.96 (t, J= 5.9 Hz, 1H), 2.65-2.51 (m, 6H), 2.49-2.39 (m, 3H), 2.32 (td, J= 9.8, 4.2 Hz, 3H), 2.12-2.02 (m, 2H), 2.01-1.59 (m, 21H), 1.54-1.37 (m, 8H), 1.33-1.21 (m, 24H), 0.89 (t, J= 6.7 Hz, 6H). LCMS (ESI): C 44 H 81 N2O5 [M+H] + Calculated value: 717.61, measured value: 717.4.

[0469] Example 5 2-(9-(5-hydroxypentyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-hexyldecanoate) (5)

[0470] [ka] Step 1: 2-(9-(5-((tert-butyldimethylsilyl)oxy)pentyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diol K2CO3 (11.7 g, 84.4 mmol) was added to a solution of 2-(3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diol (Example 3, Step 2) (3.0 g, 10 mmol) in DMF (50 mL) and MeOH (15 mL), and the mixture was stirred for 30 min. ((5-bromopentyl)oxy)(tert-butyl)dimethylsilane (2.8 g, 9.95 mmol) was added. The reaction mixture was stirred at 65 °C for 16 h, then The mixture was filtered. The filtrate was concentrated in vacuo. The residue was dissolved in EtOAc (30 mL) and washed with HO (20 mL × 4). The organic layer was dried over NaSO and filtered. The filtrate was concentrated in vacuo and purified by column chromatography to give 2-(9-(5-((tert-butyldimethylsilyl)oxy)pentyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diol (1.75 g, 40% yield) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 3.69-3.58 (m, 6H), 2.82 (p, J = 6.4 Hz, 1H), 2.72 (t, J = 5.5 Hz, 4H), 2.58-2.43 (m, 6H), 1.68-1.51 (m, 12H), 1.36 (q, J = 7.9 Hz, 2H), 0.90 (s, 9H), 0.06 (s, 6H). LCMS (ESI): C 23 H 49 N2O3Si [M+H] + Calculated value: 429.35, measured value: 429.3.

[0471] Step 2: 2-(9-(5-((tert-butyldimethylsilyl)oxy)pentyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-hexyldecanoate) This compound was prepared according to the general acylation procedure as described in Example 1 (Step 4). 2-(9-(5-((tert-butyldimethylsilyl)oxy)pentyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diol (0.800 g, 1.87 mmol) and 2-hexyldecanoic acid (A13) (1.20 g, 3.18 mmol) gave 2-(9-(5-((tert-butyldimethylsilyl)oxy)pentyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-hexyldecanoate) (1.0 g, 59% yield) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 4.31 (dd, J= 11.5, 6.3 Hz, 2H), 4.08 (dd, J= 11.5, 5.5 Hz, 2H), 3.60 (t, J= 6.4 Hz, 2H), 3.00 (q, J= 5.9 Hz, 1H), 2.65-2.48 (m, 8H), 2.40-2.22 (m, 3H), 1.98-1.91 (m, 1H), 1.74-1.40 (m, 24H), 1.36-1.19 (m, 38H), 0.92-0.84 (m, 21H), 0.05 (s, 6H).

[0472] Step 3: 2-(9-(5-hydroxypentyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-hexyldecanoate) This compound was prepared following the general tert-butyldimethylsilyl deprotection using TBAF as described in Example 5 (Step 2). 2-(9-(5-((tert-butyldimethylsilyl)oxy)pentyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-hexyldecanoate) (0.950 g, 1.05 mmol) gave 2-(9-(5-hydroxypentyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-hexyldecanoate) (0.13 g, 15% yield) as a pale yellow oil. 1 H NMR (400 MHz, CD3OD) δ 4.32 (ddd, J= 11.6, 5.9, 2.0 Hz, 2H), 4.16 (ddd, J=11.5, 5.5 1.6 Hz, 2H), 3.57 (t, J= 6.4 Hz, 2H), 3.02 (q, J= 5.8 Hz, 1H), 2.69-2.67 (m, 8H), 2.58 (m, 2H), 2.37 (tt, J= 9.3, 5.1 Hz, 2H), 1.70-1.53 ​​(m, 16H), 1.51-1.22 (m, 6H), 1.36-1.22 (m, 40H), 0.88 (t, J= 6.7 Hz, 12H). LCMS (ESI): C 49 H 95 N2O5 [M+H] + Calculated value: 791.72, measured value: 791.7.

[0473] Example 6 2-(2-(4-hydroxybutyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-hexyldecanoate) (6)

[0474] [ka] Step 1: tert-butyl 8-(2,2-dimethyl-1,3-dioxan-5-yl)-2,8-diazaspiro[4.5]decane-2-carboxylate This compound was prepared according to the general reductive amination procedure as described in Example 3 (Step 1). tert-Butyl 2,8-diazaspiro[4.5]decane-2-carboxylate (5.00 g, 18.1 mmol) gave tert-butyl 8-(2,2-dimethyl-1,3-dioxan-5-yl)-2,8-diazaspiro[4.5]decane-2-carboxylate (4.1 g, 64% yield) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 3.96 (dt, J= 11.8, 5.9 Hz, 2H), 3.82 (dt, J= 11.5, 7.2 Hz, 2H), 3.35 (dt, J = 21.8, 7.2 Hz, 2H), 3.18 (s, 1H), 3.08 (s, 1H), 2.65-2.55 (m, 3H), 2.51-2.41 (m, 2H), 1.72-1.63 (m, 3H), 1.59-1.50 (m, 3H), 1.48-1.35 (m, 15H). LCMS (ESI): C 19 H 35 N2O4 [M+H] + Calculated value: 355.25; measured value: 355.1.

[0475] Step 2: 2-(2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diol To a solution of tert-butyl 8-(2,2-dimethyl-1,3-dioxan-5-yl)-2,8-diazaspiro[4.5]decane-2-carboxylate (4.10 g, 11.6 mmol) in MeOH (50 mL) was added HCl (0.12 M, 50 mL). The mixture was stirred at 20 °C for 16 h. The mixture was dried under lyophilization and the solvent was removed to give 2-(2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diol (3.32 g, 100% yield) as a yellow oil. LCMS (ESI): C 11 H 23 N2O2 [M+H] + Calculated value: 215.17; measured value: 215.1.

[0476] Step 3: 2-(2-(4-((tert-butyldimethylsilyl)oxy)butyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diol This compound was prepared according to the general alkylation procedure as described in Example 1 (Step 3). 2-(2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diol (3.32 g, 11.6 mmol) and 1-bromo-4-(t-butyldimethylsilyloxy)butane (3.09 g, 11.6 mmol) gave 2-(2-(4-((tert-butyldimethylsilyl)oxy)butyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diol (2.7 g, 59% yield) as a pale yellow oil. LCMS (ESI): C 21 H 45 N2O3Si [M+H] + Calculated value: 401.31; measured value: 401.2.

[0477] Step 4: 2-(2-(4-((tert-butyldimethylsilyl)oxy)butyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-hexyldecanoate) This compound was prepared according to the general acylation procedure as described in Example 1 (Step 4). 2-(2-(4-((tert-butyldimethylsilyl)oxy)butyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diol (0.500 g, 1.25 mmol) and 2-hexyldecanoic acid (A13) (0.800 g, 3.12 mmol) gave 2-(2-(4-((tert-butyldimethylsilyl)oxy)butyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-hexyldecanoate) (0.85 g, 78% yield) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 4.31 (dd, J= 11.5, 6.3 Hz, 2H), 4.07 (dd, J= 11.5, 5.5 Hz, 2H), 3.65-3.60 (m, 2H), 2.99 (p, J= 5.9 Hz, 1H), 2.63-2.56 (m, 6H), 2.47-2.29 (m, 6H), 1.64-1.52 (m, 14H), 1.49-1.39 (m, 5H), 1.34- 1.21 (m, 39H), 0.93-0.85 (m, 21H), 0.06 (s, 6H).

[0478] Step 5: 2-(2-(4-hydroxybutyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-hexyldecanoate) This compound was prepared following the general tert-butyldimethylsilyl deprotection using TBAF as described in Example 5 (Step 2). 2-(2-(4-((tert-butyldimethylsilyl)oxy)butyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-hexyldecanoate) (0.800 g, 0.912 mmol) gave 2-(2-(4-hydroxybutyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-hexyldecanoate) (0.17 g, 25% yield) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 4.30 (dd, J= 11.5, 6.4 Hz, 2H), 4.05 (dd, J= 11.5, 5.5 Hz, 2H), 3.64-3.59 (m, 2H), 3.00 (p, J= 5.9 Hz, 1H), 2.84-2.73 (br s, 1H), 2.61 (t, J= 5.4 Hz, 6H), 2.34 (tt, J= 8.6, 5.4 Hz, 2H), 1.80-1.67 (m, 6H), 1.65-1.53 (m, 8H), 1.50-1.38 (m, 5H), 1.34-1.02 (m, 43H), 0.89 (t, J= 6.6 Hz, 12H). LCMS (ESI): C 47 H 91 N2O5[M+H] + Calculated value: 763.68, measured value: 763.5.

[0479] Example 7 3-((2-hexyldecanoyl)oxy)-2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl palmitate (7)

[0480] [ka] Step 1: 2-(9-(4-((tert-butyldimethylsilyl)oxy)butyl)-3,9-diazaspiro[5.5]undecan-3-yl)-3-hydroxypropyl 2-hexyldecanoate DCC (1.49 g, 7.23 mmol), DMAP (0.118 g, 0.965 mmol), and 2-hexyldecanoic acid (A13) (1.30 g, 5.06 mmol) were dissolved in 2-(9-(4-((tert-butyldimethylsilyl)oxy)butyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diol (Example 3, Step 3) (2.0 g, 4.8 mmol) in DCM (60.3 mL). ), and the mixture was stirred at room temperature for 16 hours. The reaction mixture was filtered through Celite. The filtrate was concentrated in vacuo, and the residue was purified by silica gel column chromatography to give 2-(9-(4-((tert-butyldimethylsilyl)oxy)butyl)-3,9-diazaspiro[5.5]undecan-3-yl)-3-hydroxypropyl 2-hexyldecanoate (2.6 g, 84% yield) as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ 4.25 (dd, J= 11.6, 6.4 Hz, 1H), 4.01 (dd, J=11.7, 5.6 Hz, 1H), 3.66-3.60 (m, 2H), 3.52 (dd, J= 10.4, 5.1 Hz, 1H), 3.39 (t, J= 10.3 Hz, 1H), 2.96 (dq, J= 11.4, 5.9 Hz, 1H), 2.83-2.74 (m, 2H), 2.52-2.29 (m, 9H), 1.63-1.41 (m, 17H), 1.35-1.19 (m, 20H), 0.93-0.86 (m, 15H), 0.05 (s, J= 1.3 Hz, 6H).

[0481] Step 2: 2-(9-(4-((tert-butyldimethylsilyl)oxy)butyl)-3,9-diazaspiro[5.5]undecan-3-yl)-3-((2-hexyldecanoyl)oxy)propyl palmitate DCC (0.237 g, 1.15 mmol), DMAP (0.019 g, 0.153 mmol), and palmitic acid (A17) (0.245 g, 0.957 mmol) were added to a solution of 2-(9-(4-((tert-butyldimethylsilyl)oxy)butyl)-3,9-diazaspiro[5.5]undecan-3-yl)-3-hydroxypropyl 2-hexyldecanoate (0.500 g, 0.766 mmol) in DCM (3.83 mL), and the mixture was stirred at room temperature for 5 h. The reaction mixture was filtered over Celite. The filtrate was concentrated in vacuo, and the residue was purified by silica gel column chromatography to give 2-(9-(4-((tert-butyldimethylsilyl)oxy)butyl)-3,9-diazaspiro[5.5]undecan-3-yl)-3-((2-hexyldecanoyl)oxy)propyl palmitate (0.54 g, 79% yield) as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ 4.30 (ddd, J= 10.9, 6.2, 4.2 Hz, 2H), 4.11 (ddd, J=11.7, 5.7, 2.7 Hz, 2H), 3.63 (t, J= 5.9 Hz, 2H), 3.05-2.96 (m, 2H), 2.61 (t, J= 5.4 Hz, 4H), 2.48-2.24 (m, 12H), 1.67-1.39 (m, 20H), 1.36-1.20 (m, 38H), 0.94-0.86 (m, 18H), 0.06 (s, 6H).

[0482] Step 3: 3-((2-hexyldecanoyl)oxy)-2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl palmitate TBAF (1.17 g, 4.85 mmol) was added to a solution of 2-(9-(4-((tert-butyldimethylsilyl)-oxy)butyl)-3,9-diazaspiro[5.5]undecan-3-yl)-3-((2-hexyldecanoyl)oxy)propyl palmitate (0.540 g, 0.606 mmol) in THF (3.0 mL). The mixture was stirred at room temperature for 3 hours. The above sequence was repeated with an additional batch of 2-(9-(4-((tert-butyldimethylsilyl)-oxy)butyl)-3,9-diazaspiro[5.5]undecan-3-yl)-3-((2-hexyldecanoyl)oxy)propyl palmitate (0.300 g, 0.337 mmol), and the reaction mixtures were combined. The combined reaction mixture was washed with HO (20 mL) and extracted with EtOAc (10 mL × 3). The combined organic layers were dried over NaSO and filtered. The filtrate was concentrated in vacuo, and the residue was purified by silica gel column chromatography to give 3-((2-hexyldecanoyl)oxy)-2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl palmitate (0.21 g, 42% yield) as a colorless oil. 1 H NMR (400 MHz, CD3OD) δ 4.36-4.27 (m, 2H), 4.21-4.11 (m, 2H), 3.56 (t, J= 5.9 Hz, 2H), 3.05-2.98 (m, 1H), 2.67 (t, J= 5.6 Hz, 4H), 2.58-2.51 (m, 4H), 2.45 (t, J=7.2 Hz, 2H), 2.41-2.29 (m, 3H),1.69-1.42 (m, 18H), 1.40-1.20 (m, 44H), 0.90 (t, J= 6.7 Hz, 9H). LCMS (ESI): C 48 H 93 N2O5 [M+H] + Calculated value: 777.70, measured value: 777.5.

[0483] Example 8 2-(9-(3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate) (8)

[0484] [ka] Step 1: tert-butyl (3-(9-(1,3-dihydroxypropan-2-yl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl)carbamate General alkylation procedure using 3-(boc-amino)propyl bromide K2CO3 (4.5 g, 33 mmol) was added to a solution of 2-(3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diol (Example 3, Step 2) (1.63 g, 5.43 mmol) in DMF (60 mL) and MeOH (3.0 mL). The mixture was stirred at room temperature for 1 h. 3-(Boc-amino)propyl bromide) (1.29 g, 5.43 mmol) was added. The mixture was stirred at 65 °C for an additional 3 h. The mixture was filtered, and the filtrate was concentrated in vacuo. The residue was partitioned between DCM / HO (50 mL each). The organic layer was washed with brin...

Claims

1. Formula (I) 【Chemistry 1】 or a pharmaceutically acceptable salt, N-oxide, tautomer, or stereoisomer thereof [In the formula, m, n, o, and p each independently represent 1 to 3; G １ is C １～１２ Alkylene or C ２～１２ is alkenylene, R １ is -N(R ２ ) R ３ , -OR ４ , CN, -N(R ４ ) (heteroaryl), —O(CH ２ ) ｑ OH, -(OCH ２ CH ２ ) ｒ OH, -OC(=O)R ５ , -N(R ４ ) C(=O)R ５ , -N(R ４ ) S (O) ２ R ５ , -N(R ４ )C(=O)N(R ２ ) R ３ , -OC(=O)N(R ２ ) R ３ , -N(R ４ ) C(=O) OR ５ , -N(R ４ ) C(=S)N(R ２ ) R ３ , -N(R ４ ) C(=NR ６ ) N (R ２ ) R ３ ,or 【Chemistry 2】 and R ２ and R ３ are each independently H, C １～６ Alkyl, C ３～８ cycloalkyl, or aryl, or R ２ and R ３ form a heterocyclic ring together with the nitrogen atom to which they are attached, R ４ is H, C １～６ Alkyl or C ３～８ is cycloalkyl, R ５ is C １～６ Alkyl or C １～６ C optionally substituted by alkyl ３～８ is cycloalkyl, R ６ H, CN, NO ２ , C １～６ Alkyl, OR ５ , S(O) ２ R ５ , or S(O) ２ N (R ２ ) R ３ and q is 2 to 6 r is 1 to 6; W is 【Transformation 3】 and X is N or CH; G ２ and G ３ are each independently C １～１２ Alkylene or C ２～１２ is alkenylene, L １ and L ２ are each independently —C(═O)OR ７ , -OC(=O)R ７ , -OC(=O)(CH ２ ) ｒ C(=O)OR ７ , -OC(=O)(CH ２ ) ｒ OC(=O)R ７ , -OC(=O)N(R ４ ) R ７ , -N(R ４ ) C(=O) OR ７ , -N(R ４ )C(=O)N(R ４ ) R ７ , -OC(=O)OR ７ , or -S-SR ７ and R ７ is C ６～２４ Alkyl, C ６～２４ alkenyl, or C ６～２４ alkynyl, each of which is F, C １～６ Alkoxy, C ３～８ cycloalkyl, or C ３～８ optionally substituted by cycloalkenyl; L １ and L ２ R ７ may be the same or different].

2. Formula (Ia) 【Chemistry 4】 or a pharmaceutically acceptable salt, N-oxide, tautomer, or stereoisomer thereof. [In the formula, m, n, o, and p each independently represent 1 or 2].

3. Formula (Ib) 【Transformation 5】 or a pharmaceutically acceptable salt, N-oxide, tautomer, or stereoisomer thereof. [In the formula, m, n, o, and p each independently represent 1 or 2].

4. Formula (Ic) 【Transformation 6】 or a pharmaceutically acceptable salt, N-oxide, tautomer, or stereoisomer thereof. wherein m and n each independently represent 1 or 2; o and p are each 1.

5. G １ But C １～１２ is alkylene, R １ is —OH or 【Transformation 7】 and R ２ and R ３ are each independently H, C １～６ Alkyl or C ３～８ cycloalkyl or R ２ and R ３ form a heterocyclic ring together with the nitrogen atom to which they are attached, R ４ But H, C １～６ Alkyl or C ３～８ is cycloalkyl, G ２ and G ３ are each independently, C １～１２ is alkylene, L １ and L ２ are -OC(=O)R ７ and R ７ are F and C respectively. １～６ Alkoxy, C ３～８ cycloalkyl, or C ３～８ C optionally substituted by cycloalkenyl ６～２４ Alkyl, C ６～２４ alkenyl, or C ６～２４ alkynyl, and L １ and L ２ R ７ may be the same or different.

6. R ７ but the following structure: 【Transformation 8】 6. The compound of claim 1, wherein

7. R １ The compound according to any one of claims 1 to 6, wherein is OH.

8. R １ but, 【Chemistry 9】 7. The compound of claim 1, wherein

9. G １ But C ２ ~C ５ 9. The compound of any one of claims 1 to 8, which is alkylene.

10. G １ But C ３ ~C ５ 9. The compound of any one of claims 1 to 8, which is alkylene.

11. (3-(4-hydroxybutyl)-3-azaspiro[5.5]undecane-9,9-diyl)bis(methylene)bis(2-heptylnonanoate); 2-(3-(4-hydroxybutyl)-3-azaspiro[5.5]undecan-9-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-cyclobutyldecanoate); 2-(9-(5-hydroxypentyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(2-(4-hydroxybutyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-hexyldecanoate); 3-((2-hexyldecanoyl)oxy)-2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl palmitate; 2-(9-(3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(3-(1-methylcyclopropane-1-carboxamido)propyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(9-(3-hydroxypropyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(3-(ethylsulfonamido)propyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(7-(4-hydroxybutyl)-2,7-diazaspiro[4.4]nonan-2-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(7-(4-hydroxybutyl)-2,7-diazaspiro[3.5]nonan-2-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(8-(4-hydroxybutyl)-2,8-diazaspiro[4.5]decan-2-yl)propane-1,3-diylbis(2-heptylnonanoate); 3-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)pentane-1,5-diylbis(2-hexyldecanoate); 2-(7-(5-hydroxypentyl)-2,7-diazaspiro[4.4]nonan-2-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(2-(5-hydroxypentyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(7-(5-hydroxypentyl)-2,7-diazaspiro[3.5]nonan-2-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)pentane-1,5-diylbis(2-hexyldecanoate); 2-(2-(4-hydroxybutyl)-2,7-diazaspiro[3.5]nonan-7-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(5-hydroxypentan-2-yl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(2-(4-hydroxybutyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-octyldecanoate); 2-(9-(5-hydroxypentyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptyldecanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-butyldecanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(3-hexylundecanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-pentyldecanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-(cyclobutylmethyl)decanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptyltetradecanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-(cyclopentylmethyl)decanoate); 2-(7-(4-hydroxybutyl)-2,7-diazaspiro[4.4]nonan-2-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(7-(4-hydroxybutyl)-2,7-diazaspiro[3.5]nonan-2-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-(cyclopent-3-en-1-ylmethyl)decanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-(2-cyclobutylethyl)decanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-(cyclohexylmethyl)decanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(4,5-dibutylnonanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(3,3-dibutylnonanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(4-heptylundecanoate); 2-(7-(5-hydroxypentyl)-2,7-diazaspiro[4.4]nonan-2-yl)propane-1,3-diylbis(2-heptylnonanoate); 3-(decanoyloxy)-2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl 2-hexyldecanoate; 3-((2-hexyldecanoyl)oxy)-2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl (Z)-dodec-5-enoate; 3-((2-(cyclobutylmethyl)decanoyl)oxy)-2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl 2-hexyldecanoate; 3-((2-hexyldecanoyl)oxy)-2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl 2-butylundecanoate; 3-((4,5-dibutylnonanoyl)oxy)-2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl palmitate; 3-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)pentane-1,5-diylbis(2-heptylnonanoate); 2-(2-(5-hydroxypentyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(7-(5-hydroxypentyl)-2,7-diazaspiro[3.5]nonan-2-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(2-(4-hydroxybutyl)-2,7-diazaspiro[3.5]nonan-7-yl)propane-1,3-diylbis(2-hexyldecanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate); rac-(2R,3R)-8-(2-hydroxyethyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate); rac-(7R,8R)-2-(3-hydroxypropyl)-2-azaspiro[4.4]nonane-7,8-diylbis(2-heptylnonanoate); rac-(2R,3R)-8-(3-hydroxypropyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate); rac-(2R,3R)-8-(3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate); rac-(2R,3R)-8-(5-hydroxypentyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate); (2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate); (2S,3S)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-octyldecanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptyltetradecanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-hexyldecanoate); rac-O,O'-((2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diyl)di(pentadecan-8-yl)disuccinate; rac-O' １ , O １ -((2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diyl)7,7'-di(pentadecan-8-yl)di(heptanedioate); rac-(((2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diyl)bis(oxy))bis(6-oxohexane-6,1-diyl)bis(2-heptylnonanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-pentyldecanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptyldecanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-hexylundecanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-hexylnonanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-butyldecanoate); rac-(2R,3R)-3-((2-ethylnonanoyl)oxy)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decan-2-yl 2-hexyldecanoate; and Bis(3-pentyloctyl) 3-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)pentanedioate; 2. The compound of claim 1, selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

12. 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(7-(4-hydroxybutyl)-2,7-diazaspiro[3.5]nonan-2-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(2-(4-hydroxybutyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(9-(5-hydroxypentyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate); rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-hexyldecanoate); 2-(2-(4-hydroxybutyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(9-(3-hydroxypropyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate); 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(3-hexylundecanoate); 2-(7-(4-hydroxybutyl)-2,7-diazaspiro[4.4]nonan-2-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(7-(5-hydroxypentyl)-2,7-diazaspiro[3.5]nonan-2-yl)propane-1,3-diylbis(2-hexyldecanoate); 2-(2-(4-hydroxybutyl)-2,7-diazaspiro[3.5]nonan-7-yl)propane-1,3-diylbis(2-hexyldecanoate); and rac-(2R,3R)-8-(5-hydroxypentyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate); 2. The compound of claim 1, selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

13. 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate), or a pharmaceutically acceptable salt thereof.

14. 2-(7-(4-hydroxybutyl)-2,7-diazaspiro[3.5]nonan-2-yl)propane-1,3-diylbis(2-heptylnonanoate), or a pharmaceutically acceptable salt thereof.

15. 2-(2-(4-hydroxybutyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-heptylnonanoate), or a pharmaceutically acceptable salt thereof.

16. 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-hexyldecanoate), or a pharmaceutically acceptable salt thereof.

17. 2-(9-(5-hydroxypentyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate), or a pharmaceutically acceptable salt thereof.

18. rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate), or a pharmaceutically acceptable salt thereof.

19. rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-hexyldecanoate), or a pharmaceutically acceptable salt thereof.

20. 2-(2-(4-hydroxybutyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-hexyldecanoate), or a pharmaceutically acceptable salt thereof.

21. 2-(9-(3-hydroxypropyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate), or a pharmaceutically acceptable salt thereof.

22. 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(3-hexylundecanoate), or a pharmaceutically acceptable salt thereof.

23. 2-(7-(4-hydroxybutyl)-2,7-diazaspiro[4.4]nonan-2-yl)propane-1,3-diylbis(2-hexyldecanoate), or a pharmaceutically acceptable salt thereof.

24. 2-(7-(5-hydroxypentyl)-2,7-diazaspiro[3.5]nonan-2-yl)propane-1,3-diylbis(2-hexyldecanoate), or a pharmaceutically acceptable salt thereof.

25. 2-(2-(4-hydroxybutyl)-2,7-diazaspiro[3.5]nonan-7-yl)propane-1,3-diylbis(2-hexyldecanoate), or a pharmaceutically acceptable salt thereof.

26. rac-(2R,3R)-8-(5-hydroxypentyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate), or a pharmaceutically acceptable salt thereof.

27. 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate).

28. 2-(7-(4-hydroxybutyl)-2,7-diazaspiro[3.5]nonan-2-yl)propane-1,3-diylbis(2-heptylnonanoate).

29. 2-(2-(4-hydroxybutyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-heptylnonanoate).

30. 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-hexyldecanoate).

31. 2-(9-(5-hydroxypentyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate).

32. rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate).

33. rac-(2R,3R)-8-(4-hydroxybutyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-hexyldecanoate).

34. 2-(2-(4-hydroxybutyl)-2,8-diazaspiro[4.5]decan-8-yl)propane-1,3-diylbis(2-hexyldecanoate).

35. 2-(9-(3-hydroxypropyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(2-heptylnonanoate).

36. 2-(9-(4-hydroxybutyl)-3,9-diazaspiro[5.5]undecan-3-yl)propane-1,3-diylbis(3-hexylundecanoate).

37. 2-(7-(4-hydroxybutyl)-2,7-diazaspiro[4.4]nonan-2-yl)propane-1,3-diylbis(2-hexyldecanoate).

38. 2-(7-(5-hydroxypentyl)-2,7-diazaspiro[3.5]nonan-2-yl)propane-1,3-diylbis(2-hexyldecanoate).

39. 2-(2-(4-hydroxybutyl)-2,7-diazaspiro[3.5]nonan-7-yl)propane-1,3-diylbis(2-hexyldecanoate).

40. rac-(2R,3R)-8-(5-hydroxypentyl)-8-azaspiro[4.5]decane-2,3-diylbis(2-heptylnonanoate).

41. 41. A pharmaceutical composition comprising a nucleic acid, at least one pharmaceutically acceptable excipient, and a compound of any one of claims 1 to 40, or a pharmaceutically acceptable salt thereof.

42. 42. The pharmaceutical composition of claim 41, wherein the pharmaceutically acceptable excipient is selected from the group consisting of neutral lipids, steroids, and polymer-conjugated lipids.

43. 43. The pharmaceutical composition of claim 42, comprising 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), a phosphatidylethanolamine, e.g., 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM), or a combination thereof.

44. 43. The pharmaceutical composition of claim 42, wherein the steroid is cholesterol.

45. 43. The pharmaceutical composition of claim 42, wherein the polymer-conjugated lipid is a PEGylated lipid.

46. 46. ​​The pharmaceutical composition of claim 45, wherein the PEGylated lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, PEG-DMG, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), or PEG dialkyloxypropylcarbamate.

47. 42. The pharmaceutical composition of claim 41, wherein the nucleic acid is RNA.

48. 48. The pharmaceutical composition of claim 47, wherein the RNA is messenger RNA.

49. The pharmaceutical composition of claims 47 and 48, wherein the RNA is modRNA or saRNA.

50. 50. A method for administering a nucleic acid to a subject in need thereof, comprising preparing or providing a pharmaceutical composition according to any one of claims 41 to 49 and administering the pharmaceutical composition to the subject.

51. 50. A method of making a pharmaceutical composition according to any one of claims 41 to 49, comprising combining a nucleic acid, at least one pharmaceutically acceptable excipient, and a compound according to any one of claims 1 to 40.

52. 41. A compound according to any one of claims 1 to 40 for use as a component of a medicament.

53. 41. Use of a compound according to any one of claims 1 to 40 for the manufacture of a medicament.

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