TW202511244A - Lipids, pharmaceutical compositions comprising the same and methods of delivering active pharmaceutical ingredients - Google Patents

Abstract

The present application relates a lipid, a pharmaceutical composition comprising the same, and a method of delivering an active pharmaceutical ingredient to a cell or a subject.

Description

Lipids, pharmaceutical compositions containing the same, and methods for delivering active pharmaceutical ingredients

This application relates to a lipid, a pharmaceutical composition comprising the lipid, and a method for delivering an active pharmaceutical ingredient to a cell or an individual.

Therapeutics such as drug compounds, nucleic acid molecules, and other active pharmaceutical ingredients act by being taken up into the cells, tissues, and organs of an individual. Transfection of drugs and molecules into cells is often the limiting step in therapeutic action.

For example, one way to perform nucleic acid transfection is to encapsulate it in lipid nanoparticles.

PCT Publication No. WO2017/049245 discloses a lipid having a head group connected to a nitrogen atom via a methylene group, a linear lipophilic tail connected to the nitrogen atom, and a branched lipophilic tail connected to the nitrogen atom, such as compound R1 of the following formula (hereinafter also referred to as "SM-102"). PCT Publication No. WO2017/049245 discloses that compound R1 is suitable for preparing nanoparticles with favorable transfection properties. Compound R1

PCT Publication No. WO2016/210190 discloses a lipid having a head group connected to a nitrogen atom via a carbonyl group and two lipophilic tails connected to the nitrogen atom, such as compound R2 of the following formula. PCT Publication No. WO2016/210190 discloses that compound R2 is suitable for preparing nanoparticles with favorable transfection properties. Compound R2

There continues to be a need for lipid molecules and compositions for the efficient transfection of nucleic acid molecules and other agents into cells and subjects.

The present disclosure relates to a lipid suitable for preparing nanoparticles with favorable transfection properties.

Specifically, the present disclosure includes the following embodiments.

Example 1. A compound of formula I or a pharmaceutically acceptable salt thereof: Formula I wherein ^A1 is selected from the group consisting of: , the arrow indicates the connection to the carbonyl group, ^R1 and ^R2 are the same or different and are hydrogen or hydroxyl, ^R3 , ^R5 , ^R6 , ^R7 , ^R8 , ^R9 and ^R10 are the same or different and are linear C1 to C4 alkyl, ^R4 is linear C1 to C4 alkyl or -( _CH2 ) _p4 -OH, p1 and p2 are the same or different and are each independently selected from the group consisting of 1, 2, 3 and 4, p3 is an integer selected from the group consisting of 0, 1, 2, 3 and 4, p4 is an integer selected from the group consisting of 2, 3 and 4, ^X- is a pharmaceutically acceptable counter anion, each A ^A2 is independently a straight chain C4 to C10 alkyl, a straight chain C4 to C10 alkenyl or a straight chain C4 to C10 diene alkyl, m is an integer selected from the group consisting of 5, 6, 7, 8 and 9, n is an integer selected from the group consisting of 2, 3, 4, 5 and 6, ^A3 is -OC(O)- or -C(O)O-, and ^A4 is a straight chain C10-C18 alkyl, a straight chain C10-C18 alkenyl or a straight chain C10-C18 diene alkyl.

Embodiment 2. The compound of Embodiment 1 or a pharmaceutically acceptable salt thereof, wherein each A ² is independently a linear C 4 to C 10 alkyl group.

Embodiment 3. The compound or a pharmaceutically acceptable salt thereof as in Embodiment 1, wherein each ^A2 is the same.

Embodiment 4. The compound according to any one of Embodiments 1 to 3 or a pharmaceutically acceptable salt thereof, wherein m is an integer selected from the group consisting of 5, 6, 7 and 8.

Embodiment 5. The compound according to any one of Embodiments 1 to 4 or a pharmaceutically acceptable salt thereof, wherein n is an integer selected from the group consisting of 2, 3, 4 and 5.

Embodiment 6. The compound according to any one of Embodiments 1 to 5 or a pharmaceutically acceptable salt thereof, wherein the number of carbon atoms in A ⁴ -A ³ -(CH ₂ ) _n - is 15 to 21.

Embodiment 7. The compound or pharmaceutically acceptable salt thereof according to any one of Embodiments 1 to 6, wherein ^A1 is selected from the group consisting of: , where the arrow indicates the connection to the carbonyl group.

Embodiment 8. The compound of any one of Embodiments 1 to 6 or a pharmaceutically acceptable salt thereof, wherein ^A1 is selected from the group consisting of: , where the arrow indicates the connection to the carbonyl group.

Embodiment 9. The compound or pharmaceutically acceptable salt thereof according to any one of Embodiments 1 to 6, wherein ^A1 is selected from the group consisting of: , where the arrow indicates the connection to the carbonyl group.

Embodiment 10. The compound of any one of Embodiments 1 to 6 or a pharmaceutically acceptable salt thereof, wherein ^A1 is selected from the group consisting of: , where the arrow indicates the connection to the carbonyl group.

Embodiment 11. The compound of any one of Embodiments 1 to 10 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of compound numbers 1 to 30 shown in Table 1 below: Table 1 .

Embodiment 12. A pharmaceutical composition comprising: Lipid nanoparticles; and An active pharmaceutical ingredient encapsulated in the lipid nanoparticles, wherein the lipid nanoparticles comprise a compound as described in any one of Embodiments 1 to 11 or a pharmaceutically acceptable salt thereof.

Embodiment 13. The pharmaceutical composition of Embodiment 12, wherein the active pharmaceutical ingredient is selected from the group consisting of siRNA, mRNA, antisense oligonucleotide and miRNA.

Embodiment 14. A method for delivering an active pharmaceutical ingredient to a cell, comprising contacting the pharmaceutical composition of Embodiment 12 or 13 with the cell.

Embodiment 15. A method for delivering an active pharmaceutical ingredient to a subject in need thereof, comprising administering the pharmaceutical composition of Embodiment 12 or 13 to the subject.

Compound

Some embodiments of the present disclosure relate to a compound of formula I or a pharmaceutically acceptable salt thereof: Formula I wherein ^A1 is selected from the group consisting of: , the arrow indicates the connection to the carbonyl group, ^R1 and ^R2 are the same or different and are hydrogen or hydroxyl, ^R3 , ^R5 , ^R6 , ^R7 , ^R8 , ^R9 and ^R10 are the same or different and are linear C1 to C4 alkyl, ^R4 is linear C1 to C4 alkyl or -( _CH2 ) _p4 -OH, p1 and p2 are the same or different and are each independently selected from the group consisting of 1, 2, 3 and 4, p3 is an integer selected from the group consisting of 0, 1, 2, 3 and 4, p4 is an integer selected from the group consisting of 2, 3 and 4, ^X- is a pharmaceutically acceptable counter anion, each A ^A2 is independently a straight chain C4 to C10 alkyl, a straight chain C4 to C10 alkenyl or a straight chain C4 to C10 diene alkyl, m is an integer selected from the group consisting of 5, 6, 7, 8 and 9, n is an integer selected from the group consisting of 2, 3, 4, 5 and 6, ^A3 is -OC(O)- or -C(O)O-, ^A4 is a straight chain C10-C18 alkyl, a straight chain C10-C18 alkenyl or a straight chain C10-C18 diene alkyl.

In some embodiments, R ¹ and R ² are hydroxyl.

In some embodiments, R ³ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ and R ¹⁰ are the same or different and are methyl or ethyl.

In some embodiments, R ⁴ is methyl, ethyl, or -(CH ₂ ) ₂ -OH.

In some embodiments, p1 and p2 are the same or different and are integers independently selected from the group consisting of 1, 2, and 3. In some embodiments, p1 and p2 are the same or different and are 1 or 2. In some embodiments, p1 and p2 are 1.

In some embodiments, p3 is an integer independently selected from the group consisting of 0, 1, 2, and 3. In some embodiments, p3 is an integer independently selected from the group consisting of 0, 1, and 2. In some embodiments, p3 is 0 or 1.

In some embodiments, each A ² is independently a straight chain C4 to C9 alkyl, a straight chain C4 to C9 alkenyl, or a straight chain C4 to C9 dienyl. In some embodiments, each A ² is independently a straight chain C8 to C9 alkyl, a straight chain C8 to C9 alkenyl, or a straight chain C8 to C9 dienyl. In some embodiments, each A ² is independently a straight chain C8 to C10 alkyl, a straight chain C8 to C10 alkenyl, or a straight chain C8 to C10 dienyl. In some embodiments, each A ² is independently a straight chain C4 to C10 alkyl. In some embodiments, each A ² is independently a straight chain C4 to C9 alkyl. In some embodiments, each A ² is independently a straight chain C7 or C8 alkyl. In some embodiments, each A ² in the compound is the same. For example, in some embodiments, each A ² is n-octyl.

In some embodiments, m is an integer selected from the group consisting of 5, 6, 7, and 8. In some embodiments, m is 5, 6, or 7. In some embodiments, m is 7 or 8. In some embodiments, m is 7.

In some embodiments, n is an integer selected from the group consisting of 2, 3, 4, and 5.

In some embodiments, ^A4 is a straight chain C11-C18 alkyl, a straight chain C11-C18 alkenyl, or a straight chain C11-C18 dienyl. In some embodiments, ^A4 is a straight chain C10-C17 alkyl, a straight chain C10-C17 alkenyl, or a straight chain C10-C17 dienyl. In some embodiments, ^A4 is a straight chain C11-C17 alkyl, a straight chain C11-C17 alkenyl, or a straight chain C11-C17 dienyl.

In some embodiments, the number of carbon atoms in A ⁴ -A ³ -(CH ₂ ) _n - is 15 to 21. In some embodiments, the number of carbon atoms in A ⁴ -A ³ -(CH ₂ ) _n - is 16 to 20.

In some embodiments, ^A1 is selected from the group consisting of: , (where the arrow indicates the connection to the carbonyl group).

In some embodiments, ^A1 is selected from the group consisting of: , (where the arrow indicates the connection to the carbonyl group).

In some embodiments, ^A1 is selected from the group consisting of: , (where the arrow indicates the connection to the carbonyl group).

In some embodiments, ^A1 is selected from the group consisting of , (where the arrow indicates the connection to the carbonyl group).

In some embodiments, ^A1 is selected from the group consisting of , (where the arrow indicates the connection to the carbonyl group).

In some embodiments, ^A1 is selected from the group consisting of , (where the arrow indicates the connection to the carbonyl group).

In some embodiments, X- ^is a pharmaceutically acceptable counter anion selected from the group consisting of chloride, bromide, fluoride, iodide, nitrate, sulfate, methylsulfate, phosphate, acetate, benzoate, citrate, glutamate, and lactate.

As used herein, the term "linear alkyl" refers to a straight chain alkyl group of a saturated aliphatic group, which may be of any length unless otherwise specified. The term "linear C1 to C4 alkyl" includes methyl, ethyl, n-propyl and n-butyl. The term "linear C4 to C10 alkyl" includes n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl and n-decyl. As used herein, the term "linear C10-C18 alkyl" includes n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl and n-octadecyl.

As used herein, the term "linear alkenyl" refers to a straight chain alkyl group having one carbon-carbon double bond and, unless otherwise specified, may be of any length. The term "linear C4 to C10 alkenyl" includes 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-oc ... 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl and 9-decenyl. The term "straight chain C10-C18 alkenyl" includes 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl, 11-dodecenyl, 12-dodecenyl, 13-dodecenyl, 14-dodecenyl, 15-dodecenyl, 16-dodecenyl, 17-dodecenyl, 18-dodecenyl, 19-dodecenyl, 20-21-24-26-28-29- dienyl, 1-tridecenyl, 1-tridecenyl, 2-tridecenyl, 3-tridecenyl, 4-tridecenyl, 5-tridecenyl, 6-tridecenyl, 7-tridecenyl, 8-tridecenyl, 9-tridecenyl, 10-tridecenyl, 11-tridecenyl, 12-tridecenyl, 1-tetradecenyl, 2-tetradecenyl, 3-tetradecenyl, 4-tetradecenyl, 5-tetradecenyl, 6-tetradecenyl, 7-tetradecenyl, 8-tetradecenyl, 9-tetradecenyl, 10-tetradecenyl, 11-tetradecenyl, 12-tetradecenyl, 13-tetradecenyl, 1-pentadecenyl, 2-pentadecenyl, 3-pentadecenyl, 4-pentadecenyl, 1-hexadecenyl, 2-hexadecenyl, 3-hexadecenyl, 4-hexadecenyl, 5-hexadecenyl, 6-hexadecenyl, 7-hexadecenyl, 8-hexadecenyl, 9-hexadecenyl, 10-hexadecenyl, 11-hexadecenyl, 12-hexadecenyl, 13-hexadecenyl, 14-hexadecenyl, and 15-hexadecenyl, 1-heptadecenyl, 2-heptadecenyl, 3-heptadecenyl, 4-heptadecenyl, 5-heptadecenyl, 6-heptadecenyl, 7-heptadecenyl, 8-heptadecenyl, 9-heptadecenyl, 10-heptadecenyl, 11-heptadecenyl, 12-heptadecenyl, 13-heptadecenyl, 14-heptadecenyl, 15-heptadecenyl, 16-heptadecenyl, 1-octadecenyl, 2-octadecenyl, 3-octadecenyl, 4-octadecenyl, 5-octadecenyl, 6-octadecenyl, 7-octadecenyl, 8-octadecenyl, 9-octadecenyl, 10-octadecenyl, 11-octadecenyl, 12-octadecenyl, 13-octadecenyl, 14-octadecenyl, 15-octadecenyl, 16-octadecenyl and 17-octadecenyl.

As used herein, the term "linear alkadienyl" refers to a straight chain alkyl group having two carbon-carbon double bonds and, unless otherwise specified, may be of any length. The term "straight chain C4 to C10 alkenyl" includes 1,3-butadienyl, 1,3-pentadienyl, 1,4-pentadienyl, 2,4-pentadienyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, 2,5-hexadienyl, 3,5-hexadienyl, 1,3-heptadienyl, 1,4-heptadienyl, 1,5-heptadienyl, 1,6-heptadienyl, 2,4-heptadienyl, 2,5-heptadienyl, 2,6-heptadienyl, 3,5-heptadienyl, 3,6-heptadienyl, 4,6- heptadienyl, 1,3-octadienyl, 1,4-octadienyl, 1,5-octadienyl, 1,6-octadienyl, 1,7-octadienyl, 2,4-octadienyl, 2,5-octadienyl, 2,6-octadienyl, 2,7-octadienyl, 3,5-octadienyl, 3,6-octadienyl, 3,7-octadienyl, 4,6-octadienyl, 4,7-octadienyl, 5,7-octadienyl, 1,3-nonadienyl, 1,4-nonadienyl, 1,5-nonadienyl, 1,6-nonadienyl, 1,7-nonadienyl, 1,8-nonadienyl, 2,4-nonadienyl, 2,5-nonadienyl, 2,6-nonadienyl, 2,7-nonadienyl, 2,8-nonadienyl, 3,5-nonadienyl, 3,6-nonadienyl, 3,7-nonadienyl, 3,8-nonadienyl, 4,6-nonadienyl, 4,7-nonadienyl, 4,8-nonadienyl, 5,7-nonadienyl, 5,8-nonadienyl, 6,8-nonadienyl, 1,3-decadienyl, 1,4-decadienyl, 1,5-decadienyl, 1,6-decadienyl, 1,7-decadienyl, 1,8-decadienyl, 1,9- Decadienyl, 2,4-decadienyl, 2,5-decadienyl, 2,6-decadienyl, 2,7-decadienyl, 2,8-decadienyl, 2,9-decadienyl, 3,5-decadienyl, 3,6-decadienyl, 3,7-decadienyl, 3,8-decadienyl, 3,9-decadienyl, 4,6-decadienyl, 4,7-decadienyl, 4,8-decadienyl, 4,9-decadienyl, 5,7-decadienyl, 5,8-decadienyl, 5,9-decadienyl, 6,8-decadienyl, 6,9-decadienyl and 7,9-decadienyl. The term "straight chain C10-C18 diene alkyl" includes 1,3-decadienyl, 1,4-decadienyl, 1,5-decadienyl, 1,6-decadienyl, 1,7-decadienyl, 1,8-decadienyl, 1,9-decadienyl, 2,4-decadienyl, 2,5-decadienyl, 2,6-decadienyl, 2,7-decadienyl, 2,8-decadienyl, 2,9-decadienyl, 3,5-decadienyl, 3,6-decadienyl, 3,7-decadienyl, 3,8-decadienyl, 3,9-decadienyl, 4,6-decadienyl, 4,7-decadienyl, 4,8-decadienyl, 4,9-decadienyl, 5,7-decadienyl, 5 ,8-decadienyl, 5,9-decadienyl, 6,8-decadienyl, 6,9-decadienyl, 7,9-decadienyl, 1,3-undecadienyl, 1,4-undecadienyl, 1,5-undecadienyl, 1,6-undecadienyl, 1,7-undecadienyl, 1,8-undecadienyl, 1,9-undecadienyl, 1,10-undecadienyl, 2,4-undecadienyl, 2,5-undecadienyl, 2,6-undecadienyl, 2,7-undecadienyl, 2,8-undecadienyl, 2,9-undecadienyl, 2,10-undecadienyl, 3,5-undecadienyl, 3,6-undecadienyl, 1,3-dodecadienyl, 3,7-dodecadienyl, 3,8-dodecadienyl, 3,9-dodecadienyl, 3,10-dodecadienyl, 4,6-dodecadienyl, 4,7-dodecadienyl, 4,8-dodecadienyl, 4,9-dodecadienyl, 4,10-dodecadienyl, 5,7-dodecadienyl, 5,8-dodecadienyl, 5,9-dodecadienyl, 5,10-dodecadienyl, 6,8-dodecadienyl, 6,9-dodecadienyl, 6,10-dodecadienyl, 7,9-dodecadienyl, 7,10-dodecadienyl, 8,10-dodecadienyl, 1,3 ... dodecadienyl, 1,4-dodecadienyl, 1,5-dodecadienyl, 1,6-dodecadienyl, 1,7-dodecadienyl, 1,8-dodecadienyl, 1,9-dodecadienyl, 1,10-dodecadienyl, 1,11-dodecadienyl, 2,4-dodecadienyl, 2,5-dodecadienyl, 2,6-dodecadienyl, 2,7-dodecadienyl, 2,8-dodecadienyl, 2,9-dodecadienyl, 2,10-dodecadienyl, 2,11-dodecadienyl, 3,5-dodecadienyl, 3,6-dodecadienyl, 3,7-dodecadienyl, 3,8-dodecadienyl, 3,9-dodecadienyl, 3,10-dodecadienyl, 3,11-dodecadienyl, 4,6-dodecadienyl, 4,7-dodecadienyl, 4,8-dodecadienyl, 4,9-dodecadienyl, 4,10-dodecadienyl, 4,11-dodecadienyl, 5,7-dodecadienyl, 5,8-dodecadienyl, 5,9-dodecadienyl, 5,10-dodecadienyl, 5,11-dodecadienyl, 6,8-dodecadienyl, 6,9-dodecadienyl, 6,10-dodecadienyl, 6,11-dodecadienyl, 7,9-dodecadienyl, 7,10-dodecadienyl 1,7-tridecadienyl, 1,8-tridecadienyl, 1,9-tridecadienyl, 1,10-tridecadienyl, 1,11-tridecadienyl, 1,12-tridecadienyl, 2,4-tridecadienyl, 2,5-tridecadienyl, 2,6-tridecadienyl, 2,7-tridecadienyl, 2,8-tridecadienyl, 2,9-tridecadienyl , 2,10-tridecadienyl, 2,11-tridecadienyl, 2,12-tridecadienyl, 3,5-tridecadienyl, 3,6-tridecadienyl, 3,7-tridecadienyl, 3,8-tridecadienyl, 3,9-tridecadienyl, 3,10-tridecadienyl, 3,11-tridecadienyl, 3,12-tridecadienyl, 4,6-tridecadienyl, 4,7-tridecadienyl, 4,8-tridecadienyl, 4,9-tridecadienyl, 4,10-tridecadienyl, 4,11-tridecadienyl, 4,12-tridecadienyl, 5,7-tridecadienyl, 5,8-tridecadienyl, tridecadienyl, 5,9-tridecadienyl, 5,10-tridecadienyl, 5,11-tridecadienyl, 5,12-tridecadienyl, 6,8-tridecadienyl, 6,9-tridecadienyl, 6,10-tridecadienyl, 6,11-tridecadienyl, 6,12-tridecadienyl, 7,9-tridecadienyl, 7,10-tridecadienyl, 7,11-tridecadienyl, 7,12-tridecadienyl, 8,10-tridecadienyl, 8,11-tridecadienyl, 8,12-tridecadienyl, 9,11-tridecadienyl, 9,12-tridecadienyl, 10,12-tridecadienyl, 1,3-tetradecadienyl, 1,4-tetradecadienyl, 1,5-tetradecadienyl, 1,6-tetradecadienyl, 1,7-tetradecadienyl, 1,8-tetradecadienyl, 1,9-tetradecadienyl, 1,10-tetradecadienyl, 1,11-tetradecadienyl, 1,12-tetradecadienyl, 1,13-tetradecadienyl, 2,4-tetradecadienyl, 2,5-tetradecadienyl, 2,6-tetradecadienyl, 2,7-tetradecadienyl, 2,8-tetradecadienyl, 2,9-tetradecadienyl, 2,10-tetradecadienyl, 2,11-tetradecadienyl, 2,12-tetradecadienyl, 2,13-tetradecadienyl, 3,5-tetradecadienyl, 3,6-tetradecadienyl, 3,7-tetradecadienyl, 3,8-tetradecadienyl, 3,9-tetradecadienyl, 3,10-tetradecadienyl, 3,11-tetradecadienyl, 3,12-tetradecadienyl, 3,13-tetradecadienyl 4,6-tetradecadienyl, 4,7-tetradecadienyl, 4,8-tetradecadienyl, 4,9-tetradecadienyl, 4,10-tetradecadienyl, 4,11-tetradecadienyl, 4,12-tetradecadienyl, 4,13-tetradecadienyl, 5,7-tetradecadienyl, 5,8-tetradecadienyl, 5,9-tetradecadienyl, 5,10-tetradecadienyl, 5,11-tetradecadienyl, 5,12-tetradecadienyl, 5,13-tetradecadienyl Tetradecadienyl, 6,8-tetradecadienyl, 6,9-tetradecadienyl, 6,10-tetradecadienyl, 6,11-tetradecadienyl, 6,12-tetradecadienyl, 6,13-tetradecadienyl, 7,9-tetradecadienyl, 7,10-tetradecadienyl, 7,11-tetradecadienyl, 7,12-tetradecadienyl, 7,13-tetradecadienyl, 8,10-tetradecadienyl, 8,11-tetradecadienyl, 8,12-tetradecadienyl , 8,13-tetradecadienyl, 9,11-tetradecadienyl, 9,12-tetradecadienyl, 9,13-tetradecadienyl, 10,12-tetradecadienyl, 10,13-tetradecadienyl, 11,13-tetradecadienyl, 1,3-pentadecadienyl, 1,4-pentadecadienyl, 1,5-pentadecadienyl, 1,6-pentadecadienyl, 1,7-pentadecadienyl, 1,8-pentadecadienyl, 1,9-pentadecadienyl, 1,10 -pentadecadienyl, 1,11-pentadecadienyl, 1,12-pentadecadienyl, 1,13-pentadecadienyl, 1,14-pentadecadienyl, 2,4-pentadecadienyl, 2,5-pentadecadienyl, 2,6-pentadecadienyl, 2,7-pentadecadienyl, 2,8-pentadecadienyl, 2,9-pentadecadienyl, 2,10-pentadecadienyl, 2,11-pentadecadienyl, 2,12-pentadecadienyl, 2,13-pentadecadienyl, 2,14-pentadecadieyl, 3,5-pentadecadieyl, 3,6-pentadecadieyl, 3,7-pentadecadieyl, 3,8-pentadecadieyl, 3,9-pentadecadieyl, 3,10-pentadecadieyl, 3,11-pentadecadieyl, 3,12-pentadecadieyl, 3,13-pentadecadieyl, 3,14-pentadecadieyl, 4,6-pentadecadieyl, 4,7-pentadecadieyl, 4,8-pentadecadieyl, 4,9-pentadecadieyl 4,10-pentadecadienyl, 4,11-pentadecadienyl, 4,12-pentadecadienyl, 4,13-pentadecadienyl, 4,14-pentadecadienyl, 5,7-pentadecadienyl, 5,8-pentadecadienyl, 5,9-pentadecadienyl, 5,10-pentadecadienyl, 5,11-pentadecadienyl, 5,12-pentadecadienyl, 5,13-pentadecadienyl, 5,14-pentadecadienyl, 6,8-pentadecadienyl, 6,9-pentadecadienyl pentadecadienyl, 6,10-pentadecadienyl, 6,11-pentadecadienyl, 6,12-pentadecadienyl, 6,13-pentadecadienyl, 6,14-pentadecadienyl, 7,9-pentadecadienyl, 7,10-pentadecadienyl, 7,11-pentadecadienyl, 7,12-pentadecadienyl, 7,13-pentadecadienyl, 7,14-pentadecadienyl, 8,10-pentadecadienyl, 8,11-pentadecadienyl, 8,12-pentadecadienyl 8,13-pentadecadienyl, 8,14-pentadecadienyl, 9,11-pentadecadienyl, 9,12-pentadecadienyl, 9,13-pentadecadienyl, 9,14-pentadecadienyl, 10,12-pentadecadienyl, 10,13-pentadecadienyl, 10,14-pentadecadienyl, 11,13-pentadecadienyl, 11,14-pentadecadienyl, 12,14-pentadecadienyl, 1,3-hexadecadienyl, 1,4-decadienyl, Hexadecadienyl, 1,5-hexadecadienyl, 1,6-hexadecadienyl, 1,7-hexadecadienyl, 1,8-hexadecadienyl, 1,9-hexadecadienyl, 1,10-hexadecadienyl, 1,11-hexadecadienyl, 1,12-hexadecadienyl, 1,13-hexadecadienyl, 1,14-hexadecadienyl, 1,15-hexadecadienyl, 2,4-hexadecadienyl, 2,5-hexadecadienyl, 2,6-hexadecadienyl, 2,7- Hexadecadienyl, 2,8-hexadecadienyl, 2,9-hexadecadienyl, 2,10-hexadecadienyl, 2,11-hexadecadienyl, 2,12-hexadecadienyl, 2,13-hexadecadienyl, 2,14-hexadecadienyl, 2,15-hexadecadienyl, 3,5-hexadecadienyl, 3,6-hexadecadienyl, 3,7-hexadecadienyl, 3,8-hexadecadienyl, 3,9-hexadecadienyl, 3,10-hexadecadienyl, 3, 11-hexadecadienyl, 3,12-hexadecadienyl, 3,13-hexadecadienyl, 3,14-hexadecadienyl, 3,15-hexadecadienyl, 4,6-hexadecadienyl, 4,7-hexadecadienyl, 4,8-hexadecadienyl, 4,9-hexadecadienyl, 4,10-hexadecadienyl, 4,11-hexadecadienyl, 4,12-hexadecadienyl, 4,13-hexadecadienyl, 4,14-hexadecadienyl, 4,15-hexadecadienyl 5,7-hexadecadienyl, 5,8-hexadecadienyl, 5,9-hexadecadienyl, 5,10-hexadecadienyl, 5,11-hexadecadienyl, 5,12-hexadecadienyl, 5,13-hexadecadienyl, 5,14-hexadecadienyl, 5,15-hexadecadienyl, 6,8-hexadecadienyl, 6,9-hexadecadienyl, 6,10-hexadecadienyl, 6,11-hexadecadienyl, 6,12-hexadecadienyl, 6,1 3-hexadecadienyl, 6,14-hexadecadienyl, 6,15-hexadecadienyl, 7,9-hexadecadienyl, 7,10-hexadecadienyl, 7,11-hexadecadienyl, 7,12-hexadecadienyl, 7,13-hexadecadienyl, 7,14-hexadecadienyl, 7,15-hexadecadienyl, 8,10-hexadecadienyl, 8,11-hexadecadienyl, 8,12-hexadecadienyl, 8,13-hexadecadienyl, 8,14-hexadecadienyl Hexadecadienyl, 8,15-hexadecadienyl, 9,11-hexadecadienyl, 9,12-hexadecadienyl, 9,13-hexadecadienyl, 9,14-hexadecadienyl, 9,15-hexadecadienyl, 10,12-hexadecadienyl, 10,13-hexadecadienyl, 10,14-hexadecadienyl, 10,15-hexadecadienyl, 11,13-hexadecadienyl, 11,14-hexadecadienyl, 11,15-hexadecadienyl, 1 2,14-hexadecadienyl, 12,15-hexadecadienyl, 13,15-hexadecadienyl, 1,3-heptadecadienyl, 1,4-heptadecadienyl, 1,5-heptadecadienyl, 1,6-heptadecadienyl, 1,7-heptadecadienyl, 1,8-heptadecadienyl, 1,9-heptadecadienyl, 1,10-heptadecadienyl, 1,11-heptadecadienyl, 1,12-heptadecadienyl, 1,13-heptadecadienyl, 1,14-decadienyl heptadecadienyl, 1,15-heptadecadienyl, 1,16-heptadecadienyl, 2,4-heptadecadienyl, 2,5-heptadecadienyl, 2,6-heptadecadienyl, 2,7-heptadecadienyl, 2,8-heptadecadienyl, 2,9-heptadecadienyl, 2,10-heptadecadienyl, 2,11-heptadecadienyl, 2,12-heptadecadienyl, 2,13-heptadecadienyl, 2,14-heptadecadienyl, 2,15-heptadecadienyl, 2, 16-heptadecadienyl, 3,5-heptadecadienyl, 3,6-heptadecadienyl, 3,7-heptadecadienyl, 3,8-heptadecadienyl, 3,9-heptadecadienyl, 3,10-heptadecadienyl, 3,11-heptadecadienyl, 3,12-heptadecadienyl, 3,13-heptadecadienyl, 3,14-heptadecadienyl, 3,15-heptadecadienyl, 3,16-heptadecadienyl, 4,6-heptadecadienyl, 4,7-heptadecadienyl 4,8-heptadecadienyl, 4,9-heptadecadienyl, 4,10-heptadecadienyl, 4,11-heptadecadienyl, 4,12-heptadecadienyl, 4,13-heptadecadienyl, 4,14-heptadecadienyl, 4,15-heptadecadienyl, 4,16-heptadecadienyl, 5,7-heptadecadienyl, 5,8-heptadecadienyl, 5,9-heptadecadienyl, 5,10-heptadecadienyl, 5,11-heptadecadienyl, 5,12-heptadecadienyl heptadecadienyl, 5,13-heptadecadienyl, 5,14-heptadecadienyl, 5,15-heptadecadienyl, 5,16-heptadecadienyl, 6,8-heptadecadienyl, 6,9-heptadecadienyl, 6,10-heptadecadienyl, 6,11-heptadecadienyl, 6,12-heptadecadienyl, 6,13-heptadecadienyl, 6,14-heptadecadienyl, 6,15-heptadecadienyl, 6,16-heptadecadienyl, 7,9-heptadecadienyl yl, 7,10-heptadecadienyl, 7,11-heptadecadienyl, 7,12-heptadecadienyl, 7,13-heptadecadienyl, 7,14-heptadecadienyl, 7,15-heptadecadienyl, 7,16-heptadecadienyl, 8,10-heptadecadienyl, 8,11-heptadecadienyl, 8,12-heptadecadienyl, 8,13-heptadecadienyl, 8,14-heptadecadienyl, 8,15-heptadecadienyl, 8,16-heptadecadienyl, 9,11-heptadecadienyl, 9,12-heptadecadienyl, 9,13-heptadecadienyl, 9,14-heptadecadienyl, 9,15-heptadecadienyl, 9,16-heptadecadienyl, 10,12-heptadecadienyl, 10,13-heptadecadienyl, 10,14-heptadecadienyl, 10,15-heptadecadienyl, 10,16-heptadecadienyl, 11,13-heptadecadienyl, 11,14-heptadecadienyl, 11,15-heptadecadienyl, heptadecadienyl, 11,16-heptadecadienyl, 12,14-heptadecadienyl, 12,15-heptadecadienyl, 12,16-heptadecadienyl, 13,15-heptadecadienyl, 13,16-heptadecadienyl, 14,16-heptadecadienyl, 1,3-octadecadienyl, 1,4-octadecadienyl, 1,5-octadecadienyl, 1,6-octadecadienyl, 1,7-octadecadienyl, 1,8-octadecadienyl, 1,9-octadecadienyl 1,10-octadecadienyl, 1,11-octadecadienyl, 1,12-octadecadienyl, 1,13-octadecadienyl, 1,14-octadecadienyl, 1,15-octadecadienyl, 1,16-octadecadienyl, 1,17-octadecadienyl, 2,4-octadecadienyl, 2,5-octadecadienyl, 2,6-octadecadienyl, 2,7-octadecadienyl, 2,8-octadecadienyl, 2,9-octadecadienyl, 2,1 0-octadecadienyl, 2,11-octadecadienyl, 2,12-octadecadienyl, 2,13-octadecadienyl, 2,14-octadecadienyl, 2,15-octadecadienyl, 2,16-octadecadienyl, 2,17-octadecadienyl, 3,5-octadecadienyl, 3,6-octadecadienyl, 3,7-octadecadienyl, 3,8-octadecadienyl, 3,9-octadecadienyl, 3,10-octadecadienyl, 3,11-octadecadienyl 3,12-octadecadienyl, 3,13-octadecadienyl, 3,14-octadecadienyl, 3,15-octadecadienyl, 3,16-octadecadienyl, 3,17-octadecadienyl, 4,6-octadecadienyl, 4,7-octadecadienyl, 4,8-octadecadienyl, 4,9-octadecadienyl, 4,10-octadecadienyl, 4,11-octadecadienyl, 4,12-octadecadienyl, 4,13-octadecadienyl, 4,14-octadecadienyl, -octadecadienyl, 4,15-octadecadienyl, 4,16-octadecadienyl, 4,17-octadecadienyl, 5,7-octadecadienyl, 5,8-octadecadienyl, 5,9-octadecadienyl, 5,10-octadecadienyl, 5,11-octadecadienyl, 5,12-octadecadienyl, 5,13-octadecadienyl, 5,14-octadecadienyl, 5,15-octadecadienyl, 5,16-octadecadienyl, 5,17-octadecadienyl, 6,8-octadecadienyl, 6,9-octadecadienyl, 6,10-octadecadienyl, 6,11-octadecadienyl, 6,12-octadecadienyl, 6,13-octadecadienyl, 6,14-octadecadienyl, 6,15-octadecadienyl, 6,16-octadecadienyl, 6,17-octadecadienyl, 7,9-octadecadienyl, 7,10-octadecadienyl, 7,11-octadecadienyl, 7,12-octadecadienyl, 7,1 3-octadecadienyl, 7,14-octadecadienyl, 7,15-octadecadienyl, 7,16-octadecadienyl, 7,17-octadecadienyl, 8,10-octadecadienyl, 8,11-octadecadienyl, 8,12-octadecadienyl, 8,13-octadecadienyl, 8,14-octadecadienyl, 8,15-octadecadienyl, 8,16-octadecadienyl, 8,17-octadecadienyl, 9,11-octadecadienyl, 9,12- Octadecadienyl, 9,13-octadecadienyl, 9,14-octadecadienyl, 9,15-octadecadienyl, 9,16-octadecadienyl, 9,17-octadecadienyl, 10,12-octadecadienyl, 10,13-octadecadienyl, 10,14-octadecadienyl, 10,15-octadecadienyl, 10,16-octadecadienyl, 10,17-octadecadienyl, 11,13-octadecadienyl, 11,14-octadecadienyl , 11,15-octadecadienyl, 11,16-octadecadienyl, 11,17-octadecadienyl, 12,14-octadecadienyl, 12,15-octadecadienyl, 12,16-octadecadienyl, 12,17-octadecadienyl, 13,15-octadecadienyl, 13,16-octadecadienyl, 13,17-octadecadienyl, 14,16-octadecadienyl, 14,17-octadecadienyl and 15,17-octadecadienyl.

As used herein, the term "pharmaceutically acceptable" means suitable for use in subjects, such as mammals, such as humans.

The pharmaceutically acceptable counter anions of the present disclosure include, but are not limited to, chloride, bromide, fluoride, iodide, nitrate, sulfate, methylsulfate, phosphate, acetate, benzoate, citrate, glutamate and lactate.

The pharmaceutically acceptable salts disclosed herein include, but are not limited to, salts containing chloride, bromide, fluoride, iodide, nitrate, sulfate, methylsulfate, phosphate, acetate, benzoate, citrate, glutamate and/or lactate. The pharmaceutically acceptable salts of the compounds of formula I disclosed herein can be synthesized by conventional chemical methods. For example, the salts of the compounds are prepared by ion exchange chromatography or by reacting the free base in the compound with a stoichiometric amount or excess of the desired salt-forming inorganic or organic acid in a suitable solvent or various combinations of solvents.

In general, compounds may contain one or more chiral centers. Compounds containing one or more chiral centers may include those described as "isomers," "stereoisomers," "non-mirror isomers," "mirror isomers," "optical isomers," or "racemic mixtures." Conventions for stereochemical nomenclature, such as Cahn, Ingold, and Prelog's stereoisomer nomenclature, and methods for determining stereochemistry and separating stereoisomers are known in the art. See, for example, Michael B. Smith and Jerry March, March's Advanced Organic Chemistry, 5th edition, 2001. The compounds and structures disclosed herein, including chemical schemes, are intended to encompass all possible isomers, chemically reasonable positional isomers, stereoisomers, non-mirror isomers, mirror isomers and/or optical isomers of a given compound or structure, including racemic or any other mixtures thereof.

The compounds of Formula I disclosed herein can be prepared by at least one of the techniques described herein or known organic synthesis techniques.

For example, a compound of formula I can be obtained as shown in Scheme I below. In Scheme I, the definitions of variables n, m, ^A1 , ^A2 , ^A3 , ^A4 and ^A5 are the same as those of Formula I described herein. Compound a1 can be reacted with compound a2 to obtain compound a3. Step 1 can be carried out in an organic solvent (e.g., dichloromethane) in the presence of, for example, ^EDC.HCl and DMAP. Step 1 can be carried out at 0°C to 50°C for 1 hour to 24 hours. Compound a4 can be reacted with compound a5 to obtain compound a6. Other acid-unstable protecting groups can also be used to replace the Boc group in compound a5. Step 2 can be carried out in an organic solvent (e.g., dichloromethane) in the presence of, for example, ^EDC.HCl , triethylamine and DMAP. Step 2 can be carried out at 0°C to 50°C for 1 hour to 24 hours. The Boc group of compound a6 can be deprotected to obtain compound a7. Step 3 can be carried out in an organic solvent (e.g., dichloromethane) in the presence of an acid (e.g., methanesulfonic acid). Step 3 can be carried out at 0°C to 50°C for 1 hour to 24 hours. Compound a7 can react with compound a3 to obtain compound a8. Step 4 can be carried out in an organic solvent (e.g., acetonitrile, cyclopropyl methyl ether, or a mixed solvent thereof) in the presence of a base (e.g., potassium carbonate). KI can be added to promote the reaction. Step 4 can be carried out at 0°C to 100°C for 1 hour to 24 hours. Compound a8 can react with compound a9 to obtain a compound of formula I. Step 5 can be carried out in an organic solvent such as dichloromethane in the presence of, for example, ^EDC.HCl , a combination of triethylamine and DMAP or a combination of HATU and triethylamine. Step 5 can be carried out at 0°C to 50°C for 1 hour to 24 hours.

Compounds of formula I can also be obtained as shown in the following Scheme II. In Scheme II, the definitions of variables n, m, p1, p2, ^R1 , ^R2 , ^R3 , ^R4 , ^A1 , ^A2 , ^A3 , ^A4 and ^A5 are the same as those of Formula I as described herein. Compound a8, which can be prepared according to Scheme I, can be reacted with compound b1 to obtain compound b2. Step 1 can be carried out in an organic solvent (e.g., dichloromethane) in the presence of a base, such as triethylamine. Step 1 can be carried out at 0°C to 50°C for 5 minutes to 24 hours. Compound b2 can be reacted with compound b3 to obtain a compound of formula I. Step 2 can be carried out in an organic solvent (e.g., acetonitrile, THF or a mixed solvent thereof) in the presence of a base, such as DBU. Step 2 may be performed at 0°C to 100°C for 1 hour to 24 hours.

Compounds of formula I can also be obtained as shown in Scheme III. In Scheme III, the definitions of variables n, m, ^A1 , ^A2 , ^A3 , ^A4 and ^A5 are the same as those of formula I as described herein. Compound a3, which can be prepared according to Scheme I, can be reacted with compound c1. Step 1 can be carried out in an organic solvent (e.g., acetonitrile, cyclopropyl methyl ether or a mixed solvent thereof) in the presence of a base (e.g., potassium carbonate). Step 1 can be carried out at 0°C to 100°C for 1 hour to 24 hours. The resulting product of step 1 can be reacted with _Boc2O to obtain compound c1. Step 2 can be carried out in an organic solvent (e.g., dichloromethane) in the presence of a base (e.g., triethylamine). Step 2 can be carried out at 0°C to 50°C for 1 hour to 24 hours. Other acid-unstable protecting groups may also be used to replace the Boc group in compound c1. Compound c1 may react with compound c2. Step 3 may be carried out in an organic solvent (e.g., dichloromethane) in the presence of a base (e.g., triethylamine). Step 3 may be carried out at 0°C to 50°C for 10 minutes to 24 hours. The resulting product of step 3 may be freed from the protecting group to obtain compound c3. Step 4 may be carried out in an organic solvent (e.g., dichloromethane) in the presence of an acid (e.g., methanesulfonic acid). Step 4 may be carried out at 0°C to 50°C for 1 hour to 24 hours. Step 5 may be carried out according to the procedure of step 5 in process I or steps 1 and 2 in process II.

When ^A1 of the compound of formula I includes a tertiary amine, the amine can be alkylated with an alkylating agent (e.g., MeI). The reaction can be carried out at 0°C to 50°C for 1 hour to 24 hours. Pharmaceutical Compositions

Some embodiments of the present disclosure relate to a pharmaceutical composition comprising: lipid nanoparticles, and an active pharmaceutical ingredient encapsulated in the lipid nanoparticles, wherein the lipid nanoparticles comprise a compound of formula I or a pharmaceutically acceptable salt thereof as described elsewhere herein.

As used herein, the term "lipid nanoparticle" (also referred to as "LNP" or "liposome") refers to a nanoparticle comprising lipid molecules.

As used herein, the term "encapsulate" refers to the ability of a nanoparticle to carry an active pharmaceutical ingredient within its structure or on its surface such that the active pharmaceutical ingredient is not removed by a solvent or mobile phase external to the particle.

The term "lipid" is used broadly herein to include substances that are soluble in organic solvents but slightly soluble or insoluble in water.

The "ionizable cationic lipids" of the present disclosure are characterized by the weak alkalinity of their lipid head groups, which affects the surface charge of the lipid in a pH-dependent manner, making it positively charged at acidic pH but close to charge neutrality at physiological pH. In some embodiments, the compounds of formula I of the present disclosure are ionizable cationic lipids. The "cationic lipids" of the present disclosure are characterized by monovalent or multivalent cationic charges on their head groups, which make them positively charged at neutral pH. In some embodiments, the compounds of formula I of the present disclosure are cationic lipids. In certain embodiments, cationic and ionizable cationic lipids are capable of complexing with hydrophilic bioactive molecules to produce hydrophobic complexes that partition into the organic phase of a two-phase aqueous/organic system. It is contemplated that both monovalent and multivalent cationic lipids can be used to form hydrophobic complexes with bioactive molecules.

In some embodiments, the lipid nanoparticles comprise structured lipids, such as sterol lipids. In some embodiments, the structured lipids are sterol lipids, such as β-sterol, stigmasterol, ergosterol, ergocalciferol, cholesterol, or derivatives thereof.

In some embodiments, the lipid nanoparticles include stabilizer lipids, such as phospholipids. In some embodiments, the stabilizer lipids are phospholipids, such as diphytanoyl phosphatidyl ethanolamine (DPhPE) and 1,2-diphytanoyl- sn -Glycero-3-Phosphocholine (DPhPC). Examples of stabilizer lipids include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-3-phosphocholine (DPPC), and 1,2-dioleoyl-sn-3-phosphocholine (DPPC). sn-glycero-3-phosphocholine; DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

In some embodiments, lipid nanoparticles include lipids for reducing immunogenicity, such as PEGylated lipids. The PEG region of the PEGylated lipid can have any molecular weight. In some embodiments, the PEG region can have a molecular weight of 200, 300, 350, 400, 500, 550, 750, 1000, 1500, 2000, 3000, 3500, 4000 or 5000 Da. In some embodiments, PEGylated lipids include DSPE-PEG, DMPE-PEG, DPPE-PEG, DOPE-PEG and DMG-PEG.

In some embodiments, the lipid nanoparticles comprise 40-60 mol% of a compound of formula I as described elsewhere herein or a pharmaceutically acceptable salt thereof; 30-50 mol% of a sterol lipid relative to the total lipid mass of the lipid nanoparticles; 5-15 mol% of a phospholipid selected from the group consisting of DSPC, DOPC and DOPE relative to the total lipid mass of the lipid nanoparticles; and 1-5 mol% of a lipid for reducing immunogenicity, which is DMG-PEG, relative to the total lipid mass of the lipid nanoparticles. The cationic lipids, sterols, phospholipids selected from the group consisting of DSPC, DOPC and DOPE, and DMG-PEG in the lipid nanoparticles according to the present disclosure The molar ratio of (compound of formula I or its pharmaceutically acceptable salt/sterol lipid/phospholipid/DMG-PEG) includes but is not limited to (60/31/8/1), (60/31/7.5/1.5), (60/31/7/2), (60/31/6.5/2.5), (60/31/6/3), (60/31/5.5/3.5), (60/31/5/4), (60/31/4.5/4.5), (60/31/4/5), (60/31.5/7. 5/1), (60/30.5/7.5/2), (60/30/7.5/2.5), (60/29.5/7.5/3), (60/29/7.5/3.5), (60/28.5/7.5/4), (60/2 8/7.5/4.5), (60/27.5/7.5/5), (59/32/7.5/1.5), (58/33/7.5/1.5), (57/34/7.5/1.5), (56/35/7.5/1.5) , (55/36/7.5/1.5), (60/29/10/1), (60/28.5/10/1.5), (60/28/10/2), (60/27.5/10/2.5), (60/27/10/3) , (60/26.5/10/3.5), (60/26/10/4), (60/25.5/10/4.5), (60/25/10/5), (60/27/12/1), (60/27/11.5/1.5) , (60/27/11/2), (60/27/10.5/2.5), (60/27/9.5/3.5), (60/27/9/4), (60/27/8.5/4.5), (60/27/8/5), (59 /28/10/3), (58/29/10/3), (57/30/10/3), (56/31/10/3), (55/32/10/3), (50/39/10/1), (50/38.5/10/1.5 ), (50/38/10/2), (50/37.5/10/2.5), (50/37/10/3), (50/36.5/10/3.5), (50/36/10/4), (50/35.5/10/4. 5), (50/35/10/5), (50/38.5/10.5/1), (50/38.5/9.5/2), (50/38.5/9/2.5), (50/38.5/8.5/3), (50/38.5/ 8/3.5), (50/38.5/7.5/4), (50/38.5/7/4.5), (50/38.5/6.5/5), (51/37.5/10/1.5), (52/36.5/10/1.5), ( 53/35.5/10/1.5), (54/34.5/10/1.5), (55/33.5/10/1.5), (49/39.5/10/1.5), (48/40.5/10/1.5), (47/41 .5/10/1.5), (46/42.5/10/1.5), (45/43.5/10/1.5), (51/34/10/5), (52/33/10/5), (53/32/10/5), (54/31 /10/5), (55/30/10/5), (40/44/15/1), (40/43.5/15/1.5), (40/43/15/2), (40/42.5/15/2.5), (40/42/15/ 3), (40/41.5/15/3.5), (40/41/15/4), (40/40.5/15/4.5), (40/40/15/5), (41/39/15/5), (42/38/15/5), (43/37/15/5), (44/36/15/5), (45/35/15/5), (41/41/14/4), (42/42/13/3), (43/43/12/2) and (44/44/11/1).

In some embodiments, the lipid nanoparticles comprise cationic lipids and/or ionizable cationic lipids other than the compound of Formula I.

In some embodiments, the average particle size of the lipid nanoparticles is 50 to 250 nm (e.g., 50 nm to 200 nm, 50 nm to 175 nm, or 50 nm to 150 nm). In some embodiments, the polydispersity index (PDI) of the lipid nanoparticles is less than 0.2. The average particle size and PDI of LNPs can be evaluated by dynamic light scattering (DLS).

In some embodiments, the active pharmaceutical ingredient comprises a nucleic acid. In some embodiments, the nucleic acid comprises an interfering RNA molecule, such as siRNA, aiRNA, miRNA, or a mixture thereof. In certain other instances, the nucleic acid comprises single-stranded or double-stranded DNA, RNA, or a DNA/RNA hybrid (such as an antisense oligonucleotide), a ribonuclease, a plasmid, an immunostimulatory oligonucleotide, or a mixture thereof. In some instances, the nucleic acid comprises an mRNA molecule.

In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. As used herein, a "pharmaceutically acceptable carrier" (also referred to as an "excipient") is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle. A pharmaceutically acceptable carrier may be liquid or solid and may be selected according to the intended mode of administration so as to provide the desired volume, consistency, and other relevant transport and chemical properties. Typical pharmaceutically acceptable carriers include, but are not limited to, water, saline solutions, binders (such as polyvinylpyrrolidone or hydroxypropylmethylcellulose), fillers (such as lactose and other sugars, gelatin or calcium sulfate), lubricants (such as starch, polyethylene glycol or sodium acetate), disintegrants (such as starch or sodium starch hydroxyacetate) and wetting agents (such as sodium lauryl sulfate).

Pharmaceutical compositions can be prepared in a variety of forms suitable for a variety of administration routes and methods. For example, pharmaceutical compositions can be prepared in liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, pastes and elixirs), injectable forms, solid dosage forms (e.g., capsules, tablets, pills, powders and granules), dosage forms for topical and/or transdermal administration (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and patches), suspensions, powders and other forms.

The lipid nanoparticles disclosed herein can be synthesized by any suitable procedure, including adaptation of procedures known in the art, and loaded with encapsulated cargo. In some embodiments, lipid nanoparticles can be prepared by immersion injection procedures. Some examples of procedures for preparing lipid nanoparticles are given in U.S. Publication 2013/0115274. Some examples for preparing liposomes are given in Szoka, Ann. Rev. Biophys. Bioeng. 9:467 (1980); Liposomes, Marc J. Ostro, ed., Marcel Dekker, Inc., New York, 1983, Chapter 1. In general, lipid nanoparticles can be synthesized by mixing lipid components in an organic solvent with a buffered aqueous solution containing an active nucleic acid agent. Liposomes can be sized by filtering or extrusion. The liposomal suspension or solution can be further transformed by filtration. In some embodiments, lipid nanoparticles can be synthesized by injecting an ethanol solution of lipid into a buffer solution including an active pharmaceutical ingredient in the same manner as described in U.S. Publication No. 2013-0022665, PCT Publication No. WO2019/090359, and PCT Publication No. WO2020/102668. In some embodiments, lipid nanoparticles can be synthesized by combining a lipid solution with an active pharmaceutical ingredient solution using a microfluidic mixing device, such as NanoAssemblr ^TM (Precision Nano Systems). Methods for delivering active pharmaceutical ingredients to cells or individuals

Some embodiments of the present disclosure relate to a method of delivering an active pharmaceutical ingredient to a cell, comprising contacting a pharmaceutical composition as described herein with the cell. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vivo. In some embodiments, the cell is ex vivo.

Some embodiments of the present disclosure relate to a method of delivering an active pharmaceutical ingredient to an individual in need thereof, comprising administering to the individual a pharmaceutical composition as described above. For example, the active pharmaceutical ingredient may be an siRNA that effectively blocks the GSTp gene as described in Example 22 below. Individuals include, but are not limited to, mammals, such as humans, monkeys, rats, and mice. The pharmaceutical composition may be administered by any means known in the art, including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, or respiratory (aerosol) administration. Example abbreviations EDC ^. HCl: N -(3-Dimethylaminopropyl)- N′ -ethylcarbodiimide hydrochloride DMAP: 4-Dimethylaminopyridine DCM: Dichloromethane MSA: Methanesulfonic acid ACN: Acetonitrile _Boc2O : Di-Tri-butyl pyrocarbonate DBU: 2,3,4,6,7,8,9,10 - Octahydropyrimidol[1,2- a ]azepine cis -DHP.HCl: Cis-3,4-dihydroxypyrrolidine hydrochloride CPME: Cyclopentyl methyl ether HATU: 1-[Bis(dimethylamino)methylene]-1 H -1,2,3 - triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate Example 1: Synthesis of Compound 1 in Table 1 (8-(2-(dimethylamino)-N-(6-oxo-6-(undecyloxy)hexyl)acetamido)octanoic acid heptadecan-9-yl ester)

Compound 1 was prepared according to Scheme 1 shown in FIG1 .

Step 1: To a solution of heptadecan-9-ol (2 g, 7.7 mmol, 1 eq.) and 8-bromooctanoic acid (2.2 g, 10 mmol, 1.3 eq.) in anhydrous dichloromethane (DCM, 40 ml) was added ^EDC.HCl (1.8 g, 9.7 mmol, 1.25 eq.) and DMAP (95 mg, 0.77 mmol, 0.1 eq.). The clear solution was stirred at room temperature overnight (18 h). The next day, the reaction was stopped and diluted with DCM. The organics were washed sequentially with saturated sodium bicarbonate solution (25 mL), water (25 mL), and brine (25 mL). The separated organic phase was dried over sodium sulfate and filtered. The organic phase was concentrated and the crude product was purified by ISCO MPLC using a silica gel column (80 g) and eluting the column with a (0-20)% hexane-ethyl acetate gradient. The fractions were combined and concentrated to give intermediate 1 (2.7 g, 75% yield) as an oil.

Step 2: To a solution of undecan-1-ol (8 g, 46.4 mmol, 1 eq.) and 6-N-Boc aminohexanoic acid (12.8 g, 55.7 mmol, 1.2 eq.) in anhydrous DCM (150 ml) was added ^EDC.HCl (10.6 g, 55.7 mmol, 1.2 eq.), triethylamine (12.9 mL, 92.8 mmol) and DMAP (0.5 g, 4.64 mmol, 0.1 eq.). The clear reaction solution was stirred at room temperature under _N2 gas overnight. IPC (LC/MS) showed the corresponding product peak (m/z=385 (M+H)) along with saturated sodium bicarbonate solution (50 mL) and brine (50 mL). The organic phase was dried over sodium sulfate, filtered, and the solvent was evaporated by rotary evaporator to give the crude product. 5 g of the crude product was taken out for the next reaction and the remaining material was dissolved in anhydrous DCM (100 mL). Et ₃ N and Boc ₂ O were added to the above solution and stirred overnight. LC/MS confirmed the complete conversion of de-Boc amine to the desired product of Boc-amine. The organic layer was washed with water and saline solution. The organic phase was dried over Na ₂ SO ₄ and filtered. The organic phase was concentrated and the crude product was purified by ISCO MPLC using a silica gel column (220 g) and the column was eluted with a (0-100)% hexane-ethyl acetate gradient. The fractions were combined and concentrated to give intermediate 2 (12.5 g) as an oil. The product was kept in the freezer to form the product as a white solid.

Step 3: Intermediate 2 (3.4 g, 8.8 mmol) was dissolved in DCM (40 mL) and methanesulfonic acid (1.2 mL, 20.7 mmol, 2 eq.) was added. The clear solution was stirred at room temperature overnight. The next day, LC/MS confirmed complete conversion of the starting material to the product. The reaction _mixture was diluted with DCM (25 mL) and washed with saturated sodium bicarbonate solution (25 mL). The organic phase was dried over _Na2SO4 and filtered. The organic phase was concentrated and dried to give the free amine intermediate 3 in quantitative yield. This material was used in the next step without further purification.

Step 4: A solution of intermediate 1 (1.8 g, 3.8 mmol, 1 eq.) and intermediate 3 (2.2 g, 7.6 mmol, 2 eq.) in a mixture of acetonitrile/cyclopropyl methyl ether ( ₄₀ mL, 1:1) was treated with potassium carbonate (134 mg, 10.8 mmol, 2 eq.) under N2 atmosphere. The reaction flask was heated at 72-75 °C for 5 h. LC/MS showed the formation of the desired peak as well as a small peak corresponding to the dialkylated byproduct. The reaction mixture was cooled to room temperature and filtered through a pad of celite and washed with DCM (20 mL). The obtained product was concentrated and purified by ISCO MPLC system using a silica column (80 g). The column was eluted with a (0-20)% DCM/MeOH gradient. The fractions were combined and concentrated by rotary evaporator under reduced pressure to afford intermediate 4 (2.2 g, 84% yield) as a solid.

Step 5: A solution of intermediate 4 (2.2 g, 3.3 mmol, 1 eq) in anhydrous DCM (30 mL) was stirred and cooled at 0 °C. Chloroacetyl chloride (0.26 mL, 3.3 mmol, 1 eq) was added followed by slow addition of Et ₃ N (1.3 mL, 9.9). After 10 min, LC/MS confirmed the completion of the reaction. The reaction mixture was diluted with DCM (25 mL) and washed with saturated NaHCO ₃ solution (25 mL) and brine (25 mL). The organic phase was dried over Na ₂ SO ₄ and filtered. The organic phase was concentrated and purified by ISCO MPLC using a silica gel column (80 g) and eluting the column with a (0-50)% hexane/ethyl acetate gradient. The fractions were combined and concentrated under reduced pressure using a rotary evaporator to give intermediate 5 (2.0 g, 83% yield) as an oil.

Step 6: To a solution of intermediate 5 (1 g, 1.3 mmol, 1 eq) in LC/ _MS grade acetonitrile (20 mL) was added dimethylamine (3.3 mL, 6.7 mmol, 5 eq, 2 M in THF) and DBU (0.4 mL, 2.6 mmol, 2 eq) sequentially under N2 gas. The clear solution was stirred at room temperature overnight. The next day, LC/MS confirmed complete conversion to the product. The reaction mixture was concentrated under reduced pressure and diluted with DCM (55 mL) and washed with saturated _NaHCO3 solution (25 mL) and brine (25 mL) _. The organic phase was dried over _Na2SO4 and filtered. The organic phase was concentrated and the crude product was purified by MPLC/ISCO using a silica column (40 g). The column was eluted with a gradient of (0-100)% hexane-EtOAc and (0-20)% DCM-MeOH. The fractions were combined and concentrated using a rotary evaporator to give Compound 1 (720 mg, 71% yield) as an oil. ESI MS (m/z): 751.74 (M+H) ⁺ . Example 2: Synthesis of Compound 2 of Table 1 (2-((8-(heptadecan-9-yloxy)-8-oxooctyl)(6-oxo-6-(undecanyloxy)hexyl)amino)-N,N,N-trimethyl-2-oxoethane-1-ammonium chloride)

Compound 2 was prepared according to Scheme 2 shown in FIG2 .

Compound 1 (210 mg, 0.27 mmol, 1 eq) was placed in a 15 mL flash bottle and MeI (1.5 mL, excess) was added. The resulting clear solution was stirred at room temperature overnight. The next day, LC/MS showed complete conversion of the starting material to the methyl salt. Excess MeI was evaporated and the intermediate was purified by ISCO MPLC using a 25 g silica column. The column was eluted with a (0-20)% DCM-MeOH gradient. The pure fractions were concentrated to give the intermediate as iodine ions counter ions. This material was dissolved in DCM (4 mL) and passed through a column of Amberlyst A26 (5.5 g, activated with HCl). The column was eluted with 100% DCM and all fractions were concentrated. The residue was redissolved in a minimum volume of DCM and passed through the column. The column was again eluted with DCM and all fractions were concentrated. The product was dried under high vacuum to give compound 2 (130 mg, 60% yield) as a chloride counter ion. ESI MS (m/z): 765.85 (M) ⁺ . Example 3: Synthesis of compound 3 of Table 1 (8-(2-((2-(dimethylamino)ethyl)thio)-N-(6-oxo-6-(undecyloxy)hexyl)acetamido)octanoic acid heptadecan-9-yl ester

Compound 3 was prepared according to Scheme 3 shown in FIG3 .

Step 5: Steps 1 to 4 of Scheme 3 were carried out according to the same procedure as described in Steps 1 to 4 of Scheme 1 above. To a mixture of intermediate 4 (800 mg, 1.2 mmol, 1 eq) and {(2-dimethylamino)ethylthio}acetic acid (391 mg, 2.4 mmol, 2 eq) in anhydrous _DCM (20 mL) in DCM (20 mL) was added _Et3N (0.33 mL, 2.4 mmol), DMAP (14 mg, 0.12 mmol, 0.1 eq) and EDC.HCl (287 mg, 1.56 mmol, 1.2 eq) sequentially at room temperature under N2 gas. The resulting mixture was stirred at room temperature overnight. The next day, LC/MS confirmed the formation of the product. The reaction mixture was diluted with DCM (30 mL) and washed with saturated aqueous NaHCO ₃ (20 mL) and brine (20 mL). The organic phase was dried over Na ₂ SO ₄ and filtered. The organic phase was concentrated and purified by ISCO MPLC using a silica gel column (40 g) and the column was eluted with a (0-100)% hexane/ethyl acetate and (0-20%) DCM-MeOH gradient. The fractions were combined and concentrated under reduced pressure using a rotary evaporator to give compound 3 (405 mg, 43% yield) as an oil. ESI MS (m/z): 811.71 (M+H) ⁺ . Example 4: Synthesis of Compound 4 in Table 1 (2-((2-((8-(heptadecan-9-yloxy)-8-oxooctyl)(6-oxo-6-(undecyloxy)hexyl)amino)-2-oxoethyl)thio)-N,N,N-trimethylethyl-1-ammonium chloride)

Compound 4 was prepared according to Scheme 4 shown in FIG4 .

Compound 3 (120 mg, 0.14 mmol, 1 eq) was weighed into a 15 mL flash bottle, followed by the addition of MeI (1.5 mL, excess). The resulting clear solution was stirred overnight at room temperature. The next day, LC/MS showed a peak corresponding to the product along with another peak corresponding to m/z=834. Excess MeI was evaporated, and the intermediate was purified by ISCO MPLC using a 25 g silica column. The column was eluted with a (0-20)% DCM-MeOH gradient. The pure fraction was concentrated to obtain the intermediate as an iodine ion counter ion. The intermediate was dissolved in DCM (4 mL) and passed through a column of Amberlyst A26 (5 g, activated with HCl). The column was eluted with 100% DCM and all fractions were concentrated. The resulting residue was redissolved in a minimum volume of DCM and passed through the column. The column was again eluted with DCM and all fractions were concentrated. The product was dried under high vacuum to give the desired product compound 4 (82 mg, 67% yield) as a chloride counter ion. ESI MS (m/z): 825.78 (M) ⁺ . Example 5: Synthesis of Compound 5 of Table 1 (8-(2-(cis-3,4-dihydroxypyrrolidin-1-yl)-N-(6-oxo-6-(undecyloxy)hexyl)acetamido)octanoic acid heptadecan-9-yl ester

Compound 5 was prepared according to Scheme 5 shown in FIG5 .

Step 6: Steps 1 to 5 of Scheme 5 were carried out according to the same procedure as described in Steps 1 to 5 of Scheme 1 above. Intermediate 5 (600 mg, 0.81 mmol, 1 eq) and cis -DHP.HCl (225 mg, 1.6 mmol, 2 eq) were suspended in LC/MS grade acetonitrile (40 mL). DBU (0.23 mL, 1.6 mmol, 2 eq) was added and the reaction flask was heated at 45 °C and stirred under _N2 gas overnight. The next day, LC/MS confirmed complete conversion to the product. The heating bath was removed and the reaction flask was cooled to room temperature. The reaction mixture was concentrated under reduced pressure using a rotary evaporator and the crude product was purified by MPLC/ISCO using a silica column (40 g). The column was eluted with a gradient of (0-100)% hexane-EtOAc and (0-20)% DCM-MeOH. The fractions were combined and concentrated using a rotary evaporator to give compound 5 (455 mg, 70% yield) as an oil. ESI MS (m/z): 809.72 (M+H) ⁺ . Example 6: Synthesis of compound 6 of Table 1 (1-(2-((8-(heptadecan-9-yloxy)-8-oxooctyl(6-oxo-6-(undecyloxy)hexyl)amino)-2-oxoethyl)-cis-3,4-dihydroxy-1-methylpyrrolidin-1-ium chloride)

Compound 6 was prepared according to Scheme 6 shown in FIG6 .

Compound 5 (120 mg, 0.14 mmol, 1 eq) was placed in a 15 mL flash bottle and MeI (1.3 mL, excess) was added. The resulting clear solution was stirred overnight at room temperature. The next day, LC/MS showed that the starting material was completely converted to methyl iodide. Excess MeI was evaporated and the residue was dissolved in DCM (4 mL). This solution was passed through a column of Amberlyst A26 (5 g, activated with HCl). The column was eluted with 100% DCM and all elutions were concentrated. The resulting residue was redissolved in a minimum volume of DCM and passed through the above column. The column was again eluted with DCM and all elutions were concentrated. The product was dried under high vacuum to obtain compound 6 (116 mg, 89% yield) as a chloride counter ion. ESI MS (m/z): 823.73 (M) ⁺ . Example 7: Synthesis of compound 7 in Table 1 (8-(2-((2-hydroxyethyl)(methyl)amino)-N-(6-oxo-6-(undecyloxy)hexyl)acetamido)octanoic acid heptadecan-9-yl ester)

Compound 7 was prepared according to Scheme 7 shown in FIG7 .

Step 6: Steps 1 to 5 of Scheme 7 were carried out according to the same procedure as described in Steps 1 to 4 of Scheme ₁ above. To a mixture of intermediate 5 (450 mg, 0.60 mmol, 1 eq.) and N-methyl N-hydroxyethylamine (91 mg, 1.21 mmol, 2 eq.) in LC/MS grade acetonitrile (20 mL) was added DBU (0.17 mL, 1.2 mmol, 2 eq.) under N2 gas. The clear solution was stirred at room temperature overnight. The next day, LC/MS confirmed complete conversion to the product. The product was concentrated under reduced pressure and diluted with DCM (40 mL), and washed with saturated _NaHCO3 solution (2 mL) and brine (20 mL). The organic phase was dried _{over Na2SO4} and filtered. The organic phase was concentrated and the crude product was purified by MPLC/ISCO using a silica column (40 g). The column was eluted with a gradient of (0-100)% hexane-EtOAc and (0-20)% DCM-MeOH. The fractions were combined and concentrated using a rotary evaporator to give compound 7 (204 mg, 43% yield) as an oil. ESI MS (m/z): 781.77 (M+H) ⁺ . Example 8: Synthesis of Compound 8 of Table 1 (2-(2-(dimethylamino)-N-(8-(heptadecan-9-yloxy)-8-oxooctyl)acetamido)ethyl tetradecanoate)

Compound 8 was prepared according to Scheme 8 shown in FIG8 .

Step 1: To a stirred solution of heptadecan-9-ol (10 g, 38.9 mmol, 1 eq.) and 8-bromooctanoic acid (10.4 g, 46.7 mmol, 1.2 eq.) in DCM (100 mL) were added triethylamine (10.8 mL, 77.9 mmol, 2 eq.), DMAP (470 mg, 3.89 mmol, 0.1 eq.) and ^EDC.HCl (8.9 g, 46.7 mmol, 1.2 eq.). The clear reaction solution was stirred at room temperature under nitrogen for 5 h. The reaction mixture was diluted with DCM (50 mL) and subsequently washed with saturated sodium bicarbonate solution (70 mL), water (50 mL) and brine (50 mL). The organic phase was dried over sodium sulfate and filtered. The organic phase was concentrated and the crude product was purified by ISCO/MPLC using a silica gel column (220 g). The column was eluted with a (0-30)% hexane-ethyl acetate gradient. The fractions were combined and concentrated under reduced pressure using a rotary evaporator to give intermediate 1 (11 g, 61% yield).

Step 2: Intermediate 1 (6 g, 12.9 mmol, 1 eq) and ethanolamine (5.9 g, 97.4 mmol, 7.5 eq) were suspended in a mixture of acetonitrile/CPME (60 mL, 1:1). K ₂ CO ₃ (1.8 g, 12.9 mmol, 1 eq) was added to the above mixture. The reaction mixture was heated to 70 °C and stirred for 3 hours. LC/MS confirmed the completion of the reaction. The heating bath was removed and the reaction mixture was cooled to room temperature. The solid was filtered off and washed with acetonitrile. The filtrate was concentrated and the residue was suspended in DCM (50 5 L). The organic layer was washed with a saturated aqueous solution of NaHCO ₃ (25 mL), water (25 mL) and brine (20 mL). The organic phase was dried _{over Na2SO4} and filtered. The organic phase was concentrated to give the mono-N-alkylated product in quantitative yield. This intermediate was dissolved in DCM (50 mL) followed by the addition of triethylamine (3.6 mL, 25.8 mmol, 2 eq) and _Boc2O (3.1 g, 14.1 mmol, 1.1 eq). The mixture was stirred at room temperature overnight. The next day, LC/MS showed the reaction was complete. The reaction mixture was diluted with DCM (30 mL) followed by washing with saturated sodium bicarbonate solution (40 mL) and brine (40 mL). The organic phase was dried over sodium sulfate and filtered. The organic phase was concentrated and the crude product was purified by ISCO/MPLC using a silica gel column (120 g). The column was eluted with a (0-100)% hexane-ethyl acetate gradient. The fractions were combined and concentrated under reduced pressure using a rotary evaporator to give intermediate 2 (4.6 g, 66.6% yield) as an oil.

Step 3: To a solution of intermediate 2 (2.1 g, 3.8 mmol, 1 eq.) in anhydrous DCM (25 ml) was added _Et3N (1.0 mL, 7.7 mmol, 2 eq.) and myristic acid chloride (1.16 mL, 4.2 mmol, 1.1 eq.) at room temperature. After 2 hours, LC/MS confirmed the completion of the reaction. Stop the reaction and dilute with DCM (20 mL). Wash the organics with saturated sodium bicarbonate solution (20 mL), water (20 mL), and brine solution (20 mL) in sequence. Dry the organic phase over sodium sulfate and filter. The organic phase was concentrated and the crude product was purified by ISCO/MPLC using a silica gel column (80 g) and eluting the column with a (0-50)% hexane-ethyl acetate gradient. The fractions were combined and concentrated under reduced pressure using a rotary evaporator to give intermediate 3 (2.2 g, 76% yield) as an oil.

Step 4: To a solution of intermediate 3 (1.7 g, 2.2 mmol, 1.0 equiv) in DCM (25 ml) was added methanesulfonic acid (MSA, 0.29 mL, 4.4 mmol, 2.0 equiv) under _N2 gas. The clear solution was stirred at room temperature overnight. The next day, LC/MS confirmed complete cleavage of the Boc group. The reaction flask was cooled with an ice bath, followed by the addition of chloroacetyl chloride (0.2 mL, 2.6 mmol, 1.15 equiv) followed by the dropwise addition of triethylamine (1.6 mL, 11.2 mmol, 5.0 equiv). After 15 minutes, LC/MS confirmed the completion of the reaction. The reaction mixture was diluted with DCM (15 mL) and washed with water (20 mL) and brine (20 mL). The organic phase was dried over _Na2SO4 and filtered. The organic phase was concentrated and purified by ISCO MPLC system using _a silica column (80 g). The column was eluted with a (0-50)% hexane-ethyl acetate gradient. The fractions were combined and concentrated via rotary evaporator to give intermediate 4 (1.6 g, 87% yield) as a solid.

Step 5: To a solution of intermediate 4 (480 mg, 0.65 mmol, 1 eq) in LC/MS grade acetonitrile (40 mL) was added dimethylamine (1.64 mL, 3.2 mmol, 5 eq, 2 M in THF) and DBU (2 eq) at room temperature. The reaction mixture was stirred under _N2 gas at room temperature overnight. The next day, LC _/ MS confirmed consumption of starting material and complete conversion to product. The reaction mixture was concentrated and the residue was suspended in DCM. The product was washed with saturated aqueous _NaHCO3 , water and brine. The organic phase was dried over _Na2SO4 and filtered. The organic phase was concentrated and purified by ISCO/MPLC system using a silica column (40 g). The column was eluted with a gradient of (0-100)% hexane-ethyl acetate and (0-20)% DCM-MeOH. The fractions were combined and concentrated by rotary evaporator to give compound 8 (325 mg, 68% yield) as an oil. ESI MS (m/z): 737.72 (M+H) ⁺ . Example 9: Synthesis of compound 9 of Table 1 (tetradecanoic acid 2-(2-(cis-3,4-dihydroxypyrrolidin-1-yl)-N-(8-(heptadecan-9-yloxy)-8-oxooctyl)acetamido)ethyl ester)

Compound 9 was prepared according to Scheme 9 shown in FIG9 .

To a solution of intermediate 4 of Example 8 (520 mg, 0.71 mmol, 1 eq.) in LC/MS grade acetonitrile (40 mL) was added cDHP.HCl (199 mg, 1.42 mmol, 2 eq.) and DBU (2 eq.) at room temperature. The reaction mixture was stirred at 45 °C under _N2 gas overnight. The next day, LC/MS confirmed consumption of the starting material and complete conversion to the product. The reaction mixture was concentrated and the residue was suspended in DCM. The product was washed with saturated aqueous _NaHCO3 , water and brine. The organic phase was dried _{over Na2SO4} and filtered. The organic phase was concentrated and purified by ISCO/MPLC system using a silica column (40 g). The column was eluted with a gradient of (0-100)% hexane-ethyl acetate and (0-20)% DCM-MeOH. The fractions were combined and concentrated by rotary evaporator to give compound 9 (450 mg, 79% yield) as an oil. ESI MS (m/z): 795.74 (M+H) ⁺ . Example 10: Synthesis of compound 10 of Table 1 (tetradecanoic acid 2-(N-(8-(heptadecan-9-yloxy)-8-oxooctyl)-1-methylpiperidin-4-carboxamido)ethyl ester)

Compound 10 was prepared according to Scheme 10 shown in FIG10 .

Step 4: Steps 1 to 3 of Scheme 10 were carried out according to the same procedure as described in Steps 1 to 3 of Scheme 8 above. To a solution of intermediate 3 (500 mg, 0.66 mmol, 1.0 equiv) in DCM (20 ml) was added methanesulfonic acid (MSA, 86 µL, 1.32 mmol, 2.0 equiv) under _N2 gas. The clear solution was stirred overnight at room temperature. The next day, LC/MS confirmed complete cleavage of the Boc group. The reaction flask was cooled with an ice bath, followed by addition of a saturated aqueous solution of _NaHCO3 . The reaction mixture was allowed to warm to room temperature slowly. The organic phase was then separated. The aqueous phase was extracted with DCM (20 mL). The combined organic layers were dried _{over Na2SO4} and filtered. The organic phase was concentrated to give the crude amine intermediate which was used in the next step without further purification. This amine intermediate was dissolved in DCM (20 mL) and cooled to 0°C. N-Methylpiperidinic acid (142 mg, 0.99 mmol, 1.5 eq), HATU (290 mg, 0.76 mmol, 1.15 eq) and triethylamine (0.18 mL, 1.32 mmol, 2 eq) were added sequentially to the above mixture. After 15 min, the cooling was removed and the reaction was stirred at room temperature for 2 h. LC/MS confirmed the formation of the product. The reaction mixture was diluted with DCM (20 mL) and washed with saturated aqueous NaHCO ₃ (25 mL), water (20 mL) and brine (20 mL). The organic phase was dried over _Na2SO4 and filtered. The organic phase was concentrated and purified by ISCO/MPLC system using a silica column (40 g). The column was eluted with a gradient of ₍ 0-100)% hexane-ethyl acetate and (0-20)% DCM-MeOH. The fractions were combined and concentrated by rotary evaporator to give compound 10 (300 mg, 59% yield) as an oil. ESI MS (m/z): 777.73 (M+H) ⁺ . Example 11: Synthesis of Compound 11 of Table 1 (tetradecanoic acid 4-(N-(8-(heptadecan-9-yloxy)-8-oxooctyl)-1-methylpiperidinyl-4-carboxamido)butyl ester)

Compound 11 was prepared according to Scheme 11 shown in FIG11 .

Step 2: Step 1 of Scheme 11 was carried out according to the same procedure as described in Step 1 of Scheme 8 above. Intermediate 1 (3 g, 6.4 mmol, 1 eq) and aminobutanol (1.7 g, 19.4 mmol, 3 eq) were suspended in a mixture of acetonitrile/CPME (40 mL, 1:1). K ₂ CO ₃ (0.89 g, 6.4 mmol, 1 eq) was added to the above mixture. The reaction mixture was heated to 70 °C and stirred for 5 hours. LC/MS confirmed the completion of the reaction. The heating bath was removed and the reaction mixture was cooled to room temperature. The solid was filtered off and washed with acetonitrile. The reaction mixture was concentrated and the residue was suspended in DCM (50 mL). The organic layer was washed with saturated aqueous NaHCO ₃ solution (25 mL), water (25 mL) and brine (20 mL). The organic phase was dried over Na ₂ SO ₄ and filtered. The organic phase was concentrated to give the mono-N-alkylated product in quantitative yield. This intermediate was dissolved in DCM (25 mL) followed by the addition of Boc ₂ O (1.6 g, 7.1 mmol, 1.1 eq) and triethylamine (1.8 mL, 12.9 mmol, 2 eq). The reaction mixture was stirred at room temperature overnight. The next day, LC/MS showed the reaction was complete. The reaction mixture was diluted with DCM (30 mL) followed by washing with saturated sodium bicarbonate solution (40 mL) and brine (40 mL). The organic phase was dried over sodium sulfate and filtered. The organic phase was concentrated and the crude product was purified by ISCO/MPLC using a silica gel column (120 g). The column was eluted with a (0-60)% hexane-ethyl acetate gradient. The fractions were combined and concentrated under reduced pressure using a rotary evaporator to give intermediate 2 (2.0 g, 54% yield) as an oil.

Step 3: To a solution of intermediate 2 (2.0 g, 3.5 mmol, 1 eq.) in anhydrous DCM (25 ml) was added _Et3N (0.97 mL, 7.0 mmol, 2 eq.) and myristic acid chloride (1.05 mL, 3.8 mmol, 1.1 eq.) at room temperature. After 1 hour, LC/MS confirmed the completion of the reaction. Stop the reaction and dilute with DCM (20 mL). Wash the organics with saturated sodium bicarbonate solution (20 mL), water (20 mL), and brine solution (20 mL). Dry the organic phase over sodium sulfate and filter. The organic phase was concentrated and the crude product was purified by ISCO/MPLC using a silica gel column (80 g) and eluting the column with a (0-50)% hexane-ethyl acetate gradient. The fractions were combined and concentrated under reduced pressure using a rotary evaporator to give intermediate 3 (2.4 g, 89% yield) as an oil.

Step 4: To a solution of intermediate 3 (500 mg, 0.64 mmol, 1.0 eq) in DCM (15 ml) was added methanesulfonic acid (MSA, 86 L, 1.32 mmol, 2.0 eq) under _N2 gas. The clear solution was stirred overnight at room temperature. The next day, LC/MS confirmed complete cleavage of the Boc group. The reaction flask was cooled with an ice bath, followed by the addition of N-methylpiperidinic acid (183 mg, 1.28 mmol, 2 eq), HATU (365 mg, 1.28 mmol, 1.5 eq) and triethylamine (0.26 mL, 1.92 mmol, 3 eq). After 15 min, the ice bath was removed and the reaction was stirred for 30 min. LC/MS confirmed the formation of the product. The reaction mixture was diluted with DCM (20 mL) and washed with saturated aqueous NaHCO ₃ (20 mL), water (20 mL) and brine (20 mL). The organic phase was dried over Na ₂ SO ₄ and filtered. The organic phase was concentrated and purified by ISCO/MPLC system using a silica column (40 g). The column was eluted with a gradient of (0-100)% hexane-ethyl acetate and (0-20)% DCM-MeOH. The fractions were combined and concentrated by rotary evaporator to give compound 11 (356 mg, 69% yield) as an oil. ESI MS (m/z): 805.8 (M+H) ⁺ . Example 12: Synthesis of Compound 12 in Table 1 (tetradecanoic acid 4-(2-(dimethylamino)-N-(8-(heptadecan-9-yloxy)-8-oxooctyl)acetamido)butyl ester)

Compound 12 was prepared according to Scheme 12 shown in FIG12 .

Step 4: Step 1 of Scheme 12 was carried out according to the same procedure as described in Step 1 of Scheme 8 above. Steps 2 and 3 of Scheme 12 were carried out according to the same procedure as described in Steps 2 and 3 of Scheme 11 above. To a solution of intermediate 3 (1.8 g, 2.3 mmol, 1.0 eq.) in DCM (25 ml) was added methanesulfonic acid (MSA, 0.3 mL, 4.6 mmol, 2.0 eq.) under _N2 gas. The clear solution was stirred at room temperature overnight. The next day, LC/MS confirmed complete cleavage of the Boc group. The reaction flask was cooled with an ice bath, followed by the addition of chloroacetyl chloride (0.2 mL, 2.7 mmol, 1.1 eq.) followed by the dropwise addition of triethylamine (1.6 mL, 11.5 mmol, 5.0 eq.). After 15 minutes, LC/MS confirmed the completion of the reaction. The reaction mixture was diluted with DCM (20 mL) and washed with water (20 mL) and brine (20 mL). The organic phase was dried _{over Na2SO4} and filtered. The organic phase was concentrated and purified by ISCO MPLC system using a silica column (80 g). The column was eluted with a (0-50)% hexane-ethyl acetate gradient. The fractions were combined and concentrated via rotary evaporator to give intermediate 4 (1.5 g, 88% yield) as a solid.

Step 5: To a solution of intermediate 4 (600 mg, 0.79 mmol, 1 eq.) in LC/MS grade acetonitrile (40 mL) was added dimethylamine (1.9 mL, 3.9 mmol, 5 eq., 2 M in THF) and DBU (2 eq.) at room temperature. The reaction mixture was stirred under _N2 gas at room temperature overnight. The next day, LC/MS confirmed consumption of the starting material and complete conversion to the product. The reaction mixture was concentrated and the residue was suspended in DCM and washed with saturated aqueous _NaHCO3 , water and brine. The organic phase was dried _{over Na2SO4} and filtered. The organic phase was concentrated and purified by ISCO/MPLC system using a silica column (40 g). The column was eluted with a gradient of (0-100)% hexane-ethyl acetate and (0-20)% DCM-MeOH. The fractions were combined and concentrated by rotary evaporator to give compound 12 (436 mg, 72% yield) as an oil. ESI MS (m/z): 765.3 (M+H) ⁺ . Example 13: Synthesis of compound 13 of Table 1 (tetradecanoic acid 4-(2-(cis-3,4-dihydroxypyrrolidin-1-yl)-N-(8-(heptadecan-9-yloxy)-8-oxooctyl)acetamido)butyl ester

Compound 13 was prepared according to Scheme 13 shown in FIG13 .

To a solution of intermediate 4 of Example 12 (580 mg, 0.76 mmol, 1 eq.) in LC/MS grade acetonitrile (40 mL) was added cDHP.HCl (212 mg, 1.53 mmol, 2 eq.) and DBU (2 eq.) at room temperature. The reaction mixture was stirred at 45 °C under _N2 gas overnight. The next day, LC/MS confirmed consumption of the starting material and complete conversion to the product. The reaction mixture was concentrated and the residue was suspended in DCM and washed with saturated _{aqueous NaHCO3} , water and brine. The organic phase was dried over _Na2SO4 and filtered. The organic phase was concentrated and purified by ISCO/MPLC system using a silica column (40 g). The column was eluted with a gradient of (0-100)% hexane-ethyl acetate and (0-20)% DCM-MeOH. The fractions were combined and concentrated by rotary evaporator to give compound 13 (476 mg, 75% yield) as a viscous oil. ESI MS (m/z): 823.79 (M+H) ⁺ . Example 14: Synthesis of compound 14 of Table 1 (tetradecanoic acid 3-(2-(dimethylamino)-N-(8-(heptadecan-9-yloxy)-8-oxooctyl)acetamido)propyl ester)

Compound 14 was prepared according to Scheme 14 shown in FIG14 .

Step 2: Step 1 of Scheme 14 was carried out according to the same procedure as described in Step 1 of Scheme 8 above. Intermediate 1 (2.8 g, 6.06 mmol, 1 eq) and aminopropanol (1.4 g, 18.1 mmol, 3 eq) were suspended in a mixture of acetonitrile/CPME (40 mL, 1:1). K ₂ CO ₃ (0.83 g, 12.9 mmol, 1 eq) was added to the above mixture. The reaction mixture was heated to 70 °C and stirred for 5 hours. LC/MS confirmed the completion of the reaction. The heating bath was removed and the reaction mixture was cooled to room temperature. The solid was filtered off and washed with acetonitrile. The reaction mixture was concentrated and filtered and the residue was suspended in DCM (40 mL). The organic layer was washed with saturated aqueous NaHCO ₃ solution (25 mL), water (25 mL) and brine (20 mL). The organic phase was dried over Na ₂ SO ₄ and filtered. The organic phase was concentrated to give the mono-N-alkylated product in quantitative yield. This intermediate was dissolved in DCM (30 mL) followed by the addition of Boc ₂ O (1.6 g, 7.2 mmol, 1.2 eq) and triethylamine (1.7 mL, 12.1 mmol, 2 eq). The reaction mixture was stirred at room temperature overnight. The next day, LC/MS showed the reaction was complete. The reaction mixture was diluted with DCM (30 mL) followed by washing with saturated sodium bicarbonate solution (40 mL) and brine (40 mL). The organic phase was dried over sodium sulfate and filtered. The organic phase was concentrated and the crude product was purified by ISCO/MPLC using a silica gel column (120 g). The column was eluted with a (0-100)% hexane-ethyl acetate gradient. The fractions were combined and concentrated under reduced pressure using a rotary evaporator to give intermediate 2 (2.0 g, 61% yield) as an oil.

Step 3: To a solution of intermediate 2 (2.0 g, 3.6 mmol, 1 eq.) in anhydrous DCM (25 ml) was added _Et3N (0.97 mL, 7.2 mmol, 2 eq.) and myristic acid chloride (1.08 mL, 3.9 mmol, 1.1 eq.) at room temperature. After 2 hours, LC/MS confirmed the completion of the reaction. The reaction was stopped and diluted with DCM (20 mL). The organics were washed sequentially with saturated sodium bicarbonate solution (20 mL), water (20 mL), and brine solution (20 mL). The organic phase was dried over sodium sulfate and filtered. The organic phase was concentrated and the crude product was purified by ISCO/MPLC using a silica gel column (80 g) and eluting the column with a (0-50)% hexane-ethyl acetate gradient. The fractions were combined and concentrated under reduced pressure using a rotary evaporator to give intermediate 3 (1.8 g, 66.6% yield) as an oil.

Step 4: To a solution of intermediate 3 (450 mg, 0.58 mmol, 1 eq.) in DCM (30 ml) was added methanesulfonic acid (MSA, 2.0 eq.) under _N2 gas. The clear solution was stirred at room temperature overnight. The next day, LC/MS confirmed complete cleavage of the Boc group. The reaction flask was cooled with an ice bath, followed by the addition of a saturated aqueous solution of _NaHCO3 . The reaction mixture was allowed to warm up to room temperature slowly. The organic phase was then separated. The aqueous phase was extracted with DCM (20 mL). The combined organic layers were dried _{over Na2SO4} and filtered. The organic phase was concentrated to give the crude amine intermediate, which was used in the next step without further purification. This amine intermediate was dissolved in DCM (20 mL) and cooled to 0 °C. N,N'-dimethylglycine HCl (163 mg, 1.17 mmol, 2 eq), HATU (1.15 eq) and triethylamine (2 eq) were added to the above mixture in sequence. After 15 min, the cooling was removed and the reaction was stirred at room temperature for required time. The reaction mixture was diluted with DCM (20 mL) and washed with saturated aqueous NaHCO ₃ solution (20 mL), water (20 mL) and brine (20 mL). The organic phase was dried over Na ₂ SO ₄ and filtered. The organic phase was concentrated and purified by ISCO/MPLC system using a silica column (40 g). The column was eluted with a gradient of (0-100)% hexane-ethyl acetate and (0-20)% DCM-MeOH. The fractions were combined and concentrated by rotary evaporator to give compound 14 (275 mg, 63% yield) as an oil. ESI MS (m/z): 751.83 (M+H) ⁺ . Example 15: Synthesis of compound 15 of Table 1 (tetradecanoic acid 3-(N-(8-(heptadecan-9-yloxy)-8-oxooctyl)-2-(1-methylpiperidin-4-yl)acetamido)propyl ester)

Compound 15 was prepared according to Scheme 15 shown in FIG15 .

To a solution of intermediate 3 (450 mg, 0.58 mmol, 1 eq.) of Example 14 in DCM (30 ml) was added methanesulfonic acid (MSA, 2.0 eq.) under _N2 gas. The clear solution was stirred at room temperature overnight. The next day, LC/MS confirmed complete cleavage of the Boc group. The reaction flask was cooled with an ice bath, followed by addition of a saturated aqueous solution of _NaHCO3 . The reaction mixture was allowed to warm up to room temperature slowly. The organic phase was then separated. The aqueous phase was extracted with DCM (20 mL). The combined organic layers were dried _{over Na2SO4} and filtered. The organic phase was concentrated to give the crude amine intermediate, which was used in the next step without further purification. This amine intermediate was dissolved in DCM (20 mL) and cooled to 0°C. N-Methylpiperidine 4-acetic acid (227 mg, 1.17 mmol, 2 eq.), HATU (1.15 eq.) and triethylamine (2 eq.) were added to the above mixture in sequence. After 15 min, the cooling was removed and the reaction was stirred at room temperature for the required time. The reaction mixture was diluted with DCM (20 mL) and washed with saturated aqueous NaHCO ₃ (20 mL), water (20 mL) and brine (20 mL). The organic phase was dried over Na ₂ SO ₄ and filtered. The organic phase was concentrated and purified by ISCO/MPLC system using a silica column (40 g). The column was eluted with a gradient of (0-100)% hexane-ethyl acetate and (0-20)% DCM-MeOH. The eluted fractions were combined and concentrated by rotary evaporator to obtain compound 15 (290 mg, 61% yield) as a viscous oil. ESI MS (m/z): 805.88 (M+H) ⁺ . Example 16: Synthesis of compound 16 in Table 1 (tetradecanoic acid 3-(N-(8-(heptadecan-9-yloxy)-8-oxooctyl)-1-methylpiperidin-4-carboxamido)propyl ester)

Compound 16 was prepared according to Scheme 16 shown in FIG16 .

To a solution of intermediate 3 (450 mg, 0.58 mmol, 1 eq.) of Example 14 in DCM (30 ml) was added methanesulfonic acid (MSA, 2.0 eq.) under _N2 gas. The clear solution was stirred at room temperature overnight. The next day, LC/MS confirmed complete cleavage of the Boc group. The reaction flask was cooled with an ice bath, followed by addition of a saturated aqueous solution of _NaHCO3 . The reaction mixture was allowed to warm up to room temperature slowly. The organic phase was then separated. The aqueous phase was extracted with DCM (20 mL). The combined organic layers were dried _{over Na2SO4} and filtered. The organic phase was concentrated to give the crude amine intermediate, which was used in the next step without further purification. This amine intermediate was dissolved in DCM (20 mL) and cooled to 0°C. N-Methylpiperidine 4-carboxylic acid (167 mg, 1.17, 2 eq.), HATU (1.15 eq.) and triethylamine (2 eq.) were added to the above mixture in sequence. After 15 min, the cooling was removed and the reaction was stirred at room temperature for the required time. The reaction mixture was diluted with DCM (20 mL) and washed with saturated aqueous NaHCO ₃ (20 mL), water (20 mL) and brine (20 mL). The organic phase was dried over Na ₂ SO ₄ and filtered. The organic phase was concentrated and purified by ISCO/MPLC system using a silica column (40 g). The column was eluted with a gradient of (0-100)% hexane-ethyl acetate and (0-20)% DCM-MeOH. The eluted fractions were combined and concentrated by rotary evaporator to obtain compound 16 (292 mg, 63% yield) as an oil. ESI MS (m/z): 790.87 (M+H)+. Example 17: Synthesis of compound 17 in Table 1 (tetradecanoic acid 3-(2-(cis-3,4-dihydroxypyrrolidin-1-yl)-N-(8-(heptadecan-9-yloxy)-8-oxooctyl)acetamido)propyl ester)

Compound 17 was prepared according to Scheme 17 shown in FIG17 .

Step 4: Step 1 of Scheme 17 was carried out according to the same procedure as described in Step 1 of Scheme 8 above. Steps 2 and 3 of Scheme 17 were carried out according to the same procedure as described in Steps 2 and 3 of Scheme 14 above. To a solution of intermediate 3 (450 g, 0.58 mmol, 1.0 eq.) in DCM (15 ml) was added methanesulfonic acid (MSA, 80 mL, 1.17 mmol, 2.0 eq.) under _N2 gas. The clear solution was stirred at room temperature overnight. The next day, LC/MS confirmed complete cleavage of the Boc group. The reaction flask was cooled with an ice bath, followed by the addition of chloroacetyl chloride (52 µL, 0.64 mmol, 1.1 eq.) followed by the dropwise addition of triethylamine (0.4 mL, 2.9 mmol, 5.0 eq.). After 15 minutes, the ice bath was removed and the reaction was cooled to room temperature. After 1 hour of stirring, LC/MS confirmed the completion of the reaction. The reaction mixture was diluted with DCM (20 mL) and washed with water (20 mL) and brine (20 mL). The organic phase was dried _{over Na2SO4} and filtered. The organic phase was concentrated and purified by ISCO MPLC system using a silica column (40 g). The column was eluted with a (0-50)% hexane-ethyl acetate gradient. The fractions were combined and concentrated via a rotary evaporator to give intermediate 4 (425 mg, 97% yield) as a solid.

Step 5: Intermediate 4 (425 mg, 0.57 mmol, 1 eq) and cis-DHP.HCl (159 mg, 1.14 mmol, 2 eq) were suspended in LC/MS grade acetonitrile (20 mL). DBU (144 µL, 1.14 mmol, 2 eq) was added to the above mixture. The reaction flask was heated at 45 °C and stirred under _N2 gas for 4 hours. LC/MS confirmed the consumption of starting material and the formation of product. The heating bath was removed and the reaction was stirred at room temperature overnight. The reaction mixture was concentrated and the residue was suspended in DCM (30 mL) and washed with saturated aqueous _NaHCO3 (15 mL), water (15 mL) and brine (15 mL). The organic phase was dried over _Na2SO4 and filtered. The organic phase was concentrated and purified by ISCO/MPLC system using _a silica column (40 g). The column was eluted with a gradient of (0-100)% hexane-ethyl acetate and (0-20)% DCM-MeOH. The fractions were combined and concentrated by rotary evaporator to give compound 17 (307 mg, 67% yield) as a viscous oil. ESI MS (m/z): 809.77 (M+H) ⁺ . Example 18: Synthesis of compound 18 of Table 1 ((9Z,12Z)-octadec-9,12-dienoic acid 2-(2-(dimethylamino)-N-(8-(heptadecan-9-yloxy)-8-oxooctyl)acetamido)ethyl ester)

Compound 18 was prepared according to Scheme 18 shown in FIG18 .

Step 3: Steps 1 and 2 of Scheme 18 were carried out according to the same procedure as described in Steps 1 and 2 of Scheme 8 above. To a mixture of intermediate 2 (2.5 g, 4.6 mmol, 1 eq.) and linoleic acid (2.6 g, 9.2 mmol, 2 eq.) in anhydrous DCM (20 mL) were added EDC.HCl (1.7 g, 9.2 mmol, 1.5 eq.), _Et3N (1.28 mL, 9.2 mmol, 2 eq.) and DMAP (220 mg, 1.8 mmol, 0.4 eq.) in sequence under nitrogen. The clear solution was stirred at room temperature overnight. The next day, LC/MS confirmed the completion of the reaction. The reaction was stopped and diluted with DCM (30 mL). The organics were washed sequentially with saturated sodium bicarbonate solution (30 mL) and brine solution (30 mL). The organic phase was dried over sodium sulfate and filtered. The organic phase was concentrated and the crude product was purified by ISCO/MPLC using a silica gel column (80 g) and eluting the column with a (0-50)% hexane-ethyl acetate gradient. The fractions were combined and concentrated under reduced pressure using a rotary evaporator to give intermediate 3 (3.15 g, 87% yield) as an oil.

Step 4: To a solution of intermediate 3 (1.2 g, 1.5 mmol, 1.0 equiv) in DCM (20 ml) was added methanesulfonic acid (MSA, 0.2 mL, 3.0 mmol, 2.0 equiv) under _N2 gas. The clear solution was stirred at room temperature overnight. The next day, LC/MS confirmed the complete cleavage of the Boc group. The reaction flask was cooled with an ice bath, followed by the addition of chloroacetyl chloride (138 µL, 1.7 mmol, 1.15 equiv) followed by the dropwise addition of triethylamine (1.0 mL, 7.5 mmol, 5.0 equiv). After 30 min, LC/MS confirmed the completion of the reaction. The reaction mixture was diluted with DCM (20 mL) and washed with water (20 mL) and brine (20 mL). The organic phase was dried over _Na2SO4 and filtered. The organic phase was concentrated and purified by ISCO MPLC system using _a silica column (40 g). The column was eluted with a (0-40)% hexane-ethyl acetate gradient. The fractions were combined and concentrated via rotary evaporator to give intermediate 4 (1.03 g, 88% yield) as a solid.

Step 5: To a solution of intermediate 4 (500 mg, 0.64 mmol, 1 eq) in LC/MS grade acetonitrile (40 mL) was added dimethylamine (1.6 mL, 3.2 mmol, 5 eq, 2 M in THF) and DBU (2 eq) at room temperature. The reaction mixture was stirred under _N2 gas at room temperature overnight. The next day, LC/MS confirmed consumption of starting material and complete conversion to product. The reaction mixture was concentrated and the residue was suspended in DCM and washed with saturated aqueous _NaHCO3 , water and brine. The organic phase was dried _{over Na2SO4} and filtered. The organic phase was concentrated and purified by ISCO/MPLC system using a silica column (40 g). The column was eluted with a gradient of (0-100)% hexane-ethyl acetate and (0-20)% DCM-MeOH. The fractions were combined and concentrated by rotary evaporator to give compound 18 (335 mg, 66% yield) as an oil. ESI MS (m/z): 789.83 (M+H) ⁺ . Example 19: Synthesis of compound 19 ((9Z,12Z)-octadecane-9,12-dienoic acid 2-(2-(cis-3,4-dihydroxypyrrolidin-1-yl)-N-(8-(heptadecan-9-yloxy)-8-oxooctyl)acetamido)ethyl ester of Table 1)

Compound 19 was prepared according to Scheme 19 shown in FIG19 .

To a solution of intermediate 4 of Example 18 (500 mg, 0.64 mmol, 1 eq.) in LC/MS grade acetonitrile (40 mL) was added cDHP.HCl (177 mg, 1.28 mmol, 2 eq.) and DBU (2 eq.) at room temperature. The reaction mixture was stirred at 45 °C under _N2 gas overnight. The next day, LC/MS confirmed consumption of the starting material and complete conversion to the product. The reaction mixture was concentrated, and the residue was suspended in DCM and washed with saturated aqueous _NaHCO3 , water, and brine. The organic phase was dried _{over Na2SO4} and filtered. The organic phase was concentrated and purified by ISCO/MPLC system using a silica column (40 g). The column was eluted with a gradient of (0-100)% hexane-ethyl acetate and (0-20)% DCM-MeOH. The fractions were combined and concentrated by rotary evaporator to give compound 19 (400 mg, 74% yield) as a viscous oil. ESI MS (m/z): 847.89 (M+H) ⁺ . Example 20: Synthesis of compound 20 ((9Z,12Z)-octadecane-9,12-dienoic acid 2-(N-(8-(heptadecan-9-yloxy)-8-oxooctyl)-2-(1-methylpiperidin-4-yl)acetamido)ethyl ester) of Table 1

Compound 20 was prepared according to Scheme 20 shown in FIG. 20 .

Step 4: Steps 1 and 2 of Scheme 20 were carried out according to the same procedure as described in Steps 1 and 2 of Scheme 8 above. Step 3 of Scheme 20 was carried out according to the same procedure as described in Step 3 of Example 18 above. To a solution of intermediate 3 (1.9 g, 2.3 mmol, 1.0 eq.) in DCM (20 ml) was added methanesulfonic acid (MSA, 0.32 mL, 4.7 mmol, 2.0 eq.) under _N2 gas. The clear solution was stirred at room temperature overnight. The next day, LC/MS confirmed complete cleavage of the Boc group. The resulting amine intermediate was dissolved in DCM (20 mL) and cooled to 0°C. N-methylpiperidine 4-acetic acid (1.5 eq.), HATU (1.15 eq.) and triethylamine (2 eq.) were added sequentially to the above mixture. After 15 minutes, the cooling was removed and the reaction was stirred at room temperature for the required time. The reaction mixture was diluted with DCM (20 mL) and washed with saturated aqueous NaHCO ₃ (20 mL), water (20 mL) and brine (20 mL). The organic phase was dried over Na ₂ SO ₄ and filtered. The organic phase was concentrated and purified by ISCO/MPLC system using a silica column (40 g). The column was eluted with a gradient of (0-100)% hexane-ethyl acetate and (0-20)% DCM-MeOH. The fractions were combined and concentrated by rotary evaporator to give compound 20 (503 mg, 75% yield) as an oil. ESI MS (m/z): 843.4 (M+H) ⁺ . Example 21: Synthesis of Compound 21 in Table 1 ((9Z,12Z)-octadecane-9,12-dienoic acid 2-(N-(8-(heptadecan-9-yloxy)-8-oxooctyl)-1-methylpiperidin-4-carboxamido)ethyl ester)

Compound 21 was prepared according to Scheme 21 shown in FIG21 .

To a solution of intermediate 3 (1.9 g, 2.3 mmol, 1.0 eq.) of Example 20 in DCM (20 ml) was added methanesulfonic acid (MSA, 0.32 mL, 4.7 mmol, 2.0 eq.) under _N2 gas. The clear solution was stirred overnight at room temperature. The next day, LC/MS confirmed complete cleavage of the Boc group. The resulting amine intermediate was dissolved in DCM (20 mL) and cooled to 0°C. N-methylpiperidine 4-carboxylic acid (1.5 eq.), HATU (1.15 eq.) and triethylamine (2 eq.) were added sequentially to the above mixture. After 15 minutes, the cooling was removed and the reaction was stirred at room temperature for the desired time. The reaction mixture was diluted with DCM (20 mL) and washed with saturated aqueous NaHCO ₃ (20 mL), water (20 mL) and brine (20 mL). The organic phase was dried over Na ₂ SO ₄ and filtered. The organic phase was concentrated and purified by ISCO/MPLC system using a silica column (40 g). The column was eluted with a gradient of (0-100)% hexane-ethyl acetate and (0-20)% DCM-MeOH. The fractions were combined and concentrated by rotary evaporator to give compound 21 (530 mg, 82% yield) as a viscous oil. ESI MS (m/z): 829.4 (M+H) ⁺ . Example 22: Synthesis of Compound 22 in Table 1 ((9Z,12Z)-octadecane-9,12-dienoic acid 2-(2-(dimethylamino)-N-(6-oxo-6-(tridec-7-yloxy)hexyl)acetamido)ethyl ester)

Compound 22 was prepared according to Scheme 22 shown in FIG. 22 .

Step 1: DMAP (300 mg, 2.9 mmol, 0.1 eq) and ^EDC.HCl (6.8 g, 35.9 mmol, 1.2 eq) were added to a stirred solution of tridecan-7-ol (6 g, 29.9 mmol, 1 eq) and 6-bromohexanoic acid (7 g, 35.9 mmol, 1.2 eq) in DCM (70 mL). The clear reaction solution was stirred under nitrogen at room temperature overnight. The reaction mixture was diluted with DCM (50 mL) and subsequently washed with saturated sodium bicarbonate solution (40 mL), water (40 mL) and brine (40 mL). The organic phase was dried over sodium sulfate and filtered. The organic phase was concentrated to give intermediate 1 (7.2 g, 66% yield).

Step 2: Intermediate 1 (2.3 g, 6.1 mmol, 1 eq) and aminoethanol (2.6 g, 42.7 mmol, 7 eq) were suspended in a mixture of acetonitrile/CPME (60 mL, 1:1). K ₂ CO ₃ (0.84 g, 12.9 mmol, 1 eq) was added to the above mixture. The reaction mixture was heated to 70 °C and stirred for 5 hours. LC/MS confirmed the completion of the reaction. The heating bath was removed and the reaction mixture was cooled to room temperature. The solid was filtered and washed with acetonitrile. The filtrate was concentrated and the residue was suspended in DCM (60 mL). The organic layer was washed with a saturated aqueous solution of NaHCO ₃ (30 mL), water (30 mL) and brine (30 mL). The organic phase was dried _{over Na2SO4} and filtered. The organic phase was concentrated to give the mono-N-alkylated product in quantitative yield. This intermediate was dissolved in DCM (50 mL), followed by the addition of _Boc2O (1.4 g, 6.7 mmol, 1.1 eq) and triethylamine (1.7 mL, 12.2 mmol, 2 eq) to the mixture. The mixture was stirred at room temperature overnight. The next day, LC/MS showed the reaction was complete. The reaction mixture was diluted with DCM (30 mL), followed by washing with saturated sodium bicarbonate solution (40 mL) and brine (40 mL). The organic phase was dried over sodium sulfate and filtered. The organic phase was concentrated and the crude product was purified by MPLC/ISCO using a silica gel column (80 g). The column was eluted with a (0-100)% hexane-ethyl acetate gradient. The fractions were combined and concentrated under reduced pressure using a rotary evaporator to give intermediate 2 (1.3 g, 62% yield) as an oil.

Step 3: Under nitrogen, EDC.HCl (0.81 g, 4.2 mmol, 1.5 eq), _Et3N (0.79 mL, 5.6 mmol, 2 eq) and DMAP (138 mg, 1.1 mmol, 0.4 eq) were added sequentially to a mixture of intermediate 2 (1.3 g, 2.8 mmol, 1 eq) and linoleic acid (1.2 g, 4.2 mmol, 1.5 eq) in anhydrous DCM (20 mL). The clear solution was stirred at room temperature overnight. The next day, LC/MS confirmed the completion of the reaction. The reaction was stopped and diluted with DCM (30 mL). The organics were washed sequentially with saturated sodium bicarbonate solution (30 mL) and brine solution (30 mL). The organic phase was dried over sodium sulfate and filtered. The organic phase was concentrated and the crude product was purified by ISCO/MPLC using a silica gel column (80 g) and eluting the column with a (0-50)% hexane-ethyl acetate gradient. The fractions were combined and concentrated under reduced pressure using a rotary evaporator to give intermediate 3 (1.5 g, 75% yield) as an oil.

Step 4: Methanesulfonic acid (MSA, 72 μL, 1.1 mmol, 2.0 equiv) was added to a solution of intermediate 3 (400 mg, 0.55 mmol, 1.0 equiv) in DCM (20 mL) under _N2 gas. The clear solution was stirred overnight at room temperature. The next day, LC/MS confirmed the complete cleavage of the Boc group. N,N'-dimethylglycine (72 mg, 0.72 mmol, 1.3 equiv), HATU (316 mg, 0.83 mmol, 1.4 equiv) and diisopropylethylamine (0.5 mL, 2.7 mmol, 5 equiv) were added to the above reaction mixture in sequence. The resulting solution was stirred under _N2 gas at ambient temperature for 3 hours. LC/MS showed complete conversion of the reaction. The reaction mixture was diluted with DCM (20 mL) and washed with saturated aqueous NaHCO ₃ (10 mL), water (10 mL) and brine (10 mL). The organic phase was dried over Na ₂ SO ₄ and filtered. The organic phase was concentrated and purified by ISCO/MPLC system using a silica column (24 g). The column was eluted with a gradient of (0-100)% hexane-ethyl acetate and (0-20)% DCM-MeOH. The fractions were combined and concentrated by rotary evaporator to give compound 22 (145 mg, 37% yield) as an oil. ESI MS (m/z): 705.66 (M+H) ⁺ . Example 23: Synthesis of Compound 23 in Table 1 ((9Z,12Z)-octadecane-9,12-dienoic acid 2-(4-(dimethylamino)-N-(6-oxo-6-(tridec-7-yloxy)hexyl)butyrylamino)ethyl ester)

Methanesulfonic acid (MSA, 72 μL, 1.1 mmol, 2.0 equiv) was added to a solution of intermediate 3 (400 mg, 0.55 mmol, 1.0 equiv) from Example 22 in DCM (20 mL) under _N2 gas. The clear solution was stirred overnight at room temperature. The next day, LC/MS confirmed complete cleavage of the Boc group. N,N'-dimethylbutyric acid (94 mg, 0.72 mmol, 1.3 equiv), HATU (316 mg, 0.83 mmol, 1.4 equiv) and diisopropylethylamine (0.5 mL, 2.7 mmol, 5 equiv) were added to the above reaction mixture in sequence. The resulting solution was stirred under _N2 gas at ambient temperature for 3 hours. LC/MS showed complete conversion of the reaction. The reaction mixture was diluted with DCM (20 mL) and washed with saturated aqueous NaHCO ₃ (10 mL), water (10 mL) and brine (10 mL). The organic phase was dried over Na ₂ SO ₄ and filtered. The organic phase was concentrated and purified by ISCO/MPLC system using a silica column (24 g). The column was eluted with a gradient of (0-100)% hexane-ethyl acetate and (0-20)% DCM-MeOH. The fractions were combined and concentrated by rotary evaporator to give compound 23 (160 mg, 37% yield) as an oil. ESI MS (m/z): 733.71 (M+H) ⁺ . Example 24: Synthesis of Compound 24 in Table 1 ((9Z,12Z)-octadecane-9,12-dienoic acid 2-(2-((2-dimethylamino)ethyl)thio)-N-(6-oxo-6-(tridec-7-yloxy)hexyl)acetamido)ethyl ester)

Methanesulfonic acid (MSA, 72 μL, 1.1 mmol, 2.0 equiv) was added to a solution of intermediate 3 (400 mg, 0.55 mmol, 1.0 equiv) from Example 22 in DCM (20 mL) under _N2 gas. The clear solution was stirred overnight at room temperature. The next day, LC/MS confirmed complete cleavage of the Boc group. N,N'-dimethylethylthioacetic acid (117 mg, 2.7 mmol, 5 equiv), HATU (316 mg, 0.83 mmol, 1.4 equiv) and diisopropylethylamine (0.5 mL, 1.0 mmol) were added to the above reaction mixture in sequence. The resulting solution was stirred under _N2 gas at ambient temperature for 3 hours. LC/MS showed complete conversion of the reaction. The reaction mixture was diluted with DCM (20 mL) and washed with saturated aqueous NaHCO ₃ (10 mL), water (10 mL) and brine (10 mL). The organic phase was dried over Na ₂ SO ₄ and filtered. The organic phase was concentrated and purified by ISCO/MPLC system using a silica column (24 g). The column was eluted with a gradient of (0-100)% hexane-ethyl acetate and (0-20)% DCM-MeOH. The fractions were combined and concentrated by rotary evaporator to give compound 24 (170 mg, 37% yield) as an oil. ESI MS (m/z): 765.98 (M+H) ⁺ . Example 25: Synthesis of Compound 25 in Table 1 (6-(2-(dimethylamino)-N-(6-oxo-6-(undecyloxy)hexyl)acetamido)hexanoic acid tridecyl-7-yl ester)

Compound 25 was prepared according to Scheme 23 shown in FIG. 23 .

Step 1: DMAP (300 mg, 2.9 mmol, 0.1 eq) and ^EDC.HCl (6.8 g, 35.9 mmol, 1.2 eq) were added to a stirred solution of tridecan-7-ol (6 g, 29.9 mmol, 1 eq) and 6-bromohexanoic acid (7 g, 35.9 mmol, 1.2 eq) in DCM (70 mL). The clear reaction solution was stirred under nitrogen at room temperature overnight. The reaction mixture was diluted with DCM (50 mL) and subsequently washed with saturated sodium bicarbonate solution (40 mL), water (40 mL) and brine (40 mL). The organic phase was dried over sodium sulfate and filtered. The organic phase was concentrated to give intermediate 1 (7.2 g, 66% yield).

Step 2: To a solution of undecan-1-ol (5 g, 29.0 mmol, 1 eq.) and 6-N-Boc aminohexanoic acid (8 g, 34.8 mmol, 1.2 eq.) in anhydrous DCM (70 ml) was added ^EDC.HCl (8.3 g, 43.5 mmol, 1.5 eq.), triethylamine (5.8 mL, 58 mmol) and DMAP (700 mg, 2.9 mmol, 0.2 eq.). The clear reaction solution was stirred at room temperature under _N2 gas overnight. The next day, LC/MS confirmed the completion of the reaction. The reaction mixture was diluted with DCM (50 mL) and subsequently washed with saturated sodium bicarbonate solution (60 mL) and brine (60 mL). The organic phase was dried over sodium sulfate, filtered, and the solvent was evaporated via a rotary evaporator _to give the crude product. The organic phase was dried over _Na2SO4 and filtered. The organic phase was concentrated and the crude product was purified by ISCO MPLC using a silica gel column (120 g) and the column was eluted with a (0-100)% hexane-ethyl acetate gradient. The fractions were combined and concentrated to give intermediate 2 (9.3 g, 79% yield) as an oil.

Step 3: Intermediate 2 (6 g, 15.5 mmol, 1 eq) was dissolved in DCM (60 mL) and methanesulfonic acid (2.0 mL, 31.1 mmol, 2 eq) was added to the solution. The clear solution was stirred at room temperature overnight. The next day, LC/MS confirmed complete conversion of the starting material to the product. The reaction mixture was diluted with DCM (40 mL) and washed with saturated sodium bicarbonate solution (40 mL). The organic phase was dried _{over Na2SO4} and filtered. The organic phase was concentrated and dried to give the free amine intermediate in quantitative yield. This material was used in the next step without further purification. A solution of intermediate 1 (1.5 g, 3.9 mmol, 1 eq.) and the deprotected product of intermediate ₂ (2.2 g, 7.9 mmol, 2 eq.) in a mixture of acetonitrile/cyclopropyl methyl ether (60 mL, 1:1) was treated with potassium carbonate (1.0 g, 7.9 mmol, 2 eq.) under N2 gas. The reaction flask was heated at 70°C overnight. LC/MS showed the formation of the desired peak corresponding to the monoalkylated product. The reaction mixture was cooled to room temperature and filtered through a pad of celite and washed with DCM (40 mL). The organic phase was concentrated and purified by ISCO MPLC system using a silica column (80 g). The column was eluted with a (0-100)% hexane/EtOAc gradient. The fractions were combined and concentrated by rotary evaporator under reduced pressure to afford intermediate 3 (450 mg, 20% yield) as a solid. ESI MS (m/z): 582.63 (M+H) ⁺ .

Step 4: To a mixture of intermediate 3 (150 mg, 0.25 mmol, 1 eq.) and N,N'-dimethylglycine (31 mg, 0.30 mmol, 1.2 eq.) in DCM (5 ml) was added diisopropylethylamine (184 μL, 1.0 mmol) followed by HATU (137 mg, 0.37 mmol, 1.4 eq.). The resulting solution was stirred at ambient temperature under _N2 gas overnight. The next day, LCMS showed complete conversion of the reaction. The reaction mixture was diluted with _DCM (20 mL) and washed with saturated aqueous _NaHCO3 solution (10 mL), water (10 mL) and brine (10 mL). The organic phase was dried over _Na2SO4 and filtered. The organic phase was concentrated and purified by ISCO/MPLC system using a silica column (12 g). The column was eluted with a gradient of (0-100)% hexane-ethyl acetate and (0-20)% DCM-MeOH. The fractions were combined and concentrated by rotary evaporator to give compound 25 (96 mg, 56% yield) as an oil. ESI MS (m/z): 667.66 (M+H) ⁺ . Example 26: Synthesis of compound 26 of Table 1 (6-(N-(6-oxo-6-(undecyloxy)hexyl)-2-(pyrrolidin-1-yl)acetamido)hexanoic acid tridecyl-7-yl ester)

To a mixture of intermediate 3 of Example 25 (150 mg, 0.25 mmol, 1 eq.) and pyrrolidineacetic acid (51 mg, 0.30 mmol, 1.2 eq.) in DCM (5 ml) was added diisopropylethylamine (184 μL, 1.0 mmol) followed by HATU (137 mg, 0.37 mmol, 1.4 eq.). The resulting solution was stirred overnight at ambient temperature under _N2 gas. The next day, LCMS showed complete conversion of the reaction. The reaction mixture was diluted with DCM (20 mL) and washed with saturated aqueous _NaHCO3 solution (10 mL), water (10 mL) and brine ( ₁₀ mL). The organic phase was dried over _Na2SO4 and filtered. The organic phase was concentrated and purified by ISCO/MPLC system using a silica column (12 g). The column was eluted with a gradient of (0-100)% hexane-ethyl acetate and (0-20)% DCM-MeOH. The fractions were combined and concentrated by rotary evaporator to give compound 26 (146 mg, 82% yield) as an oil. ESI MS (m/z): 693.67 (M+H) ⁺ . Example 27: Synthesis of compound 27 of Table 1 (6-(2-((2-(dimethylamino)ethyl)thio)-N-(6-oxo-6-(undecanyloxy)hexyl)acetamido)hexanoic acid tridecane-7-yl ester)

To a mixture of intermediate 3 of Example 25 (150 mg, 0.27 mmol, 1 eq.) and N,N'-dimethylethylthioacetic acid (50 mg, 0.30 mmol, 1.2 eq.) in DCM (5 ml) was added diisopropylethylamine (184 μL, 1.0 mmol) followed by HATU (137 mg, 0.37 mmol, 1.4 eq.). The resulting solution was stirred overnight at ambient temperature under _N2 gas. The next day, LCMS showed complete conversion of the reaction. The reaction mixture was diluted with _DCM (20 mL) and washed with saturated aqueous _NaHCO3 solution (10 mL), water (10 mL) and brine (10 mL). The organic phase was dried over _Na2SO4 and filtered. The organic phase was concentrated and purified by ISCO/MPLC system using a silica column (12 g). The column was eluted with a gradient of (0-100)% hexane-ethyl acetate and (0-20)% DCM-MeOH. The fractions were combined and concentrated by rotary evaporator to give compound 27 (115 mg, 61% yield) as an oil. ESI MS (m/z): 727.6 (M+H) ⁺ . Example 28: Synthesis of compound 28 of Table 1 (6-(2-(dimethylamino)-N-(4-oxo-4-(undecyloxy)butyl)acetamido)hexanoic acid tridecyl-7-yl ester)

Compound 28 was prepared according to Scheme 24 shown in FIG. 24 .

Step 1: To a stirred solution of tridecan-7-ol (6 g, 29.9 mmol, 1 eq.) and 6-bromohexanoic acid (7 g, 35.9 mmol, 1.2 eq.) in DCM (70 mL) was added DMAP (300 mg, 2.9 mmol, 0.1 eq.) and ^EDC.HCl (6.8 g, 35.9 mmol, 1.2 eq.). The clear reaction solution was stirred at room temperature under nitrogen overnight. The reaction mixture was diluted with DCM (50 mL) and subsequently washed with saturated sodium bicarbonate solution (40 mL), water (40 mL) and brine (40 mL). The organic phase was dried over sodium sulfate and filtered. The organic phase was concentrated to give intermediate 1 (7.2 g, 66% yield).

Step 2: To a solution of undecan-1-ol (3 g, 17.4 mmol, 1 eq.) and 6-N-Boc aminobutyric acid (4.2 g, 20.8 mmol, 1.2 eq.) in anhydrous DCM (50 ml) was added ^EDC.HCl (5 g, 26.4 mmol, 1.5 eq.), triethylamine (4.9 mL, 34.8 mmol) and DMAP (424 mg, 3.4 mmol, 0.2 eq.). The clear reaction solution was stirred at room temperature under _N2 gas overnight. The next day, LC/MS confirmed the completion of the reaction. The reaction mixture was diluted with DCM (30 mL) and subsequently washed with saturated sodium bicarbonate solution (40 mL) and brine (40 mL). The organic phase was dried over sodium sulfate, filtered, and the solvent was evaporated via a rotary evaporator _to give the crude product. The organic phase was dried over _Na2SO4 and filtered. The crude product was concentrated and purified by ISCO MPLC using a silica gel column (120 g) and the column was eluted with a (0-100)% hexane-ethyl acetate gradient. The fractions were combined and concentrated to give intermediate 2 (5.5 g, 88% yield) as an oil.

Step 3: Intermediate 2 (3.7 g, 10.3 mmol) was dissolved in DCM (40 mL) and methanesulfonic acid (1.3 mL, 20.6 mmol, 2 eq.) was added to the solution. The clear solution was stirred at room temperature overnight. The next day, LC/MS confirmed complete conversion of the starting material to the product. The reaction mixture was diluted with DCM (25 mL) and washed with saturated sodium bicarbonate solution (25 mL). The organic phase was dried _{over Na2SO4} and filtered. The organic phase was concentrated and dried to give the free amine intermediate in quantitative yield. This material was used in the next step without further purification. A solution of intermediate ₁ (1.0 g, 2.6 mmol, 1 eq.) and the deprotected product of intermediate 2 (1.3 g, 5.2 mmol, 2 eq.) in a mixture of acetonitrile/cyclopropyl methyl ether (50 mL, 1:1) was treated with potassium carbonate (730 mg, 5.3 mmol, 2 eq.) under N2 gas. The reaction flask was heated at 70°C overnight. LC/MS showed the formation of the desired peak corresponding to the monoalkylated product. The reaction mixture was cooled to room temperature and filtered through a pad of celite and washed with DCM (20 mL). The organic phase was concentrated and purified by an ISCO MPLC system using a silica column (80 g). The column was eluted with a (0-100)% hexane/EtOAc gradient. The solvent was concentrated by rotary evaporator under reduced pressure to afford intermediate 3 as a solid (300 mg, 21% yield). ESI MS (m/z): 554.56 (M+H) ⁺ .

Step 4: To a mixture of intermediate 3 (150 mg, 0.27 mmol, 1 eq.) and N,N'-dimethylglycine (33 mg, 0.32 mmol, 1.2 eq.) in DCM (5 ml) was added triethylamine (152 μL, 1.0 mmol) followed by HATU (144 mg, 0.37 mmol, 1.4 eq.). The resulting solution was stirred at ambient temperature under _N2 gas overnight. The next day, LCMS showed complete conversion of the reaction. The reaction mixture was diluted with DCM (20 mL) and washed with saturated aqueous _NaHCO3 solution (20 mL), water (20 mL) and brine ( ₂₀ mL). The organic phase was dried over _Na2SO4 and filtered. The organic phase was concentrated and purified by ISCO/MPLC system using a silica column (12 g). The column was eluted with a gradient of (0-100)% hexane-ethyl acetate and (0-20)% DCM-MeOH. The fractions were combined and concentrated by rotary evaporator to give compound 28 (96 mg, 55% yield) as an oil. ESI MS (m/z): 639.63 (M+H) ⁺ . Example 29: Synthesis of compound 29 of Table 1 (6-(2-((2-(dimethylamino)ethyl)thio)-N-(4-oxo-4-(undecanyloxy)butyl)acetamido)hexanoic acid tridecane-7-yl ester)

To a mixture of intermediate 3 of Example 28 (150 mg, 0.27 mmol, 1 eq.) and N,N'-dimethylethylthioacetic acid (52 mg, 0.32 mmol, 1.2 eq.) in DCM (5 ml) was added triethylamine (152 μL, 1.0 mmol) followed by HATU (144 mg, 0.37 mmol, 1.4 eq.). The resulting solution was stirred overnight at ambient temperature under _N2 gas. The next day, LCMS showed complete conversion of the reaction. The reaction mixture was diluted with DCM (20 mL) and washed with saturated aqueous _NaHCO3 solution (20 mL), water (20 mL) and brine (20 _mL ). The organic phase was dried over _Na2SO4 and filtered. The organic phase was concentrated and purified by ISCO/MPLC system using a silica column (12 g). The column was eluted with a gradient of (0-100)% hexane-ethyl acetate and (0-20)% DCM-MeOH. The fractions were combined and concentrated by rotary evaporator to give compound 29 (147 mg, 77% yield) as an oil. ESI MS (m/z: 699.63 (M+H) ⁺ . Example 30: Synthesis of compound 30 of Table 1 (tetradec-5-yl 6-(2-(dimethylamino)-N-(6-oxo-6-(undecyloxy)hexyl)acetamido)hexanoate)

Compound 30 was prepared according to Scheme 25 shown in FIG. 25 .

Step 1: To a stirred solution of tetradecan-5-ol (3.0 g, 13.9 mmol, 1 eq.) in anhydrous DCM (40 mL) was added triethylamine (3.9 mL, 27 mmol, 2 eq.). After stirring for 10 min, 6-bromohexyl chloride (3.2 g, 35.9 mmol, 1.2 eq.) was slowly added to the above mixture. The clear reaction solution was stirred at room temperature under nitrogen for 5 h. The reaction mixture was diluted with DCM (50 mL) and subsequently washed with water (40 mL) and brine (40 mL). The organic phase was dried over sodium sulfate and filtered. The organic phase was concentrated and the crude product was purified by ISCO MPLC using a silica gel column (120 g) and eluting the column with a (0-30)% hexane-ethyl acetate gradient. The organic phase was concentrated to give intermediate 1 (1.4 g, 66% yield).

Step 2: To a solution of undecan-1-ol (5 g, 29.0 mmol, 1 eq.) and 6-N-Boc aminohexanoic acid (8 g, 34.8 mmol, 1.2 eq.) in anhydrous DCM (70 ml) was added ^EDC.HCl (8.3 g, 43.5 mmol, 1.5 eq.), triethylamine (5.8 mL, 58 mmol) and DMAP (700 mg, 2.9 mmol, 0.2 eq.). The clear reaction solution was stirred at room temperature under _N2 gas overnight. The next day, LC/MS confirmed the completion of the reaction. The reaction mixture was diluted with DCM (50 mL) and subsequently washed with saturated sodium bicarbonate solution (60 mL) and brine (60 mL). The organic phase was dried over sodium sulfate, filtered, and the solvent was evaporated via a rotary evaporator _to give the crude product. The organic phase was dried over _Na2SO4 and filtered. The organic phase was concentrated and the crude product was purified by ISCO MPLC using a silica gel column (120 g) and the column was eluted with a (0-100)% hexane-ethyl acetate gradient. The fractions were combined and concentrated to give intermediate 2 (9.3 g, 79% yield) as an oil.

Step 3: Intermediate 2 (6 g, 15.5 mmol, 1 eq) was dissolved in DCM (60 mL) and methanesulfonic acid (2.0 mL, 31.1 mmol, 2 eq) was added. The clear solution was stirred at room temperature overnight. The next day, LC/MS confirmed complete conversion of the starting material to the product. The reaction mixture was diluted with DCM (40 mL) and washed with saturated _sodium bicarbonate solution (40 mL). The organic phase was dried over _Na2SO4 and filtered. The organic phase was concentrated and dried to give the free amine intermediate in quantitative yield. This material was used in the next step without further purification. A solution of intermediate ₁ (0.8 g, 2.0 mmol, 1 eq.) and the deprotected product of intermediate 2 (1.1 g, 4.0 mmol, 2 eq.) in a mixture of acetonitrile/cyclopropyl methyl ether (40 mL, 1:1) was treated with potassium carbonate (280 mg, 2.0 mmol, 2 eq.) under N2 gas. The reaction flask was heated at 70°C for 3 hours. LC/MS showed the formation of the desired peak corresponding to the monoalkylated product. The reaction mixture was cooled to room temperature and filtered through a pad of celite and washed with DCM (20 mL). The crude product was concentrated and purified by an ISCO MPLC system using a silica column (80 g). The column was eluted with a (0-100)% hexane/EtOAc gradient. The fractions were combined and concentrated by rotary evaporator under reduced pressure to give intermediate 3 (130 mg, 10% yield) as a solid.

Step 4: Compound 30: To a mixture of intermediate 3 (130 mg, 0.21 mmol, 1 eq.) and N,N'-dimethylglycine (26 mg, 0.30 mmol, 1.2 eq.) in DCM (5 ml) was added diisopropylethylamine (77 μL, 0.43 mmol) followed by HATU (116 mg, 0.30 mmol, 1.4 eq.). The resulting solution was stirred at ambient temperature under _N2 gas overnight. The next day, LCMS showed complete conversion of the reaction. The reaction mixture was diluted with DCM (20 mL) and washed with saturated aqueous _NaHCO3 solution (10 mL), water (10 mL) and brine (10 mL). The organic phase was dried _{over Na2SO4} and filtered. The organic phase was concentrated and purified by ISCO/MPLC system using a silica column (12 g). The column was eluted with a gradient of (0-100)% hexane-ethyl acetate and (0-20)% DCM-MeOH. The fractions were combined and concentrated by rotary evaporator to give compound 30 (74 mg, 50% yield) as an oil. ESI MS (m/z): 682.69 (M+H) ⁺ . Example 31: In vitro activity of LNP formulations

The sequences of the siRNA used in this study are as follows: • Sense strand SEQ ID NO: 1 (5'-＞3'): GAA GCCU U U U GAGACCC UAUU ; • Antisense strand SEQ ID NO: 2 (5'-＞3'): fUAGgGuCu C A AA AGGC UU C UU ; wherein A, G, C and U refer to ribose-A, ribose-G, ribose-C and ribose-U, respectively; lowercase letters a, u, g, c, t refer to 2'-deoxy-A, 2'-deoxy-U, 2'-deoxy-G, 2'-deoxy-C and deoxythymidine (dT=T=t), respectively; underlining indicates 2'-OMe-substitution; and lowercase letter f refers to 2'-deoxy-2'-fluoro substitution. siRNA can attenuate the GSTp gene and is disclosed in PCT Publication No. WO2016/106400, which is expressly incorporated herein by reference in its entirety.

The LNP formulation was prepared using the following composition: (Test compound/cholesterol/DSPC/DMG-PEG) = 50/38/10/2 (mol%) The test compound is a compound selected from the group consisting of compound numbers 1 to 21, R1 and R2. R1 is a commercially available product. R2 was prepared according to PCT Publication No. WO2016/210190.

LNP formulations were prepared by injecting an ethanol solution of lipids into a dsRNA buffer solution in the same manner as described in U.S. Publication No. 2013-0022665, PCT Publication No. WO2019/090359, and PCT Publication No. WO2020/102668. They are expressly incorporated herein by reference in their entirety. The average particle size (PS), polydispersity index (PDI), encapsulation efficiency (%EE) and yield of each LNP formulation are shown in Table 2. The pKa value of each LNP was determined using an analysis based on fluorescence of 2-(p-toluidinyl)-6-naphthalenesulfonic acid (TNS).

The in vitro activity of the LNP formulations was measured according to the following protocol:

Lung adenocarcinoma (A549) cells were cultured in vitro in the presence of DMEM medium (HyClone catalog number SH30243.01) with 10% FBS (Invitrogen catalog number A4766801) at 37°C in a humidified atmosphere incubator with 5% CO _2. One day before transfection, cells were seeded at 2×10 ³ cells/well in 96-well plates. The next day, siRNA/LNP complexes were prepared at room temperature and 10 µl of siRNA/LNP complexes were added on top of the cells 24 hours later. The 96-well plates were gently mixed and incubated again at 37°C in a humidified atmosphere incubator with 5% CO ₂ . The knockdown efficiency of the target gene was determined after 24, 48 and 72 hours by QRT-PCR. For total RNA isolation, at each time point, the medium was removed from each well and washed with 1× DPBS. Cell lysis: 50 μL of Cell-to-Ct Lysis Buffer (Life Technologies Catalog No. 4391851 C) was added on top of the cells and incubated at room temperature with gentle shaking/5 minutes. The cell lysate was treated with 5 μl of stop solution to stop the cell lysis reaction. At the mRNA level, the knockdown of the target gene was measured by QRT-PCR using the TAQMAN kit following the manufacturer's instructions.

The experimental results of in vitro genetic silencing studies are shown in Table 2. Table 2 Compound No. PS (nm) PDI %EE Yield (%) pKa (TNS) KD IC ₅₀ (nM) 1 101 0.04 91 100 7.5 3.6 2 81 0.00 97 88 N/A 48 3 95 0.09 63 95 7.9 84 4 92 0.00 98 90 N/A 105 5 135 0.16 82 74 6.9 30 6 170 0.32 98 58 N/A 41 7 90 0.04 83 94 6.8 280 8 143 0.16 88 56 7.4 3.2 9 152 0.04 74 46 6.5 84 10 133 0.22 95 96.5 7.8 twenty four 11 140 0.06 97 98 and above 7.9 16 12 129 0.14 94 69 7.3 0.19 13 137 0.15 73 74 6.6 37 14 145 0.15 91 61 7.4 4.4 15 115 0.14 97 76 8.1 104 16 129 0.01 97 61 8.0 28 17 137 0.15 78 52 6.6 67 18 145 0.15 91 57 7.0 0.087 19 115 0.14 74 58 6.4 20 20 122 0.14 97 83 8.1 42 twenty one 153 0.16 86 77 7.8 28 R1 91 0.12 92 76 6.9 twenty four R2 100 0.09 82 99 3.9 231 N/A = Not Applicable Example 32: Formulation Distribution Study

NanoAssemblr ^™ (Precision NanoSystems) was used to prepare LNP No. 1 (Compound No. 1/Cholesterol/DSPC/DMG-PEG=50/38.5/10/1.5 (mol%)) with Fluc mRNA (TriLink, 5moU) according to the manufacturer's instructions. A single injection of LNP No. 1 was given intravenously to Balb/c mice (n=4) at a dose of 0.5 mg/kg. Mice were anesthetized 6-8 hours after the injection of mRNA. Animals were immediately sacrificed and tissues (pancreas, spleen, liver, kidney, lung and muscle) were collected. Tissues were homogenized in CCLR lysis buffer and centrifuged. The resulting supernatant was used for luciferase activity analysis using Promega E4510 assay reagent. mRNA was mainly transfected and translated in the liver and spleen (see Figure 26). Example 33: In vitro expression of Fluc mRNA

The LNP formulation with Fluc mRNA was prepared using the following composition: (Test compound/cholesterol/DSPC/DMG-PEG2000) = 50/38/10/2 (mol%) The test compound is any one of compound numbers 1, 2 and 4 to 30, R1 and R2.

LNP formulations were prepared by injecting an ethanol solution of lipids into a Fluc mRNA (TriLink, 5 moU) buffer solution in the same manner as described in U.S. Publication No. 2013-0022665, PCT Publication No. WO2019/090359, and PCT Publication No. WO2020/102668, which are incorporated herein by reference in their entirety. The average particle size (PS), polydispersity index (PDI), mRNA encapsulation efficiency (%EE), and yield of each LNP formulation obtained are shown in Table 3 below. PS and PDI were obtained using a Malvern Zetasizer Nano-ZS ZEN 3600. %EE was obtained by Ribogreen fluorescence analysis following the GenVoy-ILM ^™ User Guide of Precision NanoSystems. Measure the in vitro expression of Fluc mRNA according to the following protocols 1 or 2: Protocol 1:

Hep3B and Panc-1 cell lines were cultured in medium supplemented with 10% HI-FBS (Gibco Ref. 10082-147). EMEM medium (ATCC Ref. 30-2003) and DMEM medium (Gibco Ref. 11965-092) were used, respectively. On day 0, cells were seeded at a density of 5000 cells/well in white opaque 96-well TC-treated plates (Greiner Ref. 655083) using 90 μL of cell mixture per well. The plates were placed in a 37°C incubator with 5% _CO2 overnight to allow cells to attach. On day 1, the mRNA/LNP complex was equilibrated to room temperature, then diluted with DPBS (Gibco Ref. 14190-144) to generate a dose-response curve and added to the plate at a volume of 10 μL/well. The plate was returned to _a 37°C incubator with 5% CO2 for 24 hours. On day 2, the Promega Luciferase Assay System (Ref. E1501) buffer and substrate were equilibrated to room temperature and combined, then added to the SpectraMax L Luminometer (Molecular Devices) syringe. According to the Promega kit guidelines, the plates were prepared by first removing the medium from each well, then gently rinsing the wells with DPBS, and finally adding 20 μL of 1X reporter dissolution buffer (reference number E397A) to each well. The plates were placed in a Luminometer and 100 μL of luciferase buffer/substrate mixture was injected into each well, and the luminescence value was obtained simultaneously. EC ₅₀ values were determined by fitting the dose-response curves using a 4-parameter logic model using GraphPad/Prism. The EC ₅₀ values of LNP formulations including any one of compounds 2, 4, 8, 14, 18, 19 and R2 were determined according to Scheme 1. Scheme 2:

Hep3B and Panc-1 cell lines were cultured in medium supplemented with 10% HI-FBS (Gibco Ref. 10082-147). EMEM medium (ATCC Ref. 30-2003) and DMEM medium (Gibco Ref. 11965-092) were used, respectively. On day 0, cells were seeded at a density of 1500 cells/well in white opaque 384-well TC-treated plates (USA Scientific Ref. 5678-1080) by adding 30 μL of the cell mixture per well using Multidrop Combi+ (Thermo Scientific). The plates were placed in a 37°C incubator with 5% _CO2 overnight to allow the cells to attach. On day 1, the mRNA/LNP complex was equilibrated to room temperature, then diluted with DPBS (Gibco reference number 14190-144) to generate a dose-response curve and added to the plate at a volume of 3.3 μL/well. The plate was returned to _a 37°C incubator with 5% CO2 for 24 hours. On day 2, the ONE-Glo EX Luciferase Assay System (Promega reference number E8130) was equilibrated to room temperature and combined. The plate was prepared according to the Promega kit guidelines by adding 33.3 μL of the reagent mixture to each well. The plate was placed on a plate shaker for 3 minutes to ensure cell lysis. Once lysis was complete, the plate was added to a Luminometer (Molecular Devices) and the luminescence value was obtained. _EC50 values were determined by fitting the dose-response curves with a 4-parameter logic model using GraphPad/Prism. _EC50 values for LNP formulations including any of compounds 1, 5, 6, 7, 9, 10, 11, 12, 13, 15, 16, 17, 20-30 and R1 were determined according to Scheme 2. The experimental results are shown in Table 3 below. Table 3 LNP Test compound PS (nm) PDI %EE Yield (%) Hep3B EC ₅₀ (nM) PANC-1 EC ₅₀ (nM) LNP 1a Compound 1 195 0.133 94 100 0.12 0.58 LNP 2a Compound 2 162 0.2 92 twenty three 0.15 0.07 LNP 4a Compound 4 152 0.22 92 25 0.30 0.19 LNP 5a Compound 5 220 0.16 92 91 0.61 0.28 LNP 6a Compound 6 187 0.17 103 92 0.45 0.55 LNP 7a Compound 7 148 0.24 69 88 0.09 0.12 LNP 8a Compound 8 144 0.144 62 34 0.10 0.16 LNP 9a Compound 9 152 0.043 91 66 0.37 0.16 LNP 10a Compound 10 168 0.117 96 71 0.25 0.42 LNP 11a Compound 11 172 0.101 95 72 0.30 0.35 LNP 12a Compound 12 221 0.131 85 69 0.34 0.41 LNP 13a Compound 13 171 0.071 92 71 1.05 0.16 LNP 14a Compound 14 90 0.186 90 33 0.08 0.10 LNP 15a Compound 15 164 0.11 96 74 0.55 0.49 LNP 16a Compound 16 195 0.18 95 70 0.46 0.10 LNP 17a Compound 17 199 0.167 78 73 1.18 0.18 LNP 18a Compound 18 89 0.115 96 60 0.09 0.09 LNP 19a Compound 19 93 0.14 96 54 0.27 0.37 LNP 20a Compound 20 196 0.198 97 72 0.08 0.37 LNP 21a Compound 21 176 0.16 96 73 0.53 0.07 LNP 22a Compound 22 182 0.158 80 76 0.12 0.24 LNP 23a Compound 23 223 0.129 98 78 0.23 0.38 LNP 24a Compound 24 217 0.143 82 76 0.40 0.04 LNP 25a Compound 25 171 0.133 72 80 0.12 0.07 LNP 26a Compound 26 211 0.155 65 66 0.12 0.35 LNP 27a Compound 27 148 0.133 86 73 0.76 0.16 LNP 28a Compound 28 206 0.135 73 65 0.17 0.50 LNP 29a Compound 29 147 0.152 83 67 0.11 0.22 LNP 30a Compound 30 206 0.135 68 69 0.23 0.20 LNP R1a Compound R1 218 0.158 92 98 0.28 0.73 LNP R2a Compound R2 109 0.068 44 33 0.45 0.38

When LNP 1a, 2a, 7a, 8a, 10a, 14a, 18a, 19a, 20a, 22a, 23a, 25a, 26a, 28a, 29a or 30a was applied to HEP3B cells, the _EC50 value was lower than when any of the control LNPs (LNP R1a and LNP R2a) was applied to the same cells. When LNP 2a, 4a, 5a, 7a, 8a, 9a, 11a, 13a, 14a, 16a, 17a, 18a, 19a, 20a, 21a, 22a, 24a, 25a, 26a, 27a, 29a or 30a was applied to PANC-1 cells, the EC50 value was lower than when any of the control LNPs (LNP R1a and LNP R2a) was applied to the same cells. When LNP 6a, 12a or 15a was applied to PANC-1 cells, the EC50 value was lower than when LNP R1a was applied to the same cells.

As used herein, the term "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

The above description discloses several methods and materials of the present invention. The present invention is sensitive to changes in methods and materials and changes in manufacturing methods and equipment. Such modifications will become apparent to those skilled in the art from consideration of the present disclosure or practice of the invention disclosed herein. Therefore, it is not intended that the present invention be limited to the specific embodiments disclosed herein, but that it encompasses all modifications and alternatives within the true scope and spirit of the present invention.

All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and become part of this specification. To the extent that the publications, patents, or patent applications incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to replace and/or take precedence over any such conflicting materials.

FIG1 shows a reaction scheme of the method for preparing Compound 1 as described in Example 1.

FIG2 shows a reaction scheme for the method of preparing Compound 2 as described in Example 2.

FIG3 shows a reaction scheme for the method of preparing Compound 3 as described in Example 3.

FIG4 shows a reaction scheme for the method of preparing Compound 4 as described in Example 4.

FIG5 shows a reaction scheme for the method of preparing Compound 5 as described in Example 5.

Figure 6 shows the reaction flow of the method for preparing compound 6 as described in Example 6.

FIG. 7 shows a reaction scheme for the method of preparing Compound 7 as described in Example 7.

FIG8 shows a reaction scheme for the method of making Compound 8 as described in Example 8.

FIG9 shows a reaction scheme for the method of making Compound 9 as described in Example 9.

FIG. 10 shows a reaction scheme for the method of making compound 10 as described in Example 10.

FIG. 11 shows a reaction scheme for the method of preparing Compound 11 as described in Example 11.

FIG. 12 shows a reaction scheme for the method of making Compound 12 as described in Example 12.

Figure 13 shows the reaction flow of the method for preparing compound 13 as described in Example 13.

FIG. 14 shows a reaction scheme for the method of making Compound 14 as described in Example 14.

FIG. 15 shows a reaction scheme for the method of making Compound 15 as described in Example 15.

FIG. 16 shows a reaction scheme for the method of making Compound 16 as described in Example 16.

FIG. 17 shows a reaction scheme for the method of making Compound 17 as described in Example 17.

FIG. 18 shows a reaction scheme for the method of making Compound 18 as described in Example 18.

FIG. 19 shows a reaction scheme for the method of making Compound 19 as described in Example 19.

FIG. 20 shows the reaction flow of the method for preparing compound 20 as described in Example 20.

FIG. 21 shows a reaction scheme for the method of making Compound 21 as described in Example 21.

FIG. 22 shows a reaction scheme for the method of making Compound 22 as described in Example 22.

FIG. 23 shows a reaction scheme for the method of making Compound 25 as described in Example 25.

FIG. 24 shows a reaction scheme for the method of making compound 28 as described in Example 28.

FIG. 25 shows a reaction scheme for a method of making compound 30 as described in Example 30.

FIG. 26 shows a graph showing the distribution of Fluc mRNA after administration of LNP No. 1.

These and other embodiments are described in more detail below.

Claims (17)

A compound of formula I or a pharmaceutically acceptable salt thereof: Formula I wherein ^A1 is selected from the group consisting of: , wherein the arrow indicates the connection to the carbonyl group, ^R1 and ^R2 are the same or different and are hydrogen or hydroxyl, ^R3 , ^R5 , ^R6 , ^R7 , ^R8 , ^R9 and ^R10 are the same or different and are linear C1 to C4 alkyl, ^R4 is a linear C1 to C4 alkyl or -( _CH2 ) _p4 -OH, p1 and p2 are the same or different and are each independently selected from the group consisting of 1, 2, 3 and 4, p3 is an integer selected from the group consisting of 0, 1, 2, 3 and 4, p4 is an integer selected from the group consisting of 2, 3 and 4, ^X- is a pharmaceutically acceptable counter anion, each A ^A2 is independently a straight chain C4 to C10 alkyl, a straight chain C4 to C10 alkenyl or a straight chain C4 to C10 diene alkyl, m is an integer selected from the group consisting of 5, 6, 7, 8 and 9, n is an integer selected from the group consisting of 2, 3, 4, 5 and 6, ^A3 is -OC(O)- or -C(O)O-, and ^A4 is a straight chain C10-C18 alkyl, a straight chain C10-C18 alkenyl or a straight chain C10-C18 diene alkyl. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein each A ² is independently a linear C 4 to C 10 alkyl group. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein each ^A2 is the same. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein m is an integer selected from the group consisting of 5, 6, 7 and 8. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein n is an integer selected from the group consisting of 2, 3, 4 and 5. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the number of carbon atoms in A ⁴ -A ³ -(CH ₂ ) _n - is 15 to 21. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein each A ² is independently a linear C4 to C10 alkyl group, m is an integer selected from the group consisting of 5, 6, 7 and 8, n is an integer selected from the group consisting of 2, 3, 4 and 5, and the number of carbon atoms in A ⁴ -A ³ -(CH ₂ ) _n - is 15 to 21. The compound of claim 1, wherein ^A1 is selected from the group consisting of: , where the arrow indicates the connection to the carbonyl group. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein ^A1 is selected from the group consisting of: , where the arrow indicates the connection to the carbonyl group. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein ^A1 is selected from the group consisting of: , where the arrow indicates the connection to the carbonyl group. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein ^A1 is selected from the group consisting of: , where the arrow indicates the connection to the carbonyl group. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein each ^A2 is independently a linear C4 to C10 alkyl group, m is an integer selected from the group consisting of 5, 6, 7 and 8, n is an integer selected from the group consisting of 2, 3, 4 and 5, the number of carbon atoms in the ^A4 - ^A3- ( _CH2 ) _n- is 15 to 21, and ^A1 is selected from the group consisting of: , where the arrow indicates the connection to the carbonyl group. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of compound numbers 1 to 30 shown in Table 1 below: Table 1 . A pharmaceutical composition comprising: Lipid nanoparticles; and an active pharmaceutical ingredient encapsulated in the lipid nanoparticles, wherein the lipid nanoparticles comprise a compound as claimed in claim 1 or a pharmaceutically acceptable salt thereof. The pharmaceutical composition of claim 14, wherein the active pharmaceutical ingredient is selected from the group consisting of siRNA, mRNA, antisense oligonucleotide and miRNA. A method for delivering an active pharmaceutical ingredient to a cell, comprising contacting the cell with the pharmaceutical composition of claim 14. A method for delivering an active pharmaceutical ingredient to a subject in need thereof, comprising administering the pharmaceutical composition of claim 14 to the subject.