Lipid Amines

Abstract

Kind Code: A1 Lipid amine compounds are provided that are useful in the preparation of lipid nanoparticle compositions for the delivery of therapeutic or prophylactic payloads to cells. TIFF2024542001000695.tif68164

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing dates of U.S. Provisional Application No. 63 / 273,421, filed October 29, 2021, U.S. Provisional Application No. 63 / 308,180, filed February 9, 2022, and U.S. Provisional Application No. 63 / 395,243, filed August 4, 2022, the contents of which are incorporated herein by reference in their entireties.

[0002] Lipid amine compounds are provided that are useful in the preparation of lipid nanoparticle compositions for delivery of therapeutic or prophylactic payloads to cells. [Background technology]

[0003] The delivery of biologically active payloads, such as nucleic acids and proteins, into cells has the potential to be used to treat a variety of diseases and / or pathologies. However, effective targeted delivery of such payloads presents a continuing medical challenge. In particular, the delivery of nucleic acids into cells is made difficult by the relative instability and poor cell permeability of such species.

[0004] While lipid nanoparticles have provided an effective delivery vehicle for payloads into cells and intracellular compartments, improvements in safety, efficacy, and specificity are still needed. Thus, there is a need to develop lipid nanoparticle compositions to facilitate the delivery of therapeutic and prophylactic agents, such as nucleic acids, to cells. Summary of the Invention

[0005] As used herein, the structure of formula A1: [ka] or a salt thereof, wherein the constituent members are defined herein.

[0006] Also provided herein is a lipid nanoparticle composition comprising a lipid amine of formula A1, or a salt thereof.

[0007] Also provided herein is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a lipid nanoparticle composition comprising a lipid amine of formula A1, or a salt thereof.

[0008] Also provided herein are methods of delivering a payload to a cell, comprising contacting the cell with a lipid nanoparticle composition described herein.

[0009] Also provided herein are methods for delivering a therapeutic or prophylactic payload to a patient, comprising administering to the patient a lipid nanoparticle composition described herein.

[0010] Also provided herein is a process for preparing a lipid nanoparticle composition, the process comprising contacting a lipid nanoparticle core with a lipid amine compound of formula A1, or a salt thereof.

[0011] Also provided herein are products of any of the processes described herein.

[0012] Each of the limitations may encompass various embodiments. As such, it is contemplated that each of the limitations including any one element or combination of elements may be included in each of the described aspects. The invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. It is capable of other embodiments and of being practiced and carried out in various ways. DETAILED DESCRIPTION OF THE INVENTION

[0013] As used herein, the structure of formula A1: [ka] or a salt thereof, wherein Z is N or CH; R 1 is C 1-14 Alkyl, C 1-14 Alkenyl, or C 1-14 is hydroxyalkyl; R 2 and R 3 are C 2-20 is alkyl, (I C 2-20 Alkyl is -NR 8 R 9 , OH, and halo, wherein at least one substituent is —NR 8 R 9 and; (ii) C 2-20 1, 2, 3, or 4 non-terminal carbons of the alkyl are optionally replaced with O; (iii)C 2-20 One, two, three, or four non-terminal carbons of an alkyl can be NR 10 is optionally replaced by; (iv) C 2-20 1, 2, 3, or 4 non-terminal carbons of the alkyl are optionally replaced with C(=O); (v) C 2-20 One, two, three, or four non-terminal carbons of an alkyl can be CR a R b is arbitrarily replaced by R a and R b are C atoms along with the C atoms to which they are bonded. 3-6 Forms a cycloalkyl group; R 2 and R 3 are the same or different; or R 2 and R 3 together with the N atom to which they are attached form one, two, or three ring-forming NR 10 A 7- to 18-membered heterocycloalkyl group containing a C 1-4 Alkyl, -NR 8 R 9optionally substituted with 1, 2, or 3 substituents independently selected from , OH, and halo; or R 2 , R 3 , and R 6 are, together with the atoms to which they are bonded and any intervening atoms, C 1-4 Alkyl, -NR 8 R 9 forming a 7- to 18-membered bridged heterocycloalkyl group optionally substituted with 1, 2, or 3 substituents independently selected from , OH, and halo; R 4 , R 5 , R 6 , and R 7 H, halo, and C 1-4 are each independently selected from alkyl; or R 4 and R 5 C together with the carbon atoms to which they are attached 3-7 Forms a cycloalkyl group; or R 6 and R 7 C together with the carbon atoms to which they are attached 3-7 Forms a cycloalkyl group; R 8 , R 9 , and R 10 is H and C 1-4 are each independently selected from alkyl; j is 0 or 1; k is 0, 1, 2, 3, 4, 5, or 6; l is 0 or 1; m is 0, 1, 2, 3, 4, 5, or 6; n is 0 or 1; If j is 0, then l is 1, j and l cannot both be 0) is.

[0014] As used herein, the structure of formula A1: [ka] or a salt thereof, wherein Z is N or CH; R 1 is C 1-14 Alkyl, C 1-14 Alkenyl, or C 1-14 is hydroxyalkyl; R 2 and R 3 is C 2-20 is alkyl, (I C 2-20 Alkyl is -NR 8 R 9 , OH, and halo, wherein at least one substituent is —NR 8 R 9 and; (ii) C 2-20 1, 2, 3, or 4 non-terminal carbons of the alkyl are optionally replaced with O; (iii)C 2-20 One, two, three, or four non-terminal carbons of an alkyl can be NR 10 is optionally replaced by; R 2 and R 3 are the same or different; or R 2 and R 3 together with the N atom to which they are attached form one, two, or three ring-forming NR 10 A 7- to 18-membered heterocycloalkyl group containing a C 1-4 optionally substituted with 1, 2, or 3 substituents independently selected from alkyl, -NR8R9, OH, and halo; or R 2 , R 3 , and R 6 are, together with the atoms to which they are bonded and any intervening atoms, C 1-4 forming a 7-18 membered bridged heterocycloalkyl group optionally substituted with 1, 2, or 3 substituents independently selected from alkyl, -NR8R9, OH, and halo; R 4 , R5 , R 6 , and R 7 H, halo, and C 1-4 are each independently selected from alkyl; or R 4 and R 5 C together with the carbon atoms to which they are attached 3-7 Forms a cycloalkyl group; or R 6 and R 7 C together with the carbon atoms to which they are attached 3-7 Forms a cycloalkyl group; R 8 , R 9 , and R 10 is H and C 1-4 are each independently selected from alkyl; j is 0 or 1; k is 0, 1, 2, 3, 4, 5, or 6; l is 0 or 1; m is 0, 1, 2, 3, 4, 5, or 6; n is 0 or 1; If j is 0, then l is 1, j and l cannot both be 0) is provided.

[0015] In some embodiments, the compound is [ka] [ka] [ka] It's surprising.

[0016] In some embodiments, Z is N. In some embodiments, Z is CH.

[0017] In some embodiments, R 1 is C 1-14In some embodiments, R 1 is C 3-12 In some embodiments, R 1 is C 6-12 In some embodiments, R 1 is C 8-10 In some embodiments, R 1 is C alkyl. In some embodiments, R 1 is C 10 It is alkyl.

[0018] In some embodiments, R 1 is C 1-14 In some embodiments, R 1 is C 3-12 In some embodiments, R 1 is C 6-12 In some embodiments, R 1 is C 8-10 In some embodiments, R 1 is C hydroxyalkyl. In some embodiments, R 1 is C 10 It is a hydroxyalkyl.

[0019] In some embodiments, R 1 is C 1-14 In some embodiments, R is alkenyl. 1 is C 3-12 In some embodiments, R is alkenyl. 1 is C 6-12 In some embodiments, R is alkenyl. 1 is C 8-10 In some embodiments, R is alkenyl. 1 is C alkenyl. In some embodiments, R 1 is C 10 It is alkenyl.

[0020] In some embodiments, R 1teeth, [ka] is.

[0021] In some embodiments, R 1 teeth, [ka] is.

[0022] In some embodiments, R 1 teeth, [ka] is.

[0023] In some embodiments, R 1 teeth, [ka] is.

[0024] In some embodiments, when j is 1, then l is 0.

[0025] In some embodiments, when j is 0, then l is 1.

[0026] In some embodiments, when one of j and l is 1, then the other is 0.

[0027] In some embodiments, j is 0. In some embodiments, j is 1.

[0028] In some embodiments, k is 0, 1, 2, 3, or 4. In some embodiments, k is 0, 2, 3, or 4. In some embodiments, k is 0. In some embodiments, k is 1. In some embodiments, k is 2. In some embodiments, k is 3. In some embodiments, k is 4. In some embodiments, k is 5. In some embodiments, k is 6.

[0029] In some embodiments, l is 0. In some embodiments, l is 1.

[0030] In some embodiments, m is 0, 1, 2, or 4. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6.

[0031] In some embodiments, n is 0. In some embodiments, n is 1.

[0032] In some embodiments, j is 0, k is 0, l is 1, m is 1, and n is 1. In some embodiments, j is 0, k is 0, l is 1, m is 2, and n is 1. In some embodiments, j is 0, k is 0, l is 1, m is 4, and n is 1. In some embodiments, j is 1, k is 0, l is 0, m is 0, and n is 0. In some embodiments, j is 1, k is 1, l is 0, m is 0, and n is 0. In some embodiments, j is 1, k is 1, l is 0, m is 0, and n is 1. In some embodiments, j is 1, k is 1, l is 0, m is 2, and n is 0. In some embodiments, j is 1, k is 1, l is 1, m is 1, and n is 1. In some embodiments, j is 1, k is 2, l is 0, m is 0, and n is 0. In some embodiments, j is 1, k is 2, l is 0, m is 0, and n is 1. In some embodiments, j is 1, k is 3, l is 0, m is 0, and n is 1. In some embodiments, j is 1, k is 4, l is 0, m is 0, and n is 1.

[0033] In some embodiments, k is 1 and R 4 and R 5 and both are H. In some embodiments, k is 1 and R 4 and R 5 One of them is C 1-4 alkyl, and R 4 and R 5 and the other is H. In some embodiments, k is 1 and R 4 and R 5 is methyl, and R 4 and R 5 and the other is H. In some embodiments, k is 2 and each R 4 and R 5is H. In some embodiments, k is 2 and one R 4 is C 1-4 alkyl, and the remaining R 4 and R 5 The substituent is H. In some embodiments, k is 2 and one R 4 is methyl, and the remaining R 4 and R 5 The substituent is H. In some embodiments, k is 3 and each R 4 and R 5 is H. In some embodiments, k is 4 and each R 4 and R 5 is H.

[0034] In some embodiments, m is 1 and R 6 and R 7 and both of R are H. In some embodiments, m is 2 and each R 6 and R 7 is H. In some embodiments, m is 4 and each R 6 and R 7 is H. In some embodiments, m is 2 and one R 6 is R 2 and R 3 together with the atom to which they are attached and any intervening atoms, form a 7- to 18-membered bridged heterocycloalkyl group, and other R 6 is H and R 7 Both are H.

[0035] In some embodiments, j is 0, k is 0, l is 1, m is 1, and R 6 and R 7 are H and n is 1. In some embodiments, j is 0, k is 0, l is 0, m is 0, and each R 6 and R 7 is H and n is 1. In some embodiments, j is 0, k is 0, l is 0, m is 0, and each R 6 and R 7is H and n is 1. In some embodiments, j is 1, k is 1, and each R 4 and R 5 is H, l is 0, m is 0, and n is 0. In some embodiments, j is 1, k is 1, and R 4 and R 5 One of them is C 1-4 alkyl, and R 4 and R 5 and the other of R is H, l is 0, m is 0, and n is 0. In some embodiments, j is 1, k is 1, and each R 4 and R 5 is H, l is 0, m is 0, and n is 1. In some embodiments, j is 1, k is 1, and R 4 and R 5 One of them is C 1-4 alkyl, and R 4 and R 5 and the other of R is H, l is 0, m is 0, and n is 1. In some embodiments, j is 1, k is 2, and each R 4 and R 5 is H, l is 0, m is 0, and n is 0. In some embodiments, j is 1, k is 2, and one R 4 is C 1-4 alkyl, and the remaining R 4 and R 5 The substituents are H, l is 0, m is 0, and n is 0. In some embodiments, j is 1, k is 2, and each R 4 and R 5 is H, l is 0, m is 0, and n is 1. In some embodiments, j is 1, k is 3, and each R 4 and R 5 is H, l is 0, m is 0, and n is 1. In some embodiments, j is 1, k is 4, and each R 4 and R 5is H, l is 0, m is 0, and n is 1. In some embodiments, j is 1, k is 1, and each R 4 and R 5 is H, l is 1, m is 1, and R 6 and R 7 are H and n is 1. In some embodiments, j is 1, k is 1, and each R 4 and R 5 is H, l is 0, m is 2, and R 6 One of them is R 2 and R 3 together with the atom to which they are attached and any intervening atoms, form a 7- to 18-membered bridged heterocycloalkyl group, and other R 6 is H and R 7 are H and n is 0.

[0036] In some embodiments, j is 1, k is 1, and R 4 and R 5 one of which is methyl), and R 4 and R 5 and the other of R is H, l is 0, m is 0, and n is 0. In some embodiments, j is 1, k is 1, and R 4 and R 5 is methyl, and R 4 and R 5 and the other of R is H, l is 0, m is 0, and n is 1. In some embodiments, j is 1, k is 2, and R 4 One of the R 4 and R 5 The substituent is H, l is 0, m is 0, and n is 0.

[0037] In some embodiments, R 2 and R 3 is C 2-10 alkyl, each independently selected from C 2-10 Alkyl is -NR 8 R 9, OH, and halo, wherein at least one substituent is —NR 8 R 9 is.

[0038] In some embodiments, R 2 and R 3 is C 2-10 are each independently selected from alkyl, (I C 2-10 Alkyl is -NR 8 R 9 , OH, and halo, wherein at least one substituent is —NR 8 R 9 and; (ii) C 2-10 Optionally, 1, 2, 3, or 4 non-terminal carbons of the alkyl are replaced with O.

[0039] In some embodiments, R 2 and R 3 is C 2-10 are each independently selected from alkyl, (I C 2-10 Alkyl is -NR 8 R 9 , OH, and halo, wherein at least one substituent is —NR 8 R 9 and; (iii)C 2-10 One, two, three, or four non-terminal carbons of an alkyl can be NR 10 is arbitrarily replaced by .

[0040] In some embodiments, R 2 and R 3 is C 2-10 are each independently selected from alkyl, (I C 2-10 Alkyl is -NR 8 R 9, OH, and halo, wherein at least one substituent is —NR 8 R 9 and; (iv) C 2-10 One, two, three, or four non-terminal carbons of the alkyl are optionally replaced with C(=O).

[0041] In some embodiments, R 2 and R 3 is C 2-10 are each independently selected from alkyl, (I C 2-20 Alkyl is -NR 8 R 9 , OH, and halo, wherein at least one substituent is —NR 8 R 9 and; (v) C 2-20 One, two, three, or four non-terminal carbons of an alkyl can be CR a R b is arbitrarily replaced by R a and R b are C atoms along with the C atoms to which they are bonded. 3-6 Forms a cycloalkyl group.

[0042] In some embodiments, R 2 and R 3 is C 2-10 are each independently selected from alkyl, (I C 2-10 Alkyl is -NR 8 R 9 , OH, and halo, wherein at least one substituent is —NR 8 R 9 and; (ii) C 2-10 one or two non-terminal carbons of the alkyl are optionally replaced with O; (iii)C 2-10One or two non-terminal carbons of an alkyl can be NR 10 is optionally replaced by; (iv) C 2-10 one or two non-terminal carbons of the alkyl are optionally replaced with C(=O); (v) C 2-10 One or two non-terminal carbons of the alkyl are CR a R b is arbitrarily replaced by R a and R b are C atoms along with the C atoms to which they are bonded. 3-6 Forms a cycloalkyl group.

[0043] In some embodiments, R 2 and R 3 is C 2-10 are each independently selected from alkyl, (I C 2-10 Alkyl is -NR 8 R 9 , OH, and halo, wherein at least one substituent is —NR 8 R 9 and; (ii) C 2-10 One or two non-terminal carbons of the alkyl are optionally replaced with O.

[0044] In some embodiments, R 2 and R 3 is C 2-10 are each independently selected from alkyl, (I C 2-10 Alkyl is -NR 8 R 9 , OH, and halo, wherein at least one substituent is —NR 8 R 9 and; (iii)C 2-10 One or two non-terminal carbons of an alkyl can be NR 10 is arbitrarily replaced by .

[0045] In some embodiments, R 2 and R 3 is C 2-10 are each independently selected from alkyl, (I C 2-10 Alkyl is -NR 8 R 9 , OH, and halo, wherein at least one substituent is —NR 8 R 9 and; (iv) C 2-10 One or two non-terminal carbons of the alkyl are optionally replaced with C(=O).

[0046] In some embodiments, R 2 and R 3 is C 2-10 are each independently selected from alkyl, (I C 2-20 Alkyl is -NR 8 R 9 , OH, and halo, wherein at least one substituent is —NR 8 R 9 and; (v) C 2-10 One or two non-terminal carbons of the alkyl are CR a R b is arbitrarily replaced by R a and R b are C atoms along with the C atoms to which they are bonded. 3-6 Forms a cycloalkyl group.

[0047] In some embodiments, R 2 and R 3 is C 2-10 are each independently selected from alkyl, (I C 2-10 Alkyl is -NR 8 R 9, OH, and halo, wherein at least one substituent is —NR 8 R 9 and; (ii) C 2-10 one or two non-terminal carbons of the alkyl are optionally replaced with O; (iii)C 2-10 One or two non-terminal carbons of an alkyl can be NR 10 is arbitrarily replaced by .

[0048] In some embodiments, R 2 and R 3 is C 4-10 are each independently selected from alkyl, (I C 4-10 Alkyl is -NR 8 R 9 , OH, and halo, wherein at least one substituent is —NR 8 R 9 and; (ii) C 4-10 one or two non-terminal carbons of the alkyl are optionally replaced with O; (iii)C 4-10 One or two non-terminal carbons of an alkyl can be NR 10 is optionally replaced by; (iv) C 4-10 one or two non-terminal carbons of the alkyl are optionally replaced with C(=O); (v) C 4-10 One or two non-terminal carbons of the alkyl are CR a R b is arbitrarily replaced by R a and R b are C atoms along with the C atoms to which they are bonded. 3-6 Forms a cycloalkyl group.

[0049] In some embodiments, R 2 and R 3 is C 4-10are each independently selected from alkyl, (I C 4-10 Alkyl is -NR 8 R 9 , OH, and halo, wherein at least one substituent is —NR 8 R 9 and; (ii) C 4-10 one or two non-terminal carbons of the alkyl are optionally replaced with O; (iii)C 4-10 One or two non-terminal carbons of an alkyl can be NR 10 is arbitrarily replaced by .

[0050] In some embodiments, R 2 and R 3 One of them is C 2-5 is alkyl, C 2-5 Alkyl is -NR 8 R 9 , OH, and halo, wherein at least one substituent is —NR 8 R 9 and; R 2 and R 3 The other is C 7-10 is alkyl, (I C 7-10 Alkyl is -NR 8 R 9 , OH, and halo, wherein at least one substituent is —NR 8 R 9 and; (ii) C 7-10 1, 2, 3, or 4 non-terminal carbons of the alkyl are optionally replaced with O; (iii)C 7-10 One, two, three, or four non-terminal carbons of an alkyl can be NR 10 is optionally replaced by; (iv) C7-10 1, 2, 3, or 4 non-terminal carbons of the alkyl are optionally replaced with C(=O); (v) C 7-10 One, two, three, or four non-terminal carbons of an alkyl can be CR a R b is arbitrarily replaced by R a and R b are C atoms along with the C atoms to which they are bonded. 3-6 Forms a cycloalkyl group.

[0051] In some embodiments, R 2 and R 3 One of them is C 2-5 is alkyl, (I C 2-5 Alkyl is -NR 8 R 9 , OH, and halo, wherein at least one substituent is —NR 8 R 9 and; R 2 and R 3 The other is C 7-10 is alkyl, (I C 7-10 Alkyl is -NR 8 R 9 , OH, and halo, wherein at least one substituent is —NR 8 R 9 and; (ii) C 7-10 1, 2, 3, or 4 non-terminal carbons of the alkyl are optionally replaced with O; (iii)C 7-10 One, two, three, or four non-terminal carbons of an alkyl can be NR 10 is arbitrarily replaced by .

[0052] In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9C replaced by 2-20 In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 C replaced by 2-20 alkyl, C 2-20 One non-terminal carbon of an alkyl is NR 10 In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 C replaced by 2-20 alkyl, C 2-20 One non-terminal carbon of the alkyl is replaced with O. In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 and C substituted by two halo 2-20 alkyl, C 2-20 One non-terminal carbon of an alkyl is NR 10 In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 and C substituted by two -F 2-20 alkyl, C 2-20 One non-terminal carbon of an alkyl is NR 10 In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 and C substituted by two halo 2-20 In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 and C substituted by two -F 2-20 In some embodiments, R 2 and R 3 One of them is a single -NR 8 R9 and C substituted by 1 halo 2-20 alkyl, C 2-20 One non-terminal carbon of an alkyl is NR 10 In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 and C substituted by one -F 2-20 alkyl, C 2-20 One non-terminal carbon of an alkyl is NR 10 In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 and C substituted by 1 halo 2-20 In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 and C substituted by one -F 2-20 In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 and C replaced by one OH 2-20 It is alkyl.

[0053] In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 C replaced by 2-20 alkyl, C 2-20 One non-terminal carbon of the alkyl is replaced with C(=O). In some embodiments, R 2 and R 3 One of them is two -NR 8 R 9 C replaced by 2-20 alkyl, C 2-20 One non-terminal carbon of the alkyl is replaced with C(=O). In some embodiments, R2 and R 3 One of them is a single -NR 8 R 9 C replaced by 2-20 alkyl, C 2-20 One non-terminal carbon of an alkyl is NR 10 has been replaced by C 2-20 One non-terminal carbon of the alkyl is CR a R b is replaced by R a and R b are C atoms along with the C atoms to which they are bonded. 3-6 Forms a cycloalkyl group. In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 C replaced by 2-20 alkyl, C 2-20 One non-terminal carbon of the alkyl is CR a R b is replaced by R a and R b are C atoms along with the C atoms to which they are bonded. 3-6 Forms a cycloalkyl group.

[0054] In some embodiments, R 2 and R 3 On the other hand, 1 -NR 8 R 9 C replaced by 2-20 Alkyl, 1 -NR 8 R 9 C replaced by 2-20 Alkyl, C 2-20 One non-terminal carbon of the alkyl is NR 10 which has been replaced by 1 -NR 8 R 9 C replaced by 2-20 Alkyl, C 2-20 one non-terminal carbon of an alkyl is replaced by O, 1 -NR 8 R9 and C substituted by two halo 2-20 Alkyl, C 2-20 One non-terminal carbon of the alkyl is NR 10 which has been replaced by 1 -NR 8 R 9 and C substituted by 1 halo 2-20 Alkyl, C 2-20 One non-terminal carbon of the alkyl is NR 10 which has been replaced by 1 -NR 8 R 9 C replaced by 2-20 Alkyl, C 2-20 one non-terminal carbon of an alkyl is replaced by C(=O); 1 -NR 8 R 9 C replaced by 2-20 Alkyl, C 2-20 two non-terminal carbons of an alkyl replaced by C(=O), and 1 -NR 8 R 9 C replaced by 2-20 Alkyl, C 2-20 One non-terminal carbon of the alkyl is NR 10 has been replaced by C 2-20 One non-terminal carbon of the alkyl is CR a R b is replaced by R a and R b However, together with the C atoms to which they are bonded, 3-6 Forming a cycloalkyl group Selected from R 2 and R 3 The other is, 1 -NR 8 R 9 C replaced by 2-20 Alkyl, 1 -NR 8 R 9 C replaced by 2-20 Alkyl, C 2-20One non-terminal carbon of the alkyl is NR 10 which has been replaced by 1 -NR 8 R 9 and C substituted by two halo 2-20 Alkyl, 1 -NR 8 R 9 and C substituted by 1 halo 2-20 Alkyl, 1 -NR 8 R 9 and C replaced by one OH 2-20 alkyl, and 1 -NR 8 R 9 C replaced by 2-20 Alkyl, C 2-20 One non-terminal carbon of the alkyl is CR a R b is replaced by R a and R b However, together with the C atoms to which they are bonded, 3-6 Forming a cycloalkyl group is selected from.

[0055] In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 C replaced by 2-20 Alkyl, one -NR 8 R 9 C replaced by 2-20 Alkyl, C 2-20 One non-terminal carbon of the alkyl is NR 10 , one -NR 8 R 9 C replaced by 2-20 Alkyl, C 2-20 One non-terminal carbon of alkyl is replaced by O, one -NR 8 R 9 and C substituted by two halo 2-20 Alkyl, C 2-20One non-terminal carbon of the alkyl is NR 10 and one -NR 8 R 9 and C substituted by 1 halo 2-20 Alkyl, C 2-20 One non-terminal carbon of the alkyl is NR 10 is selected from those replaced by R 2 and R 3 The other is one -NR 8 R 9 C replaced by 2-20 Alkyl, one -NR 8 R 9 C replaced by 2-20 Alkyl, C 2-20 One non-terminal carbon of the alkyl is NR 10 , one -NR 8 R 9 and C substituted by two halo 2-20 Alkyl, one -NR 8 R 9 and C substituted by 1 halo 2-20 alkyl and one -NR 8 R 9 and C replaced by one OH 2-20 alkyl.

[0056] In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 C replaced by 2-10 In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 C replaced by 2-10 alkyl, C 2-10 One non-terminal carbon of an alkyl is NR 10 In some embodiments, R 2 and R 3 One of them is a single -NR 8 R9 C replaced by 2-10 alkyl, C 2-10 One non-terminal carbon of the alkyl is replaced with O. In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 and C substituted by two halo 2-10 alkyl, C 2-10 One non-terminal carbon of an alkyl is NR 10 In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 and C substituted by two -F 2-10 alkyl, C 2-10 One non-terminal carbon of an alkyl is NR 10 In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 and C substituted by two halo 2-10 In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 and C substituted by two -F 2-10 In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 and C substituted by 1 halo 2-10 alkyl, C 2-10 One non-terminal carbon of an alkyl is NR 10 In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 and C substituted by one -F 2-10 alkyl, C 2-10 One non-terminal carbon of an alkyl is NR 10In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 and C substituted by 1 halo 2-10 In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 and C substituted by one -F 2-10 In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 and C replaced by one OH 2-10 It is alkyl.

[0057] In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 C replaced by 2-10 alkyl, C 2-10 One non-terminal carbon of the alkyl is replaced with C(=O). In some embodiments, R 2 and R 3 One of them is two -NR 8 R 9 C replaced by 2-10 alkyl, C 2-10 One non-terminal carbon of the alkyl is replaced with C(=O). In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 C replaced by 2-10 alkyl, C 2-10 One non-terminal carbon of an alkyl is NR 10 has been replaced by C 2-10 One non-terminal carbon of the alkyl is CR a R b is replaced by R a and R b are C atoms along with the C atoms to which they are bonded.3-6 Forms a cycloalkyl group. In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 C replaced by 2-10 alkyl, C 2-10 One non-terminal carbon of the alkyl is CR a R b is replaced by R a and R b are C atoms along with the C atoms to which they are bonded. 3-6 Forms a cycloalkyl group.

[0058] In some embodiments, R 2 and R 3 On the other hand, 1 -NR 8 R 9 C replaced by 2-10 Alkyl, 1 -NR 8 R 9 C replaced by 2-10 Alkyl, C 2-10 One non-terminal carbon of the alkyl is NR 10 which has been replaced by 1 -NR 8 R 9 C replaced by 2-10 Alkyl, C 2-10 one non-terminal carbon of an alkyl is replaced by O, 1 -NR 8 R 9 and C substituted by two halo 2-10 Alkyl, C 2-10 One non-terminal carbon of the alkyl is NR 10 which has been replaced by 1 -NR 8 R 9 and C substituted by 1 halo 2-10 Alkyl, C 2-10 One non-terminal carbon of the alkyl is NR 10 which has been replaced by 1 -NR8 R 9 C replaced by 2-10 Alkyl, C 2-10 one non-terminal carbon of an alkyl is replaced by C(=O); 1 -NR 8 R 9 C replaced by 2-10 Alkyl, C 2-10 two non-terminal carbons of an alkyl replaced by C(=O), and 1 -NR 8 R 9 C replaced by 2-10 Alkyl, C 2-10 One non-terminal carbon of the alkyl is NR 10 has been replaced by C 2-10 One non-terminal carbon of the alkyl is CR a R b is replaced by R a and R b However, together with the C atoms to which they are bonded, 3-6 Forming a cycloalkyl group Selected from R 2 and R 3 The other is, 1 -NR 8 R 9 C replaced by 2-10 Alkyl, 1 -NR 8 R 9 C replaced by 2-10 Alkyl, C 2-10 One non-terminal carbon of the alkyl is NR 10 which has been replaced by 1 -NR 8 R 9 and C substituted by two halo 2-10 Alkyl, 1 -NR 8 R 9 and C substituted by 1 halo 2-10 Alkyl, 1 -NR 8 R 9 and C replaced by one OH2-10 alkyl, and 1 -NR 8 R 9 C replaced by 2-10 Alkyl, C 2-10 One non-terminal carbon of the alkyl is CR a R b is replaced by R a and R b However, together with the C atoms to which they are bonded, 3-6 Forming a cycloalkyl group is selected from.

[0059] In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 C replaced by 2-10 Alkyl, one -NR 8 R 9 C replaced by 2-10 Alkyl, C 2-10 One non-terminal carbon of the alkyl is NR 10 , one -NR 8 R 9 C replaced by 2-10 Alkyl, C 2-10 One non-terminal carbon of alkyl is replaced by O, one -NR 8 R 9 and C substituted by two halo 2-10 Alkyl, C 2-10 One non-terminal carbon of the alkyl is NR 10 and one -NR 8 R 9 and C substituted by 1 halo 2-10 Alkyl, C 2-10 One non-terminal carbon of the alkyl is NR 10 is selected from those replaced by R 2 and R 3 The other is one -NR 8 R 9 C replaced by 2-10Alkyl, one -NR 8 R 9 C replaced by 2-10 Alkyl, C 2-10 One non-terminal carbon of the alkyl is NR 10 , one -NR 8 R 9 and C substituted by two halo 2-10 Alkyl, one -NR 8 R 9 and C substituted by 1 halo 2-10 alkyl and one -NR 8 R 9 and C replaced by one OH 2-10 alkyl.

[0060] In some embodiments, R 2 and R 3 On the other hand, 1 -NR 8 R 9 C replaced by 5-10 Alkyl, 1 -NR 8 R 9 C replaced by 5-10 Alkyl, C 5-10 One non-terminal carbon of the alkyl is NR 10 which has been replaced by 1 -NR 8 R 9 C replaced by 5-10 Alkyl, C 5-10 one non-terminal carbon of an alkyl is replaced by O, 1 -NR 8 R 9 and C substituted by two halo 5-10 Alkyl, C 5-10 One non-terminal carbon of the alkyl is NR 10 which has been replaced by 1 -NR 8 R 9 and C substituted by 1 halo 5-10 Alkyl, C 5-10One non-terminal carbon of the alkyl is NR 10 has been replaced by, and 1 -NR 8 R 9 C replaced by 5-10 Alkyl, C 2-10 One non-terminal carbon of the alkyl is NR 10 has been replaced by C 2-10 One non-terminal carbon of the alkyl is CR a R b is replaced by R a and R b However, together with the C atoms to which they are bonded, 3-6 Forming a cycloalkyl group Selected from R 2 and R 3 The other is, 1 -NR 8 R 9 C replaced by 3-6 Alkyl, 1 -NR 8 R 9 C replaced by 3-6 Alkyl, C 3-6 One non-terminal carbon of the alkyl is NR 10 which has been replaced by 1 -NR 8 R 9 and C substituted by two halo 3-6 Alkyl, 1 -NR 8 R 9 and C substituted by 1 halo 3-6 Alkyl, 1 -NR 8 R 9 and C replaced by one OH 3-6 Alkyl, 1 -NR 8 R 9 C replaced by 3-6 Alkyl, C 2-10 one non-terminal carbon of an alkyl is replaced by C(=O); 1 -NR 8 R 9C replaced by 3-6 Alkyl, C 2-10 two non-terminal carbons of an alkyl replaced by C(=O), and 1 -NR 8 R 9 C replaced by 3-6 Alkyl, C 2-10 One non-terminal carbon of the alkyl is CR a R b is replaced by R a and R b However, together with the C atoms to which they are bonded, 3-6 Forming a cycloalkyl group is selected from.

[0061] In some embodiments, R 2 and R 3 One of them is a single -NR 8 R 9 C replaced by 5-10 Alkyl, one -NR 8 R 9 C replaced by 5-10 Alkyl, C 5-10 One non-terminal carbon of the alkyl is NR 10 , one -NR 8 R 9 C replaced by 5-10 Alkyl, C 5-10 One non-terminal carbon of alkyl is replaced by O, one -NR 8 R 9 and C substituted by two halo 5-10 Alkyl, C 5-10 One non-terminal carbon of the alkyl is NR 10 and one -NR 8 R 9 and C substituted by 1 halo 5-10 Alkyl, C 5-10 One non-terminal carbon of the alkyl is NR 10 is selected from those replaced by R 2 and R 3The other is one -NR 8 R 9 C replaced by 3-6 Alkyl, one -NR 8 R 9 C replaced by 3-6 Alkyl, C 3-6 One non-terminal carbon of the alkyl is NR 10 , one -NR 8 R 9 and C substituted by two halo 3-6 Alkyl, one -NR 8 R 9 and C substituted by 1 halo 3-6 alkyl and one -NR 8 R 9 and C replaced by one OH 3-6 alkyl.

[0062] In some embodiments, R 2 and R 3 One of the groups has at least one -NR 8 R 9 group and optionally substituted by one or two groups selected from OH and halo.

[0063] In some embodiments, R 2 and R 3 On the other hand, [ka] [ka] is selected from.

[0064] In some embodiments, R 2 and R 3 On the other hand, [ka] [ka] is selected from.

[0065] In some embodiments, R 2 and R 3 On the other hand, [ka] is selected from.

[0066] In some embodiments, R 2 and R 3 On the other hand, [ka] is selected from.

[0067] In some embodiments, R 2 and R 3 On the other hand, [ka] is selected from.

[0068] In some embodiments, R 2 and R 3 On the other hand, [ka] is selected from.

[0069] In some embodiments, R 2 and R 3 On the other hand, [ka] Selected from; R 2 and R 3 The other is, [ka] is selected from.

[0070] In some embodiments, R 2 and R 3 On the other hand, [ka] Selected from; R 2 and R 3 The other is, [ka] is selected from.

[0071] In some embodiments, R 2 and R 3 On the other hand, [ka] Selected from; R 2 and R 3 The other is, [ka] is selected from.

[0072] In some embodiments, R 2 and R 3 On the other hand, [ka] Selected from; R 2 and R 3 The other is, [ka] is selected from.

[0073] In some embodiments, R 2 and R 3 together with the N atom to which they are attached form one, two, or three ring-forming NR 10 A 7- to 18-membered heterocycloalkyl group containing a C 1-4Alkyl, -NR 8 R 9 , OH, and halo.

[0074] In some embodiments, R 2 and R 3 together with the N atom to which they are attached form one, two, or three ring-forming NR 10 A 7- to 12-membered heterocycloalkyl group containing a C 1-4 Alkyl, -NR 8 R 9 , OH, and halo.

[0075] In some embodiments, R 2 and R 3 together with the N atom to which they are attached form one, two, or three ring-forming NR 10 The 8- to 10-membered heterocycloalkyl group includes a C 1-4 Alkyl, -NR 8 R 9 , OH, and halo.

[0076] In some embodiments, R 2 and R 3 together with the N atom to which they are attached form an 8- to 10-membered heterocycloalkyl group containing one, two, or three ring-forming NCH3 or NH groups, and the 8- to 10-membered heterocycloalkyl group is 1-4 Alkyl, -NR 8 R 9 , OH, and halo.

[0077] In some embodiments, R 2 and R 3together with the N atom to which they are attached form an 8- to 10-membered heterocycloalkyl group containing 1, 2, or 3 ring-forming NCH3 or NH groups.

[0078] In some embodiments, R 2 and R 3 together with the N atom to which they are attached form the formula: [ka] Form.

[0079] In some embodiments, R 2 , R 3 , and R 6 are, together with the atoms to which they are bonded and any intervening atoms, C 1-4 Forms a 7-18 membered bridged heterocycloalkyl group optionally substituted with 1, 2, or 3 substituents independently selected from alkyl, -NR8R9, OH, and halo.

[0080] In some embodiments, R 2 , R 3 , and R 6 are, together with the atoms to which they are bonded and any intervening atoms, C 1-4 Forms a 7- to 13-membered bridged heterocycloalkyl group optionally substituted with 1, 2, or 3 substituents independently selected from alkyl, -NR8R9, OH, and halo.

[0081] In some embodiments, R 2 , R 3 , and R 6 are, together with the atoms to which they are bonded and any intervening atoms, C 1-4 Forms a 7-10 membered bridged heterocycloalkyl group optionally substituted with 1, 2, or 3 substituents independently selected from alkyl, -NR8R9, OH, and halo.

[0082] In some embodiments, R 2 , R 3 , and R 6together with the atom to which they are attached and any intervening atoms, form a 7- to 10-membered bridged heterocycloalkyl group.

[0083] In some embodiments, R 2 , R 3 , and R 6 together with the atoms to which they are attached and any intervening atoms, form the formula: [ka] Form a 7- to 18-membered bridged heterocycloalkyl group having the following structure.

[0084] In some embodiments, R 4 and R 5 are each independently H or C 1-4 In some embodiments, R 4 and R 5 are each independently H or methyl. In some embodiments, R 4 and R 5 and R are H. In some embodiments, R 4 and R 5 Both are C 1-4 In some embodiments, R 4 and R 5 and R are both methyl. 4 and R 5 One of the two is H and the other is R 4 and R 5 The other is C 1-4 In some embodiments, R 4 and R 5 One of the two is H and the other is R 4 and R 5 The other is methyl.

[0085] In some embodiments, R 6 and R 7 are each independently H or C 1-4 In some embodiments, R 6 and R 7are each independently H or methyl. In some embodiments, R 6 and R 7 and R are H. In some embodiments, R 6 and R 7 Both are C 1-4 In some embodiments, R 6 and R 7 and R are both methyl. 6 and R 7 One of the two is H and the other is R 6 and R 7 The other is C 1-4 In some embodiments, R 6 and R 7 One of the two is H and the other is R 6 and R 7 The other is methyl.

[0086] In some embodiments, R 8 , R 9 , and R 10 are each independently selected from H and methyl. In some embodiments, R 8 and R 9 are both H. In some embodiments, R 8 and R 9 are both C 1-4 In some embodiments, R 8 and R 9 are both methyl. In some embodiments, R 8 and R 9 One of the two is H and the other is R 8 and R 9 The other is C 1-4 In some embodiments, R 8 and R 9 One of the two is H and the other is R 8 and R 9 and the other is methyl. In some embodiments, R 10 is H or methyl. In some embodiments, R 10 is H. In some embodiments, R 10is methyl.

[0087] In some embodiments, R a and R b together with the C atom to which they are attached form a C cycloalkyl group, e.g., cyclopropyl. In some embodiments, R a and R b together with the C atom to which they are attached form a C cycloalkyl group, e.g., cyclobutyl. In some embodiments, R a and R b together with the C atom to which they are attached form a C cycloalkyl group, e.g., cyclopentyl. In some embodiments, R a and R b together with the C atom to which they are attached form a C6 cycloalkyl group, for example cyclopentyl.

[0088] In some embodiments, Z is N or CH; R 1 is C 1-14 Alkyl, C 1-14 Alkenyl, or C 1-14 is hydroxyalkyl; R 2 and R 3 are C 2-20 is alkyl, (I C 2-20 Alkyl is -NR 8 R 9 , OH, and halo, and at least one substituent is —NR 8 R 9 and; (ii) C 2-20 one non-terminal carbon of the alkyl is optionally replaced with O; (iii)C 2-20 One non-terminal carbon of an alkyl is NR 10 is optionally replaced by; (iv) C 2-20 one non-terminal carbon of the alkyl is optionally replaced with C(=O); (v) C 2-20 One non-terminal carbon of the alkyl is CR a R b is arbitrarily replaced by R a and R b are C atoms along with the C atoms to which they are bonded. 3-6 Forms a cycloalkyl group; R 2 and R 3 are the same or different; or R 2 and R 3 together with the N atom to which they are attached, form two ring-forming NR 10 forming a 7- to 18-membered heterocycloalkyl group containing a group; R 4 is H and C 1-4 alkyl; R 5 , R 6 , and R 7 are H, respectively; R 8 , R 9 , and R 10 is H and C 1-4 are each independently selected from alkyl; j is 0 or 1; k is 0, 1, 2, 3, or 4; l is 0 or 1; m is 0, 1, 2, or 4; n is 0 or 1; If j is 0, then l is 1, j and l are not both 0.

[0089] In some embodiments, Z is N or CH; R 1 is C 1-14 Alkyl, C 1-14 Alkenyl, or C 1-14 is hydroxyalkyl; R 2 and R 3 are C 2-20is alkyl, (I C 2-20 Alkyl is -NR 8 R 9 , OH, and halo, and at least one substituent is —NR 8 R 9 and; (ii) C 2-20 one non-terminal carbon of the alkyl is optionally replaced with O; (iii)C 2-20 One non-terminal carbon of an alkyl is NR 10 is optionally replaced by; R 2 and R 3 are the same or different; or R 2 and R 3 together with the N atom to which they are attached, form two ring-forming NR 10 forming a 7- to 18-membered heterocycloalkyl group containing a group; R 4 is H and C 1-4 alkyl; R 5 , R 6 , and R 7 are H, respectively; R 8 , R 9 , and R 10 is H and C 1-4 are each independently selected from alkyl; j is 0 or 1; k is 0, 1, 2, 3, or 4; l is 0 or 1; m is 0, 1, 2, or 4; n is 0 or 1; j and l are not both 0, If j is 0, then l is 1.

[0090] In some embodiments, Z is N; R 1 is C1-14 Alkyl, C 1-14 Alkenyl, or C 1-14 is hydroxyalkyl; R 2 and R 3 are C 2-20 is alkyl, (I C 2-20 Alkyl is -NR 8 R 9 , OH, and halo, and at least one substituent is —NR 8 R 9 and; (ii) C 2-20 one non-terminal carbon of the alkyl is optionally replaced with O; (iii)C 2-20 One non-terminal carbon of an alkyl is NR 10 is optionally replaced by; R 2 and R 3 are the same or different; or R 2 and R 3 together with the N atom to which they are attached, form two ring-forming NR 10 forming a 7- to 18-membered heterocycloalkyl group containing a group; R 4 is H and C 1-4 alkyl; R 5 , R 6 , and R 7 are H, respectively; R 8 , R 9 , and R 10 is H and C 1-4 are each independently selected from alkyl; j is 0 or 1; k is 0, 1, 2, 3, or 4; l is 0 or 1; m is 0, 1, or 4; n is 0 or 1; j and l are not both 0, If j is 0, then l is 1.

[0091] In some embodiments, the compound of formula A1 is a compound of formula A2: [ka] or a salt thereof.

[0092] In some embodiments, the compound of formula A1 is a compound of formulas A3-A15: [ka] [ka] or a salt thereof.

[0093] In some embodiments, the compound of formula A1 is [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] or a salt of any of the foregoing.

[0094] In some embodiments, the compound of formula A1 is [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] or a salt of any of the foregoing.

[0095] In some embodiments, the compound of formula A1 is [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] or a salt of any of the foregoing.

[0096] It will be understood that certain features described herein, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features described herein, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0097] Lipid Nanoparticle Composition Further provided are lipid nanoparticle (LNP) compositions comprising a lipid amine disclosed herein, e.g., a lipid amine of Formula A1. In some embodiments, the lipid nanoparticle composition further comprises, in addition to the lipid amine, at least one of an ionizable lipid, a phospholipid, a structural lipid, and a PEG-lipid. In some embodiments, the lipid nanoparticles of the lipid nanoparticle composition are loaded with a payload. In some embodiments, the lipid amine is primarily disposed on the outer surface of the lipid nanoparticles of the lipid nanoparticle composition. In some embodiments, the lipid nanoparticle composition has a zeta potential greater than neutral at physiological pH.

[0098] In some embodiments, the lipid nanoparticle composition comprises: (i) ionizable lipids; (ii) phospholipids, (iii) structured lipids; (iv) optionally a PEG-lipid; (v) optionally a payload for delivery to a cell, and (vi) a lipid amine disclosed herein, e.g., a lipid amine of formula A1 Includes:

[0099] The lipid nanoparticle composition may further comprise additional components, including, but not limited to, a helper lipid, a stabilizer, a salt, a buffer, and a solvent. The helper lipid is a non-cationic lipid. The helper lipid may comprise at least one fatty acid chain of at least 8 carbons and at least one polar head group. In some embodiments, the lipid nanoparticle core has a neutral charge at neutral pH.

[0100] In some embodiments, the weight ratio of lipid amine to payload in the lipid nanoparticle composition is about 0.1:1 to about 15:1, about 0.2:1 to about 10:1, about 1:1 to about 10:1, about 1:1 to about 8:1, about 1:1 to about 7:1, about 1:1 to about 6:1, about 1:1 to about 5:1, about 1:1 to about 4:1, or about 1.25:1 to about 3.75:1. In some embodiments, the weight ratio of lipid amine to payload is about 1.25:1, about 2.5:1, or about 3.75:1. In some embodiments, the molar ratio of lipid amine to payload is about 0.1:1 to about 20:1, about 1.5:1 to about 10:1, about 1.5:1 to about 9:1, about 1.5:1 to about 8:1, about 1.5:1 to about 7:1, about 1.5:1 to about 6:1, or about 1.5:1 to about 5:1. In some embodiments, the molar ratio of lipid amine to payload is about 1.5:1, about 2:1, about 3:1, about 4:1, or about 5:1.

[0101] In some embodiments, the lipid nanoparticle composition is characterized as having a zeta potential of about 5 mV to about 20 mV. In some embodiments, the lipid nanoparticle composition has a zeta potential of about 5 mV to about 15 mV. In some embodiments, the lipid nanoparticle composition has a zeta potential of about 5 mV to about 10 mV. Zeta potential measures the surface charge of a colloidal dispersion. The magnitude of the zeta potential indicates the degree of electrostatic repulsion between similarly charged adjacent particles in the dispersion. Zeta potential can be measured with a Wyatt Technologies Mobius Zeta Potential instrument. This instrument characterizes mobility and zeta potential using the principles of "massively parallel phase analysis light scattering," or MP-PALS. This measurement is more sensitive and induces less stress than ISO Method 13099-1:2012, which uses only one detection angle and requires higher voltages for operation. In some embodiments, the zeta potential of the lipids in the empty lipid nanoparticle compositions described herein is measured using an instrument employing the principles of MP-PALS. The zeta potential can be measured with a Malvern Zetasizer (Nano ZS).

[0102] In some embodiments, greater than about 80%, greater than about 90%, or greater than about 95% of the lipid amines are on the surface of the lipid nanoparticles of the lipid nanoparticle composition.

[0103] In some embodiments, the lipid nanoparticle compositions have a polydispersity value of less than about 0.4, less than about 0.3, or less than about 0.2. In some embodiments, the LNPs have a polydispersity value of about 0.1 to about 1, about 0.1 to about 0.5, or about 0.1 to about 0.3.

[0104] In some embodiments, the lipid nanoparticles of the lipid nanoparticle composition have an average diameter of about 40 nm to about 150 nm, about 50 nm to about 100 nm, about 60 nm to about 120 nm, about 60 nm to about 100 nm, or about 60 nm to about 80 nm.

[0105] In some embodiments, the lipid nanoparticles of the lipid nanoparticle composition have a Laurdan's general polarization of about 0.6 or greater. In some embodiments, the LNPs have a d-spacing of greater than about 6 nm or greater than about 7 nm.

[0106] In some embodiments, at least about 50%, at least about 75%, at least about 90%, at least about 95% of the lipid nanoparticles of the lipid nanoparticle composition have a surface fluidity value above the threshold polarization level.

[0107] Ionizable lipids As used herein, the term "ionizable lipid" has its ordinary meaning in the art and may refer to a lipid containing one or more charged moieties. In some embodiments, an ionizable lipid may be positively or negatively charged. For example, an ionizable lipid may be positively charged at lower pH, in which case it may be referred to as a "cationic lipid." In certain embodiments, an ionizable lipid molecule may contain an amine group and may be referred to as an ionizable amino lipid. As used herein, a "charged moiety" is a chemical moiety that carries a formal charge, such as monovalent (+1 or -1), divalent (+2 or -2), trivalent (+3 or -3), etc. A charged moiety may be anionic (i.e., negatively charged) or cationic (i.e., positively charged). Examples of positively charged moieties include amine groups (e.g., primary, secondary, and / or tertiary amines), ammonium groups, pyridinium groups, guanidine groups, and imidazolium groups. In certain embodiments, the charged moiety comprises an amine group. Examples of negatively charged groups or precursors thereof include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, etc. The charge of a charged moiety may, in some cases, vary depending on environmental conditions; for example, a change in pH may alter the charge of the moiety and / or may render the moiety charged or uncharged. Generally, the charge density of a molecule may be selected as desired.

[0108] It should be understood that the term "charged" or "charged site" does not refer to a "partial negative charge" or a "partial positive charge" on a molecule. The terms "partial negative charge" and "partial positive charge" are given their ordinary meaning in the art. A "partial negative charge" can occur when a functional group contains a bond that is polarized such that electron density is attracted from one atom of the bond, creating a partial negative charge on the atom. Those skilled in the art will typically recognize bonds that can be polarized in this way.

[0109] In some embodiments, the LNP comprises about 30 mol% to about 60 mol%, about 35 mol% to about 55 mol%, about 40 mol% to about 50 mol%, or about 45 mol% to about 50 mol% ionizable lipids.

[0110] In some embodiments, the ionizable lipid is an ionizable amino lipid. In one embodiment, the ionizable amino lipid can have a positively charged hydrophilic head and a hydrophobic tail connected via a linker structure.

[0111] In some embodiments, the ionizable lipid is a compound of formula (I): [ka] or an N-oxide or salt thereof, wherein R 1 teeth, [ka] and; [ka] indicates the point of attachment; R aα , R aβ , R aγ , and R aδ is H, C 2-12 Alkyl, and C 2-12 alkenyl; R 2 and R 3 is C 1-14 Alkyl and C 2-14 alkenyl; R 4 is -(CH2) n OH and [ka] is selected from n is selected from 1, 2, 3, 4, and 5; [ka] indicates the point of attachment, R 10 is N(R)2; Each R is C 1-6 Alkyl, C 2-3 alkenyl, and H; n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R 5 is C 1-3 Alkyl, C 2-3 alkenyl, and H; Each R 6 is C 1-3 Alkyl, C 2-3 alkenyl, and H; M and M' are each independently selected from -C(O)O- and -OC(O)-; R' is C 1-12 Alkyl or C 2-12 is alkenyl; l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13. is.

[0112] In some embodiments, the ionizable lipid is a compound of formula (I), or an N-oxide or salt thereof, wherein: R 1 teeth, [ka] and; [ka] indicates the point of attachment; R aα , R aβ , R aγ , and R aδ are H, respectively; R 2 and R 3 are C 1-14 is alkyl; R 4 is -(CH2) n OH; n is 2; Each R 5 is H; Each R 6 is H; M and M' are each -C(O)O-; R' is C 1-12 is alkyl; l is 5; m is 7) is.

[0113] In some embodiments, the ionizable lipid is a compound of formula (I), or an N-oxide or salt thereof, wherein: R 1 teeth, [ka] and; [ka] indicates the point of attachment; R aα , R aβ , R aγ , and R aδ are H, respectively; R 2 and R 3 are C 1-14 is alkyl; R 4 is -(CH2) n OH; n is 2; Each R 5 is H; Each R 6 is H; M and M' are each -C(O)O-; R' is C 1-12 is alkyl; l is 3; m is 7) is.

[0114] In some embodiments, the ionizable lipid is a compound of formula (I), or an N-oxide or salt thereof, wherein: R 1 teeth, [ka] and; [ka] indicates the point of attachment; R aα is C 2-12 is alkyl; R aβ , R aγ , and R aδ are H, respectively; R 2 and R 3 are C 1-14 is alkyl; R 4 teeth, [ka] and; R 10 is -NH(C 1-6 alkyl); n2 is 2; Each R 5 is H; Each R 6 is H; M and M' are each -C(O)O-; R' is C 1-12 is alkyl; l is 5; m is 7) is.

[0115] In some embodiments, the ionizable lipid is a compound of formula (I), or an N-oxide or salt thereof, wherein: R 1 teeth, [ka] and; [ka] indicates the point of attachment; R aα , R aβ , and R aδ are H, respectively; R aγ is C 2-12 is alkyl; R 2 and R 3 are C 1-14 is alkyl; R 4 is -(CH2) n OH; n is 2; Each R 5 is H; Each R 6 is H; M and M' are each -C(O)O-; R' is C 1-12 is alkyl; l is 5; m is 7) is.

[0116] In some embodiments, the ionizable lipid is [ka] or an N-oxide or salt thereof.

[0117] In some embodiments, the ionizable lipid is the compound: [ka] or an N-oxide or salt thereof.

[0118] In some embodiments, the ionizable lipid is the compound: [ka] or an N-oxide or salt thereof.

[0119] In some embodiments, the ionizable lipid is the compound: [ka] or an N-oxide or salt thereof.

[0120] In some embodiments, the ionizable lipid is the compound: [ka] or an N-oxide or salt thereof.

[0121] In some embodiments, the ionizable lipid is a compound of formula (I): [ka] or an N-oxide or salt thereof, wherein R 1 teeth, [ka] and; [ka] indicates the point of attachment; R aβ , R aγ , and R aδ is H, C 2-12 Alkyl, and C 2-12 alkenyl; R 2 and R 3 is C 1-14 Alkyl and C 2-14 alkenyl; R 4 is -(CH2) n OH and [ka] is selected from [ka] indicates the point of attachment; n is selected from 1, 2, 3, 4, and 5; R 10 is N(R)2; Each R is C 1-6 Alkyl, C 2-3 alkenyl, and H; n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R 5 is C 1-3 Alkyl, C 2-3 alkenyl, and H; Each R 6 is C 1-3 Alkyl, C 2-3 alkenyl, and H; M and M' are each independently selected from -C(O)O- and -OC(O)-; R' is C 1-12 Alkyl or C 2-12 is alkenyl; l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13. is.

[0122] In some embodiments, the ionizable lipid is a compound of formula (I): [ka] or an N-oxide or salt thereof, wherein R 1 teeth, [ka] and; [ka] indicates the point of attachment; R aα , R aβ , R aγ , and R aδ is H, C 2-12 Alkyl, and C 2-12 alkenyl; R 2 and R 3 is C 1-14 Alkyl and C 2-14 alkenyl; R 4 is -(CH2) n OH, and n is selected from 1, 2, 3, 4, and 5; Each R 5 is C 1-3 Alkyl, C 2-3 alkenyl, and H; Each R 6 is C 1-3 Alkyl, C 2-3 alkenyl, and H; M and M' are each independently selected from -C(O)O- and -OC(O)-; R' is C 1-12 Alkyl or C 2-12 is alkenyl; l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13. is.

[0123] In some embodiments, the ionizable lipid is a compound of formula (I), or an N-oxide or salt thereof, wherein: R 1 teeth, [ka] and; [ka] indicates the point of attachment; R aβ , R aγ , and Raδ are H, respectively; R 2 and R 3 are C 1-14 is alkyl; R 4 is -(CH2) n OH; n is 2; Each R 5 is H; Each R 6 is H; M and M' are each -C(O)O-; R' is C 1-12 is alkyl; l is 5; m is 7) is.

[0124] In some embodiments, the ionizable lipid is a compound of formula (I), or an N-oxide or salt thereof, wherein: R 1 teeth, [ka] and; [ka] indicates the point of attachment; R aβ , R aγ , and R aδ are H, respectively; R 2 and R 3 are C 1-14 is alkyl; R 4 is -(CH2) n OH; n is 2; Each R 5 is H; Each R 6 is H; M and M' are each -C(O)O-; R' is C 1-12 is alkyl; l is 3; m is 7) is.

[0125] In some embodiments, the ionizable lipid is a compound of formula (I), or an N-oxide or salt thereof, wherein: R 1 teeth, [ka] and; [ka] indicates the point of attachment; R aβ and R aδ is H; R aγ is C 2-12 is alkyl; R 2 and R 3 are C 1-14 is alkyl; R 4 is -(CH2) n OH; n is 2; Each R 5 is H; Each R 6 is H; M and M' are each -C(O)O-; R' is C 1-12 is alkyl; l is 5; m is 7) is.

[0126] In some embodiments, the ionizable lipid is a compound of formula (I): [ka] or an N-oxide or salt thereof, wherein R 1 teeth, [ka] and; [ka] indicates the point of attachment; R aα , R aβ , R aγ , and R aδ is H, C 2-12 Alkyl, and C 2-12 alkenyl; R 2 and R 3 is C 1-14 Alkyl and C 2-14 alkenyl; R 4 teeth, [ka] and [ka] indicates the point of attachment; R 10 is N(R)2; Each R is C 1-6 Alkyl, C 2-3 alkenyl, and H; n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R 5 is C 1-3 Alkyl, C 2-3 alkenyl, and H; Each R 6 is C 1-3 Alkyl, C 2-3 alkenyl, and H; M and M' are each independently selected from -C(O)O- and -OC(O)-; R' is C 1-12 Alkyl or C 2-12 is alkenyl; l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13. is.

[0127] In some embodiments, R 1 teeth, [ka] and; [ka] indicates the point of attachment; R aβ , R aγ , and R aδ are H, respectively; R aα is C 2-12 is alkyl; R 2 and R 3 are C 1-14 is alkyl; R 4 teeth, [ka] and; [ka] indicates the point of attachment; R 10 is -NH(C 1-6 alkyl); n2 is 2; Each R 5 is H; Each R 6 is H; M and M' are each -C(O)O-; R' is C 1-12 is alkyl; l is 5; m is 7.

[0128] In some embodiments, the ionizable lipid of formula (I) is [ka] or an N-oxide or salt thereof.

[0129] In some embodiments, the ionizable lipid is a compound of formula (II): [ka] or an N-oxide or salt thereof, wherein R' a is R' 分岐状 or R' 環状 and; R' 分岐状 teeth, [ka] and R' 環状 teeth, [ka] and; R' b teeth, [ka] and; [ka] indicates the point of attachment; R aγ and R aδ is H, C 1-12 Alkyl, and C 2-12 alkenyl; R aγ and R aδ At least one of the 1-12 Alkyl and C 2-12 alkenyl; R bγ and R bδ is H, C 1-12 Alkyl, and C 2-12 alkenyl; R bγ and Rbδ At least one of the 1-12 Alkyl and C 2-12 alkenyl; R 2 and R 3 is C 1-14 Alkyl and C 2-14 alkenyl; R 4 is -(CH2) n OH and [ka] is selected from [ka] indicates the point of attachment; n is selected from 1, 2, 3, 4, and 5; R 10 is N(R)2; Each R is C 1-6 Alkyl, C 2-3 alkenyl, and H; n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R' is independently C 1-12 Alkyl or C 2-12 is alkenyl; Y a is C 3-6 It is a carbocyclic ring; R*” a is C 1-15 Alkyl and C 2-15 alkenyl; s is 2 or 3; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9 is.

[0130] In some embodiments, the ionizable lipid is a compound of formula (II): [ka] or an N-oxide or salt thereof, wherein R' a is R' 分岐状 or R' 環状 and; R' 分岐状 teeth, [ka] and R' b teeth, [ka] and; [ka] indicates the point of attachment; R aγ and R aδ is H, C 1-12 Alkyl, and C 2-12 alkenyl; R aγ and R aδ At least one of the 1-12 Alkyl and C 2-12 alkenyl; R bγ and R bδ is H, C 1-12 Alkyl, and C 2-12 alkenyl; R bγ and R bδ At least one of the 1-12 Alkyl and C 2-12 alkenyl; R 2 and R 3 is C 1-14 Alkyl and C 2-14 alkenyl; R 4 is -(CH2) n OH and [ka] is selected from [ka] indicates the point of attachment; n is selected from 1, 2, 3, 4, and 5; R 10 is N(R)2; Each R is C 1-6 Alkyl, C 2-3 alkenyl, and H; n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R' is independently C 1-12 Alkyl or C 2-12 is alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9 is.

[0131] In some embodiments, the ionizable lipid is a compound of formula (II): [ka] or an N-oxide or salt thereof, wherein R' a is R' 分岐状 or R' 環状 and; R' 分岐状 teeth, [ka] and R' b teeth, [ka] and; [ka] indicates the point of attachment; R aγ and R bγ is C 1-12 Alkyl and C 2-12alkenyl; R 2 and R 3 is C 1-14 Alkyl and C 2-14 alkenyl; R 4 is -(CH2) n OH and [ka] is selected from [ka] indicates the point of attachment; n is selected from 1, 2, 3, 4, and 5; R 10 is N(R)2; Each R is C 1-6 Alkyl, C 2-3 alkenyl, and H; n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R' is independently C 1-12 Alkyl or C 2-12 is alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9 is.

[0132] In some embodiments, the ionizable lipid is a compound of formula (II): [ka] or an N-oxide or salt thereof, wherein R' a is R' 分岐状 or R' 環状 and; R' 分岐状 teeth, [ka] and R'b teeth, [ka] and; [ka] indicates the point of attachment; R aγ is C 1-12 Alkyl and C 2-12 alkenyl; R 2 and R 3 is C 1-14 Alkyl and C 2-14 alkenyl; R 4 is -(CH2) n OH and [ka] is selected from [ka] indicates the point of attachment; n is selected from 1, 2, 3, 4, and 5; R 10 is N(R)2; Each R is C 1-6 Alkyl, C 2-3 alkenyl, and H; n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; R' is C 1-12 Alkyl or C 2-12 is alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9 is.

[0133] In some embodiments, the ionizable lipid is a compound of formula (II): [ka] or an N-oxide or salt thereof, wherein R' a is R' 分岐状 or R' 環状 and; R' 分岐状 teeth, [ka] and R' b teeth, [ka] and; [ka] indicates the point of attachment; R aγ and R bγ is C 1-12 Alkyl and C 2-12 alkenyl; R 4 is -(CH2) n OH and [ka] is selected from [ka] indicates the point of attachment; n is selected from 1, 2, 3, 4, and 5; R 10 is N(R)2; Each R is C 1-6 Alkyl, C 2-3 alkenyl, and H; n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R' is independently C 1-12 Alkyl or C 2-12 is alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9 is.

[0134] In some embodiments, the ionizable lipid is a compound of formula (II): [ka] or an N-oxide or salt thereof, wherein R' a is R' 分岐状 or R' 環状 and; R' 分岐状 teeth, [ka] and R' b teeth, [ka] and; [ka] indicates the point of attachment; R aγ is C 1-12 Alkyl and C 2-12 alkenyl; R 2 and R 3 is C 1-14 Alkyl and C 2-14 alkenyl; R 4 is -(CH2) n OH, and n is selected from 1, 2, 3, 4, and 5; R' is C 1-12 Alkyl or C 2-12 is alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9 is.

[0135] In some embodiments, m and l are each independently selected from 4, 5, and 6. In some embodiments, m and l are each 5.

[0136] In some embodiments, each R' is independently C 1-12 In some embodiments, each R' is independently C 2-5 It is alkyl.

[0137] In some embodiments, R' b teeth, [ka] and R 2 and R 3 are each independently 1-14 It is alkyl.

[0138] In some embodiments, R' b teeth, [ka] and R 2 and R 3 are each independently 6-10 It is alkyl.

[0139] In some embodiments, R' b teeth, [ka] and R 2 and R 3 are each C8 alkyl.

[0140] In some embodiments, R' 分岐状 teeth, [ka] and R' b teeth, [ka] and R aγ is C 1-12 alkyl, and R 2 and R 3 are each independently 6-10 It is alkyl.

[0141] In some embodiments, R' 分岐状 teeth, [ka] and R' b teeth, [ka] and R aγ is C 2-6 alkyl, and R 2 and R 3 are each independently 6-10 In some embodiments, R' is alkyl. 分岐状 teeth, [ka] and R' b teeth, [ka] and R aγ is C 2-6 alkyl, and R 2 and R 3 are each C8 alkyl.

[0142] In some embodiments, R' 分岐状 teeth, [ka] and R' b teeth, [ka] and R aγ and R bγ are C1-12 It is alkyl.

[0143] In some embodiments, R' 分岐状 teeth, [ka] and R' b teeth, [ka] and R aγ and R bγ are C 2-6 It is alkyl.

[0144] In some embodiments, m and l are each independently selected from 4, 5, and 6, and each R′ is independently selected from C 1-12 In some embodiments, m and l are each 5 and each R' is independently C 2-5 It is alkyl.

[0145] In some embodiments, R' 分岐状 teeth, [ka] and R' b teeth, [ka] wherein m and l are each independently selected from 4, 5, and 6; and each R′ is independently selected from C 1-12 alkyl, and R aγ and R bγ are C 1-12 It is alkyl.

[0146] In some embodiments, R' 分岐状 teeth, [ka] and R' b teeth, [ka] wherein m and l are each 5; and each R' is independently C 2-5 alkyl, and R aγ and R bγ are C 2-6 It is alkyl.

[0147] In some embodiments, R' 分岐状 teeth, [ka] and R' b teeth, [ka] wherein m and l are each independently selected from 4, 5, and 6; and R′ is C 1-12 is alkyl, aγ is C 1-12 alkyl, and R 2 and R 3 are each independently 6-10 It is alkyl.

[0148] In some embodiments, R' 分岐状 teeth, [ka] and R' b teeth, [ka] m and l are each 5; R' is C 2-5 alkyl, and R aγ is C 2-6 alkyl, and R 2 and R 3 are each C8 alkyl.

[0149] In some embodiments, R 4 teeth, [ka] and R 10 is NH(C 1-6 alkyl), and n2 is 2.

[0150] In some embodiments, R 4 teeth, [ka] and R 10 is NH(CH3) and n2 is 2.

[0151] In some embodiments, R' 分岐状 teeth, [ka] and R' b teeth, [ka] m and l are each independently selected from 4, 5, and 6; each R' is independently selected from C 1-12 alkyl; R aγ and R bγ are C 1-12 alkyl; R 4 teeth, [ka] and R 10 is NH(C 1-6 alkyl), and n2 is 2.

[0152] In some embodiments, R' 分岐状 teeth, [ka] and R' b teeth, [ka] wherein m and l are each 5; and each R' is independently C 2-5 alkyl, and R aγ and R bγ are C 2-6 alkyl, and R 4 teeth, [ka] and R 10 is NH(CH3) and n2 is 2.

[0153] In some embodiments, R' 分岐状 teeth, [ka] and R' b teeth, [ka] wherein m and l are each independently selected from 4, 5, and 6; and R′ is C 1-12 alkyl, and R 2 and R 3 are each independently 6-10 alkyl, and R aγ is C 1-12 alkyl, and R 4 teeth, [ka] and R 10 is NH(C 1-6 alkyl), and n2 is 2.

[0154] In some embodiments, R' 分岐状 teeth, [ka] and R' b teeth, [ka] m and l are each 5; R' is C 2-5 alkyl, and R aγ is C 2-6 alkyl, and R 2 and R 3 are each C8 alkyl, and R 4 teeth, [ka] and R 10 is NH(CH3) and n2 is 2.

[0155] In some embodiments, R 4 is -(CH2) n OH and n is 2, 3, or 4. In some embodiments, R 4 is -(CH2) n OH and n is 2.

[0156] In some embodiments, R' 分岐状 teeth, [ka] and R' b teeth, [ka] wherein m and l are each independently selected from 4, 5, and 6; and each R′ is independently selected from C 1-12 alkyl, and R aγ and R bγ are C 1-12 alkyl, and R 4 is -(CH2) n OH, and n is 2, 3, or 4.

[0157] In some embodiments, R' 分岐状 teeth, [ka] and R' b teeth, [ka] wherein m and l are each 5; and each R' is independently C 2-5 alkyl, and R aγ and R bγ are C 2-6 alkyl, and R 4 is -(CH2) n OH and n is 2.

[0158] In some embodiments, the ionizable lipid is a compound of formula (II): [ka] or an N-oxide or salt thereof, wherein R' a is R' 分岐状 or R' 環状 and; R' 分岐状 teeth, [ka] and R' b teeth, [ka] and; [ka] indicates the point of attachment; R aγ is C 1-12 is alkyl; R 2 and R 3 are each independently 1-14 is alkyl; R 4 is -(CH2) n OH, and n is selected from 1, 2, 3, 4, and 5; R' is C 1-12 is alkyl; m is selected from 4, 5, and 6; l is selected from 4, 5, and 6 is.

[0159] In some embodiments, m and l are each 5 and n is 2, 3, or 4.

[0160] In some embodiments, R' is C 2-5 alkyl, and R aγ is C 2-6 alkyl, and R 2 and R 3 are C 6-10 It is alkyl.

[0161] In some embodiments, m and l are each 5, n is 2, 3, or 4, and R′ is C 2-5 alkyl, and R aγ is C 2-6 alkyl, and R 2 and R 3 are C 6-10 It is alkyl.

[0162] In some embodiments, the ionizable lipid is a compound of formula (II-g): [ka] or an N-oxide or salt thereof, wherein R aγ is C 2-6 is alkyl; R' is C 2-5 is alkyl; R 4 is -(CH2) n OH and [ka] is selected from [ka] indicates the point of attachment, n is selected from 3, 4, and 5; R10 is -NH(C 1-6 alkyl); n2 is selected from 1, 2, and 3 is.

[0163] In some embodiments, the ionizable lipid is a compound of formula (II-h): [ka] or an N-oxide or salt thereof, wherein R aγ and R bγ are each independently 2-6 is alkyl; Each R' is independently C 2-5 is alkyl; R 4 is -(CH2) n OH and [ka] is selected from [ka] indicates the point of attachment, n is selected from 3, 4, and 5; R 10 is -NH(C 1-6 alkyl); n2 is selected from 1, 2, and 3 is.

[0164] In some embodiments, R 4 teeth, [ka] and R 10 is NH(CH3) and n2 is 2) is.

[0165] In some embodiments, R 4 is -(CH2)2OH.

[0166] In some embodiments, the ionizable lipid is a compound having formula (III): [ka] or an N-oxide or salt thereof, wherein R1, R2, R3, R4, and R5 are C 5-20 Alkyl, C 5-20 alkenyl, independently selected from -R"MR', -R*YR", -YR", and -R*OR"; each M is independently selected from —C(O)O—, —OC(O)—, —OC(O)O—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)—, an aryl group, and a heteroaryl group; X 1 , X 2 , and X 3 are each independently selected from a bond, —CH—, —(CH)—, —CHR—, —CHY—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)—CH—, —CH—C(O)—, —C(O)O—CH—, —OC(O)—CH—, —CH—C(O)O—, —CH—OC(O)—, —CH(OH)—, —C(S)—, and —CH(SH)—; Each Y is independently C 3-6 It is a carbocyclic ring; Each R* is C 1-12 Alkyl and C 2-12 alkenyl; Each R is C 1-3 Alkyl and C 3-6 independently selected from: Each R' is C 1-12 Alkyl, C 2-12 alkenyl, and H; Each R” is C 3-12 Alkyl and C 3-12 alkenyl; i)X 1 , X2 , and X 3 is not —CH—; and / or ii) at least one of R1, R2, R3, R4, and R5 is -R"MR' is.

[0167] In some embodiments, R1, R2, R3, R4, and R5 are each C 5-20 is alkyl; X 1 is -CH2-; X 2 and X 3 are -C(O)-, respectively.

[0168] In some embodiments, the compound of formula (III) is [ka] is.

[0169] phospholipids Phospholipids, as defined herein, are any lipids containing a phosphate group. Phospholipids are a subset of non-cationic lipids. The LNP core may contain one or more phospholipids, for example, one or more (poly)unsaturated lipids. The phospholipids may be organized into one or more lipid bilayers. Typically, a phospholipid may contain a phospholipid moiety and one or more fatty acid moieties. The phospholipid moiety may be selected from the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid moiety may be selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Non-naturally occurring species, including naturally occurring species with modifications and substitutions including branching, oxidation, cyclization, and alkynes, are also contemplated. For example, phospholipids can be functionalized or crosslinked with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced with triple bonds). Under appropriate reaction conditions, the alkyne group can undergo copper-catalyzed cycloaddition upon exposure to azide. Such reactions can be useful in functionalizing the lipid bilayer of nanoparticle compositions to facilitate membrane permeation or cellular recognition, or in conjugating nanoparticle compositions to useful components, such as targeting or imaging moieties (e.g., dyes).

[0170] In some embodiments, the LNP comprises about 5 mol% to about 15 mol%, about 8 mol% to about 13 mol%, or about 10 mol% to about 12 mol% phospholipid.

[0171] In some embodiments, the phospholipid is a compound of formula (IV): [ka] or a salt thereof (wherein Each R 1are independently H or optionally substituted alkyl; or optionally two R 1 are joined together with the intervening atoms to form an optionally substituted monocyclic cycloalkyl or an optionally substituted monocyclic heterocyclyl; or optionally, three R 1 are joined together with the intervening atoms to form an optionally substituted bicyclic cycloalkyl or an optionally substituted bicyclic heterocyclyl; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is a group having the formula: [ka] is of; L 2 Each instance of is independently a bond or an optionally substituted C 1-6 alkylene and optionally substituted C 1-6 One methylene unit of alkylene is -O-, -N(R N )-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, or -NR N C(O)N(R N )- is optionally replaced by; R 2 Each instance of may independently be an optionally substituted C 1-30 Alkyl, optionally substituted C 1-30 alkenyl, or optionally substituted C 1-30 alkynyl; optionally R 2 one or more methylene units in each of the groups independently represent an optionally substituted cycloalkylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, -N(R N )-, -O-, -S-, -C(O)-, -C(O)N(R N )-, -NR NC(O)-, -NR N C(O)N(R N )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, -C(O)S-, -SC(O)-, -C(=NR N )-, -C(=NR N )N(R N )-, -NR N C(=NR N )-, -NR N C(=NR N )N(R N )-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NR N C(S)N(R N )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R N )S(O)-, -S(O)N(R N )-, -N(R N )S(O)N(R N )-, -OS(O)N(R N )-, -N(R N )S(O)O-, -S(O)2-, -N(R N )S(O)2-, -S(O)2N(R N )-, -N(R N )S(O)2N(R N )-, -OS(O)2N(R N )-, or -N(R N )S(O)2O- is replaced by; R N each instance of is independently hydrogen, an optionally substituted alkyl, or a nitrogen protecting group; Ring B is an optionally substituted cycloalkyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl; p is 1 or 2; provided that the compound has the formula: [ka] (In the formula, R 2 Each instance of is independently an unsubstituted alkyl, an unsubstituted alkenyl, or an unsubstituted alkynyl. is.

[0172] In some embodiments, the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (4ME 16:0 PC), 1,2-diphytanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (4ME 16:0 PG), 1,2-diphytanoyl-sn-glycero-3-phospho-L-serine (sodium salt) (4ME 16:0 PS), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin.

[0173] In some embodiments, the phospholipid is DSPC, DOPE, or a combination thereof. In some embodiments, the phospholipid is DSPC. In some embodiments, the phospholipid is DOPE. In some embodiments, the phospholipid is 4ME 16:0 PE, 4ME 16:0 PC, 4ME 16:0 PG, 4ME 16:0 PS, or a combination thereof.

[0174] In some embodiments, the phospholipid is N-lauroyl-D-erythro-sphinganylphosphorylcholine.

[0175] alternative lipids In certain embodiments, alternative lipids are used in place of phospholipids. Non-limiting examples of such alternative lipids include: [ka] [ka]

[0176] structured lipids The LNP core may include one or more structural lipids. The incorporation of structural lipids into lipid nanoparticles may help reduce aggregation of other lipids in the particle. The structural lipid may be selected from the group including, but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a sterol. As defined herein, "sterol" is a subgroup of steroids consisting of steroid alcohols.

[0177] In some embodiments, the structured lipid is alpha-tocopherol. In certain embodiments, the structured lipid is a steroid. In certain embodiments, the structured lipid is cholesterol. In certain embodiments, the structured lipid is a cholesterol analog. In some embodiments, the structured lipid is β-sitosterol. In certain embodiments, the structured lipid is cholesteryl hemisuccinate. Cholesteryl hemisuccinate has the following structure: [ka] It has.

[0178] In some embodiments, the LNPs comprise about 20 mol% to about 60 mol%, about 30 mol% to about 50 mol%, or about 35 mol% to about 40 mol% structured lipids. In some embodiments, the LNPs comprise about 35 mol% structured lipids. In some embodiments, the LNPs comprise about 40 mol% structured lipids.

[0179] PEG and PEG-modified lipids The LNP core may include one or more molecules containing polyethylene glycol (PEG), such as a PEG-modified lipid. Such species may alternatively be referred to as a PEGylated lipid. A PEG-lipid is a lipid modified with polyethylene glycol. The PEG-lipid may be selected from the non-limiting group including PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified diacylglycerol, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof.

[0180] In some embodiments, the PEG lipid is a compound of formula (V): [ka] or a salt thereof (wherein R 3 -OR O and; R Ois hydrogen, an optionally substituted alkyl, or an oxygen protecting group; r is an integer between 1 and 100, inclusive; L 1 is an arbitrarily substituted C 1-10 alkylene and optionally substituted C 1-10 At least one methylene of the alkylene is independently an optionally substituted cycloalkylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, —O—, —N(R N )-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, or -NR N C(O)N(R N )- has been replaced by; D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is a group having the formula: [ka] is of; L 2 Each instance of is independently a bond or an optionally substituted C 1-6 alkylene and optionally substituted C 1-6 One methylene unit of alkylene is -O-, -N(R N )-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, or -NR N C(O)N(R N )- is optionally replaced by; R 2Each instance of may independently be an optionally substituted C 1-30 Alkyl, optionally substituted C 1-30 alkenyl, or optionally substituted C 1-30 alkynyl; optionally R 2 one or more methylene units in each of the groups independently represent an optionally substituted cycloalkylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, -N(R N )-, -O-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -NR N C(O)N(R N )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, -C(O)S-, -SC(O)-, -C(=NR N )-, -C(=NR N )N(R N )-, -NR N C(=NR N )-, -NR N C(=NR N )N(R N )-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NR N C(S)N(R N )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R N )S(O)-, -S(O)N(R N )-, -N(R N )S(O)N(R N )-, -OS(O)N(R N )-, -N(R N )S(O)O-, -S(O)2-, -N(R N )S(O)2-, -S(O)2N(R N )-, -N(R N )S(O)2N(R N )-, -OS(O)2N(R N )-, or -N(R N)S(O)2O- is replaced by; R N each instance of is independently hydrogen, an optionally substituted alkyl, or a nitrogen protecting group; Ring B is an optionally substituted cycloalkyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl; p is 1 or 2) is.

[0181] In some embodiments, the PEG lipid is PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE. In some embodiments, the PEG lipid is PEG-DMG. In some embodiments, the PEG lipid is PEG-DMG 2k. In some embodiments, the PEG lipid has the structure: [ka] It has.

[0182] DMG-PEG 2k has the following structure: [ka] It has.

[0183] In some embodiments, the PEG-modified lipid is a modified form of PEG-DMG.

[0184] In one embodiment, the PEG-lipid may be a PEGylated lipid described in International Publication No. WO2012099755, the contents of which are incorporated herein by reference in their entirety.

[0185] Any of the PEG-lipids described herein can be modified to include one or more hydroxyl groups on the PEG chain (OH-PEG-lipid) or one or more hydroxyl groups on the lipid (PEG-lipid-OH). In some embodiments, the PEG-lipid is an OH-PEG-lipid. In some embodiments, the OH-PEG-lipid includes a hydroxyl group at the end of the PEG chain. In some embodiments, the PEG-lipids described herein can be modified to include one or more alkyl groups on the PEG chain (alkyl-PEG-lipid). In some embodiments, the alkyl-PEG-lipid is a methoxy-PEG-lipid.

[0186] In some embodiments, the LNP comprises about 0.1 mol% to about 5.0 mol%, about 0.5 mol% to about 5.0 mol%, about 1.0 mol% to about 5.0 mol%, about 1.0 mol% to about 2.5 mol%, about 0.5 mol% to about 2.0 mol%, or about 1.0 mol% to about 1.5 mol% PEG-lipid. In some embodiments, the LNP comprises about 1.5 mol% or about 3.0 mol% PEG-lipid.

[0187] Some of the LNPs provided herein contain zero or low levels of PEG-lipids. Some LNPs contain less than 0.5 mol% PEG-lipids.

[0188] In some embodiments, PEG is used as a stabilizer. In some embodiments, the PEG stabilizer is a PEG-lipid. In some embodiments, the LNPs contain less than 0.5 mol% of a PEG stabilizer.

[0189] Payload molecules The lipid nanoparticle compositions of the present disclosure can be used to deliver a variety of different payloads to cells. The payload can be a therapeutic or prophylactic agent capable of mediating a therapeutic or prophylactic effect in such cells (e.g., directly or via a bystander effect). Typically, the payload delivered by the composition is a nucleic acid, although non-nucleic acid agents, such as small molecules, chemotherapeutic agents, peptides, polypeptides, and other biological molecules, are also encompassed by the present disclosure. Nucleic acids that can be delivered include DNA-based molecules (i.e., containing deoxyribonucleotides) and RNA-based molecules (i.e., containing ribonucleotides). Furthermore, the nucleic acid can be a naturally occurring form of the molecule or a chemically modified form of the molecule (e.g., containing one or more modified nucleotides).

[0190] In one embodiment, the therapeutic or prophylactic agent is an agent that enhances (i.e., increases, stimulates, upregulates) protein expression. Non-limiting examples of types of therapeutic or prophylactic agents that can be used to enhance protein expression include RNA, mRNA, dsRNA, CRISPR / Cas9 technology, ssDNA, and DNA (e.g., expression vectors).

[0191] In one embodiment, the therapeutic or prophylactic agent is an agent that reduces (i.e., decreases, inhibits, downregulates) protein expression. Non-limiting examples of types of therapeutic or prophylactic agents that can be used to reduce protein expression include mRNA incorporating microRNA binding site(s) (miR binding sites), microRNA (miRNA), antagomir, small (short) interfering RNA (siRNA) (including shortmers and Dicer-substrate RNA), RNA interference (RNAi) molecules, antisense RNA, ribozymes, small hairpin RNA (shRNA), locked nucleic acid (LNA), and CRISPR / Cas9 technology.

[0192] In one embodiment, the therapeutic or prophylactic agent is a peptide therapeutic. In one embodiment, the therapeutic or prophylactic agent is a polypeptide therapeutic. In some embodiments, the therapeutic or prophylactic agent comprises a secreted protein; a membrane-bound protein; or an intracellular protein, or an mRNA encoding a peptide, polypeptide, or biologically active fragment thereof.

[0193] In some embodiments, at least about 50%, at least about 75%, at least about 90%, or at least about 95% of the payload is encapsulated within the lipid nanoparticle, or about 50% to about 99%, about 65% to about 99%, about 75% to about 95%, or about 80% to about 95% of the payload is encapsulated within the lipid nanoparticle.

[0194] cell LNPs can be used to deliver payload molecules to a population of cells. In some embodiments, the LNPs are contacted with the population of cells. In some embodiments, about 10% or more, 15% or more, 20% or more, or 30% or more of the cell population accumulate the LNPs when the LNPs are contacted with the cell population. In some embodiments, about 1% to about 75%, about 5% to about 50%, about 10% to about 40%, or about 15% to about 25% of the cell population accumulate the LNPs when the LNPs are contacted with the cell population.

[0195] In some embodiments, about 5% or more, about 10% or more, or about 20% or more of the cells in a population of cells express the payload when the LNP is contacted with the population of cells, hi some embodiments, about 0.5% to about 50%, about 1% to about 40%, about 3% to about 20%, or about 5% to about 15% of the cells in a population of cells express the payload when the LNP is contacted with the population of cells.

[0196] In some embodiments, the cell population is an epithelial cell population. In some embodiments, the cell population is a respiratory epithelial cell population. In some embodiments, the respiratory epithelial cell population is a pulmonary cell population. In some embodiments, the respiratory epithelial cell population is a nasal cell population. In some embodiments, the respiratory epithelial cell population is an alveolar epithelial cell population. In some embodiments, the respiratory epithelial cell population is a bronchial epithelial cell population. In some embodiments, the respiratory epithelial cell population is an HBE population. In some embodiments, the cell population is a HeLa population.

[0197] Pharmaceutical Compositions and Formulations The present disclosure provides pharmaceutical compositions and formulations comprising any of the LNPs described herein.

[0198] The pharmaceutical composition or formulation may optionally contain one or more additional active substances, e.g., therapeutically and / or prophylactically active substances. The pharmaceutical composition or formulation of the present disclosure may be sterile and / or pyrogen-free. General considerations in the formulation and / or manufacture of pharmaceutical agents are described, for example, in Remington: The Science and Practice of Pharmacy 21 st ed., Lippincott Williams & Wilkins, 2005, incorporated herein by reference in its entirety. In some embodiments, the compositions are administered to a human, human patient, or subject. For purposes of this disclosure, the phrase "active ingredient" generally refers to nanoparticles containing a polynucleotide or polypeptide payload delivered as described herein.

[0199] The formulations and pharmaceutical compositions described herein may be prepared by any method known or later developed in the art of pharmacology. Typically, such preparative methods include bringing the nanoparticles into association with an excipient and / or one or more other accompanying ingredients, and then, as necessary or desired, dividing, shaping, and / or packaging the product into the desired single or multiple dose units.

[0200] Pharmaceutical compositions or formulations according to the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a "unit dose" refers to a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient. The amount of the active ingredient is typically equal to the dosage of the active ingredient that would be administered to a subject, and / or a convenient fraction of such a dosage, such as, for example, one-half or one-third of such a dosage.

[0201] The relative amounts of the active ingredient, pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition according to the present disclosure may vary depending on the identity, size, and / or condition of the subject being treated and also depending on the route by which the composition is administered.

[0202] Although the description of pharmaceutical compositions and formulations provided herein is primarily directed to pharmaceutical compositions and formulations suitable for administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other animal, e.g., non-human animals, e.g., non-human mammals.

[0203] As used herein, pharmaceutically acceptable additives include, but are not limited to, any solvent, dispersion medium, or other liquid vehicle, dispersing or suspending aid, diluent, granulating and / or dispersing agent, surfactant, isotonicity agent, thickening or emulsifying agent, preservative, binder, lubricant or oil, color, sweetening or flavoring agent, stabilizer, antioxidant, antibacterial or antifungal agent, osmolality adjusting agent, pH adjusting agent, buffer, chelating agent, cryoprotectant, and / or bulking agent, that are suitable for the particular dosage form desired. Various additives for formulating pharmaceutical compositions and techniques for preparing compositions are known in the art (see, The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006, the entire contents of which are incorporated herein by reference)).

[0204] Exemplary diluents include, but are not limited to, calcium or sodium carbonate, calcium phosphate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, and the like, and / or combinations thereof.

[0205] Exemplary granulating and / or dispersing agents include, but are not limited to, starch, pregelatinized starch, or microcrystalline starch, alginic acid, guar gum, agar, poly(vinyl-pyrrolidone), (povidone), cross-linked poly(vinyl-pyrrolidone) (crospovidone), cellulose, methylcellulose, carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscarmellose), magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, and the like, and / or combinations thereof.

[0206] Exemplary surfactants and / or emulsifiers include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monooleate [TWEEN® 80], sorbitan monopalmitate [SPAN® 40], glyceryl monooleate, polyoxyethylene esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [BRIJ® 30]), PLUORINC® F68, POLOXAMER® 188, and the like, and / or combinations thereof.

[0207] Exemplary binders include, but are not limited to, starch, gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol), amino acids (e.g., glycine), natural and synthetic gums (e.g., acacia, sodium alginate), ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, and the like, and combinations thereof.

[0208] Oxidation is a potential degradation pathway for mRNA, especially for liquid mRNA formulations.To prevent oxidation, antioxidants can be added to the formulation.Exemplary antioxidants include, but are not limited to, alpha-tocopherol, ascorbic acid, ascorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, m-cresol, methionine, butylated hydroxytoluene, monothioglycerol, sodium or potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, etc., and combinations thereof.

[0209] Exemplary chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, edetate disodium, fumaric acid, malic acid, phosphoric acid, edetate sodium, tartaric acid, edetate trisodium, and the like, and combinations thereof.

[0210] Exemplary antibacterial or antifungal agents include, but are not limited to, benzalkonium chloride, benzethonium chloride, methylparaben, ethylparaben, propylparaben, butylparaben, benzoic acid, hydroxybenzoic acid, potassium or sodium benzoate, potassium or sodium sorbate, sodium propionate, sorbic acid, and the like, and combinations thereof.

[0211] Exemplary preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, ascorbic acid, butylated hydroxyanisole, ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), and the like, and combinations thereof.

[0212] In some embodiments, the pH of the polynucleotide solution is maintained at pH 5 to pH 8 to improve stability. Exemplary buffers for controlling pH can include, but are not limited to, sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium malate, sodium carbonate, and / or combinations thereof.

[0213] Exemplary lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium or magnesium lauryl sulfate, and the like, and combinations thereof.

[0214] The pharmaceutical compositions described herein may contain a cryoprotectant to stabilize the polynucleotides described herein during freezing. Exemplary cryoprotectants include, but are not limited to, mannitol, sucrose, trehalose, lactose, glycerol, dextrose, and the like, and combinations thereof.

[0215] The pharmaceutical compositions described herein may include bulking agents in the lyophilized polynucleotide formulation to produce a "pharmaceutical elegant" cake and stabilize the lyophilized polynucleotide during long-term (e.g., 36 months) storage. Exemplary bulking agents of the present disclosure may include, but are not limited to, sucrose, trehalose, mannitol, glycine, lactose, raffinose, and combinations thereof.

[0216] The composition may be in liquid or solid form. In some embodiments, the composition or formulation is in liquid form. In some embodiments, the composition is suitable for inhalation. The composition may be administered to the pulmonary tract. Aerosolized pharmaceutical formulations may be preferably delivered to the lungs using a number of commercially available devices.

[0217] The compositions can be administered to the respiratory tract by any suitable method, such as intranasal instillation, intratracheal instillation, and intratracheal injection. In some embodiments, the compositions or nanoparticles are administered intranasally, intrabronchially, or by pulmonary administration. For example, the compositions and nanoparticles are administered by a nebulizer or inhaler.

[0218] In some embodiments, the composition is delivered to the lungs by inhalation of an aerosolized pharmaceutical formulation. Inhalation can occur through the subject's nose and / or mouth. Administration can occur by self-administration of the formulation while inhaling, or by administration of the formulation via the ventilator to a ventilated subject. Exemplary devices for delivering formulations to the lungs include, but are not limited to, dry powder inhalers, pressurized metered dose inhalers, nebulizers, and electrohydrodynamic aerosol devices.

[0219] Liquid formulations can be administered to a patient's lungs using a pressurized metered dose inhaler (pMDI). pMDIs typically include at least two components: a canister in which the liquid formulation is held under pressure in combination with one or more propellants, and a container used to hold and actuate the canister. The canister may contain single or multiple doses of the formulation. The canister may include a valve, typically a metering valve, through which the contents of the canister may be expelled. Aerosolized medication is dispensed from a pMDI by applying force to the canister to push it into the container, thereby opening the valve and causing medication particles to be carried out of the valve through the container outlet. Upon expulsion from the canister, the liquid formulation is atomized, forming an aerosol. pMDIs typically use one or more propellants to pressurize the contents of the canister, propelling the liquid formulation out of the container outlet and forming an aerosol. Any suitable propellant may be utilized. The propellant may take a variety of forms. For example, the propellant may be a compressed gas or a liquefied gas.

[0220] Liquid formulations can also be administered using a nebulizer. A nebulizer is a liquid aerosol generator that converts a liquid formulation into a mist or cloud of small droplets, preferably with a diameter less than 5 microns mass median aerodynamic diameter, that can be inhaled into the lower respiratory tract. This process is called nebulization. The droplets carry one or more active agents to the nose, upper respiratory tract, or deep lung when the aerosol cloud is inhaled. Any type of nebulizer, including but not limited to pneumatic (jet) nebulizers and electromechanical nebulizers, can be used to administer the formulation to a patient. Compression (jet) nebulizers use a pressurized gas supply as the driving force for nebulization of the liquid formulation. The compressed gas is delivered through a nozzle or jet to generate low pressure that entrains the surrounding liquid formulation and shears it into a thin film or filament. The film or filament is unstable and breaks down into small droplets, which are carried into the inhaled air by the compressed gas flow. A baffle inserted into the droplet plume eliminates larger droplets and returns them to the bulk liquid reservoir. Electromechanical nebulizers use electrically generated mechanical force to atomize liquid formulations. For example, electromechanical driving force can be applied by vibrating the liquid formulation at ultrasonic frequencies or by forcing bulk liquid through small holes in a thin membrane. The force generates a thin liquid film or filament flow, which breaks into small droplets to form a slow-moving aerosol stream that can be entrained in the inspiratory airflow. Liquid formulations can also be administered using electrohydrodynamic (EHD) aerosol devices. EHD aerosol devices use electrical energy to aerosolize liquid drug solutions or suspensions.

[0221] Dry powder inhalers (DPIs) typically use a mechanism such as a burst of gas to generate a cloud of dry powder in a container that can then be inhaled by the subject. In DPIs, the administered dose is stored in the form of a non-pressurized dry powder, and upon actuation of the inhaler, the powder particles are inhaled by the subject. In some cases, a compressed gas (i.e., a propellant) can be used to dispense the powder, similar to pressurized metered dose inhalers (pMDIs). In some cases, DPIs can be breath-actuated, meaning that the aerosol is generated in precise response to inspiration. Typically, dry powder inhalers administer doses of less than tens of milligrams per inhalation to avoid inducing coughing. Examples of DPIs include the Turbohaler® inhaler (Astrazeneca, Wilmington, Del.), Clickhaler® inhaler (Innovata, Ruddington, Nottingham, UKL), Diskus® inhaler (Glaxo, Greenford, Middlesex, UK), EasyHaler® (Orion, Expoo, FI), Exubera® inhaler (Pfizer, New York, NY), Qdose® inhaler (Microdose, Monmouth Junction, NJ), and Spiros® inhaler (Dura, San Diego, Calif.).

[0222] Pharmaceutical compositions, in some embodiments, are administered in an amount effective to produce a desired biological effect, e.g., a therapeutic or prophylactic effect, as measured by the alleviation of one or more symptoms, e.g., by virtue of expression of a normal gene product to replenish or replace a defective protein or to reduce expression of an undesirable protein. Formulations can be administered in an amount effective to deliver a payload, e.g., to deliver LNPs to the apical membrane of respiratory and non-respiratory epithelial cells.

[0223] How to use The lipid amine compounds can be used to prepare lipid nanoparticle compositions that can be loaded with a payload and administered to cells, e.g., cells in a patient, for the treatment of disease. Accordingly, provided herein are methods of delivering a payload to a cell, e.g., by contacting the cell with a lipid nanoparticle composition disclosed herein.

[0224] In some embodiments, the cells are epithelial cells. In some embodiments, the cells are airway epithelial cells. In some embodiments, the cells are respiratory epithelial cells. The respiratory epithelial cells can be, for example, lung cells, nasal cells, alveolar epithelial cells, or bronchial epithelial cells. In some embodiments, the cells are HBE cells or HeLa populations. In some embodiments, the cells are in a patient.

[0225] In some embodiments, the payload is a polynucleotide or polypeptide. Polynucleotides include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA including LNA with a β-D-ribonucleotide structure, α-LNA (a diastereomer of LNA) with an α-L-ribonucleotide structure, 2′-amino-LNA with a 2′-amino functionalization, and 2′-amino-α-LNA with a 2′-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), or a hybrid or combination thereof. In some embodiments, the polynucleotide is mRNA, rRNA, or tRNA. In some embodiments, the polynucleotide is mRNA.

[0226] The lipid nanoparticle composition can be administered to a patient by intranasal, intrabronchial, or pulmonary administration, for example, the composition and nanoparticles can be administered by a nebulizer or inhaler.

[0227] As will be appreciated by those skilled in the art, the lipid amines disclosed herein have additional uses. For example, the lipid amines can be used to treat inflammatory diseases. The lipid amines can also be used as antibacterial agents.

[0228] Kits and Devices The present disclosure provides a variety of kits for conveniently and / or effectively using the claimed nanoparticles of the present disclosure. Typically, the kits will contain components in amounts and / or numbers sufficient to enable a user to perform multiple treatments of a subject(s) and / or to perform multiple experiments.

[0229] In one aspect, the present disclosure provides a kit comprising the nanoparticles of the present disclosure.

[0230] The kit may further include packaging and instructions and / or a delivery agent for forming the formulation. The delivery agent may include saline, a buffer, a lipidoid, or any delivery agent disclosed herein. In one embodiment, such a kit further includes an administration device, such as a nebulizer or inhaler.

[0231] Process for preparing LNPs The present disclosure also provides a process for preparing a lipid nanoparticle composition, comprising contacting a lipid nanoparticle core disclosed herein with a lipid amine disclosed herein.

[0232] In some embodiments, the process for preparing a lipid nanoparticle composition comprises: (a) a nucleic acid payload, (1) ionizable lipids, (2) phospholipids, (3) structural lipids, and (4) optionally, PEG-lipid to form a loaded lipid nanoparticle (fLNP) core; and (c) contacting the fLNP core with a lipid amine Includes:

[0233] In some embodiments, the process for preparing nanoparticles comprises: (a) (1) ionizable lipids, (2) phospholipids, (3) structural lipids, and (4) optionally, PEG-lipid mixing a lipid solution containing the (b) contacting the eLNP core with a nucleic acid payload to form an fLNP; and (c) contacting the fLNP core with a lipid amine Includes:

[0234] In some embodiments, the mixture further comprises an aqueous buffer solution. In some embodiments, the aqueous buffer solution has a pH of about 3.5 to about 4.5. In further embodiments, the aqueous buffer solution has a pH of about 4. In some embodiments, the aqueous buffer solution has a pH of about 4.6 to about 6.5. In some embodiments, the aqueous buffer solution has a pH of about 5.

[0235] In some embodiments, the aqueous buffer solution may comprise an acetate buffer, a citrate buffer, a phosphate buffer, or a Tris buffer. In some embodiments, the aqueous buffer solution comprises an acetate buffer or a citrate buffer. In further embodiments, the aqueous buffer solution is an acetate buffer, e.g., a sodium acetate buffer.

[0236] In some embodiments, the aqueous buffer solution has a buffer concentration greater than about 30 mM. In some embodiments, the aqueous buffer solution has a buffer concentration greater than about 40 mM. In some embodiments, the aqueous buffer solution has a buffer concentration of about 30 mM to about 100 mM. In some embodiments, the aqueous buffer solution has a buffer concentration of about 40 mM to about 75 mM. In further embodiments, the aqueous buffer solution has a buffer concentration of about 33 mM, about 37.5 mM, or about 45 mM.

[0237] In some embodiments, the aqueous buffer solution can have an ionic strength of about 15 mM or less, about 10 mM or less, or about 5 mM or less, hi some embodiments, the aqueous buffer solution has an ionic strength of about 0.1 mM to about 15 mM, about 0.1 mM to about 10 mM, or about 0.1 mM to about 5 mM.

[0238] In some embodiments, the lipid solution has a lipid concentration of about 5 to about 100 mg / mL, about 15 to about 35 mg / mL, about 20 to about 30 mg / mL, or about 24 mg / mL.

[0239] The lipid solution may further comprise an organic solvent, such as an alcohol, e.g., ethanol. The organic solvent may be present in an amount of about 1% to about 50%, about 5% to about 40%, or about 10% to about 33% by volume. In further embodiments, the solvent is 100% ethanol or greater than 95% ethanol by volume.

[0240] In some embodiments, the lipid solution contains about 30 mol% to about 60 mol%, about 35 mol% to about 55 mol%, or about 40 mol% to about 50 mol% ionizable lipids based on total lipids. In some embodiments, the lipid solution contains about 5 mol% to about 15 mol%, about 8 mol% to about 13 mol%, or about 10 mol% to about 12 mol% phospholipids based on total lipids. In some embodiments, the lipid solution contains about 30 mol% to about 50 mol%, about 35 mol% to about 45 mol%, or about 37 mol% to about 42 mol% structured lipids based on total lipids. In some embodiments, the lipid solution contains about 0.1 mol% to about 2 mol%, about 0.1 mol% to about 1 mol%, or about 0.25 mol% to about 0.75 mol% PEG-lipids based on total lipids.

[0241] In some embodiments, the lipid solution contains about 40 mol% to about 50 mol% ionizable lipids; about 10 mol% to about 12 mol% phospholipids; about 37 mol% to about 42 mol% structural lipids; and Approximately 0.25 mol% to approximately 0.75 mol% PEG-lipid Includes:

[0242] In some embodiments, the lipid solution contains approximately 49 mol% ionizable lipids; about 11 mol% to about 12 mol% phospholipids; Approximately 39 mol% structural lipids; and Approximately 0.5 mol% PEG-lipid Includes:

[0243] Mixing the lipid solution and buffer solution results in precipitation of lipid nanoparticles and preparation of the empty lipid nanoparticle compositions described herein. Precipitation can be performed, for example, by dropwise ethanol precipitation using a high-energy mixer (e.g., a T-junction, a confined impinging jet, a microfluidic mixer, or a vortex mixer) to controllably introduce lipids (in ethanol) into a suitable antisolvent (i.e., water) and induce liquid supersaturation and spontaneous precipitation within the lipid particles. In some embodiments, mixing is performed in a multi-port vortex mixer. In some embodiments, mixing is performed in a microfluidic mixer, as described in WO 2014 / 172045. The mixing step can be performed at ambient temperature or at a temperature, for example, below about 30°C, below about 28°C, below about 26°C, below about 25°C, below about 24°C, below about 22°C, or below about 20°C.

[0244] In some embodiments, mixing involves nanoprecipitation. Nanoprecipitation is a unit operation in which nanoparticles self-assemble from their individual lipid components through dynamic mixing and subsequent maturation and continuous dilution. This unit operation involves three individual steps: mixing of aqueous and organic inputs, maturation of nanoparticles, and dilution after a controlled residence time. Due to the continuous nature of these steps, they are considered one unit operation. The unit operation involves the continuous in-line combination of three liquid streams with one in-line maturation step: mixing of aqueous buffer with lipid stock solution, maturation via a controlled residence time, and dilution of nanoparticles. The nanoprecipitation itself occurs in a scalable mixer designed to allow continuous, high-energy combination of an aqueous solution with a lipid stock solution dissolved in ethanol. Both the aqueous solution and the lipid stock solution flow continuously through the mixing hardware simultaneously throughout the operation. The ethanol content, which keeps the lipids dissolved, is rapidly reduced, causing all the lipids to precipitate out of each other. Thus, the particles self-assemble in the mixing chamber. One of the objectives of the unit operation is to exchange the solution into a sufficiently aqueous buffer that is free of ethanol and to reach a target concentration of nanoparticles, which can be achieved first by reaching a target treatment concentration once the ethanol has been completely removed, then by using diafiltration, and then (if necessary) a final concentration step.

[0245] In some embodiments, the lipid nanoparticle core contacted with the lipid amine comprises a PEG-lipid. In some embodiments, the lipid nanoparticle core contacted with the lipid amine is substantially free of a PEG-lipid. In some embodiments, the PEG-lipid is added to the lipid nanoparticle together with the lipid amine before contacting with the lipid amine or after contacting with the lipid amine. In some embodiments, the PEG-lipid is used as a stabilizer.

[0246] In some embodiments, the contacting in step (b) occurs at a pH of about 3.5 to about 6.5. In some embodiments, the combining occurs at a pH of about 5. In some embodiments, the pH of the empty lipid nanoparticle composition is adjusted to about 4.5 to about 5.5 before combining the empty lipid nanoparticle composition with the payload. In some embodiments, the pH of the empty lipid nanoparticle composition is adjusted to about 5 before combining the empty lipid nanoparticle composition with the payload.

[0247] The nucleic acid payload can be provided as a nucleic acid solution comprising (i) a nucleic acid, e.g., DNA or RNA (e.g., mRNA), and (ii) a buffer capable of maintaining an acidic pH, e.g., a pH of about 3 to about 6, about 4 to about 6, or about 5 to about 6. In some embodiments, the pH of the nucleic acid solution is about 5.

[0248] In some embodiments, the buffer of the nucleic acid solution is an acetate buffer, a citrate buffer, a phosphate buffer, or a Tris buffer. In some embodiments, the buffer is an acetate buffer or a citrate buffer. In further embodiments, the buffer is an acetate buffer, for example, a sodium acetate buffer. The buffer concentration of the nucleic acid solution can be about 5 mM to about 140 mM. In some embodiments, the buffer concentration is about 20 mM to about 100 mM, about 30 mM to about 70 mM, or about 40 mM to about 50 mM. In some embodiments, the buffer concentration is about 42.5 mM.

[0249] The nucleic acid solution may contain nucleic acid at a concentration of about 0.05 to about 5.0 mg / mL, 0.05 to about 2.0 mg / mL, about 0.05 to about 1.0 mg / mL, about 0.1 to about 0.5 mg / mL, or about 0.2 to about 0.3 mg / mL. In some embodiments, the nucleic acid concentration is about 0.25 mg / mL.

[0250] A high-energy mixer (e.g., a T-junction, a confined impinging jet, a microfluidic mixer, a vortex mixer) can be used for the contacting in step (b). In some embodiments, the combining is performed in a multi-port vortex mixer. In some embodiments, the combining is performed in a microfluidic mixer, as described in WO2014 / 172045. The combining step can be carried out at ambient temperature or at a temperature, for example, less than about 30°C, less than about 28°C, less than about 26°C, less than about 25°C, less than about 24°C, less than about 22°C, or less than about 20°C.

[0251] In some embodiments, contacting the LNP core with the lipid amine comprises dissolving the lipid amine in a non-ionic additive. In some embodiments, the non-ionic additive is selected from macrogol 15-hydroxystearate (HS15), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG-2K), PL1, polyoxyethylene sorbitan monooleate [TWEEN® 80], and d-α-tocopherol polyethylene glycol succinate (TPGS). In some embodiments, the non-ionic additive is macrogol 15-hydroxystearate (HS15).

[0252] In some embodiments, the contacting of the lipid nanoparticle core with the lipid amine comprises dissolving the lipid amine in a buffer solution. In some embodiments, the buffer is acetate buffer, citrate buffer, phosphate buffer, or Tris buffer. In some embodiments, the buffer solution is phosphate buffered saline (PBS). In some embodiments, the buffer solution is a Tris-based buffer. In some embodiments, the buffer solution concentration is about 5 mM to about 100 mM, about 5 mM to about 50 mM, about 10 mM to about 30 mM, or about 20 mM.

[0253] In some embodiments, the lipid amine solution has a pH of about 7 to about 8, or about 7.5. In some embodiments, the lipid amine solution has a concentration of about 0.1 to about 50 mg / mL, about 1 to about 30 mg / mL, about 1 to about 10 mg / mL, or about 2 to about 3 mg / mL.

[0254] In some embodiments, the lipid nanoparticle composition undergoes maturation via a controlled residence time after loading and before neutralization, in some embodiments, the residence time is about 5 to about 120 seconds, about 10 to about 90 seconds, about 20 to about 70 seconds, about 30 to about 60 seconds, about 30 seconds, about 45 seconds, or about 60 seconds.

[0255] In some embodiments, the lipid nanoparticle composition undergoes maturation via a controlled residence time after neutralization and before addition of the cationic agent, hi some embodiments, the residence time is about 1 to about 30 seconds, about 2 to about 20 seconds, about 5 to about 15 seconds, about 7 to about 12 seconds, or about 10 seconds.

[0256] In some embodiments, the process for preparing a lipid nanoparticle composition comprises: diluting the composition with a dilution buffer; adjusting the pH of the composition; adding one or more surface acting agents to the composition; filtering the composition; concentrating the composition; exchanging the buffer solution of the composition; adding a cryoprotectant to the composition; and Adding an osmolality modifying agent to the composition The method further comprises one or more additional steps selected from:

[0257] In some embodiments, the process for preparing lipid nanoparticle compositions can further include one, two, three, four, five, six, seven, or all of the steps listed above. Some steps can be repeated. The steps can be, but do not have to be, performed in the order listed. Each step refers to the action on the composition resulting from the previous step. For example, if the process includes the step of adding one or more surface acting agents to the composition, then the surface acting agents are added to the composition resulting from the previous step, and the previous step can be any of the steps listed above.

[0258] In some embodiments, one or more additional steps include adjusting the pH of the composition to a pH of about 7 to about 8. In some embodiments, the pH is adjusted to a pH of about 7.5.

[0259] In some embodiments, one or more additional steps is to add an additional surface acting agent (e.g., in addition to the lipid amine) to the loaded lipid nanoparticles, which may be disposed within and / or on the surface of the nanoparticles (e.g., by coating, adsorption, covalent bonding, or other process). Surface-acting agents may include, but are not limited to, PEG derivatives (e.g., PEG-DMG), lipid amines (e.g., sterol amines and related), anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants, such as dimethyldioctadecylammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., acetylcysteine, artemisia, bromelain, papain, clerodendrum, bromhexine, carbocysteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4, dornase alfa, neltenexin, and erdosteine), and DNase (e.g., rhDNase). In some embodiments, the additional surface-acting agent is a PEG lipid, e.g., PEG-DMG. In some embodiments, an additional surfactant is provided with the lipid amine. In some embodiments, the additional surfactant is present with the lipid amine in a lipid amine solution. In some embodiments, the additional surfactant is a PEG-lipid having a concentration of about 0.1 to about 50 mg / mL, about 1 to about 10 mg / mL, or about 1 to about 3 mg / mL.

[0260] In some embodiments, one or more additional steps include adding an osmolality-modifying agent to the composition. The osmolality-modifying agent can be a salt or a sugar. In some embodiments, the osmolality-modifying agent is a sugar. The sugar can be selected from, but is not limited to, glucose, fructose, galactose, sucrose, lactose, maltose, and dextrose. In some embodiments, the osmolality-modifying agent is a salt. The salt can be an inorganic salt, such as sodium chloride, potassium chloride, calcium chloride, or magnesium chloride. In some embodiments, the inorganic salt is sodium chloride. In some embodiments, the salt is 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt. The salt can be provided as a salt solution having a salt concentration of about 100 to about 500 mM, about 200 to about 400 mM, about 250 to about 350 mM, or about 300 mM. The pH of the salt solution can be about 7 to about 8. The salt solution may further contain a buffer solution such as acetate buffer, citrate buffer, phosphate buffer, or Tris buffer, with a buffer concentration of, for example, about 0.1 mM to about 100 mM, about 0.5 mM to about 90 mM, about 1.0 mM to about 80 mM, about 2 mM to about 70 mM, about 3 mM to about 60 mM, about 4 mM to about 50 mM, about 5 mM to about 40 mM, about 6 mM to about 30 mM, about 7 mM to about 20 mM, about 8 mM to about 15 mM, or about 9 mM to about 12 mM.

[0261] Cryoprotectants can be added to the loaded nanoparticle composition by the addition of an aqueous cryoprotectant solution, which can include an aqueous buffer having a buffer concentration of about 0.1 mM to about 100 mM, about 0.5 mM to about 90 mM, about 1.0 mM to about 80 mM, about 2 mM to about 70 mM, about 3 mM to about 60 mM, about 4 mM to about 50 mM, about 5 mM to about 40 mM, about 6 mM to about 30 mM, about 7 mM to about 20 mM, about 8 mM to about 15 mM, or about 9 mM to about 12 mM. In some embodiments, the buffer concentration is about 1 to about 20 mM, about 1 to about 10 mM, or about 5 mM. In some embodiments, the buffer in the cryoprotectant solution comprises acetate buffer, citrate buffer, phosphate buffer, or Tris buffer. In some embodiments, the buffer is acetate buffer or citrate buffer. In further embodiments, the buffer is an acetate buffer, e.g., sodium acetate. In some embodiments, the pH of the cryoprotectant solution is about 7 to about 8, e.g., about 7.5. In some embodiments, the cryoprotectant solution comprises about 40% to about 90%, about 50% to about 85%, about 60% to about 80%, or about 70% sucrose by weight.

[0262] In some embodiments, the process further includes diluting the composition with a dilution buffer. The dilution buffer can be an aqueous buffer solution having a buffer concentration of about 0.1 mM to about 100 mM, about 0.5 mM to about 90 mM, about 1.0 mM to about 80 mM, about 2 mM to about 70 mM, about 3 mM to about 60 mM, about 4 mM to about 50 mM, about 5 mM to about 40 mM, about 6 mM to about 30 mM, about 7 mM to about 20 mM, about 8 mM to about 15 mM, or about 9 mM to about 12 mM. In some embodiments, the buffer concentration is about 30 mM to about 75 mM, about 30 mM to about 60 mM, or about 30 mM to about 50 mM. In some embodiments, the dilution buffer comprises an acetate buffer, a citrate buffer, a phosphate buffer, or a Tris buffer. In some embodiments, the dilution buffer comprises an acetate buffer or a citrate buffer. In further embodiments, the dilution buffer is an acetate buffer, e.g., sodium acetate. In some embodiments, the pH of the dilution buffer is about 3 to about 7, about 3 to about 6, about 3 to about 5, about 4, about 5, about 5.5, or about 6. In some embodiments, the dilution buffer comprises the same buffer as in the aqueous buffer solution used during combination of the empty lipid nanoparticle composition with the nucleic acid solution.

[0263] In some embodiments, the process further comprises any one or more of the following steps: filtering the composition; concentrating the composition; and exchanging the buffer solution of the composition. The filtering, concentrating, and buffer solution exchange steps can be achieved by tangential flow filtration (TFF). Residual organic solvents can be removed by the filtering step.

[0264] In some embodiments, buffer exchange can change the composition of the loaded lipid nanoparticle composition by increasing or decreasing the buffer concentration, by changing the buffer composition, or by changing the pH.

[0265] In some embodiments, the concentration step may increase the concentration of loaded lipid nanoparticles in the composition.

[0266] In some embodiments, the process for preparing a loaded lipid nanoparticle composition further comprises adjusting the pH of the composition to at least about 7 to about 8 (e.g., about pH 7.5); and adding an osmolality-modifying agent (e.g., an inorganic salt) to the composition.

[0267] In some embodiments, the process for preparing a loaded lipid nanoparticle composition further comprises at least the steps of adjusting the pH of the composition to a pH of about 7 to about 8 (e.g., about pH 7.5); adding a surface acting agent to the composition; and adding an osmolality modifying agent (e.g., an inorganic salt) to the composition.

[0268] In some embodiments, the process for preparing a lipid nanoparticle composition comprises: (i) adjusting the pH of the composition to a pH of about 7 to about 8; (ii) adding one or more surface acting agents to the composition; (iii) concentrating the composition; (iv) adding an inorganic salt to the composition; and (v) diluting the composition It may further include:

[0269] synthesis As will be appreciated by those skilled in the art, the compounds provided herein (including salts and stereoisomers thereof) can be prepared using known organic synthesis techniques and can be synthesized according to any of a number of possible synthetic routes, for example, those provided in the following schemes.

[0270] The reaction for preparing the compounds described herein can be carried out in a suitable solvent that can be easily selected by those skilled in the art of organic synthesis.A suitable solvent can be substantially non-reactive with the starting material (reactant), intermediate, or product at the temperature at which the reaction is carried out (for example, a temperature that can range from the freezing temperature of the solvent to the boiling point of the solvent).A given reaction can be carried out in one solvent or a mixture of multiple solvents.Depending on the specific reaction step, a suitable solvent for a specific reaction step can be selected by those skilled in the art.

[0271] The expressions "ambient temperature" or "room temperature" or "rt", as used herein, are understood in the art and typically refer to a temperature that is about the temperature of the room in which a reaction is carried out, e.g., a temperature of about 20°C to about 30°C, e.g., the reaction temperature.

[0272] Preparation of the compounds described herein can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one skilled in the art. Protecting group chemistry is described, for example, in T.W. Greene and P.G.M.Wuts, Protective Groups in Organic Synthesis, 3 rd Ed., Wiley & Sons, Inc., New York (1999).

[0273] The reaction can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry, or by chromatographic methods such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS), or thin layer chromatography (TLC). Compounds can be purified by those skilled in the art by a variety of methods, including high performance liquid chromatography (HPLC) and normal phase silica chromatography.

[0274] Compounds of formula A1 can be prepared, for example, using the process shown in the following scheme:

[0275] Scheme 1 [ka] Compounds of formula A1 can be prepared via the synthetic route outlined in Scheme 1. The appropriate reaction between cholesteryl chloroformate and an amine can be carried out under suitable conditions to produce a precursor to or compound of formula A1.

[0276] Scheme 2 [ka] Compounds of formula A1 can be prepared via the synthetic route outlined in Scheme 2. A suitable reaction between cholesterol or a cholesterol derivative (e.g., stigmasterol) and 4-nitrophenyl chloroformate can be carried out under suitable conditions (e.g., using triethylamine and 4-dimethylaminopyridine). The product of the reaction can be reacted with an amine under suitable conditions (e.g., using triethylamine) to produce a precursor to or compound of formula A1.

[0277] Scheme 3 [ka] The compound of formula A1 can be prepared via the synthetic route outlined in Scheme 3. A suitable reaction between cholesterol hemisuccinate or a cholesterol derivative hemisuccinate and an activating agent can be carried out under suitable conditions. The product of the reaction can be reacted with an amine under suitable conditions to produce a precursor to or compound of formula A1.

[0278] Scheme 4 [ka] Compounds of formula A1 can be prepared via the synthetic route outlined in Scheme 4. The appropriate reaction between a compound of formula A1, HCHO, NaBH3CN, and AcONa can be carried out under suitable conditions to produce a compound of formula A1.

[0279] Scheme 5 [ka] Precursors to compounds of formula A1 can be prepared via the synthetic route outlined in Scheme 5. A suitable reaction between cholesterol or a cholesterol derivative (e.g., stigmasterol) can be carried out under suitable conditions (e.g., using triethylamine and 4-dimethylaminopyridine). The product of the reaction can be reacted with an amine under suitable conditions (e.g., using triethylamine) to obtain a precursor to compounds of formula A1.

[0280] Scheme 6 [ka] Precursors to compounds of formula A1 can be prepared via the synthetic route outlined in Scheme 5. A suitable reaction between cholesterol or a cholesterol derivative (e.g., stigmasterol) and a boc-hemiester can be carried out under suitable conditions. The product of the reaction can be reacted under suitable conditions to obtain precursors to compounds of formula A1.

[0281] Scheme 7 [ka] Intermediates for the synthesis of compounds of formula A1 can be prepared via the synthetic route outlined in Scheme 7. The appropriate reaction between spermidine or spermine and (E)-N-((tert-butoxycarbonyl)oxy)benzimidoyl cyanide (BOC-ON) can be carried out under suitable conditions to afford intermediates for the synthesis of compounds of formula A1.

[0282] Scheme 8 [ka] Intermediates for the synthesis of compounds of Formula A1 can be prepared via the synthetic route outlined in Scheme 8. The appropriate reaction between intermediate 1 and acrylonitrile can be carried out under suitable conditions to provide intermediate 2. Intermediate 2 can be reacted with benzyl bromide under suitable conditions (e.g., KCO and KI) to provide intermediate 3. Intermediate 3 can be reacted with BocO under suitable conditions (e.g., NaBH and NiCl) to provide intermediate 4. The benzyl group of intermediate 4 can be removed under suitable conditions (e.g., H and Pd / C) to provide intermediate 5.

[0283] Scheme 9 [ka] Intermediates for the synthesis of compounds of Formula A1 can be prepared via the synthetic route outlined in Scheme 9. A suitable reaction between 1,4-butanediol and acrylonitrile can be carried out under suitable conditions (e.g., Triton B, etc.) to provide intermediate 6. Intermediate 6 can be reacted with methanesulfonyl chloride under suitable conditions (e.g., triethylamine, etc.) to provide intermediate 7. Intermediate 7 can be reacted with N-Boc-1,3-diaminopropane under suitable conditions (e.g., KCO and KI, etc.) to provide intermediate 8. Intermediate 8 can be reacted with benzyl bromide under suitable conditions (e.g., KCO and KI, etc.) to provide intermediate 9. Intermediate 9 can be reacted with BocO under suitable conditions (e.g., NaBH and NiCl, etc.) to provide intermediate 10. The benzyl group of intermediate 10 can be removed under suitable conditions (e.g., H and Pd / C, etc.) to provide intermediate 11.

[0284] Scheme 10 [ka] Intermediates for the synthesis of compounds of Formula A1 can be prepared via the synthetic route outlined in Scheme 10. A suitable reaction between N-Boc-1,3-diaminopropane and 2-nitrobenzenesulfonyl chloride under suitable conditions (e.g., triethylamine, etc.) provides intermediate 12. Intermediate 12 can be reacted with tert-butyl N-(6-bromohexyl)carbamate under suitable conditions (e.g., KCO and KI, etc.) to provide intermediate 13. The 2-nitrobenzenesulfonyl group can be removed under suitable conditions (e.g., KCO and thiophenol, etc.) to provide intermediate 14.

[0285] Scheme 11 [ka] Intermediates for the synthesis of compounds of formula A1 can be prepared via the synthetic route outlined in Scheme 11. The appropriate reaction between thiocholesterol and 2,2'-dipyridyl disulfide under suitable conditions provides intermediate 15. Intermediate 15 can be reacted with methyl trifluoromethanesulfonate (methyl triflate) under suitable conditions to provide intermediate 16. Intermediate 16 can be reacted with an appropriate mercaptocarboxylic acid to provide intermediate 17.

[0286] Scheme 12 [ka] Compounds of formula A1 can be prepared via the synthetic route outlined in Scheme 12. A suitable reaction between intermediate 17 and an amine can be carried out under suitable conditions (e.g., using a coupling agent) to produce a precursor to or compound of formula A1.

[0287] Scheme 13 [ka] Intermediates for the synthesis of compounds of formula A1 can be prepared via the synthetic route outlined in Scheme 13. An appropriate reaction between benzylamine and an alkyl halide under suitable conditions (e.g., KCO and KI) provides intermediate 18. The benzyl group of intermediate 18 can be removed under suitable conditions (e.g., H and Pd / C) to provide intermediate 19.

[0288] Scheme 14 [ka] Compounds of formula A1 or precursors for the synthesis of compounds of formula A1 can be prepared via the synthetic route outlined in Scheme 14. The appropriate reaction between cholesterol chloroacetate and an amine under suitable conditions (e.g., using K2CO3 and KI) provides intermediate 20. Intermediate 20 can be reacted with a suitable carboxylic acid under suitable conditions to provide a precursor compound of formula A1 or a compound of formula A1. In some embodiments, R Y teeth, [ka] is.

[0289] Scheme 15 [ka] Precursors to compounds of formula A1 can be prepared via the synthetic route outlined in Scheme 15. A suitable reaction between intermediate 21 and nosyl chloride can be carried out under suitable conditions (e.g., triethylamine, etc.) to provide intermediate 22. Intermediate 22 can be reacted with an alkyl bromide under suitable conditions (e.g., KCO and KI, etc.) to provide intermediate 23. In some embodiments, R Z teeth, [ka] is.

[0290] Scheme 16 [ka] Precursors to compounds of formula A1 can be prepared via the synthetic route outlined in Scheme 16. A suitable reaction between cholesterol and a carboxylic acid can be carried out under suitable conditions in the presence of a coupling agent. The products of said reaction can be reacted under suitable conditions to give compounds of formula A1 or precursors to compounds of formula A1. In some embodiments, R X teeth, [ka] is.

[0291] Scheme 17 [ka] Intermediates for the synthesis of compounds of Formula A1 can be prepared via the synthetic route outlined in Scheme 17. The appropriate reaction between 8-bromooctanoic acid, oxalyl chloride, and N,O-dimethylhydroxylamine can be carried out under suitable conditions (e.g., catalytic DMF in DCM, etc.) to provide intermediate 24. Intermediate 24 can be reacted with methylmagnesium bromide and HCl under suitable conditions to provide intermediate 25. Intermediate 25 can be reacted with ammonium acetate and sodium cyanoborohydride under suitable conditions to provide intermediate 26. Intermediate 26 can be reacted with BOC anhydride under suitable conditions (e.g., triethylamine (TEA), etc.) to provide intermediate 27. Intermediate 27 can be reacted with tert-butyl (4-((2-nitrophenyl)sulfonamido)butan-2-yl)carbamate (prepared similarly to intermediate 12) under suitable conditions (e.g., K2CO3, BnBr, and thiophenol, etc.) to give intermediate 28.

[0292] Scheme 18 [ka] Intermediates for the synthesis of compounds of formula A1 can be prepared via the synthetic route outlined in Scheme 18. Appropriate reaction between intermediates 29 and 30 under suitable conditions (e.g., sodium triacetoxyborohydride in dry MeOH, etc.) provides intermediate 31.

[0293] Scheme 19 [ka] Intermediates for the synthesis of compounds of Formula A1 can be prepared via the synthetic route outlined in Scheme 19. A suitable reaction between intermediate 29 and 2-nitrobenzenesulfonyl chloride can be carried out under suitable conditions (e.g., triethylamine in DCM, etc.) to provide intermediate 32. Intermediate 30A can be reacted with p-toluenesulfonyl chloride under suitable conditions to provide intermediate 33. Intermediate 32 can be reacted with intermediate 33 under suitable conditions (e.g., KCO, BnBr, and thiophenol, etc.) to provide intermediate 31.

[0294] Scheme 20 [ka] Intermediates for the synthesis of compounds of Formula A1 can be prepared via the synthetic route outlined in Scheme 20. A suitable reaction between cholesterol or a cholesterol derivative (e.g., sitosterol) and thionyl chloride under suitable conditions provides intermediate 34. Intermediate 34 can be reacted with sodium thiocyanate under suitable conditions to give intermediate 35. Intermediate 35 can be reacted with lithium aluminum hydride under suitable conditions to give thiocholesterol or a thiocholesterol derivative (e.g., thiositosterol).

[0295] Scheme 21 [ka] Intermediates for the synthesis of compounds of Formula A1 can be prepared via the synthetic route outlined in Scheme 21. The appropriate reaction between thiocholesterol or a thiocholesterol derivative (e.g., thiositosterol) and 2,2'-dipyridyl disulfide under suitable conditions provides intermediate 15A. Intermediate 15A can be reacted with methyl trifluoromethanesulfonate (methyl triflate) under suitable conditions to provide intermediate 16A. Intermediate 16A can be reacted with an appropriate mercaptocarboxylic acid to provide intermediate 17A.

[0296] Scheme 22 [ka] Compounds of formula A1 can be prepared via the synthetic route outlined in Scheme 22. A suitable reaction between intermediate 17A and an amine can be carried out under suitable conditions (e.g., using a coupling agent) to generate a precursor to a compound of formula A1.

[0297] definition In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning indicated below. Additional definitions are set forth throughout this application.

[0298] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The term "a" (or "an"), as well as the terms "one or more" and "at least one," may be used interchangeably herein. In certain embodiments, the term "a" or "an" means "single." In other embodiments, the term "a" or "an" includes "two or more" or "plural."

[0299] The term "and / or" is to be interpreted as a specific disclosure of each of the two specified features or components (with or without the other). Thus, when the term "and / or" is used herein in phrases such as "A and / or B," it is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, when the term "and / or" is used in phrases such as "A, B, and / or C," it is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0300] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0301] Wherever an embodiment is described herein using the word "comprising," other similar embodiments are also provided that are described in terms of "consisting of" and / or "consisting essentially of."

[0302] Units, prefixes, and symbols are indicated in their accepted form in the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. When a range of values ​​is described, it is to be understood that each intervening integer value, and each fraction thereof, between the stated upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range may be independently included or excluded from the range, and each range is encompassed within the disclosure when either limit is included, neither limit is included, or both limits are included. When a value is expressly recited, it is to be understood that values ​​that are approximately the same quantity or amount as the recited value are also within the scope of the disclosure. When a combination is disclosed, each subcombination of elements of that combination is also specifically disclosed and is within the scope of the disclosure. Conversely, when different elements or groups of elements are individually disclosed, combinations of those elements are also disclosed. Where any element of the present disclosure is disclosed as having multiple alternatives, instances of the disclosure in which each alternative is excluded alone or in any combination with the other alternatives are also hereby disclosed; multiple elements of the present disclosure may have such an exclusion, and all combinations of elements with such an exclusion are hereby disclosed.

[0303] The term "about," when used in connection with numerical values ​​throughout the specification and claims, is familiar to those skilled in the art and indicates an acceptable interval of accuracy, such as ±10%.

[0304] As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms. In some embodiments, the animal is a transgenic animal, a genetically engineered animal, or a clone.

[0305] As used herein, the term "compound" is meant to include all stereoisomers and isotopes of the depicted structure. As used herein, the term "stereoisomer" refers to any geometric isomer (e.g., cis and trans isomers), enantiomer, or diastereomer of a compound. The present disclosure encompasses all stereoisomers of the compounds described herein, including stereomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and enantiomeric and stereoisomeric mixtures, e.g., racemates. Enantiomeric and stereoisomeric mixtures of compounds and means for resolving them into their component enantiomers or stereoisomers are well known. "Isotopes" refer to atoms having the same atomic number but different mass numbers due to different numbers of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deuterium. Furthermore, compounds, salts, or complexes of the present disclosure can be prepared in combination with solvents or water molecules to form solvates and hydrates by conventional methods.

[0306] As used herein, the term "contacting" means establishing a physical connection between two or more entities. For example, contacting a cell (e.g., a mammalian cell) with a nanoparticle composition means that the cell and the nanoparticle are caused to share a physical connection. Methods for contacting cells with external entities, both in vivo and ex vivo, are well known in the biological arts. For example, contacting a nanoparticle composition with a cell located within a mammal can be performed by an administration route (e.g., intravenous, intramuscular, intradermal, and subcutaneous) and can involve various amounts of the nanoparticle composition. Moreover, multiple cells can be contacted by the nanoparticle composition.

[0307] A further example of contacting is between a nanoparticle and a lipid amine. Contacting a nanoparticle (e.g., payload-filled or empty) and a lipid amine can mean that the surface of the nanoparticle is placed in physical contact with the lipid amine so that the lipid amine can form an interaction with the nanoparticle. In some embodiments, contacting a nanoparticle and a lipid amine results in the insertion of the lipid amine into the nanoparticle, e.g., starting at the surface of the nanoparticle. In some embodiments, the terms "layering," "coating," and "post-addition" and "addition" can be used to mean "contacting" with respect to contacting a nanoparticle with a lipid amine.

[0308] As used herein, the term "delivering" means providing an entity to a destination. For example, delivering a polynucleotide to a subject can involve administering a nanoparticle composition comprising the polynucleotide to the subject (e.g., by intravenous, intramuscular, intradermal, or subcutaneous routes). Administering a nanoparticle composition to a mammal or mammalian cells can involve contacting one or more cells with the nanoparticle composition.

[0309] As used herein, "delivery agent" refers to any substance that facilitates, at least in part, the in vivo, in vitro, or ex vivo delivery of a polynucleotide to a targeted cell.

[0310] As used herein, the term "diastereomers" means stereoisomers that are not mirror images of each other and are not superimposable with respect to one another.

[0311] As used herein, the term "effective amount" of an agent is that amount sufficient to achieve a beneficial or desired result, e.g., a clinical result; thus, "effective amount" depends on the context in which it is applied. For example, in the context of administering an agent to treat a protein deficiency, an effective amount of the agent is the amount of mRNA that expresses the protein sufficient to ameliorate, reduce, eliminate, or prevent signs and symptoms associated with the protein deficiency, e.g., when compared to the severity of the symptoms observed without administration of the agent. The term "effective amount" can be used interchangeably with "effective dose," "therapeutically effective amount," or "therapeutically effective dose."

[0312] As used herein, the term "enantiomer" means each individual optically active form of a compound of the present disclosure having an optical purity or enantiomeric excess (as determined by standard methods in the art) of at least 80% (i.e., at least 90% of one enantiomer and at most 10% of the other enantiomer), at least 90%, or at least 98%.

[0313] As used herein, the term "enclose" means to surround, enclose, enclose, or package.

[0314] As used herein, "encapsulation efficiency" refers to the amount of polynucleotide that becomes part of a nanoparticle composition relative to the initial total amount of polynucleotide used in preparing the nanoparticle composition. For example, if 97 mg of polynucleotide is encapsulated in the nanoparticle composition out of a total of 100 mg of polynucleotide initially provided in the composition, the encapsulation efficiency can be given as 97%. As used herein, "encapsulation" can refer to complete, substantial, or partial surrounding, enclosing, surrounding, or packaging.

[0315] As used herein, "epithelial cells" include cells derived from epithelia. Exemplary epithelial cells are respiratory epithelial cells, nasal epithelial cells, alveolar epithelial cells, pulmonary epithelial cells, or bronchial epithelial cells. In some embodiments, the epithelial cells are human bronchial epithelial (HBE) cells. In some embodiments, the epithelial cells are in vitro cells. In some embodiments, the epithelial cells are in vivo cells.

[0316] As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an mRNA template from a DNA sequence (e.g., by transcription); (2) processing of the mRNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end processing); (3) translation of the mRNA into a polypeptide or protein; and (4) post-translational modification of the polypeptide or protein.

[0317] As used herein, the term "ex vivo" refers to an event that occurs outside an organism (e.g., an animal, plant, or microorganism, or cells or tissues thereof). An ex vivo event can occur in an environment that is minimally modified from the natural (e.g., in vivo) environment.

[0318] As used herein, the term "helper lipid" refers to a compound or molecule that contains a lipidic portion (for insertion into a lipid layer, e.g., a lipid bilayer) and a polar portion (for interaction with the physiological solution at the surface of the lipid layer). Typically, the helper lipid is a phospholipid. The function of the helper lipid is to "complement" the amino lipid, increasing the fusogenicity of the bilayer and / or, for example, helping to facilitate endosomal escape of nucleic acids delivered to cells. Helper lipids are also considered essential structural components for the surface of LNPs.

[0319] As used herein, the term "in vitro" refers to events that occur not within a living organism (e.g., an animal, plant, or microorganism) but in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc.

[0320] As used herein, the term "in vivo" refers to an event that takes place within an organism (e.g., an animal, plant, or microorganism, or cells or tissues thereof).

[0321] The term "ionizable amino lipid" includes, for example, those lipids described throughout this specification that have one, two, three, or more fatty acid or fatty alkyl chains and at least one pH-adjustable amino head group (e.g., alkylamino or dialkylamino head group). Ionizable amino lipids are typically protonated (i.e., positively charged) at a pH below the pKa of the amino head group, and are substantially uncharged at a pH above the pKa. Such ionizable amino lipids include, but are not limited to, DLin-MC3-DMA (MC3) and (13Z,165Z)-N,N-dimethyl-3-nonidocosa-13-16-dien-1-amine (L608).

[0322] As used herein, the term "isomer" refers to any tautomer, stereoisomer, enantiomer, or diastereomer of any compound of the present disclosure. It is recognized that the compounds of the present disclosure may possess one or more chiral centers and / or double bonds and therefore exist as stereoisomers, e.g., double bond isomers (i.e., geometric E / Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis / trans isomers). In accordance with the present disclosure, the chemical structures depicted herein, and therefore the compounds of the present disclosure, encompass all of the corresponding stereoisomers, i.e., stereomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure), as well as both enantiomeric and stereoisomeric mixtures, e.g., racemates. Enantiomeric and stereoisomeric mixtures of the compounds of the present disclosure can typically be resolved into their component enantiomers or stereoisomers by well-known methods, such as chiral-phase gas chromatography, chiral-phase high-performance liquid chromatography, crystallizing the compounds as chiral salt complexes, or crystallizing the compounds in chiral solvents. Enantiomers and stereoisomers can also be obtained from stereomerically or enantiomerically pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.

[0323] As used herein, a "lipid nanoparticle core" refers to a lipid nanoparticle to which additional layers of components, such as lipid amines and / or PEG-lipids or other lipids, can be added after addition. In some embodiments, the lipid nanoparticle core comprises (i) an ionizable lipid, (ii) a phospholipid, (iii) a structural lipid, and (iv) optionally a PEG-lipid. In further embodiments, the lipid nanoparticle core comprises (i) an ionizable lipid, (ii) a phospholipid, (iii) a structural lipid, and (iv) a PEG-lipid. In some embodiments, the lipid nanoparticle core can contain a payload.

[0324] As used herein, "linker" or "linker structure" refers to a group of atoms, e.g., 10 to 1,000 atoms, and may include atoms or groups, such as, but not limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. The linker may be attached at one end to a nucleoside or nucleotide modified on the nucleobase or sugar moiety and at the second end to a payload, e.g., a detectable or therapeutic agent. The linker may be of sufficient length so as not to interfere with incorporation into a nucleic acid sequence. Linkers may be used for any useful purpose, such as to form polynucleotide multimers (e.g., via linkage of two or more chimeric polynucleotide molecules or IVT polynucleotides) or polynucleotide conjugates, and to administer payloads as described herein. Examples of linkers that can be incorporated into the chemical group include, but are not limited to, alkyl, alkenyl, alkynyl, amide, amino, ether, thioether, ester, alkylene, heteroalkylene, aryl, or heterocyclyl, each of which can be optionally substituted as described herein. Examples of linkers include, but are not limited to, unsaturated alkanes, polyethylene glycols (e.g., ethylene or propylene glycol monomer units, e.g., diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol, or tetraethylene glycol), and dextran polymers and their derivatives. Other examples include, but are not limited to, cleavable sites within the linker that can be cleaved using reducing agents or photolysis, such as disulfide bonds (-SS-) or azo bonds (-N=N-). Non-limiting examples of selectively cleavable bonds include amide bonds, which can be cleaved, for example, by the use of tris(2-carboxyethyl)phosphine (TCEP) or other reducing agents, and / or photolysis, and ester bonds, which can be cleaved, for example, by acidic or basic hydrolysis.

[0325] As used herein, "pulmonary cells" include cells derived from the lung. Pulmonary cells can be, for example, pulmonary epithelial cells, airway basal cells, exocrine bronchial cells, pulmonary neuroendocrine cells, alveolar cells, or airway epithelial cells. In some embodiments, the pulmonary cells are in vitro cells. In some embodiments, the pulmonary cells are in vivo cells.

[0326] The term "nucleic acid," in its broadest sense, includes any compound and / or substance comprising a polymer of nucleotides. These polymers are often referred to as polynucleotides. Exemplary nucleic acids or polynucleotides of the present disclosure include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA including LNA having a β-D-ribo configuration, α-LNA (a diastereomer of LNA) having an α-L-ribo configuration, 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino-α-LNA having a 2'-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), or hybrids or combinations thereof.

[0327] As used herein, "patient" refers to a subject who may seek or need treatment, who needs treatment, who is undergoing treatment, who will receive treatment, or who is under the care of a trained professional for a particular disease or condition.

[0328] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, substances, compositions, and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0329] The phrase "pharmaceutically acceptable excipient," as used herein, refers to any ingredient other than the compounds described herein (e.g., a vehicle capable of suspending or dissolving an active compound) that has substantially non-toxic and non-inflammatory properties in patients. Excipients may include, for example, anti-adherents, antioxidants, binders, coating agents, compression aids, disintegrants, dyes (colorants), softeners, emulsifiers, excipients (diluents), film-forming or coating agents, flavors, fragrances, glidants (flow improvers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water for hydration.

[0330] The pharmaceutically acceptable salts of the present disclosure include conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. Typically, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent, or a mixture of the two; usually, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17 th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety.

[0331] The term "solvate" as used herein refers to a compound of the present disclosure in which molecules of a suitable solvent are incorporated into the crystal lattice. The suitable solvent is physiologically tolerable at the administered dosage. For example, a solvate can be prepared by crystallization, recrystallization, or precipitation from a solution containing an organic solvent, water, or a mixture thereof. When water is the solvent, the solvate is referred to as a "hydrate."

[0332] The term "polynucleotide," as used herein, refers to a polymer of nucleotides of any length, comprising ribonucleotides, deoxyribonucleotides, their analogs, or mixtures thereof. The term refers to the primary structure of the molecule. Thus, the term includes triple-, double-, and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-, double-, and single-stranded ribonucleic acid ("RNA"). It also includes modified (e.g., by alkylation and / or capping) and unmodified forms of polynucleotides. More specifically, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA, and mRNA (spliced ​​or unspliced), any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing non-nucleotidic backbones, such as polyamides (e.g., peptide nucleic acids, "PNAs") and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers, provided the polymer contains nucleobases in a configuration that allows for base pairing and base stacking as found in DNA and RNA. In certain embodiments, a polynucleotide comprises an mRNA. In other embodiments, the mRNA is synthetic mRNA. In some embodiments, the synthetic mRNA contains at least one unnatural nucleobase. In some embodiments, all nucleobases of a given class are replaced with unnatural nucleobases (e.g., all uridines in a polynucleotide disclosed herein can be replaced with an unnatural nucleobase, e.g., 5-methoxyuridine). In some embodiments, a polynucleotide (e.g., synthetic RNA or synthetic DNA) contains only natural nucleobases, i.e., A (adenosine), G (guanosine), C (cytidine), and T (thymidine) in the case of synthetic DNA, or A, C, G, and U (uridine) in the case of synthetic RNA.

[0333] Those skilled in the art will understand that while a T base in the codon maps disclosed herein is present in DNA, the T base will be replaced by a U base in the corresponding RNA. For example, a codon-nucleotide sequence disclosed herein in DNA form, e.g., a vector or in-vitro translation (IVT) template, will have its T base transcribed as a U base in its corresponding transcribed mRNA. In this regard, both codon-optimized DNA sequences (including T) and their corresponding mRNA sequences (including U) are considered codon-optimized nucleotide sequences of the present disclosure. Those skilled in the art will also understand that equivalent codon maps can be generated by replacing one or more bases with an unnatural base. Thus, for example, a TTC codon (DNA map) would correspond to a UUC codon (RNA map), which in turn would correspond to a ΨΨC codon (RNA map with U replaced with pseudouridine).

[0334] Standard AT and GC base pairs create conditions that allow hydrogen bond formation between the N3-H and C4-oxy of thymidine and the N1 and C6-NH2 of adenosine, respectively, and between the C2-oxy, N3, and C4-NH2 of cytidine and the C2-NH2, N'-H, and C6-oxy of guanosine. Thus, for example, guanosine (2-amino-6-oxy-9-β-D-ribofuranosyl-purine) can be modified to form isoguanosine (2-oxy-6-amino-9-β-D-ribofuranosyl-purine). Such modifications result in a nucleoside base that no longer effectively forms standard base pairs with cytosine. However, modification of cytosine (1-β-D-ribofuranosyl-2-amino-4-oxy-pyrimidine) to form isocytosine (1-β-D-ribofuranosyl-2-amino-4-oxy-pyrimidine-) results in a modified nucleotide that will not base pair efficiently with guanosine, but will base pair with isoguanosine (Collins et al., U.S. Pat. No. 5,681,702). Isocytosine is available from Sigma Chemical Co. (St. Louis, Mo.); isocytidine can be prepared by the method described by Switzer et al. (1993) Biochemistry 32:10489-10496 and the references cited therein; 2'-deoxy-5-methyl-isocytidine can be prepared by the method of Tor et al., 1993, J. Am. Chem. Soc. 115:4461-4467 and the references cited therein; isoguanine nucleotides can be prepared using the method described by Switzer et al., 1993 (supra) and Mantsch et al., 1993, Biochem. 14:5593-5601, or by the method described in U.S. Patent No. 5,780,610 to Collins. Other unnatural base pairs can be synthesized by the method described in Piccirilli et al., 1990, Nature 343:33-37 for the synthesis of 2,6-diaminopyrimidine and its complement (1-methylpyrazolo-[4,3]pyrimidine-5,7-(4H,6H)-dione).Other such modified nucleotide units that form unique base pairs are known, such as those described by Leach et al. (1992) J. Am. Chem. Soc. 114:3675-3683 and Switzer et al., supra.

[0335] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer can contain modified amino acids. The term also encompasses amino acid polymers that are modified, either naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. The definition also includes, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, e.g., homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art.

[0336] As used herein, the term refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides include encoded polynucleotide products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the foregoing. Polypeptides can be monomers or multimolecular complexes, such as dimers, trimers, or tetramers. They can also include single-chain or multi-chain polypeptides. Disulfide bonds are most commonly found in multi-chain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids. In some embodiments, a "peptide" can be 50 amino acids or less in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0337] As used herein, the term "preventing" refers to partially or completely delaying the onset of an infection, disease, disorder, and / or condition; partially or completely delaying the onset of one or more signs and symptoms, characteristics, or clinical signs of a particular infection, disease, disorder, and / or condition; partially or completely delaying the onset of one or more signs and symptoms, characteristics, or signs of a particular infection, disease, disorder, and / or condition; partially or completely delaying the progression from an infection, a particular disease, disorder, and / or condition; and / or reducing the risk of developing pathology associated with an infection, disease, disorder, and / or condition.

[0338] As used herein, "prophylaxis" refers to a therapeutic agent or course of action used to prevent the spread of disease.

[0339] The term "salts" includes any anionic and cationic complexes. Pharmaceutically acceptable salts represent a subset of the non-toxic salts described above.

[0340] "Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject for which diagnosis, prognosis, or therapy is desired, particularly a mammalian subject. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, and pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and cows; primates such as apes, monkeys, orangutans, and chimpanzees; canines such as dogs and wolves; felines such as cats, lions, and tigers; equines such as horses, donkeys, and zebras; bears; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters, and guinea pigs, and the like. In certain embodiments, the mammal is a human subject. In other embodiments, the subject is a human patient. In certain embodiments, the subject is a human patient in need of treatment.

[0341] As used herein, the term "substantially" refers to a qualitative condition indicating the total or nearly total degree or extent of a feature or characteristic of interest. Those skilled in the biological arts will understand that biological and chemical characteristics rarely, if ever, reach perfection and / or progress to perfection, or achieve or avoid absolute results. As such, the term "substantially" is used herein to capture the potential lack of perfection inherent in many biological and chemical characteristics.

[0342] An individual "suffering from" a disease, disorder, and / or condition has been diagnosed with or exhibits one or more signs and symptoms of the disease, disorder, and / or condition.

[0343] An individual who is "susceptible to" a disease, disorder, and / or condition has not been diagnosed with and / or may not exhibit signs and symptoms of the disease, disorder, and / or condition, but possesses a propensity to develop the disease or its signs and symptoms. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., cancer) may be characterized by one or more of the following: (1) a genetic mutation associated with the development of the disease, disorder, and / or condition; (2) a genetic polymorphism associated with the development of the disease, disorder, and / or condition; (3) increased and / or decreased expression and / or activity of proteins and / or nucleic acids associated with the development of the disease, disorder, and / or condition; (4) habits and / or lifestyles associated with the development of the disease, disorder, and / or condition; (5) a family history of the disease, disorder, and / or condition; and (6) exposure to and / or infection by a microorganism associated with the development of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition, hi some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0344] The term "synthetic" means produced, prepared, and / or manufactured by the hand of man. Synthesis of polynucleotides or other molecules of the disclosure can be chemical or enzymatic.

[0345] The term "therapeutic or prophylactic agent" refers to an agent that has a therapeutic, diagnostic, and / or prophylactic effect and / or exerts a desired biological and / or pharmacological effect when administered to a subject. For example, an mRNA encoding a polypeptide can be a therapeutic or prophylactic agent.

[0346] As used herein, the term "therapeutically effective amount" means an amount of an agent (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) delivered that, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, is sufficient to treat, ameliorate the signs and symptoms of, diagnose, prevent, and / or delay the onset of the infection, disease, disorder, and / or condition.

[0347] Treating, Treatment, Therapy: As used herein, the term "treating" or "treatment" or "therapy" refers to partially or completely alleviating, ameliorating, ameliorating, palliating, delaying the onset of, inhibiting the progression of, reducing the severity of, and / or reducing the frequency of occurrence of one or more signs and symptoms or characteristics of a disease. For example, "treating" a disease can refer to attenuating the signs and symptoms associated with the disease, extending the patient's lifespan (increasing survival rate), reducing the severity of the disease, preventing or delaying the onset of the disease, etc. Treatment can be administered to subjects who do not show signs of the disease, disorder, and / or condition and / or to subjects who show only early signs of the disease, disorder, and / or condition for the purpose of reducing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0348] The term "n-membered" (n is an integer) typically refers to the number of ring-forming atoms at a moiety where n is the number of ring-forming atoms. For example, piperidinyl is an example of a 6-membered heterocycloalkyl ring, pyrazolyl is an example of a 5-membered heteroaryl ring, pyridyl is an example of a 6-membered heteroaryl ring, and 1,2,3,4-tetrahydro-naphthalene is an example of a 10-membered cycloalkyl group.

[0349] As used herein, the phrase "optionally substituted" means unsubstituted or substituted. The substituents are independently selected, and substitution can be at any chemically accessible position. As used herein, the term "substituted" means that a hydrogen atom has been removed and replaced with a substituent. A single divalent substituent, for example, oxo, can replace two hydrogen atoms. It should be understood that substitution at a given atom is limited by valence.

[0350] Throughout the definition, the term "C n-m " indicates a range inclusive of the endpoints, and n and m are integers indicating the number of carbons. Examples include C 1-4 , C 1-6 etc. are included.

[0351] As used herein, the term "C" when used alone or in combination with other terms n-m "Alkyl" refers to a saturated hydrocarbon group having n to m carbon atoms, which may be straight-chained or branched. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, and sec-butyl; higher homologs, e.g., 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, and 1,2,2-trimethylpropyl. In some embodiments, alkyl groups contain 1 to 6 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms.

[0352] As used herein, "C n-m"Alkenyl" refers to an alkyl group having one or more double carbon-carbon bonds and having n to m carbons. Exemplary alkenyl groups include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl, and the like. In some embodiments, the alkenyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

[0353] As used herein, "C n-m "Alkynyl" refers to an alkyl group having one or more triple carbon-carbon bonds and having n to m carbons. Exemplary alkynyl groups include, but are not limited to, ethynyl, propyn-1-yl, propyn-2-yl, and the like. In some embodiments, the alkynyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

[0354] As used herein, the term "C" when used alone or in combination with other terms n-m "Alkylene" is used to refer to a divalent alkyl linking group having n to m carbons. Examples of alkylene groups include, but are not limited to, ethane-1,1-diyl, ethane-1,2-diyl, propane-1,1-diyl, propane-1,3-diyl, propane-1,2-diyl, butane-1,4-diyl, butane-1,3-diyl, butane-1,2-diyl, 2-methyl-propane-1,3-diyl, and the like. In some embodiments, the alkylene moiety contains 2 to 6, 2 to 4, 2 to 3, 1 to 6, 1 to 4, or 1 to 2 carbon atoms.

[0355] As used herein, the term "C" when used alone or in combination with other terms n-m "Alkoxy" is used to refer to a group of formula -O-alkyl, where the alkyl group has n to m carbons. Exemplary alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), butoxy (e.g., n-butoxy and tert-butoxy), and the like. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0356] As used herein, the term "C n-m "Hydroxyalkyl" refers to an alkyl group substituted with a hydroxy (-OH) group.

[0357] As used herein, the term "C n-m "Alkylamino" refers to a group of the formula -NH(alkyl), where the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Examples of alkylamino groups include, but are not limited to, N-methylamino, N-ethylamino, N-propylamino (e.g., N-(n-propyl)amino and N-isopropylamino), N-butylamino (e.g., N-(n-butyl)amino and N-(tert-butyl)amino), and the like.

[0358] As used herein, the term "amino" refers to a group of formula -NH2.

[0359] As used herein, the term "aryl," used alone or in combination with other terms, is used to refer to an aromatic hydrocarbon group that can be monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings). n-m "Aryl" refers to an aryl group having n to m ring carbon atoms. Aryl groups include, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and the like. In some embodiments, an aryl group has 6 to 10 carbon atoms. In some embodiments, an aryl group is phenyl or naphthyl.

[0360] As used herein, "halo" refers to F, Cl, Br, or I. In some embodiments, halo is F, Cl, or Br. In some embodiments, halo is F.

[0361] As used herein, "cycloalkyl" refers to a non-aromatic cyclic hydrocarbon, including cyclized alkyl and / or alkenyl groups. Cycloalkyl groups can include mono- or polycyclic (e.g., having 2, 3, or 4 fused rings) groups and spirocycles. The ring-forming carbon atoms of a cycloalkyl group can be optionally substituted by oxo or sulfido (e.g., C(O) or C(S)). The definition of cycloalkyl also includes moieties having one or more aromatic rings fused to (i.e., having a bond in common with) the cycloalkyl ring, e.g., benzo or thienyl derivatives such as cyclopentane, cyclohexane, etc. Cycloalkyl groups containing fused aromatic rings can be bonded through any ring-forming atom, including the ring-forming atoms of the fused aromatic ring. Cycloalkyl groups can have 3, 4, 5, 6, 7, 8, 9, or 10 ring-forming carbon atoms (C 3-10 In some embodiments, cycloalkyl can have C 3-10 In some embodiments, the cycloalkyl is a monocyclic or bicyclic cycloalkyl. 3-7 Monocyclic cycloalkyl. Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcarnyl, etc. In some embodiments, the cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0362] As used herein, "heteroaryl" refers to a monocyclic or polycyclic aromatic heterocycle having at least one heteroatom ring member selected from sulfur, oxygen, and nitrogen. In some embodiments, the heteroaryl ring has 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, any ring-forming N in the heteroaryl moiety can be an N-oxide. In some embodiments, the heteroaryl is a 5-10 membered monocyclic or bicyclic heteroaryl having 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, the heteroaryl is a 5-6 membered monocyclic heteroaryl having 1 or 2 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, the heteroaryl is a 5- or 6-membered heteroaryl ring. A 5-membered heteroaryl ring is a heteroaryl having a ring with 5 ring atoms, wherein one or more (e.g., 1, 2, or 3) ring atoms are independently selected from N, O, and S. Exemplary 5-membered heteroaryls are thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl. A 6-membered heteroaryl ring is a heteroaryl having a ring with 6 ring atoms, wherein one or more (e.g., 1, 2, or 3) ring atoms are independently selected from N, O, and S. Exemplary 6-membered heteroaryls are pyridyl, pyrazinyl, pyrimidinyl, triazinyl, and pyridazinyl.

[0363] As used herein, "heterocycloalkyl" refers to a non-aromatic monocyclic or polycyclic heterocycle having one or more ring-forming heteroatoms selected from O, N, or S. Heterocycloalkyl includes monocyclic 4-, 5-, 6-, 7-, 8-, 9-, or 10-membered heterocycloalkyl groups. Heterocycloalkyl groups can also include spirocycles. Exemplary heterocycloalkyl groups include pyrrolidin-2-one, 1,3-isoxazolidin-2-one, pyranyl, tetrahydrofuran, oxetanyl, azetidinyl, morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, azepanyl, benzazapene, and the like. Ring-forming carbon atoms and heteroatoms of a heterocycloalkyl group can be optionally substituted with oxo or sulfido (e.g., C(O), S(O), C(S), or S(O), etc.). A heterocycloalkyl group can be bonded through a ring-forming carbon atom or ring-forming heteroatom. In some embodiments, a heterocycloalkyl group contains 0 to 3 double bonds. In some embodiments, a heterocycloalkyl group contains 0 to 2 double bonds. Also included within the definition of heterocycloalkyl are moieties having one or more aromatic rings fused to (i.e., having a bond in common with) a cycloalkyl ring, e.g., benzo- or thienyl derivatives such as piperidine, morpholine, azepine, etc. Heterocycloalkyl groups containing fused aromatic rings can be bonded through any ring-forming atom, including a ring-forming atom of the fused aromatic ring. In some embodiments, a heterocycloalkyl is a monocyclic 4- to 6-membered heterocycloalkyl having 1 or 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur and having one or more oxidized ring members. In some embodiments, the heterocycloalkyl is a monocyclic or bicyclic 4-10 membered heterocycloalkyl having 1, 2, 3, or 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur and having one or more oxidized ring members.

[0364] Where specified, definitions or embodiments refer to specific rings (e.g., azetidine rings, pyridine rings, etc.). Unless otherwise indicated, these rings can be bonded to any ring member as long as the atom's valence is not exceeded. For example, an azetidine ring can be bonded at any position on the ring, while a pyridin-3-yl ring can be bonded at the 3-position.

[0365] As used herein, a "bridged ring" or "bridged ring group" is a ring system having at least two connected rings that share three or more atoms. The bridged ring can be a carbocyclic ring or a heterocycloalkyl ring. Exemplary bridged rings include: [ka] Includes:

[0366] Where ranges are given, the endpoints are included. Furthermore, unless otherwise indicated or otherwise apparent from the context and the understanding of one of ordinary skill in the art, it is to be understood that values ​​expressed as ranges can assume any specific value or subrange within the ranges described in different embodiments of this disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0367] It should also be understood that any particular embodiment of the present disclosure that falls within the prior art may be expressly excluded from any one or more of the claims. Because such embodiments are deemed to be known to those skilled in the art, they may be excluded even if the exclusion is not expressly set forth herein. Any particular embodiment of the compositions of the present disclosure (e.g., any nucleic acid or protein encoded thereby; any method of production; any method of use, etc.) may be excluded from any one or more of the claims for any reason, whether related to the existence of prior art or not.

[0368] All sources cited herein, e.g., references, publications, databases, database entries, and techniques, are incorporated by reference into this application, even if not explicitly stated in the citation. In the event of a conflict between the statements of the cited sources and this application, the statements in this application shall control.

[0369] Section and table headings are not intended to be limiting. [Example]

[0370] Abbreviation: ACN: acetonitrile Aq.: Aqueous Boc2O: di-tert-butylpyrocarbonate DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene BnBr: benzyl bromide DCC: N,N'-dicyclohexylcarbodiimide DCM: dichloromethane DMAP: 4-dimethylaminopyridine DMF: dimethylformamide EtOAc: ethyl acetate h: hour(s) LCMS: Liquid chromatography-mass spectrometry MTBE: Methyl tert-butyl ether PMA: phosphomolybdic acid Soln.:Solution rt: room temperature THF: tetrahydrofuran TLC: Thin Layer Chromatography

[0371] Example 1 Synthesis of compounds according to formula A1 Intermediate A: tert-butyl (5-((3-((tert-butoxycarbonyl)amino)butyl)amino)pentan-2-yl)carbamate [ka]

[0372] Step 1: tert-butyl (5-hydroxypentan-2-yl)carbamate [ka] To a solution of di-tert-butyl dicarbonate (2.45 mL, 10.66 mmol) in dry DCM (30 mL) stirring under nitrogen at 0 °C was added 4-aminopentan-1-ol (1.00 g, 9.69 mmol) dropwise over 10 min. The solution was gradually warmed to room temperature and stirring was continued for 3 days. After this time, the solvent was then removed in vacuo, and the resulting residue was taken up in DCM and purified on silica in hexanes with a gradient of 0-100% EtOAc. Product-containing fractions were pooled and concentrated in vacuo to give tert-butyl (5-hydroxypentan-2-yl)carbamate as an oil (0.86 g, 4.23 mmol, 43.6%). UPLC / ELSD: RT = 0.28 min. MS (ES): C 10 H 21 For NO3, m / z (MH + )204.3. 1 H NMR (300 MHz, CDCl3) δ 4.51 (br. s, 1H), 3.62 (t, 3H), 2.59 (br. s, 1H), 1.53 (m, 4H), 1.42 (s, 9H), 1.12 (d, 3H, J = 6 Hz).

[0373] Step 2: 4-((tert-butoxycarbonyl)amino)pentyl 4-methylbenzenesulfonate [ka] To a solution of tert-butyl (5-hydroxypentan-2-yl)carbamate (0.86 g, 4.23 mmol) in dry DCM (30 mL) stirred under nitrogen was added triethylamine (2.95 mL, 21.15 mmol), dimethylaminopyridine (0.10 g, 0.85 mmol), and p-toluenesulfonyl chloride (1.61 g, 8.46 mmol). The solution was stirred at room temperature overnight, during which time it turned dark red. The mixture was then further diluted with DCM and washed with water (1 × 30 mL), saturated aqueous sodium bicarbonate (1 × 30 mL), and brine (1 × 30 mL), dried over sodium sulfate, filtered, and concentrated to a dark brown oil. The oil was taken up in DCM and purified on silica in hexanes with a 0–50% EtOAc gradient. Product-containing fractions were pooled and concentrated to give 4-((tert-butoxycarbonyl)amino)pentyl 4-methylbenzenesulfonate as a light brown oil (0.97 g, 2.72 mmol, 64.3%). UPLC / ELSD: RT: 1.02 min. MS(ES): C 17 H 27 Regarding NO5S, m / z (MH + )358.4. 1 H NMR (300 MHz, CDCl3) δ 7.77 (d, 2H, J = 6 Hz), 7.36 (d, 2H, J = 6 Hz), 4.32 (br. s, 1H), 4.03 (t, 2H), 3.55 (br. s, 1H), 2.45 (s, 3H), 1.69 (br. m, 2H), 1.41 (s, 11H), 1.06 (d, 3H, J = 6 Hz).

[0374] Step 3: tert-butyl (5-((3-((tert-butoxycarbonyl)amino)butyl)amino)pentan-2-yl)carbamate [ka] To a solution of tert-butyl N-[4-(2-nitrobenzenesulfonamido)butan-2-yl]carbamate (0.97 g, 2.61 mmol) in dry DMF (20 mL) stirred at room temperature under nitrogen, 4-((tert-butoxycarbonyl)amino)pentyl 4-methylbenzenesulfonate (1.03 g, 2.87 mmol) and potassium carbonate (1.08 g, 7.82 mmol) were added. The solution was warmed to 100 °C and stirred for 48 h. The reaction was then quenched with 30 mL of water and diluted with 50 mL of EtOAc. The organic layer was separated, and the aqueous layer was extracted with EtOAc (3 × 30 mL). All organic layers were combined, washed with water (1 × 50 mL) and brine (1 × 50 mL), dried over sodium sulfate, filtered, and concentrated to a yellow oil. The oil was taken up in DCM and purified on silica in hexanes with a gradient of 0 to 100% EtOAc. Product-containing fractions were pooled and concentrated to an oil. The oil was taken up in 20 mL of DMF, to which thiophenol (1.03 mL, 10.03 mmol) and potassium carbonate (1.08 g, 7.82 mmol) were added. The solution was stirred at room temperature overnight. The next morning, the salts were removed from the mixture by centrifugation, and the supernatant was concentrated to a residue. The residue was taken up in 40 mL of DCM, washed with water (2 × 10 mL) and brine (2 × 10 mL), dried over potassium carbonate, filtered, and concentrated to an oil. The oil was taken up in DCM and purified on silica in DCM with a gradient of 0 to 50% (50:45:5 DCM / MeOH / NH4OH). Product-containing fractions were pooled and concentrated to give tert-butyl (5-((3-((tert-butoxycarbonyl)amino)butyl)amino)pentan-2-yl)carbamate as a colorless oil (0.34 g, 0.91 mmol, 34.93%). UPLC / ELSD: RT: 0.28 min. MS(ES): C 19 H 39 For N3O4, m / z (MH + )374.4. 1H NMR (300 MHz, CDCl3) δ 4.91 (br. m, 1H), 4.67 (br. m, 1H), 3.70 (br. m, 2H), 3.40 (s, 2H), 2.72 (t, 1H), 2.58 (br. m, 3H), 2.41 (br. s, 4H), 1.50 (br. m, 5H), 1.41 (s, 19H), 1.10 (d, 6H, J = 6 Hz).

[0375] A. Compound SA50: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 3-(bis(3-(dimethylamino)propyl)amino)-3-oxopropanoate dihydrochloride [ka]

[0376] Step 1: tert-Butyl ((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl)malonate [ka] To a solution of cholesterol (4.00 g, 10.14 mmol) and 3-(tert-butoxy)-3-oxopropanoic acid (2.39 mL, 15.21 mmol) in dichloromethane (20 mL) stirred under nitrogen was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.95 g, 15.21 mmol). The reaction mixture was then cooled to 0 °C, and diisopropylethylamine (5.36 mL, 30.41 mmol) was added dropwise over 20 minutes. The resulting mixture was allowed to warm gradually to room temperature and proceed overnight. The mixture was then diluted to 150 mL with dichloromethane, washed with water (1 × 70 mL), saturated aqueous sodium bicarbonate (2 × 70 mL), and brine (1 × 70 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to give a yellow oil. The oil was taken up in dichloromethane and purified on silica with a gradient of 0-25% ethyl acetate in hexanes to give tert-butyl ((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl)malonate (4.99 g, 9.44 mmol, 93.1%) as an oil. UPLC / ELSD: RT: 3.36 min. MS(ES): C 34 H 56 For O4, m / z (MH + )529.8. 1 H NMR (300 MHz, CDCl3) δ: ppm 5.41 (m, 1H), 4.67 (m, 1H), 3.27 (s, 2H), 2.38 (d, 2H), 1.91 (br. m, 10 H), 1.49 (s, 12H), 1.35 (br. m, 6H), 1.04 (br. m, 17H), 0.91 (d, 3H, J = 3 Hz), 0.87 (d, 3H, J = 3 Hz), 0.70 (s, 3H).

[0377] Step 2: 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl)oxy)-3-oxopropanoic acid [ka] To a solution of tert-butyl ((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl)malonate (4.99 g, 9.44 mmol) in dichloromethane (50 mL) stirred at 0 °C under nitrogen was added trifluoroacetic acid (10.85 mL, 141.63 mmol) dropwise over 20 min. The clear, pale yellow reaction mixture was allowed to gradually warm to room temperature and proceed overnight. The next morning, the reaction was quenched with 20 mL of 5% aqueous sodium bicarbonate solution at 0 °C. The organics were separated, washed with an additional 10 mL of 5% aqueous sodium bicarbonate, dried over sodium sulfate, filtered, and concentrated to give a white solid. The solid was taken up in dichloromethane and purified on silica with a gradient of 0-60% ethyl acetate in hexanes to give 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl)oxy)-3-oxopropanoic acid (3.12 g, 6.61 mmol, 70.0%) as a white solid. UPLC / ELSD: RT: 2.97 min. MS(ES): C 30 H 48 For O4, m / z (MH + )473.7. 1H NMR (300 MHz, CDCl3) δ: ppm 10.99 (br. s, 1H), 5.42 (m, 1H), 4.73 (m, 1H), 3.45 (s, 2H), 2.37 (d, 2H, J = 9 Hz), 1.89 (br. m, 5H), 1.35 (br. m, 18H), 1.05 (s, 5H), 0.94 (d, 4H, J = 2 Hz), 0.89 (d, 6H, J = 2 Hz), 0.70 (s, 3H).

[0378] Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 3-(bis(3-(dimethylamino)propyl)amino)-3-oxopropanoate [ka] To a solution of 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl)oxy)-3-oxopropanoic acid (3.12 g, 6.61 mmol) in dichloromethane (60 mL) stirred under nitrogen was added tetramethyldipropylenetriamine (2.30 mL, 9.81 mmol), dimethylaminopyridine (0.08 g, 0.65 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.90 g, 9.81 mmol). The reaction mixture was cooled to 0° C. and diisopropylethylamine (3.46 mL, 19.62 mmol) was added dropwise over 20 minutes. The mixture was allowed to warm gradually to room temperature and proceed overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate (1×50 mL) and brine (1×50 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was taken up in dichloromethane and purified on silica with a gradient of 0-60% (9:1 methanol / concentrated aqueous ammonium hydroxide) in dichloromethane to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 3-(bis(3-(dimethylamino)propyl)amino)-3-oxopropanoate (1.82 g, 2.84 mmol, 43.4%) as a yellow oil. UPLC / ELSD: RT: 1.85 min. MS(ES): C 40 H 71 For N3O3, m / z (MH + )643.0. 1H NMR (300 MHz, CDCl3) δ: ppm 5.39 (m, 1H), 4.67 (m, 1H), 3.54 (s, 2H), 3.35 (br. m, 4H), 2.37 (br. m, 6H), 2.22 (d, 12H, J = 3 Hz), 1.50 (br. m, 28H), 1.02 (br. s, 5H), 0.92 (d, 4H, J = 6 Hz), 0.88 (d, 6H, J = 9 Hz), 0.68 (s, 3H).

[0379] Step 4: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 3-(bis(3-(dimethylamino)propyl)amino)-3-oxopropanoate dihydrochloride [ka] To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 3-(bis(3-(dimethylamino)propyl)amino)-3-oxopropanoate) (0.22 g, 0.32 mmol) in diethyl ether (4.3 mL) and isopropanol (0.22 mL) was added hydrochloric acid (5.5 M in isopropanol, 0.37 mL, 1.85 mmol) dropwise. The mixture was cooled to 0 °C and stirred vigorously for 30 min, after which the white precipitate was filtered via vacuum filtration and washed repeatedly with cold ether. The residue was dried in vacuo to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 3-(bis(3-(dimethylamino)propyl)amino)-3-oxopropanoate dihydrochloride as a white waxy solid (0.11 g, 0.15 mmol, 46.6%). UPLC / ELSD: RT: 1.81 min. MS(ES): C 40 H 73 For Cl2N3O3, m / z (MH + )627.99. 1 H NMR (300 MHz, CD3OD) δ: ppm 5.41 (br. s, 1H), 4.64 (br. m, 1H), 3.57 (br. m, 7H), 3.33 (br. s, 2H), 3.20 (br. m, 5H), 2.93 (d, 15H, J = 6 Hz), 2.40 (d, 2H, J = 9 Hz), 2.05 (br. m, 12H), 1.55 (br. m, 14H), 1.20 (br. m, 13H), 1.07 (s, 7H), 0.98 (d, 5H, J = 6 Hz), 0.91 (d, 7H, J = 6 Hz), 0.74 (s, 3H).

[0380] B. Compound SA51: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 5-(bis(3-(dimethylamino)propyl)amino)-5-oxopentanoate dihydrochloride [ka]

[0381] Step 1: 5-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl)oxy)-5-oxopentanoic acid [ka] To a solution of cholesterol (5.00 g, 12.67 mmol) in acetone (50 mL) stirred under nitrogen was added glutaric anhydride (2.63 g, 22.81 mmol) and triethylamine (3.21 mL, 22.81 mmol). The reaction mixture was refluxed at 56 °C, turning from a white slurry to a clear, colorless solution and allowed to proceed at reflux for 3 days. The solution was then cooled to room temperature, concentrated in vacuo, and taken up in 150 mL of dichloromethane. It was then washed with 0.5 M HCl (1 × 100 mL) and saturated aqueous ammonium chloride (1 × 100 mL), dried over sodium sulfate, filtered, and concentrated to give a white solid. The solid was taken up in dichloromethane and purified on silica with a gradient of 0-50% ethyl acetate in hexanes to give 5-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl)oxy)-5-oxopentanoic acid (6.011 g, 12.00 mmol, 94.7%) as a white solid. UPLC / ELSD: RT: 2.96 min. MS(ES): C 32 H 52 For O4, m / z (MH + )501.7. 1 H NMR (300 MHz, CDCl3) δ: ppm 5.40 (m, 1H), 4.66 (m, 1H), 2.45 (br. m, 5H), 2.01 (br. m, 3H), 1.85 (br. m, 3H), 1.34 (br. m, 22H), 0.94 (d, 3H, J = 6 Hz), 0.88 (d, 6H, J = 9 Hz), 0.70 (s, 3H).

[0382] Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 5-(bis(3-(dimethylamino)propyl)amino)-5-oxopentanoate [ka] To a solution of 5-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl)oxy)-5-oxopentanoic acid (6.01 g, 11.88 mmol) in dichloromethane (100 mL) stirred under nitrogen was added tetramethyldipropylenetriamine (4.19 mL, 17.82 mmol), dimethylaminopyridine (0.15 g, 1.19 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (3.45 g, 17.83 mmol). The solution was cooled to 0° C., then diisopropylethylamine (6.29 mL, 35.65 mmol) was added dropwise. The reaction mixture was gradually warmed to room temperature and allowed to proceed overnight. The solution was further diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate (1×100 mL) and brine (1×100 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The material was taken up in dichloromethane and purified on silica with a gradient of 0-60% (9:1 methanol:aqueous ammonium hydroxide) in dichloromethane to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 5-(bis(3-(dimethylamino)propyl)amino)-5-oxopentanoate (2.30 g, 11.88 mmol, 28.9%) as an oil. UPLC / ELSD: RT: 2.02 min. MS(ES): C 42 H 75 For N3O3, m / z (MH + )671.1. 1H NMR (300 MHz, CDCl3) δ: ppm 5.36 (m, 1H), 4.60 (m, 1H), 3.33 (br. m, 5H), 2.39 (br. m, 12H), 2.23 (d, 11H, J = 6 Hz), 1.99 (br. m, 4H), 1.84 (br. m, 3H), 1.71 (br. m, 5H), 1.33 (br. m, 11H), 1.14 (br. m, 7H), 1.02 (s, 6H), 0.92 (d, 3H, J = 6 Hz), 0.87 (d, 5H, J = 9 Hz), 0.68 (s, 3H).

[0383] Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 5-(bis(3-(dimethylamino)propyl)amino)-5-oxopentanoate dihydrochloride [ka] To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 5-(bis(3-(dimethylamino)propyl)amino)-5-oxopentanoate) (0.23 g, 0.33 mmol) in diethyl ether (4.6 mL) and isopropanol (0.23 mL) was added hydrochloric acid (5.5 M in isopropanol, 0.37 mL, 1.85 mmol) dropwise. The mixture was cooled to 0 °C and stirred vigorously for 30 min, after which the white precipitate was filtered via vacuum filtration and washed repeatedly with cold ether. The residue was dried in vacuo to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 5-(bis(3-(dimethylamino)propyl)amino)-5-oxopentanoate dihydrochloride as a white waxy solid (0.12 g, 0.16 mmol, 49.5%). UPLC / ELSD: RT: 1.86 min. MS(ES): C 42 H 77 For Cl2N3O3, m / z (MH + )671.81. 1H NMR (300 MHz, CD3OD) δ: ppm 5.41 (br. s, 1H), 4.55 (br. m, 1H), 3.54 (t, 5H, J = 6 Hz), 3.24 (br. m, 6H), 2.94 (d, 14H, J = 6 Hz), 2.55 (t, 2H, J = 6 Hz), 2.43 (t, 2H, J = 6 Hz), 2.35 (d, 2H, J = 9 Hz), 2.05 (br. m, 6H), 1.90 (br. m, 6H), 1.55 (br. m, 12H), 1.19 (br. m, 10H), 1.07 (s, 7H), 0.98 (d, 4H, J = 6 Hz), 0.90 (d, 7H, J = 6 Hz), 0.74 (s, 3H).

[0384] C. Compound SA56: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 4-oxo-4-(1,4,7-triazonan-1-yl)butanoate [ka]

[0385] Step 1: Di-tert-butyl 7-(4-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl)oxy)-4-oxobutanoyl)-1,4,7-triazonane-1,4-dicarboxylate [ka] To a stirred solution of cholesteryl hemisuccinate (100 mg, 0.205 mmol), 1,4-di-tert-butyl 1,4,7-triazonane-1,4-dicarboxylate (Enamine, Monmouth Junction, NJ) (0.068 g, 0.20 mmol), and DMAP (catalyst) in DCM (1.4 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.060 g, 0.31 mmol). The reaction mixture was stirred at room temperature and monitored by TLC. Water (1.5 mL) was added over 21.5 h. After stirring for 16 h, additional water (10 mL) was added. The mixture was then extracted with DCM (2 × 15 mL). The combined organics were passed through a hydrophobic frit, dried over NaSO, and concentrated. The crude material was purified via silica gel chromatography (0-4% MeOH in DCM) to provide di-tert-butyl 7-(4-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl)oxy)-4-oxobutanoyl)-1,4,7-triazonane-1,4-dicarboxylate (130 mg, 0.163 mmol, 79.3%) as a clear oil. UPLC / ELSD: RT=3.41 min. MS(ES): C 47 H 79 N3O7 m / z = 1619.2 [2M+Na] + ; 1 H NMR (300 MHz, CDCl3): δ 5.33-5.39 (m, 1H), 4.52-4.68 (m, 1H), 3.18-3.79 (br. m, 12H), 2.49-2.71 (m, 4H), 2.24-2.39 (m, 2H), 1.74-2.06 (br. m, 5H), 0.93-1.71 (br. m, 39H), 1.01 (s, 3H), 0.91 (d, 3H, J = 6.5 Hz), 0.87 (d, 3H, J = 6.6 Hz), 0.86 (d, 3H, J = 6.5 Hz), 0.67 (s, 3H).

[0386] Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 4-oxo-4-(1,4,7-triazonan-1-yl)butanoate dihydrochloride [ka] To a solution of di-tert-butyl 7-(4-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl)oxy)-4-oxobutanoyl)-1,4,7-triazonane-1,4-dicarboxylate (123 mg, 0.154 mmol) in iPrOH (2.0 mL) was added 5-6 N HCl in iPrOH (0.18 mL). The reaction mixture was stirred at 40 °C and monitored by LCMS. At 17 h, additional iPrOH (2.0 mL) and 5-6 N HCl in iPrOH (0.06 mL) were added. At 41 h, the reaction mixture was cooled to room temperature, and ACN (4 mL) was added. The solid was collected by vacuum filtration and rinsed with ACN to provide (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 4-oxo-4-(1,4,7-triazonan-1-yl)butanoate dihydrochloride (0.082 g, 0.11 mmol, 73.8%) as a white solid. UPLC / ELSD: RT=2.30 minutes. MS(ES):C 37 H 63 For N3O3, m / z = 598.1 [M+H] + ; 1H NMR (300 MHz, CDCl3): δ 10.36 (br. s, 2H), 10.11 (br. s, 2H), 5.34-5.43 (m, 1H), 4.50-4.65 (m, 1H), 3.97-4.29 (m, 4H), 3.64-3.95 (m, 6H), 3.41-3.61 (m, 2H), 2.66-2.84 (m, 2H), 2.45-2.65 (m, 2H), 2.21-2.40 (m, 2H), 1.75-2.08 (br. m, 5H), 0.94-1.70 (br. m, 21H), 1.01 (s, 3H), 0.91 (d, 3H, J = 6.4 Hz), 0.87 (d, 3H, J = 6.5 Hz), 0.86 (d, 3H, J = 6.6 Hz), 0.68 (s, 3H).

[0387] Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 4-oxo-4-(1,4,7-triazonan-1-yl)butanoate [ka] (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 4-oxo-4-(1,4,7-triazonan-1-yl)butanoate dihydrochloride (0.054 g, 0.075 mmol) was suspended in 5% aqueous NaHCO solution (10 mL) and then extracted with DCM (3 × 10 mL). KCO (approximately 100 mg) was added to the aqueous layer. The aqueous layer was extracted with DCM (2 × 10 mL). The combined organics were passed through a hydrophobic frit, dried over NaSO, and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 4-oxo-4-(1,4,7-triazonan-1-yl)butanoate (0.023 g, 0.037 mmol, 50.0%) as a white solid. 1 H NMR (300 MHz, CDCl3): δ 5.33-5.40 (m, 1H), 4.54-4.70 (m, 1H), 3.41-3.57 (m, 4H), 2.99-3.15 (m, 4H), 2.72-2.84 (m, 4H), 2.58-2.72 (m, 4H), 2.24-2.39 (m, 2H), 1.74-2.19 (br. m, 7H), 0.94-1.70 (br. m, 21H), 1.01 (s, 3H), 0.91 (d, 3H, J = 6.5 Hz), 0.87 (d, 3H, J = 6.6 Hz), 0.86 (d, 3H, J = 6.6 Hz), 0.67 (s, 3H). UPLC / ELSD: RT=2.39 minutes. MS(ES):C 37 H 63 For N3O3, m / z = 598.6 [M+H] + .

[0388] D. Compound SA57: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 6-(bis(3-(dimethylamino)propyl)amino)-6-oxohexanoate [ka]

[0389] Step 1: 6-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl)oxy)-6-oxohexanoic acid [ka] To a solution of cholesterol (5.00 g, 12.93 mmol) in dichloromethane (50 mL) stirred under nitrogen was added adipic anhydride (1.66 g, 12.93 mmol). Pyridine (3.97 mL, 28.45 mmol) was then added dropwise over 10 minutes. The reaction mixture was heated to reflux at 40° C. and allowed to proceed overnight. The mixture was then cooled to room temperature and concentrated to a yellow oil. The oil was taken up in dichloromethane and purified on silica with a gradient of 0-30% ethyl acetate in hexanes without further workup to give 6-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl)oxy)-6-oxohexanoic acid (1.97 g, 3.83 mmol, 29.6%) as a white solid. UPLC / ELSD: RT: 3.09 min. MS(ES): C 33 H 54 For O4, m / z (MH+ )515.7. 1 H NMR (300 MHz, CDCl3) δ: ppm 12.15 (br. s, 1H), 5.39 (m, 1H), 4.63 (br. m, 1H), 2.40 (br. m, 6H), 2.00 (br. m, 2H), 1.85 (br. m, 3H), 1.70 (br. m, 4H), 1.34 (br. m, 19H), 1.03 (s, 6H), 0.93 (d, 4H, J = 6 Hz), 0.88 (d, 6H, J = 6 Hz), 0.69 (s, 3H).

[0390] Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 6-(bis(3-(dimethylamino)propyl)amino)-6-oxohexanoate [ka] To a solution of 6-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl)oxy)-6-oxohexanoic acid (1.00 g, 1.92 mmol) in dichloromethane (25 mL) stirred under nitrogen was added tetramethyldipropylenetriamine (0.68 mL, 2.89 mmol), dimethylaminopyridine (0.02 g, 0.19 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.56 g, 2.89 mmol). The resulting solution was cooled to 0 °C and diisopropylethylamine (1.02 mL, 5.77 mmol) was added dropwise. The mixture was allowed to warm gradually to room temperature and proceed overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate (1 × 10 mL) and brine (1 × 10 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was taken up in DCM and purified on silica with a gradient of 0 to 60% (8:2:0.1 dichloromethane / methanol / concentrated aqueous ammonium hydroxide) in dichloromethane. 1H NMR revealed (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 6-(bis(3-(dimethylamino)propyl)amino)-6-oxohexanoate) as a yellow oil, which was purified by dichloromethane extraction in 0-25% (8:2:0.1) dichloromethane / methanol / concentrated aqueous water. Purification again on silica using a gradient of 1000kJ / 2000kcal (ammonium hydroxide) gave (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 6-(bis(3-(dimethylamino)propyl)amino)-6-oxohexanoate) as a pale yellow oil (0.38 g, 0.54 mmol, 28.2%). UPLC / ELSD: RT: 2.11 min. MS(ES): C 43 H 77 For N3O3, m / z (MH + )685.1. 1 H NMR (300 MHz, CDCl3) δ: ppm 5.40 (m, 3H), 5.20 (m, 1H), 4.40 (br. m, 1H), 3.22 (m, 4.48), 2.58 (s, 3H), 2.38 (t, 2H, J = 9 Hz), 2.26 (s, 6H), 2.20 (d, 3H, J = 9 Hz), 2.14 (br. s, 9H), 1.49 (br. m, 24H), 0.95 (br. m, 7H), 0.85 (s, 5H), 0.75 (d, 4H, J = 6 Hz), 0.70 (d, 5H, J = 9 Hz), 0.51 (s, 3H).

[0391] E. Compound SA58: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 5-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-5-oxopentanoate trihydrochloride [ka]

[0392] Step 1: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazanonadecan-19-oate [ka] 5-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl)oxy)-5-oxopentanoic acid (0.76 g, 1.49 mmol) in dichloromethane (20 mL) under stirring under nitrogen To a solution of tert-butyl(3-((tert-butoxycarbonyl)amino)propyl)(4-((3-((tert-butoxycarbonyl)amino)propyl)amino)butyl)carbamate (0.75 g, 1.49 mmol), dimethylaminopyridine (0.02 g, 0.15 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.43 g, 2.24 mmol) were added. The resulting solution was cooled to 0° C., and diisopropylethylamine (0.79 mL, 4.48 mmol) was added dropwise. The mixture was allowed to warm gradually to room temperature and proceed overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate (1×10 mL) and brine (1×10 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was taken up in DCM and purified on silica with a gradient of 0–60% ethyl acetate in hexanes to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazanonadecan-19-oate (0.71 g, 0.72 mmol, 48.2%) as a pale yellow oil. UPLC / ELSD: RT: 3.37 minutes. MS(ES):C 57 H 100 For N4O9, m / z (MH + )986.4. 1H NMR (300 MHz, CDCl3) δ: ppm 5.39 (m, 2H), 4.64 (br. m, 1H), 3.27 (br. m, 11H), 2.38 (br. m, 6H), 1.86 (br. m, 13H), 1.46 (br. d, 32H), 1.15 (br. m, 11H), 1.03 (s, 5H), 0.94 (d, 3H, J = 9 Hz), 0.88 (d, 5H, J = 9 Hz), 0.70 (s, 3H).

[0393] Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 5-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-5-oxopentanoate trihydrochloride [ka] (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 9-(t) in 2-propanol (10 mL) under stirring under nitrogen To a solution of (tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazanonadecan-19-oate (0.71 g, 0.72 mmol) was added hydrochloric acid (5.5 M in 2-propanol, 1.44 mL, 7.20 mmol) dropwise. The mixture was heated to 45° C. and stirred overnight. The solution was then cooled to room temperature, and acetonitrile (5 mL) was added to the mixture. It was then sonicated to remove precipitated solids from the sides of the flask. After sonication and stirring for 30 minutes, the solid was filtered by vacuum filtration, washed repeatedly with acetonitrile, and dried in vacuo to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 5-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-5-oxopentanoate trihydrochloride as a pale purple solid (0.39 g, 0.47 mmol, 65.6%). UPLC / ELSD: RT: 1.68 min. MS(ES): C 42 H 79 For Cl3N4O3, m / z (MH + )686.1. 1H NMR (300 MHz, CD3OD) δ: ppm 5.40 (s, 1H), 4.90 (br. s, 9H), 4.55 (br. s, 1H), 3.33 (br. m, 12H), 2.32 (br. 6H), 2.16 (br. m, 2H), 2.05 (s, 5H), 1.91 (br. m, 10H), 1.54 (br. m, 7H), 1.39 (br. m, 4H), 1.17 (d, 8H, J = 6 Hz), 1.06 (s, 5H), 0.97 (d, 4H, J = 6 Hz), 0.90 (d, 6H, J = 6 Hz), 0.73 (s, 3H).

[0394] F. Compound SA59: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 3-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-3-oxopropanoate trihydrochloride [ka]

[0395] Step 1: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazaheptadecan-17-oate [ka] 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl)oxy)-3-oxopropanoic acid (0.70 g, 1.47 mmol) in dichloromethane (20 mL) under stirring under nitrogen To a solution of tert-butyl(3-((tert-butoxycarbonyl)amino)propyl)(4-((3-((tert-butoxycarbonyl)amino)propyl)amino)butyl)carbamate (0.74 g, 1.47 mmol), dimethylaminopyridine (0.02 g, 0.15 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.43 g, 2.20 mmol) were added. The resulting solution was cooled to 0° C., and diisopropylethylamine (0.78 mL, 4.40 mmol) was added dropwise. The mixture was allowed to gradually warm to room temperature and proceed overnight. The solution was then diluted with dichloromethane, washed with water (3×20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was taken up in DCM and purified on silica with a gradient of 0–60% ethyl acetate in hexanes to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazaheptadecan-17-oate (1.13 g, 1.18 mmol, 80.4%) as a pale yellow oil. UPLC / ELSD: RT: 3.29 minutes. MS(ES):C 55 H 96 For N4O9, m / z (MH + )958.4. 1H NMR (300 MHz, CDCl3) δ: ppm 5.39 (m, 2H). 4.70 (br. m, 2H), 3.26 (br. m, 13H), 2.37 (d, 2H, J = 6 Hz), 1.86 (br. m, 16H), 1.45 (br. s, 28H), 1.23 (br. m, 12H), 1.03 (s, 4H), 0.94 (d, 3H, J = 6 Hz), 0.88 (d, 5H, J = 6 Hz). 0.69 (s, 3H).

[0396] Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 3-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-3-oxopropanoate trihydrochloride [ka] (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 9-(te To a solution of (rt-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazaheptadecan-17-oate (1.13 g, 1.18 mmol) was added hydrochloric acid (5.5 M in 2-propanol, 2.36 mL, 11.79 mmol) dropwise. The mixture was heated to 40° C. and allowed to proceed overnight. Acetonitrile (5 mL) was then added and the solution was sonicated until all solids had moved down the sides of the flask. After 30 minutes of stirring following sonication, the solid was filtered by vacuum filtration, washed repeatedly with acetonitrile, and dried under vacuum to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 3-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-3-oxopropanoate trihydrochloride as a pale purple solid (0.52 g, 0.64 mmol, 54.4%). UPLC / ELSD: RT: 1.57 min. MS(ES): C 40 H 75 For N4O3, m / z (MH + )657.2. 1 H NMR (300 MHz, CD3OD) δ: ppm 5.42 (m, 1H), 4.88 (br. s, 11H), 4.60 (m, 1H), 3.33 (br. m, 16H), 2.39 (d, 2H, J = 3 Hz), 1.55 (br. m, 40H), 0.96 (d, 4H, J = 6 Hz), 0.90 (d, 6H, J = 6 Hz), 0.74 (s, 3H).

[0397] G. Compound SA60: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(8-aminooctyl)(3-aminopropyl)carbamate dihydrochloride [ka]

[0398] Step 1: tert-butyl N-{8-[(2-cyanoethyl)amino]octyl}carbamate [ka] To a foil-covered, stirred suspension of tert-butyl N-(8-aminooctyl)carbamate (2.50 g, 10.2 mmol) in water (100 mL) was added acrylonitrile (1.00 mL, 15.3 mmol). The suspension was stirred at room temperature and monitored by TLC. At 26 h, the reaction mixture was diluted with 5% aqueous NaHCO solution (200 mL) and then extracted with EtOAc (3 × 100 mL). The combined organics were washed with brine, dried over NaSO, and concentrated. The crude material was purified via silica gel chromatography (0–10% MeOH in DCM) to provide tert-butyl N-{8-[(2-cyanoethyl)amino]octyl}carbamate (1.833 g, 6.163 mmol, 60.2%) as a yellow oil. UPLC / ELSD: RT = 0.28 min. MS(ES):m / z=197.9[(M+H)-(CH3)2C=CH2-CO2] + C 16 H 31 N3O2; 1H NMR (300 MHz, CDCl3): δ 4.50 (br. s, 1H), 3.09 (dt, 2H, 6.6, 6.5 Hz), 2.92 (t, 2H, J = 6.6 Hz), 2.62 (t, 2H, J = 7.1 Hz), 2.51 (t, 2H, J = 6.7 Hz), 1.18-1.58 (m, 13H), 1.44 (s, 9H).

[0399] Step 2: tert-butyl N-{8-[benzyl(2-cyanoethyl)amino]octyl}carbamate [ka] A mixture of tert-butyl N-{8-[(2-cyanoethyl)amino]octyl}carbamate (0.870 g, 2.92 mmol), potassium carbonate (0.808 g, 5.85 mmol), benzyl bromide (0.40 mL, 3.4 mmol), and potassium iodide (0.097 g, 0.58 mmol) in ACN (17.5 mL) was stirred at 65 °C. The reaction was monitored by LCMS. At 3 h, the reaction mixture was cooled to room temperature, filtered through a pad of Celite, rinsed with MTBE, and concentrated. The residue was taken up in 5% aqueous NaHCO solution (50 mL) and then extracted with MTBE (3 × 30 mL). The combined organics were washed with brine, dried over NaSO, and concentrated. The crude material was purified via silica gel chromatography (0-40% EtOAc in hexanes) to provide tert-butyl N-{8-[benzyl(2-cyanoethyl)amino]octyl}carbamate (0.783 g, 2.02 mmol, 69.1%) as a clear oil. UPLC / ELSD: RT = 0.44 min. MS (ES): C 23 H 37 For N3O2, m / z = 331.9 [(M+H)-(CH 3)2 C=CH2] + . 1H NMR (300 MHz, CDCl3): δ 7.21-7.39 (m, 5H), 4.49 (br. s, 1H), 3.60 (s, 2H), 3.09 (dt, 2H, J = 6.5, 6.2 Hz), 2.78 (t, 2H, J = 6.9 Hz), 2.48 (t, 2H, J = 7.4 Hz), 2.39 (t, 2H, J = 7.0 Hz), 1.38-1.54 (m, 4H), 1.44 (s, 9H), 1.19-1.36 (m, 8H).

[0400] Step 3: tert-butyl N-{3-[benzyl({8-[(tert-butoxycarbonyl)amino]octyl})amino]propyl}carbamate [ka] To a stirred solution of tert-butyl N-{8-[benzyl(2-cyanoethyl)amino]octyl}carbamate (0.492 g, 1.27 mmol) in MeOH (8.8 mL) were added di-tert-butyl dicarbonate (0.693 g, 3.17 mmol) and nickel(II) chloride hexahydrate (0.030 g, 0.13 mmol). The reaction mixture was cooled to 0 °C in an ice bath, and then NaBH (0.336 g, 8.89 mmol) was added portionwise over 30 min to give a black suspension (Caution: Vigorous gas evolution occurs during addition). The reaction mixture was stirred at room temperature and monitored by LCMS. At 17.3 h, diethylenetriamine (0.15 mL, 1.4 mmol) was added dropwise, and the reaction mixture was stirred at room temperature. After 30 min, additional diethylenetriamine (0.15 mL) was added. After 1.5 h, the reaction mixture was concentrated, taken up in 5% aqueous NaHCO3 solution, and extracted with EtOAc (3x). The combined organics were washed with 5% aqueous NaHCO3 solution and brine, dried over Na2SO4, and concentrated. The crude material was purified via silica gel chromatography (20-65% EtOAc in hexanes) to provide tert-butyl N-{3-[benzyl({8-[(tert-butoxycarbonyl)amino]octyl})amino]propyl}carbamate (0.512 g, 1.04 mmol, 82.0%) as a clear oil. UPLC / ELSD: RT = 0.92 min. MS (ES): C 28 H 49 N3O4 m / z = 492.5 [M+H] + ; 1 H NMR (300 MHz, CDCl3): δ 7.19-7.35 (m, 5H), 5.52 (br. s, 1H), 4.49 (br. s, 1H), 3.51 (s, 2H), 3.00-3.22 (m, 4H), 2.46 (t, 2H, J = 6.2 Hz), 2.36 (t, 2H, J = 7.4 Hz), 1.56-1.68 (m, 2H), 1.36-1.55 (m, 22H), 1.18-1.33 (m, 8H).

[0401] Step 4: tert-butyl N-[3-({8-[(tert-butoxycarbonyl)amino]octyl}amino)propyl]carbamate [ka] A solution of tert-butyl N-{3-[benzyl({8-[(tert-butoxycarbonyl)amino]octyl})amino]propyl}carbamate (0.496 g, 1.01 mmol) and 10% Pd / C (0.429 g, 0.202 mmol) in ethanol (10 mL) was stirred under a balloon of H. The reaction was monitored by TLC. At 3 h, the reaction mixture was diluted with EtOAc (20 mL), filtered through a pad of Celite, and rinsed with EtOAc. The filtrate was concentrated, taken up in EtOAc, and filtered using a 0.45 μm syringe filter. The filtered organics were concentrated to provide tert-butyl N-[3-({8-[(tert-butoxycarbonyl)amino]octyl}amino)propyl]carbamate (0.323 g, 0.805 mmol, 79.8%) as an off-white solid. UPLC / ELSD: RT=0.59 min. MS(ES):C 21 H 43 N3O4 m / z = 402.0 [M+H] + ; 1 H NMR (300 MHz, CDCl3): δ 5.17 (br. s, 1H), 4.50 (br. s, 1H), 3.20 (dt, 2H, J = 6.0, 6.0 Hz), 3.09 (dt, 2H, J = 6.5, 6.4 Hz), 2.67 (t, 2H, J = 6.6 Hz), 2.58 (t, 2H, J = 7.1 Hz), 1.89 (br. s, 1H), 1.59-1.74 (m, 2H), 1.37-1.55 (m, 22H), 1.21-1.37 (m, 8H).

[0402] Step 5: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(8-((tert-butoxycarbonyl)amino)octyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate [ka] Cholesterol 4-nitrophenyl carbonate (0.300 g, 0.544 mmol), tert-butyl N-[3-({8-[(tert-butoxycarbonyl)amino]octyl}amino)propyl]carbamate (0.240 g, 0.598 mmol), and triethylamine (0.12 mL, 0.85 mmol) were combined in CHCl (4.8 mL). The reaction mixture was stirred at 50 °C and monitored by TLC. At 20.25 h, tert-butyl N-[3-({8-[(tert-butoxycarbonyl)amino]octyl}amino)propyl]carbamate (77 mg) and triethylamine (0.04 mL) were added. The reaction mixture was stirred at 60 °C. At 95 h, the reaction mixture was cooled to room temperature, diluted with DCM (20 mL), and washed with water (25 mL). The aqueous layer was extracted with DCM (2 × 20 mL). The combined organics were passed through a hydrophobic frit, dried over NaSO, and concentrated. The crude material was purified via silica gel chromatography (0–30% in hexanes) to provide (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(8-((tert-butoxycarbonyl)amino)octyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate (0.398 g, 0.489 mmol, 89.9%) as a clear oil. UPLC / ELSD: RT=3.47 minutes. MS(ES):C 49 H87 For N3O6, m / z = 836.5 [M+Na] + ; 1 H NMR (300 MHz, CDCl3): δ 5.22-5.43 (m, 2H), 4.40-4.84 (m, 2H), 3.00-3.39 (br. m, 8H), 2.21-2.44 (m, 2H), 1.73-2.07 (br. m, 5H), 0.93-1.71 (br. m, 53H), 1.02 (s, 3H), 0.91 (d, 3H, J = 6.4 Hz), 0.86 (d, 3H, J = 6.6 Hz), 0.86 (d, 3H, J = 6.6 Hz), 0.67 (s, 3H).

[0403] Step 6: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(8-aminooctyl)(3-aminopropyl)carbamate dihydrochloride [ka] To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(8-((tert-butoxycarbonyl)amino)octyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate (0.395 g, 0.485 mmol) in iPrOH (2.5 mL) was added 5-6 N HCl in iPrOH (0.7 mL). The reaction mixture was stirred at 40 °C and monitored by LCMS. At 17.5 h, the reaction mixture was cooled to room temperature. ACN (5 mL) was added, the suspension was stirred for 15 min, the solid was rinsed with 2:1 ACN:iPrOH and collected by vacuum filtration to provide (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(8-aminooctyl)(3-aminopropyl)carbamate dihydrochloride (0.249 g, 0.356 mmol, 73.4%) as a white solid. UPLC / ELSD: RT=1.97 min. MS(ES): C 39 H 73 m / z = 614.4 [M+H] for Cl2N3O2 + ; 1 H NMR (300 MHz, CDCl3): δ 8.00-8.64 (br. m, 6H), 5.33-5.44 (m, 1H), 4.39-4.56 (m, 1H), 2.93-3.54 (br. m, 8H), 2.20-2.43 (m, 2H), 1.69-2.16 (br. m, 10H), 0.93-1.66 (br. m, 30H), 1.02 (s, 3H), 0.91 (d, 3H, J = 6.3 Hz), 0.87 (d, 3H, J = 6.5 Hz), 0.86 (d, 3H, J = 6.5 Hz), 0.68 (s, 3H).

[0404] H. Compound SA61: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(4-(3-aminopropoxy)butyl)(3-aminopropyl)carbamate dihydrochloride [ka]

[0405] Step 1: 3-(4-hydroxybutoxy)propanenitrile [ka] To a solution of 1,4-butanediol (8.0 mL, 91 mmol) and benzyltrimethylammonium hydroxide (0.20 mL, 1.3 mmol, 40 wt% in water) was added acrylonitrile (3.0 mL, 46 mmol). The reaction mixture was stirred at room temperature while covered with foil and monitored by TLC. At 2 h, the reaction mixture was diluted with water (150 mL) and extracted with 1:1 hexane / MTBE (50 mL) and EtOAc (3 × 50 mL). The combined organics were washed with water and brine, dried over MgSO4, and concentrated. The crude material was purified via silica gel chromatography (50–100% in hexane) to provide 3-(4-hydroxybutoxy)propanenitrile (1.374 g, 9.596 mmol, 21.0%) as a yellow oil. UPLC / ELSD: RT = 0.20 min. 1 H NMR (300 MHz, CDCl3): δ 3.67 (t, 2H, J = 6.0 Hz), 3.66 (t, 2H, J = 6.4 Hz), 3.50-3.57 (m, 2H), 2.60 (t, 2H, 6.4 Hz), 1.60-1.76 (m, 5H).

[0406] Step 2: 4-(2-cyanoethoxy)butyl methanesulfonate [ka] A stirred solution of 3-(4-hydroxybutoxy)propanenitrile (1.00 g, 6.98 mmol) and triethylamine (1.5 mL, 11 mmol) in DCM (10 mL) was cooled to 0° C. in an ice bath, and then methanesulfonyl chloride (0.60 mL, 7.8 mmol) was added dropwise. The reaction was monitored by TLC. The reaction mixture was gradually brought to room temperature. At 2 h, the reaction mixture was cooled to 0° C. in an ice bath, and additional methanesulfonyl chloride (0.06 mL) was added. At 2 h 10 min, water (10 mL) was added, and the reaction mixture was stirred at room temperature for 5 min. After this time, 5% aqueous NaHCO3 solution (50 mL) was added, and then the reaction mixture was extracted with DCM (3×30 mL). The combined organics were washed with water and brine, dried over MgSO and concentrated to provide 4-(2-cyanoethoxy)butyl methanesulfonate (1.556 g, 7.032 mmol, quantitative) as a yellow oil, which was used in the next step without further purification. 1 H NMR (300 MHz, CDCl3): δ 4.28 (t, 2H, J = 6.4 Hz), 3.64 (t, 2H, J = 6.2 Hz), 3.54 (t, 2H, J = 5.9 Hz), 3.01 (s, 3H), 2.59 (t, 2H, J = 6.2 Hz), 1.81-1.93 (m, 2H), 1.66-1.78 (m, 2H).

[0407] Step 3: tert-butyl N-(3-{[4-(2-cyanoethoxy)butyl]amino}propyl)carbamate [ka] A solution of tert-butyl N-(3-aminopropyl)carbamate (4.272 g, 24.52 mmol), 4-(2-cyanoethoxy)butyl methanesulfonate (1.550 g, 7.005 mmol), and EtOH (16 mL) was stirred at 65 °C. The reaction was monitored by TLC. At 4 h, the reaction mixture was cooled to room temperature. At 21.5 h, the reaction mixture was concentrated and then taken up in a mixture of EtOAc (75 mL) and water (75 mL). The layers were separated, and the aqueous was extracted with EtOAc (50 mL). The combined organics were washed with water (3x) and brine, dried over Na2SO4, and concentrated. The crude material was purified via silica gel chromatography (0-20% (5% concentrated aqueous NH4OH in MeOH) in DCM) to provide tert-butyl N-(3-{[4-(2-cyanoethoxy)butyl]amino}propyl)carbamate (1.396 g, 4.662 mmol, 66.6%) as a yellow oil. UPLC / ELSD: RT = 0.22 min. MS (ES): C 15 H 29 For N3O3, m / z = 243.8 [(M+H)-t-Bu] + ; 1 H NMR (300 MHz, CDCl3): δ 5.19 (br. s, 1H), 3.64 (t, 2H, J = 6.3 Hz), 3.50 (t, 2H, J = 6.0 Hz), 3.20 (dt, 2H, J = 6.2, 5.9 Hz), 2.67 (t, 2H, J = 6.6 Hz), 2.61 (t, 2H, J = 6.7 Hz), 2.59 (t, 2H, J = 6.4 Hz), 1.48-1.70 (m, 6H), 1.44 (s, 9H), 1.10 (br. s, 1H).

[0408] Step 4: tert-butyl N-(3-{benzyl[4-(2-cyanoethoxy)butyl]amino}propyl)carbamate [ka] To a mixture of tert-butyl N-(3-{[4-(2-cyanoethoxy)butyl]amino}propyl)carbamate (1.380 g, 4.609 mmol), potassium carbonate (1.274 g, 9.218 mmol), and potassium iodide (0.150 g, 0.904 mmol) in ACN (20 mL) was added benzyl bromide (0.63 mL, 5.3 mmol). The reaction mixture was stirred at 65 °C and monitored by TLC. At 2.5 h, the reaction mixture was cooled to room temperature, filtered through a pad of Celite, rinsed with ACN, and the filtrate was concentrated. The residue was taken up in 5% aqueous NaHCO solution (ca. 50 mL) and then extracted with MTBE (2 × 25 mL) and EtOAc (25 mL). The combined organics were washed with brine, dried over NaSO, and concentrated. The crude material was purified via silica gel chromatography (30-70% in hexanes) to provide tert-butyl N-(3-{benzyl[4-(2-cyanoethoxy)butyl]amino}propyl)carbamate (1.372 g, 3.522 mmol, 76.4%) as a yellow oil. UPLC / ELSD: RT=0.29 min. MS(ES): C 22 H 35 For N3O3, m / z = 390.0 [M+H] + . 1 H NMR (300 MHz, CDCl3): δ 7.20-7.39 (m, 5H), 5.41 (br. s, 1H), 3.60 (t, 2H, J = 6.4 Hz), 3.51 (s, 2H), 3.38-3.47 (m, 2H), 3.15 (dt, 2H, J = 5.8, 5.6 Hz), 2.56 (t, 2H, J = 6.4 Hz), 2.47 (t, 2H, J = 6.3 Hz), 2.35-2.43 (m, 2H), 1.51-1.69 (m, 6H), 1.44 (s, 9H).

[0409] Step 5: tert-butyl N-{3-[benzyl(4-{3-[(tert-butoxycarbonyl)amino]propoxy}butyl)amino]propyl}carbamate [ka] To a stirred solution of tert-butyl N-(3-{benzyl[4-(2-cyanoethoxy)butyl]amino}propyl)carbamate (1.357 g, 3.484 mmol) in MeOH (23 mL) were added di-tert-butyl dicarbonate (1.901 g, 8.709 mmol) and nickel(II) chloride hexahydrate (0.083 g, 0.35 mmol). The reaction mixture was cooled to 0° C. in an ice bath, and then NaBH (0.923 g, 24.4 mmol) was added portionwise over 40 min (Caution: Vigorous gas evolution occurs during addition). The reaction mixture was stirred at room temperature and monitored by LCMS. At 17.25 h, the reaction mixture was cooled to 0° C. in an ice bath, and then NaBH (500 mg) was added portionwise over 30 min. The reaction mixture was stirred at room temperature. At 18.5 h, the reaction mixture was cooled to 0 °C in an ice bath, and then NaBH (100 mg) was added. The reaction mixture was stirred at 0 °C. At 19.5 h, NaBH (101 mg) was added. At 20.5 h, NaBH (102 mg) was added. At 21.5 h, BocO (850 mg) and NaBH (103 mg) were added. The reaction mixture was gradually brought to room temperature. At 40.5 h, diethylenetriamine (0.55 mL, 5.1 mmol) was added dropwise, and the reaction mixture was stirred at room temperature for 1 h. After this time, the reaction mixture was concentrated, taken up in 5% aqueous NaHCO solution, and extracted with DCM (3x). The biphasic mixture was concentrated to remove volatile organics, and then the mixture was extracted with MTBE (3x). The combined organics were washed with brine, dried over NaSO, and concentrated. The crude material was purified via silica gel chromatography (0-7% (5% concentrated aqueous NH4OH in MeOH) in DCM) to provide tert-butyl N-{3-[benzyl(4-{3-[(tert-butoxycarbonyl)amino]propoxy}butyl)amino]propyl}carbamate (0.836 g, 1.69 mmol, 48.6%) as a pale yellow oil. UPLC / ELSD: RT=0.65 min. MS(ES): C 27 H 47 For N3O5, m / z = 494.5 [M+H] + ; 1H NMR (300 MHz, CDCl3): δ 7.19-7.38 (m, 5H), 5.43 (br. s, 1H), 4.87 (br. s, 1H), 3.51 (s, 2H), 3.43 (t, 2H, J = 5.9 Hz), 3.31-3.39 (m, 2H), 3.06-3.26 (m, 4H), 2.46 (t, 2H, J = 6.2 Hz), 2.35-2.43 (m, 2H), 1.49-1.79 (br. m, 8H), 1.44 (s, 18H).

[0410] Step 6: tert-butyl N-{3-[4-({3-[(tert-butoxycarbonyl)amino]propyl}amino)butoxy]propyl}carbamate [ka] A solution of tert-butyl N-{3-[benzyl(4-{3-[(tert-butoxycarbonyl)amino]propoxy}butyl)amino]propyl}carbamate (0.825 g, 1.67 mmol) and 10% Pd / C (0.711 g, 0.334 mmol) in EtOH (10 mL) was stirred under a balloon of H. The reaction was monitored by TLC. At 18 h, the reaction mixture was diluted with EtOAc (40 mL) and then filtered through a pad of Celite, rinsing with EtOAc. The filtrate was concentrated, taken up in EtOAc, and filtered using a 0.45 μm syringe filter. The filtered organics were concentrated to provide tert-butyl N-{3-[4-({3-[(tert-butoxycarbonyl)amino]propyl}amino)butoxy]propyl}carbamate (0.636 g, 1.58 mmol, 94.3%) as a yellow oil. UPLC / ELSD: RT=0.40 min. MS(ES):C 20 H 41 For N3O5, m / z = 404.5 [M+H] + ; 1H NMR (300 MHz, CDCl3): δ 5.20 (br. s, 1H), 4.91 (br. s, 1H), 3.47 (t, 2H, J = 5.9 Hz), 3.41 (t, 2H, J = 6.1 Hz), 3.13-3.23 (m, 4H), 2.67 (t, 2H, J = 6.6 Hz), 2.61 (t, 2H, J = 6.6 Hz), 1.48-1.80 (br. m, 9H), 1.44 (s, 18H).

[0411] Step 7: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(4-(3-((tert-butoxycarbonyl)amino)propoxy)butyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate [ka] A solution of cholesterol 4-nitrophenyl carbonate (0.663 g, 1.20 mmol), tert-butyl N-{3-[4-({3-[(tert-butoxycarbonyl)amino]propyl}amino)butoxy]propyl}carbamate (0.630 g, 1.56 mmol), and triethylamine (0.50 mL, 3.6 mmol) in PhMe (10 mL) was stirred at 90 °C. The reaction was monitored by LCMS. At 18 h, the reaction mixture was cooled to room temperature and concentrated. The residue was dissolved in DCM (50 mL) and then washed with water (3 × 30 mL). The organics were passed through a hydrophobic frit, dried over NaSO, and concentrated. The crude material was purified via silica gel chromatography (20-60% in hexanes) to provide (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(4-(3-((tert-butoxycarbonyl)amino)propoxy)butyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate (0.825 g, 1.01 mmol, 84.2%) as a sticky white foam. UPLC / ELSD: RT=3.39 min. MS(ES): C 48 H 85 N3O7 m / z = 839.2 [M+Na] + ; 1 H NMR (300 MHz, CDCl3): δ 5.34-5.43 (m, 1H), 5.30 (br. s, 1H), 4.73-5.00 (m, 1H), 4.41-4.59 (m, 1H), 3.46 (t, 2H, J = 5.9 Hz), 3.41 (t, 2H, J = 5.9 Hz), 3.00-3.36 (br. m, 8H), 2.20-2.43 (m, 2H), 0.93-2.09 (br. m, 34H), 1.43 (s, 18H), 1.02 (s, 3H), 0.91 (d, 3H, J = 6.4 Hz), 0.86 (d, 6H, J = 6.5 Hz), 0.67 (s, 3H).

[0412] Step 8: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(4-(3-aminopropoxy)butyl)(3-aminopropyl)carbamate dihydrochloride [ka] To a stirred solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(4-(3-((tert-butoxycarbonyl)amino)propoxy)butyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate (0.809 g, 0.991 mmol) in iPrOH (6.0 mL) was added 5-6 N HCl in iPrOH (1.4 mL). The reaction mixture was stirred at 40 °C and monitored by LCMS. At 15.5 h, the reaction mixture was cooled to room temperature. ACN (18 mL) was added to the reaction mixture, and the suspension was stirred at room temperature for 10 minutes. After this time, the solid was collected by vacuum filtration and rinsed with 3:1 ACN / iPrOH to provide (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(4-(3-aminopropoxy)butyl)(3-aminopropyl)carbamate dihydrochloride (0.609 g, 0.828 mmol, 83.6%) as a white solid. UPLC / ELSD: RT = 2.00 min. MS (ES): C 38 H 69 For N3O3, m / z = 617.0 [M+H] + ; 1H NMR (300 MHz, CDCl3): δ 8.51-8.82 (m, 3H), 8.05 (br. s, 3H), 5.33-5.42 (m, 1H), 4.42-4.57 (m, 1H), 3.63 (t, 2H, J = 5.4 Hz), 2.97-3.58 (br. m, 10H), 2.19-2.43 (m, 2H), 0.93-2.13 (br. m, 34H), 1.02 (s, 3H), 0.91 (d, 3H, J = 6.4 Hz), 0.86 (d, 3H, J = 6.5 Hz), 0.86 (d, 3H, J = 6.5 Hz), 0.67 (s, 3H).

[0413] I. Compound SA62: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 3-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-2-methyl-3-oxopropanoate trihydrochloride [ka]

[0414] Step 1: 1-(tert-butyl) 3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl) 2-methylmalonate [ka] To a solution of cholesterol (1.85 g, 4.69 mmol) and 3-(tert-butoxy)-2-methyl-3-oxopropanoic acid (0.96 mL, 5.63 mmol) in dichloromethane (50 mL) stirred under nitrogen was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.36 g, 7.03 mmol). The reaction mixture was then cooled to 0 °C, and diisopropylethylamine (2.48 mL, 14.07 mmol) was added dropwise over 20 minutes. The resulting mixture was allowed to warm gradually to room temperature and proceed overnight. The mixture was then diluted to 150 mL with dichloromethane, washed with water (1 × 70 mL), saturated aqueous sodium bicarbonate (2 × 70 mL), and brine (1 × 70 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to give a yellow oil. The oil was taken up in dichloromethane and purified on silica with a gradient of 0-25% ethyl acetate in hexanes to give 1-(tert-butyl) 3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl) 2-methylmalonate as an oil (1.62 g, 2.99 mmol, 63.7%). UPLC / ELSD: RT: 3.41 min. MS(ES): C 35 H 58 For O4, m / z (MH + )543.8. 1 H NMR (300 MHz, CDCl3) δ: ppm 5.31 (m, 1H), 4.58 (br. m, 1H), 3.21 (q, 1H, J = 6 Hz), 2.27 (d, 2H, J = 9 Hz), 1.87 (br. m, 6H), 1.50 (br. m, 6H), 1.39 (s, 12H), 1.28 (br. m, 12H), 1.07 (br. m, 8H), 0.95 (s, 4H), 0.91 (d, 2H, J = 6 Hz), 0.86 (d, 4H, J = 6 Hz), 0.80 (d, 8H, J = 6 Hz), 0.61 (s, 3H).

[0415] Step 2: 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl)oxy)-2-methyl-3-oxopropanoic acid [ka] A solution of 1-(tert-butyl) 3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl) 2-methylmalonate (1.62 g, 2.99 mmol) in dichloromethane (50 mL) was cooled to 0° C. To this solution was added trifluoroacetic acid (3.43 mL, 44.79 mmol) dropwise over 20 minutes. The reaction mixture was allowed to warm gradually to room temperature and proceed for 5 hours, gradually turning pale pink in color. The crude reaction mixture was concentrated in vacuo to a pink solid, taken up in DCM, and purified on silica with a gradient of 0-40% ethyl acetate in hexanes to give 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl)oxy)-2-methyl-3-oxopropanoic acid as a white solid (1.05 g, 2.16 mmol, 72.2%). UPLC / ELSD: RT: 3.02 min. MS(ES): C 31 H 50 For O4, m / z (MH + )487.7. 1H NMR (300 MHz, CDCl3) δ: ppm 11.03 (br. s, 1H), 5.40 (br. d, 1H), 4.72 (br. m, 1H), 3.49 (q, 1H, J = 6 Hz), 2.38 (d, 2H, J = 9 Hz), 2.01 (br. m, 5H), 1.61 (br. m, 5H), 1.50 (d, 5H, J = 6 Hz), 1.27 (br. m, 12H), 1.04 (s, 5H), 0.95 (d, 4H, J = 6 Hz), 0.90 (d, 6H, J = 6 Hz), 0.70 (s, 3H).

[0416] Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2,16-trimethyl-4,15-dioxo-3-oxa-5,9,14-triazaheptadecan-17-oate [ka] 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl)oxy)-2-methyl-3-oxopropanoic acid (0.50 g, 1.02 m) in dichloromethane (10 mL) under stirring under nitrogen To a solution of 1,3-dimethylaminopropyl (4-((3-((tert-butoxycarbonyl)amino)propyl)amino)butyl)carbamate (0.72 g, 1.42 mmol), dimethylaminopyridine (0.01 g, 0.10 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.30 g, 1.53 mmol) were added. The resulting solution was cooled to 0° C., and diisopropylethylamine (0.54 mL, 3.05 mmol) was added dropwise. The mixture was allowed to warm gradually to room temperature and proceed overnight. The solution was then diluted with dichloromethane, washed with water (3×10 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was taken up in DCM and purified on silica with a gradient of 0–60% ethyl acetate in hexanes to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2,16-trimethyl-4,15-dioxo-3-oxa-5,9,14-triazaheptadecan-17-oate (0.18 g, 0.19 mmol, 18.2%) as a pale yellow oil. UPLC / ELSD: RT: 3.26 minutes. MS(ES):C 56 H 98 For N4O9, m / z (MH + )972.4. 1H NMR (300 MHz, CDCl3) δ: ppm 5.36 (br. s, 1H), 4.58 (br. m, 1H), 4.10 (q, 1H, J = 6 Hz), 3.36 (br. m, 13H), 2.26 (br. m, 3H), 2.01 (s, 4H), 1.80 (br. m, 10H), 1.43 (br. m, 47H), 1.23 (t, 4H, J = 9 Hz), 1.08 (br. m, 7H), 0.97 (s, 8H), 0.90 (d, 4H, J = 9 Hz), 0.84 (d, 6H, J = 6 Hz), 0.65 (s, 3H).

[0417] Step 4: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 3-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-2-methyl-3-oxopropanoate trihydrochloride [ka] (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 9-(tert-butyl)-2-(2-methyl-1H-cyclopenta[a]heterocyclyl) ... To a solution of (tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2,16-trimethyl-4,15-dioxo-3-oxa-5,9,14-triazaheptadecan-17-oate (0.18 g, 0.19 mmol) was added hydrochloric acid (5.5 M in isopropanol, 0.37 mL, 1.85 mmol) dropwise. The mixture was heated to 45° C. and stirred overnight. The solution was then cooled to room temperature, and acetonitrile (5 mL) was added to the mixture. It was then sonicated to remove precipitated solids from the sides of the flask. After sonication and stirring for 30 minutes, the solid was filtered by vacuum filtration, washed repeatedly with acetonitrile, and dried in vacuo to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 3-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-2-methyl-3-oxopropanoate trihydrochloride as a white solid (0.06 g, 0.07 mmol, 38.7%). UPLC / ELSD: RT: 1.70 min. MS(ES): C 41 H 77 For Cl3N4O3, m / z (MH + )672.1. 1H NMR (300 MHz, CD3OD) δ: ppm 5.41 (s, 1H), 4.88 (br. m, 10H), 4.58 (br. m, 1H), 3.92 (br. m, 1H), 3.56 (br. m, 4H), 3.33 (s, 3H), 3.10 (br. m, 8H), 2.34 (br. m, 2H), 2.05 (br. m, 15H), 1.54 (br. m, 8H), 1.38 (br. m, 8H), 1.17 (d, 9H, J = 6 Hz), 1.06 (s, 6H), 0.97 (d, 4H, J = 6 Hz), 0.90 (d, 6H, J = 6 Hz), 0.73 (s, 3H).

[0418] J. Compound SA63: (3S,8S,9S,10R,13S,14S,17S)-17-(2-hydroxy-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 4-(bis(3-(dimethylamino)propyl)amino)-4-oxobutanoate [ka]

[0419] Step 1: (3S,8S,9S,10R,13S,14S,17S)-17-(2-hydroxy-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-ol [ka] A mixture of magnesium turnings (2.21 g, 90.87 mmol) and iodine (1.54 g, 6.06 mmol) was purged twice with vacuum and nitrogen, then maintained under nitrogen. Dry tetrahydrofuran (50 mL) was added to the mixture and stirred under nitrogen. 1-Bromo-4-methylpentane (8.82 mL, 60.58 mmol) was added dropwise to the mixture over 10 minutes, and the reaction was allowed to proceed for 1 hour at room temperature. The reaction mixture was then refluxed at 66°C for 3 hours, during which time the gray reaction slurry turned to a clear, colorless solution with some undissolved magnesium. The reaction was then cooled to 0°C, at which point the solution became cloudy again. At 0°C, a solution of pregnenolone (5.75 g, 18.17 mmol) in dry tetrahydrofuran (25 mL) was added dropwise over 1 hour, during which time the reaction mixture solidified. The solution was then warmed to room temperature, an additional 50 mL of tetrahydrofuran was added, and the reaction was continued at 30° C. overnight, during which time the solidified mixture broke into smaller pieces with stirring in the additional solvent. The reaction was quenched the next day with saturated aqueous ammonium chloride (50 mL) and then diluted with 100 mL of ethyl acetate. The aqueous layer was separated and extracted again with 100 mL of ethyl acetate. The organic layers were then combined, washed with water (1×100 mL) and brine (1×100 mL), dried over sodium sulfate, filtered, and concentrated to dryness. The resulting residue was taken up in DCM and purified on silica with a gradient of 0-50% ethyl acetate in hexanes to give (3S,8S,9S,10R,13S,14S,17S)-17-(2-hydroxy-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-ol as a white solid (2.68 g, 6.64 mmol, 36.6%). UPLC / ELSD: RT: 2.13 min. MS(ES): C 27 H 46 For O2, m / z (MH + )403.7. 1H NMR (300 MHz, CDCl3) δ: ppm 5.36 (br. d, 1H, J = 6 Hz), 3.54 (br. m, 1H), 2.29 (br. m, 2H), 2.06 (br. m, 2H), 1.85 (br. m, 16H), 1.29 (s, 6H), 1.17 (br. m, 6H), 1.03 (s, 6H), 0.88 (d, 10H, J = 6 Hz).

[0420] Step 2: 4-(((3S,8S,9S,10R,13S,14S,17S)-17-(2-hydroxy-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl)oxy)butanoic acid [ka] To a solution of (3S,8S,9S,10R,13S,14S,17S)-17-(2-hydroxy-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-ol (0.50 g, 1.24 mmol) in dichloromethane (10 mL) stirred under nitrogen was added succinic anhydride (0.12 g, 1.24 mmol). Pyridine (0.17 mL, 1.24 mmol) was then added dropwise at room temperature, and the mixture was refluxed at 40 °C overnight, at which time all solids had dissolved. The next day, TLC revealed incomplete conversion, and dimethylaminopyridine (0.05 g, 0.41 mmol) and succinic anhydride (0.03 g, 0.25 mmol) were added, after which the reaction mixture was refluxed again overnight at 40° C. The following morning, the mixture was concentrated in vacuo to a yellow oil. The yellow oil was taken up in dichloromethane and purified on silica with a gradient of 0-30% ethyl acetate in hexanes to give 4-(((3S,8S,9S,10R,13S,14S,17S)-17-(2-hydroxy-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl)oxy)butanoic acid as a white solid (0.27 g, 0.54 mmol, 43.3%). UPLC / ELSD: RT: 2.20 min. MS(ES): C 31 H 50 Regarding O5, m / z (MH + )503.8. 1H NMR (300 MHz, CDCl3) δ: ppm 6.60 (br. s, 1H), 5.38 (br. s, 1H), 4.64 (br. m, 1H), 4.13 (q, 1H, J = 6 Hz), 2.66 (dd, 4H, J = 6 Hz), 2.33 (d, 2H, J = 6 Hz), 2.05 (br. m, 2H), 1.84 (br. m, 3H), 1.51 (br. m, 12H), 1.28 (br. m, 8H), 1.13 (br. m, 5H), 1.02 (s, 4H), 0.86 (d, 10H, J = 6 Hz).

[0421] Step 3: (3S,8S,9S,10R,13S,14S,17S)-17-(2-hydroxy-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 4-(bis(3-(dimethylamino)propyl)amino)-4-oxobutanoate [ka] To a solution of 4-(((3S,8S,9S,10R,13S,14S,17S)-17-(2-hydroxy-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl)oxy)butanoic acid (0.35 g, 0.68 mmol) in dichloromethane (10 mL) stirred under nitrogen was added tetramethyldipropylenetriamine (0.24 mL, 1.02 mmol), dimethylaminopyridine (0.01 g, 0.07 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.20 g, 1.02 mmol). The resulting solution was cooled to 0° C. and diisopropylethylamine (0.36 mL, 2.04 mmol) was added dropwise. The mixture was allowed to warm gradually to room temperature and proceed overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate (1×10 mL) and brine (1×10 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was taken up in DCM and purified on silica with a gradient of 0-60% (80:19:1 DCM / MeOH / NH4OH) in DCM to give (3S,8S,9S,10R,13S,14S,17S)-17-(2-hydroxy-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 4-(bis(3-(dimethylamino)propyl)amino)-4-oxobutanoate) as a pale yellow oil (0.07 g, 0.09 mmol, 13.6%). UPLC / ELSD: RT: 1.25 min. MS(ES): C 41 H 73 For N3O4, m / z (MH + )673.0. 1H NMR (300 MHz, CDCl3) δ: ppm 5.37 (br. s, 1H), 4.64 (br. m, 1H), 3.35 (br. t, 4H, J = 9 Hz), 2.64 (s, 4H), 2.28 (br. m, 6H), 2.22 (s, 12H), 1.83 (br. m, 4H), 1.60 (br. m, 15H), 1.28 (br. s, 7H), 1.13 (br. m, 5H), 1.02 (s, 4H), 0.89 (d, 9H, J = 6 Hz).

[0422] K. Compound SA64: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(8-(dimethylamino)octyl)(3-(dimethylamino)propyl)carbamate [ka] To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(8-aminooctyl)(3-aminopropyl)carbamate (105 mg, 0.15 mmol) and sodium acetate trihydrate (208 mg, 1.53 mmol) in 6 mL of methanol at room temperature was added formaldehyde (0.12 mL, 37 wt% in water, 1.53 mmol) and sodium cyanoborohydride (96.1 mg, 1.53 mmol). The solution was stirred at room temperature for 16 hours, after which time no starting aminosterol remained by LCMS. The mixture was diluted with 2M aqueous NaOH solution and extracted three times with DCM. The organics were combined, washed once with brine, dried (MgSO), filtered, and concentrated. The residue was purified by silica gel chromatography (0-50% (mixture of 1% concentrated aqueous NH4OH and 20% MeOH in DCM) in DCM) to give 3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(8-(dimethylamino)octyl)(3-(dimethylamino)propyl)carbamate (63.2 mg, 0.091 mmol, 60%) as a colorless oil. UPLC / ELSD: RT = 2.14 min. MS (ES): C 43 H 80 For N3O2, m / z (MH + )671.2. 1H NMR (300 MHz, CDCl3) δ: ppm 5.35 (d, 1H, J = 5 Hz);4.48 (septet, 1H, J = 5 Hz);3.19 (s, 4H);2.39-2.27 (m, 12 H);2.25 (s, 6H);2.22 (s, 0.66 (s, 6H);2.03-1.63 (m, 8H);1.58-1.04 (m, 23H);1.00 (s, 6H);0.90 (d, 3H, J = 6 Hz);0.86 (d, 3H, J = 1 Hz) 3H).

[0423] L. Compound SA65: ((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl N-(3-aminopropyl)-N-(4-((3-aminopropyl)amino)butyl)glycinate tetrahydrochloride [ka]

[0424] Step 1: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 2-chloroacetate [ka] Cholesterol (2 g, 5.17 mmol), chloroacetic acid (573 mg, 5.69 mmol), DMAP (63 mg, 0.52 mmol), and DCC (1.17 g, 5.69 mmol) were dissolved in 10 mL of DCM. The solution was stirred at room temperature for 17 hours. The mixture was filtered, and the filtrate was washed with ethyl acetate. The filtered solution was concentrated and dissolved in ethyl acetate. The organic layer was washed once with water and brine, dried (MgSO), filtered, and concentrated. The residue was purified by silica gel chromatography (0–40% ethyl acetate in hexanes) to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 2-chloroacetate (0.71 g, 1.53 mmol, 30%) as a white solid. UPLC / ELSD: RT = 3.43 min. 1 H NMR (300 MHz, CDCl3) δ: ppm 5.40 (d, 1H, J = 5 Hz);4.48 (septet, 1H, J = 4 Hz);4.03 (s, 2H);2.36 (d, 2 H, J = 8 Hz);2.06-1.77 (m, 5H);1.64-1.05 (m, 21H);1.02 (s, 3H);0.91 (d, 3H, J = 6 Hz);0.88 (s, 3H);0.86 (s, 3H);0.68 (s, 3H).

[0425] Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4-oxo-3-oxa-5,9,14-triazahexadecan-16-oate [ka] A solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 2-chloroacetate (350 mg, 0.75 mmol) and NaI (1 To a solution of tert-butyl N-{3-[(tert-butoxycarbonyl)amino]propyl}-N-[4-({3-[(tert-butoxycarbonyl)amino]propyl}amino)butyl]carbamate (378 mg, 0.75 mmol) and N,N-diisopropylethylamine (0.2 mL, 1.13 mmol) in 7.5 mL of acetonitrile was added. The solution was stirred at 60° C. for 18 h. The mixture was diluted with ethyl acetate, washed once with water and brine, dried over MgSO4, filtered, and concentrated. The residue was purified by silica gel chromatography (0 to 100% (1% NHOH, 20% MeOH in DCM mixture) in DCM) to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4-oxo-3-oxa-5,9,14-triazahexadecan-16-oate (590 mg, 0.63 mmol, 84%) as a colorless oil. UPLC / ELSD: RT=2.94 minutes. MS(ES):C 54 H 97 For N4O8, m / z (MH + )930.0. 1H NMR (300 MHz, CDCl3) δ: ppm 5.36 (d, 1H, J = 5 Hz);5.27 (br s, 1H);4.80 (br s, 1H);4.74-4.57 (m, 1H);3.40 (br s, 1H);3.31-2.99 (m, 7H);2.75 (br s, 3 H);2.31 (d, 2 H, J = 8 Hz);2.06-1.03 (m, 64H);1.00 (s, 3H);0.91 (d, 3H, J = 6 Hz);0.87 (s, 3H);0.85 (s, 3H);0.67 (s, 3H).

[0426] Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl N-(3-aminopropyl)-N-(4-((3-aminopropyl)amino)butyl)glycinate tetrahydrochloride [ka] To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4-oxo-3-oxa-5,9,14-triazahexadecan-16-oate (590 mg, 0.64 mmol) in isopropanol (15 mL) was added 5 M HCl solution in isopropanol (15 mL, 6.4 mmol). The solution was stirred at 40°C for 41 hours. The mixture was cooled to room temperature and diluted with acetonitrile (15 mL). The resulting solid was precipitated by centrifugation (5000g, 5 min). The supernatant was removed, and the pellet was dried under vacuum to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl N-(3-aminopropyl)-N-(4-((3-aminopropyl)amino)butyl)glycinate (350 mg, 0.45 mmol, 71%) as a white powder. UPLC / ELSD: RT = 1.83 min. MS (ES): C 39 H 73 For N4O2, m / z ([M-HCl-Cl - ] + )629.6. 1 H NMR (300 MHz, CD3OD) δ: ppm 5.43 (d, 1H, J = 4 Hz);4.80-4.66 (m, 1H);4.28 (s, 2H);3.49-3.33 (m, 4H);3.22-3.02 (m, 8H);2.43 (d, 2H, J = 7 Hz);2.28-1.09 (m, 29H);1.06 (s, 3H);0.95 (d, 3H, J = 6 Hz);0.89 (s, 3H);0.87 (s, 3H);0.73 (s, 3H).

[0427] M. Compound SA66: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 2-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-2-oxoacetate trihydrochloride [ka]

[0428] Step 1: 2-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl)oxy)-2-oxoacetic acid [ka] To a stirred solution of cholesterol (0.500 g, 1.29 mmol) in a mixture of EtO (6.5 mL) and DCM (2.0 mL) cooled to 0 °C in an ice bath, oxalyl chloride (0.23 mL, 2.7 mmol) was slowly added dropwise. The reaction mixture was gradually brought to room temperature and monitored by TLC. At 24 h, the reaction mixture was cooled to 0 °C in an ice bath, and then water (3.0 mL) was added dropwise (Caution: Vigorous gas evolution occurred during the addition). The mixture was stirred at room temperature for 1 h, and then the layers were separated. The aqueous layer was extracted with EtO (3x). The combined organics were washed with brine, dried over Na2SO4, and concentrated to provide 2-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl)oxy)-2-oxoacetic acid (0.534 g, 1.16 mmol, 90.0%) as a white solid. UPLC / ELSD: RT = 2.95 min. 1 H NMR (300 MHz, CDCl3): δ 5.68 (br. s, 1H), 5.38-5.46 (m, 1H), 4.75-4.89 (m, 1H), 2.35-2.61 (m, 2H), 1.70-2.11 (br. m, 6H), 0.93-1.65 (br. m, 20H), 1.04 (s, 3H), 0.92 (d, 3H, J = 6.5 Hz), 0.87 (d, 3H, J = 6.6 Hz), 0.86 (d, 3H, J = 6.5 Hz), 0.68 (s, 3H).

[0429] Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 2-chloro-2-oxoacetate [ka] To a solution of 2-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl)oxy)-2-oxoacetic acid (0.100 g, 0.218 mmol) and DMF (catalytic) in DCM (2 mL) was added oxalyl chloride (0.03 mL, 0.4 mmol) slowly dropwise. The reaction mixture was stirred at room temperature and monitored by LCMS. At 40 min, the reaction mixture was concentrated and then reconcentrated from PhMe to provide (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 2-chloro-2-oxoacetate as a yellow solid. The material was used without further purification assuming quantitative yield.

[0430] Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazahexadecan-16-oate [ka] (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 2-chloro-2-oxoacetate (0.1 mL) in toluene (2.0 mL) cooled to 0° C. in an ice bath To a stirred solution of tert-butyl N-{3-[(tert-butoxycarbonyl)amino]propyl}-N-[4-({3-[(tert-butoxycarbonyl)amino]propyl}amino)butyl]carbamate (0.150 g, 0.298 mmol) in toluene (0.75 mL) was added dropwise. The reaction mixture was stirred at room temperature and monitored by LCMS. At 30 min, the reaction mixture was stirred at 50 °C. At 17 h, the reaction mixture was cooled to room temperature and then concentrated. The residue was taken up in DCM and washed with 5% aqueous NaHCO3 solution. The organics were passed through a hydrophobic frit, dried over Na2SO4, and concentrated. The crude material was purified via silica gel chromatography (0–50% in hexanes) to provide (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazahexadecan-16-oate (0.138 g, 0.146 mmol, 67.1%) as a yellow oil. UPLC / ELSD: RT=3.33 minutes. MS(ES):C 54 H 94 m / z = 844.4 for N4O9 [(M+H)-(CH3)2C=CH2-CO2] + ; 1H NMR (300 MHz, CDCl3): δ 5.38-5.45 (m, 1H), 5.21 (br. s, 1H), 4.65-4.87 (m, 2H), 3.32-3.47 (m, 2H), 3.02-3.31 (br. m, 10H), 2.35-2.53 (m, 2H), 0.94-2.08 (br. m, 34H), 1.46 (s, 9H), 1.44 (s, 18H), 1.02 (s, 3H), 0.92 (d, 3H, J = 6.4 Hz), 0.87 (d, 3H, J = 6.6 Hz), 0.86 (d, 3H, J = 6.5 Hz), 0.68 (s, 3H).

[0431] Step 4: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 2-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-2-oxoacetate trihydrochloride [ka] To a stirred solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazahexadecan-16-oate (0.132 g, 0.140 mmol) in iPrOH (1.3 mL) was added 5-6 N HCl in iPrOH (0.28 mL). The reaction mixture was stirred at 40° C. and monitored by LCMS. At 18 hours, the reaction mixture was cooled to room temperature. ACN (3 mL) was added to the reaction mixture, and the suspension was stirred at room temperature for 1 hour. After this time, the solid was collected by vacuum filtration to provide (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 2-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-2-oxoacetate trihydrochloride (0.085 g, 0.10 mmol, 73.2%) as a white solid. UPLC / ELSD: RT=1.70 minutes. MS(ES):C 39 H 70 N4O3 m / z = 643.8 [M+H] + ; 1H NMR (300 MHz, CD3OD): δ 5.42-5.51 (m, 1H), 4.72-4.85 (m, 1H), 3.34-3.61 (br. m, 4H), 3.04-3.19 (br. m, 6H), 2.92-3.01 (m, 2H), 2.37-2.54 (m, 2H), 0.98-2.19 (br. m, 34H), 1.08 (s, 3H), 0.96 (d, 3H, J = 6.4 Hz), 0.89 (d, 3H, J = 6.6 Hz), 0.89 (d, 3H, J = 6.6 Hz), 0.74 (s, 3H).

[0432] N. Compound SA67: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(4-(3-(dimethylamino)propoxy)butyl)(3-(dimethylamino)propyl)carbamate [ka] To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(4-(3-aminopropoxy)butyl)(3-aminopropyl)carbamate (100 mg, 0.15 mmol) and sodium acetate trihydrate (197.5 mg, 1.45 mmol) in 5.8 mL of methanol at room temperature was added formaldehyde (0.11 mL, 37 wt% in water, 1.45 mmol) and sodium cyanoborohydride (91.2 mg, 1.45 mmol). The solution was stirred at room temperature for 6 hours, after which time no starting aminosterol remained by LCMS. The mixture was diluted with 2M aqueous NaOH solution and extracted three times with DCM. The organics were combined, washed once with brine, dried (MgSO), filtered, and concentrated. The residue was purified by silica gel chromatography (0-20% (mixture of 1% NH4OH and 20% MeOH in DCM) in DCM) to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(4-(3-(dimethylamino)propoxy)butyl)(3-(dimethylamino)propyl)carbamate (35.6 mg, 0.051 mmol, 35%) as a colorless oil. UPLC / ELSD: RT = 2.01 min. MS (ES): C 42 H 78 For N3O3, m / z (MH + )673.0. 1H NMR (300 MHz, CDCl3) δ: ppm 5.36 (d, 1H, J = 5 Hz);4.49 (septet, 1H, J = 5 Hz);3.48-3.37 (m, 4H);3.23 (s, 4H);2.48-2.30 (m, 12 H);2.28 (s, 6H);2.24 (s, 6H);2.04-1.66 (m, 10H);1.64-1.04 (m, 15H);1.01 (s, 6H);0.91 (d, 3H, J = 6 Hz);0.87 (d, 3H, J = 1 Hz);0.85 (d, 3H, J = 1 Hz);0.67 (s, 3H).

[0433] O. Compound SA68: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(3-(dimethylamino)propyl)(4-((3-(dimethylamino)propyl)(methyl)amino)butyl)carbamate [ka] To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(3-aminopropyl)(4-((3-aminopropyl)amino)butyl)carbamate trihydrochloride (144 mg, 0.2 mmol) and sodium acetate trihydrate (162.3 mg, 1.19 mmol) in 2 mL of methanol at room temperature was added formaldehyde (0.094 mL, 37 wt% in water, 1.19 mmol) and sodium cyanoborohydride (75 mg, 1.19 mmol). The solution was stirred at room temperature for 17 hours, after which time no starting aminosterol remained by LCMS. The mixture was diluted with 2M aqueous NaOH solution and extracted three times with DCM. The organics were combined, washed once with brine, dried (MgSO), filtered, and concentrated. The residue was purified by silica gel chromatography (0-20% (mixture of 2% concentrated aqueous NH4OH and 20% MeOH in DCM) in DCM) to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(3-(dimethylamino)propyl)(4-((3-(dimethylamino)propyl)(methyl)amino)butyl)carbamate (31.7 mg, 0.044 mmol, 22%) as a colorless oil. UPLC / ELSD: RT = 1.71 min. MS (ES): C 43 H 81 For N4O2, m / z (MH + )685.6. 1H NMR (300 MHz, CDCl3) δ: ppm 5.36 (d, 1H, J = 5 Hz);4.49 (septet, 1H, J = 5 Hz);3.47 (s, 4H);3.22 (s, 4H);2.38-2.24 (m, 12 H);2.22 (s, 6H);2.21 (s, 6H);2.20 (s, 3H);2.05-1.95 (m, 2H);1.72-1.06 (m, 23H);1.01 (s, 6H);0.91 (d, 3H, J = 6 Hz);0.87 (d, 3H, J = 1 Hz);0.85 (d, 3H, J = 1 Hz);0.67 (s, 3H).

[0434] P. Compound SA69: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(8-aminooctyl)(3-aminopropyl)carbamate dihydrochloride [ka]

[0435] Step 1: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(8-((tert-butoxycarbonyl)amino)octyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate [ka] β-Sitosterol 4-nitrophenyl carbonate (0.300 g, 0.517 mmol), tert-butyl N-[3-({8-[(tert-butoxycarbonyl)amino]octyl}amino)propyl]carbamate (0.260 g, 0.647 mmol), and triethylamine (0.22 mL, 1.6 mmol) were combined in PhMe (4.5 mL). The reaction mixture was stirred at 90 °C and monitored by LCMS. At 18.25 h, the reaction mixture was cooled to room temperature and concentrated. The residue was taken up in DCM (20 mL) and washed with water (3x). The organic layer was passed through a hydrophobic frit, dried over Na2SO4, and concentrated. The crude material was purified via silica gel chromatography (20-50% in hexanes) to provide (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(8-((tert-butoxycarbonyl)amino)octyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate (0.327 g, 0.388 mmol, 75.0%) as a white foam. UPLC / ELSD: RT=3.74 min. MS(ES): C 51 H 91 N3O6 m / z = 842.9 [M+H] + ; 1 H NMR (300 MHz, CDCl3): δ 5.15-5.47 (m, 2H), 4.40-4.86 (m, 2H), 2.98-3.41 (br. m, 8H). 2.20-2.45 (m, 2H), 1.76-2.12 (br. m, 5H), 0.89-1.75 (br. m, 54H), 1.02 (s, 3H), 0.92 (d, 3H, J = 6.4 Hz), 0.77-0.88 (m, 9H), 0.68 (s, 3H).

[0436] Step 2: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(8-aminooctyl)(3-aminopropyl)carbamate dihydrochloride [ka] To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(8-((tert-butoxycarbonyl)amino)octyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate (0.315 g, 0.374 mmol) in iPrOH (4.0 mL) was added 5-6 N HCl in iPrOH (0.53 mL). The reaction mixture was stirred at 40 °C and monitored by LCMS. At 18 h, the reaction mixture was cooled to room temperature and ACN (12 mL) was added. The solid was collected via vacuum filtration and rinsed with 3:1 ACN / iPrOH to provide (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(8-aminooctyl)(3-aminopropyl)carbamate dihydrochloride (0.236 g, 0.309 mmol, 82.5%) as a white solid. UPLC / ELSD: RT = 3.54 min. MS (ES): C 41 H 77 m / z = 342.6 for Cl2N3O2 [M+2Na] 2+ ; 1H NMR (300 MHz, CDCl3): δ 8.33 (br. s, 3H), 8.22 (br. s, 3 H), 5.31-5.42 (m, 1H), 4.38-4.53 (m, 1H), 2.92-3.53 (br. m, 8H), 2.20-2.42 (m, 2H), 1.72-2.17 (br. m, 10H), 0.94-1.71 (br. m, 31H), 1.02 (s, 3H), 0.92 (d, 3H, J = 6.3 Hz), 0.77-0.89 (m, 9H), 0.68 (s, 3H).

[0437] Q. Compound SA70 [ka] SA70 ((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(3-aminopropyl)(4-((3-aminopropyl)amino)butyl)carbamate analog olefin elimination by-product from hydroxycholesterol) R. Compound SA71: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl N-(8-aminooctyl)-N-(3-aminopropyl)glycinate trihydrochloride [ka]

[0438] Step 1: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl N-(8-((tert-butoxycarbonyl)amino)octyl)-N-(3-((tert-butoxycarbonyl)amino)propyl)glycinate [ka] Cholesteryl chloroacetate (0.227 g, 0.490 mmol), tert-butyl N-[3-({8-[(tert-butoxycarbonyl)amino]octyl}amino)propyl]carbamate (0.236 g, 0.589 mmol), potassium carbonate (0.136 g, 0.980 mmol), and potassium iodide (0.008 g, 0.05 mmol) were combined in THF (3.5 mL). The reaction mixture was stirred at 65° C. and monitored by LCMS. At 4 h, the reaction mixture was stirred at 60° C. At 93 h, the reaction mixture was cooled to room temperature. The reaction mixture was concentrated and then taken up in DCM. The organics were washed with water, passed through a hydrophobic frit, dried over Na2SO4, and concentrated. The crude material was purified via silica gel chromatography (20-50% in hexanes) to provide (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl N-(8-((tert-butoxycarbonyl)amino)octyl)-N-(3-((tert-butoxycarbonyl)amino)propyl)glycinate (0.318 g, 0.384 mmol, 78.3%) as a clear oil. UPLC / ELSD: RT=3.62 min. MS(ES): C 50 H 89 N3O6 m / z = 829.0 [M+H] + ; 1H NMR (300 MHz, CDCl3): δ 5.45 (br. s, 1H), 5.35-5.41 (m, 1H), 4.58-4.72 (m, 1H), 4.50 (br. s, 1H), 3.25 (s, 2H), 3.20 (dt, 2H, J = 5.7, 6.0 Hz), 3.09 (dt, 2H, J = 6.4, 5.8 Hz), 2.59 (t, 2H, J = 6.4 Hz), 2.50 (t, 2H, J = 7.5 Hz), 2.28-2.36 (m, 2H), 1.75-2.08 (br. m, 5H), 0.94-1.70 (br. m, 53H), 1.02 (s, 3H), 0.91 (d, 3H, J = 6.5 Hz), 0.87 (d, 3H, J = 6.5 Hz), 0.86 (d, 3H, J = 6.6 Hz), 0.68 (s, 3H).

[0439] Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl N-(8-aminooctyl)-N-(3-aminopropyl)glycinate trihydrochloride [ka] To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl N-(8-((tert-butoxycarbonyl)amino)octyl)-N-(3-((tert-butoxycarbonyl)amino)propyl)glycinate (0.310 g, 0.374 mmol) in iPrOH (4.0 mL) was added 5-6 N HCl in iPrOH (0.53 mL). The reaction mixture was stirred at 40 °C and monitored by LCMS. At 21.75 h, the reaction mixture was cooled to room temperature and ACN (12 mL) was added. The solid was collected via vacuum filtration and rinsed with 3:1 ACN / iPrOH to provide (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl N-(8-aminooctyl)-N-(3-aminopropyl)glycinate trihydrochloride (0.208 g, 0.248 mmol, 66.4%) as a white solid. UPLC / ELSD: RT = 1.83 min. MS (ES): C 40 H 73 For N3O2, m / z = 335.4 [M+2Na] 2+ ; 1 H NMR (300 MHz, CDCl3): δ 10.72 (br. s, 1H), 8.41 (br. s, 3H), 8.27 (br. s, 3H), 5.38-5.48 (m, 1H), 4.59-4.82 (m, 1H), 2.91-4.42 (br. m, 10H), 2.22-2.72 (br. m, 4H), 1.72-2.18 (br. m, 10H), 0.93-1.70 (br. m, 28H), 1.01 (s, 3H), 0.91 (d, 3H, J = 5.5 Hz), 0.86 (d, 6H, J = 6.5Hz), 0.67 (s, 3H).

[0440] S. Compound SA72: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 4-((8-aminooctyl)(3-aminopropyl)amino)-4-oxobutanoate dihydrochloride [ka]

[0441] Step 1: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 4-((8-((tert-butoxycarbonyl)amino)octyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-4-oxobutanoate [ka] To a stirred solution of (-)-cholesterol NHS succinate (0.300 g, 0.514 mmol) in THF (3.0 mL) was added tert-butyl N-[3-({8-[(tert-butoxycarbonyl)amino]octyl}amino)propyl]carbamate (0.258 g, 0.642 mmol) in THF (1.0 mL). The reaction mixture was stirred at room temperature and monitored by LCMS. At 19 h, the reaction mixture was stirred at 50° C. At 23 h, the reaction mixture was cooled to room temperature and then concentrated. The residue was taken up in DCM and washed with water. The organics were passed through a hydrophobic frit, dried over Na2SO4, and concentrated. The crude material was purified via silica gel chromatography (20-50% in hexanes) to provide (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 4-((8-((tert-butoxycarbonyl)amino)octyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-4-oxobutanoate (0.315 g, 0.362 mmol, 70.4%) as a white foam. UPLC / ELSD: RT=3.96 min. MS(ES): C 52 H 91 N3O7 m / z = 871.0 [M+H] + ; 1 H NMR (300 MHz, CDCl3): δ 5.27-5.46 (m, 2H), 4.39-4.76 (m, 2H), 2.95-3.48 (br. m, 8H), 2.53-2.72 (m, 4H), 2.24-2.39 (m, 2H), 1.75-2.06 (br. m, 5H), 0.93-1.70 (br. m, 53H), 1.01 (s, 3H), 0.91 (d, 3H, J = 6.4 Hz), 0.86 (d, 3H, J = 6.5 Hz), 0.86 (d, 3H, J = 6.6 Hz), 0.67 (s, 3H).

[0442] Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 4-((8-aminooctyl)(3-aminopropyl)amino)-4-oxobutanoate dihydrochloride [ka] To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 4-((8-((tert-butoxycarbonyl)amino)octyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-4-oxobutanoate (0.307 g, 0.347 mmol) in iPrOH (4 mL) was added 5-6 N HCl in iPrOH (0.49 mL). The reaction mixture was stirred at 40 °C and monitored by LCMS. At 21.75 h, the reaction mixture was cooled to room temperature, and then ACN (16 mL) was added. The solid was collected via vacuum filtration and rinsed with 4:1 ACN / iPrOH to provide (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 4-((8-aminooctyl)(3-aminopropyl)amino)-4-oxobutanoate dihydrochloride (0.169 g, 0.214 mmol, 61.8%) as a white solid. UPLC / ELSD: RT = 2.15 min. MS (ES): C 42 H 75 For N3O3, m / z = 336.0 [M+2H] 2+ ; 1H NMR (300 MHz, CDCl3): δ 8.01-8.61 (m, 6H), 5.31-5.42 (m, 1H), 4.51-4.69 (m, 1H), 2.92-3.68 (br. m, 8H), 2.62 (s, 4H), 2.21-2.39 (m, 2H), 1.71-2.20 (br. m, 10H), 0.94-1.70 (br. m, 30H), 1.01 (s, 3H), 0.91 (d, 3H, J = 6.4 Hz), 0.86 (d, 6H, J = 6.5 Hz), 0.67 (s, 3H).

[0443] T. Compound SA73: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl N-(3-aminopropyl)-N-(4-((3-aminopropyl)amino)butyl)alaninate trihydrochloride [ka]

[0444] Step 1: 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2,15-trimethyl-4-oxo-3-oxa-5,9,14-triazahexadecano-16-oic acid [ka] To a solution of tert-butyl (3-((tert-butoxycarbonyl)amino)propyl)(4-((3-((tert-butoxycarbonyl)amino)propyl)amino)butyl)carbamate (0.83 g, 1.65 mmol) and potassium hydroxide (0.37 g, 6.60 mmol) in methanol (10 mL) stirred under nitrogen was added 2-bromopropionic acid (0.30 mL, 3.30 mmol) dropwise at room temperature. The resulting solution was heated to 60° C. and allowed to proceed overnight. The next day, the solution was concentrated to an oil. The oil was taken up in dichloromethane and purified on silica with a gradient of 0-60% ethyl acetate in hexanes to give 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2,15-trimethyl-4-oxo-3-oxa-5,9,14-triazahexadecano-16-oic acid as an oil (0.13 g, 0.22 mmol, 13.2%). UPLC / ELSD: RT: 2.73 min. MS(ES): C 28 H 54 For N4O8, m / z (MH + )575.8. 1 H NMR (300 MHz, CDCl3) δ: ppm 7.31 (br. s, 1H), 5.72 (br. s, 1H), 3.17 (br. m, 13H), 1.90 (br. m, 2H), 1.64 (br. m, 7H), 1.40 (s, 26H).

[0445] Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2,15-trimethyl-4-oxo-3-oxa-5,9,14-triazahexadecan-16-oate [ka] To a solution of cholesterol (0.10 g, 0.26 mmol) and 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2,15-trimethyl-4-oxo-3-oxa-5,9,14-triazahexadecan-16-oic acid (0.13 g, 0.22 mmol) in dichloromethane (10 mL) stirred under nitrogen was added dimethylaminopyridine (0.01 g, 0.04 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.06 g, 0.33 mmol). The resulting solution was cooled to 0 °C, and diisopropylethylamine (0.12 mL, 0.65 mmol) was added dropwise. The mixture was allowed to warm gradually to room temperature and proceed overnight. The solution was then diluted with dichloromethane, washed with water (1 x 10 mL), saturated aqueous sodium bicarbonate (1 x 10 mL) and brine (1 x 10 mL), dried over sodium sulfate, filtered and concentrated to an oil. The oil was taken up in DCM and purified on silica with a gradient of 0–25% ethyl acetate in hexanes to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2,15-trimethyl-4-oxo-3-oxa-5,9,14-triazahexadecan-16-oate (0.05 g, 0.05 mmol, 21.9%) as a colorless oil. UPLC / ELSD: RT: 2.83 minutes. MS(ES):C 55 H 98 For N4O8, m / z (MH + ) 944.4. The compound was not analyzed by H-NMR to avoid losing valuable material required for the following reaction.

[0446] Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl N-(3-aminopropyl)-N-(4-((3-aminopropyl)amino)butyl)alaninate trihydrochloride [ka] (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl 9-(te To a solution of (rt-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2,15-trimethyl-4-oxo-3-oxa-5,9,14-triazahexadecan-16-oate (0.05 g, 0.05 mmol) was added hydrochloric acid (5.5 M in 2-propanol, 0.10 mL, 0.48 mmol) dropwise. The mixture was heated to 45° C. and stirred overnight. The solution was then cooled to room temperature, and acetonitrile (3 mL) was added to the mixture. It was then sonicated to remove precipitated solids from the sides of the flask. After sonication and stirring for 30 minutes, the solid was filtered by vacuum filtration, washed repeatedly with acetonitrile, and dried in vacuo to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl N-(3-aminopropyl)-N-(4-((3-aminopropyl)amino)butyl)alaninate trihydrochloride as a white solid (0.03 g, 0.03 mmol, 62.8%). UPLC / ELSD: RT: 1.51 min. MS(ES): C 40 H 77For Cl3N4O2, m / z (MH + )644.1. 1 H NMR (300 MHz, CD3OD) δ: ppm 5.54 (br. s, 1H), 4.50 (br. m, 1H), 3.33 (br. d, 8H), 3.12 (br. m, 9H), 2.45 (br. m, 2H), 2.00 (br. m, 15H), 1.55 (br. m, 17H), 1.19 (br. m, 14H), 0.96 (d, 4H, J = 6 Hz), 0.90 (d, 7H, J = 6 Hz), 0.74 (s, 3H).

[0447] U. Compound SA74: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(4-aminobutan-2-yl)(4-((4-aminobutan-2-yl)amino)butyl)carbamate trihydrochloride [ka]

[0448] Step 1: tert-butyl (3-((4-nitrophenyl)sulfonamido)butyl)carbamate [ka] To a solution of tert-butyl (3-aminobutyl)carbamate (1.00 g, 5.31 mmol) in dry DCM (15 mL) stirred under nitrogen was added triethylamine (0.89 mL, 6.37 mmol). The solution was cooled to 0° C., and then a solution of 4-nitrobenzenesulfonyl chloride (1.30 g, 5.84 mmol) in 5 mL of dry DCM was added dropwise over 30 minutes. The reaction was allowed to proceed at 0° C. for 1 hour, then at room temperature for an additional 3 hours. The mixture was then diluted with an additional 10 mL of DCM and washed with 1 M aqueous sodium bicarbonate (2 x 15 mL), water (1 x 15 mL), 10% aqueous citric acid (2 x 15 mL), water (1 x 15 mL), and brine (2 x 15 mL), dried over sodium sulfate, filtered, and concentrated to give tert-butyl (3-((4-nitrophenyl)sulfonamido)butyl)carbamate as a white solid (1.95 g, 5.22 mmol, 98.3%). UPLC / ELSD: RT = 0.54 min. MS (ES): C 15 H 23 For N3O6S, m / z (MH + )374.4. 1 H NMR (300 MHz, CDCl3) δ: ppm 8.07 (m, 1H), 7.78 (m, 1H), 7.68 (m, 1H), 5.23 (m, 1H), 4.81 (br. s, 1H), 3.52 (m, 1H), 3.19 (m, 1H), 3.05 (m, 1H), 1.63 (m, 2H), 1.37 (s, 9H), 0.98 (d, 3H, J = 6 Hz).

[0449] Step 2: Di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(butane-3,1-diyl)) dicarbamate [ka] To a solution of tert-butyl (3-((4-nitrophenyl)sulfonamido)butyl)carbamate (1.95 g, 5.22 mmol) in dry DMF (20 mL) stirred under nitrogen, potassium carbonate (2.10 g, 15.17 mmol) and 1,4-diiodobutane (0.33 mL, 2.49 mmol) were added. The solution was heated to 40 °C and allowed to proceed overnight. The next morning, benzyl bromide (0.25 mL, 2.06 mmol) was added, and the reaction was allowed to proceed at room temperature for 24 h. Thiophenol (0.98 mL, 9.57 mmol), potassium carbonate (1.03 g, 7.46 mmol), and an additional 5 mL of dry DMF were then added, and the reaction was again allowed to proceed overnight. The next morning, the salts were removed from the supernatant via multiple rounds of centrifugation and rinsing with DMF. The combined supernatants were concentrated in vacuo to give an oil that was taken up in 40 mL of DCM, washed with water (2 × 10 mL) and brine (2 × 5 mL), dried over potassium carbonate, filtered, and concentrated to an oil. The oil was retaken in DCM and purified via silica gel chromatography in DCM with a gradient of 0 to 60% (70:20:10 DCM / MeOH / concentrated aqueous ammonium hydroxide). Product-containing fractions were combined and concentrated to give di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(butane-3,1-diyl)) dicarbamate as a colorless oil (0.76 g, 1.77 mmol, 71.0%). UPLC / ELSD: RT = 0.42 min. MS (ES): C 22 H 46 For N4O4, m / z (MH + )431.6. 1 H NMR (300 MHz, CDCl3) δ: ppm 5.47 (m, 2H), 3.24 (br. m, 4H), 2.74 (br. m, 4H), 2.55 (m, 2H), 1.53 (m, 10H), 1.44 (s, 18H), 1.09 (d, 6H, J = 6 Hz).

[0450] Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(4-((tert-butoxycarbonyl)amino)butan-2-yl)(4-((4-((tert-butoxycarbonyl)amino)butan-2-yl)amino)butyl)carbamate [ka] To a solution of di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(butane-3,1-diyl))dicarbamate (0.49 g, 1.15 mmol) in dry toluene (10 mL) stirred under nitrogen was added triethylamine (0.48 mL, 3.43 mmol). Then, (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(4-nitrophenyl)carbonate (0.63 g, 1.15 mmol) was added, and the solution was heated to 90 °C and allowed to proceed overnight. The next morning, the reaction mixture was cooled to room temperature, and the solution was washed with water (3 × 10 mL), dried over sodium sulfate, filtered, and concentrated to give an oil. The oil was taken up in DCM and purified via silica gel chromatography in DCM with a gradient of 0–30% (80:19:1 DCM / MeOH / concentrated aqueous ammonium hydroxide). The product-containing fractions were combined and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(4-((tert-butoxycarbonyl)amino)butan-2-yl)(4-((4-((tert-butoxycarbonyl)amino)butan-2-yl)amino)butyl)carbamate as a colorless oil (0.78 g, 0.92 mmol, 80.6%). UPLC / ELSD: RT=2.62 min. MS(ES): C 50 H 90 For N4O6, m / z (MH + )844.3. 1H NMR (300 MHz, CDCl3) δ: ppm 5.28 (m, 1H), 3.14 (br. m, 5H), 2.59 (m, 4H), 2.25 (m, 3H), 1.90 (br. m, 7H), 1.46 (br. m, 22H), 1.34 (s, 23H), 1.09 (br. m, 28H), 0.83 (d, 5H, J = 6 Hz), 0.79 (d, 7H, J = 6 Hz), 0.59 (s, 3H).

[0451] Step 4: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(4-aminobutan-2-yl)(4-((4-aminobutan-2-yl)amino)butyl)carbamate trihydrochloride [ka] To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(4-((tert-butoxycarbonyl)amino)butan-2-yl)(4-((4-((tert-butoxycarbonyl)amino)butan-2-yl)amino)butyl)carbamate (0.78 g, 0.92 mmol) in isopropanol (10 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 1.85 mL, 9.23 mmol) dropwise. The solution was heated to 40 °C and allowed to proceed overnight. The next morning, dry acetonitrile (6 mL) was added to the mixture, which was sonicated and stirred for an additional 1 h. The white solid was then filtered from the solution, washed repeatedly with acetonitrile, and dried in vacuo to give SA74 (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(4-aminobutan-2-yl)(4-((4-aminobutan-2-yl)amino)butyl)carbamate trihydrochloride as a white solid (0.57 g, 0.73 mmol, 78.8%). UPLC / ELSD: RT=1.67 minutes. MS(ES):C 40 H 77 For Cl3N4O2, m / z (MH + )753.4. 1H NMR (300 MHz, CDCl3) δ: ppm 5.42 (m, 1H), 4.49 (br. m, 1H), 4.12 (br. m, 1H), 3.45 (br. m, 1H), 3.33 (s, 2H), 3.24 (br. m, 2H), 3.13 (br. m, 4H), 2.90 (br. m, 2H), 2.41 (d, 2H, J = 3 Hz), 2.32 (br. m, 1H), 1.93 (br. m, 19H), 1.43 (d, 6H, J = 6 Hz), 1.31 (d, 5H, J = 6 Hz), 1.08 (br. m, 12H), 0.97 (d, 4H, J = 6 Hz), 0.90 (d, 6H, J = 6 Hz), 0.75 (s, 3H).

[0452] V. Compound SA75: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(3-amino-2-methylpropyl)(4-((3-amino-2-methylpropyl)amino)butyl)carbamate trihydrochloride [ka]

[0453] Step 1: tert-butyl (2-methyl-3-((4-nitrophenyl)sulfonamido)propyl)carbamate [ka] To a solution of tert-butyl (3-amino-2-methylpropyl)carbamate (1.00 g, 5.31 mmol) in dry DCM (15 mL) stirred under nitrogen was added triethylamine (0.89 mL, 6.37 mmol). The solution was cooled to 0° C., and then a solution of 4-nitrobenzenesulfonyl chloride (1.30 g, 5.84 mmol) in 5 mL of dry DCM was added dropwise over 30 minutes. The reaction was allowed to proceed at 0° C. for 1 hour, then at room temperature for an additional 3 hours. The mixture was then diluted with an additional 10 mL of DCM, washed with 1 M aqueous sodium bicarbonate (2 x 15 mL), water (1 x 15 mL), 10% aqueous citric acid (2 x 15 mL), water (1 x 15 mL), and brine (2 x 15 mL), dried over sodium sulfate, filtered, and concentrated to give tert-butyl (2-methyl-3-((4-nitrophenyl)sulfonamido)propyl)carbamate as a white solid (2.20 g, 5.90 mmol, quantitative). UPLC / ELSD: RT = 0.58 min. MS (ES): C 15 H 23 For N3O6S, m / z (MH + )374.4. 1 H NMR (300 MHz, CDCl3) δ: ppm 8.12 (m, 1H), 7.84 (m, 1H), 7.74 (m, 1H), 6.20 (br. s, 1H), 4.82 (br. s, 1H), 3.19 (m, 1H), 3.04 (m, 3H), 1.84 (m, 1H), 1.41 (s, 9H), 0.91 (d, 3H, J = 6 Hz).

[0454] Step 2: Di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(2-methylpropane-3,1-diyl)) dicarbamate [ka] To a solution of tert-butyl (2-methyl-3-((4-nitrophenyl)sulfonamido)propyl)carbamate (2.20 g, 5.90 mmol) in dry DMF (20 mL) stirred under nitrogen, potassium carbonate (2.37 g, 17.14 mmol) and 1,4-diiodobutane (0.37 mL, 2.81 mmol) were added. The solution was heated to 40 °C and allowed to proceed overnight. The next morning, benzyl bromide (0.28 mL, 2.33 mmol) was added, and the reaction was allowed to proceed at room temperature for 24 h. Thiophenol (1.11 mL, 10.82 mmol), potassium carbonate (1.17 g, 8.43 mmol), and an additional 5 mL of dry DMF were then added, and the reaction was again allowed to proceed overnight. The next morning, the salts were removed from the supernatant via multiple rounds of centrifugation and rinsing with DMF. The combined supernatants were concentrated in vacuo to an oil, which was taken up in 40 mL of DCM, washed with water (2 × 10 mL) and brine (2 × 5 mL), dried over potassium carbonate, filtered, and concentrated to an oil. The oil was retaken in DCM and purified via silica gel chromatography in DCM with a gradient of 0 to 60% (70:20:10 DCM / MeOH / concentrated aqueous ammonium hydroxide). Product-containing fractions were combined and concentrated to give di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(2-methylpropane-3,1-diyl)) dicarbamate as a colorless oil (0.85 g, 1.97 mmol, 70.0%). UPLC / ELSD: RT = 0.43 min. MS (ES): C 22 H 46 For N4O4, m / z (MH + )431.6. 1 H NMR (300 MHz, CDCl3) δ: ppm 5.76 (m, 1H), 2.93 (m, 2H), 2.73 (m, 2H), 2.28 (m, 8H), 1.56 (m, 4H), 1.28 (s, 4H), 1.18 (s, 17H), 0.66 (d, 6H, J = 6 Hz).

[0455] Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(3-((tert-butoxycarbonyl)amino)-2-methylpropyl)(4-((3-((tert-butoxycarbonyl)amino)-2-methylpropyl)amino)butyl)carbamate [ka] To a solution of di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(2-methylpropane-3,1-diyl))dicarbamate (1.06 g, 2.45 mmol) in dry toluene (20 mL) stirred under nitrogen was added triethylamine (0.86 mL, 6.13 mmol). Then, (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(4-nitrophenyl)carbonate (1.13 g, 2.04 mmol) was added, and the solution was heated to 90 °C and allowed to proceed overnight. The next morning, the reaction mixture was cooled to room temperature, and the solution was washed with water (3 × 10 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was taken up in DCM and purified via silica gel chromatography in DCM with a gradient of 0 to 30% (80:19:1 DCM / MeOH / concentrated aqueous ammonium hydroxide). The product-containing fractions were combined and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(3-((tert-butoxycarbonyl)amino)-2-methylpropyl)(4-((3-((tert-butoxycarbonyl)amino)-2-methylpropyl)amino)butyl)carbamate as a colorless oil (1.08 g, 1.28 mmol, 62.8%). UPLC / ELSD: RT=2.52 min. MS(ES): C 50 H 90 For N4O6, m / z (MH + )844.3. 1H NMR (300 MHz, CDCl3) δ: ppm 5.29 (m, 1H), 4.42 (br. m, 1H), 3.07 (br. m, 5H), 2.87 (m, 3H), 2.51 (m, 4H), 2.25 (br. m, 2H), 1.79 (br. m, 7H), 1.46 (m, 8H), 1.34 (s, 18H), 1.05 (br. m, 10H), 0.94 (s, 5H), 0.82 (m, 14H), 0.59 (s, 3H).

[0456] Step 4: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(3-amino-2-methylpropyl)(4-((3-amino-2-methylpropyl)amino)butyl)carbamate trihydrochloride [ka] To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(3-((tert-butoxycarbonyl)amino)-2-methylpropyl)(4-((3-((tert-butoxycarbonyl)amino)-2-methylpropyl)amino)butyl)carbamate (1.08 g, 1.28 mmol) in isopropanol (10 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 2.57 mL, 12.83 mmol) dropwise. The solution was heated to 40 °C and allowed to proceed overnight. The next morning, dry acetonitrile (6 mL) was added to the mixture, which was sonicated and stirred for an additional 1 h. The white solid was then filtered from the solution, washed repeatedly with acetonitrile, and dried in vacuo to give SA75 (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(3-amino-2-methylpropyl)(4-((3-amino-2-methylpropyl)amino)butyl)carbamate trihydrochloride as a white solid (0.66 g, 0.85 mmol, 66.1%). UPLC / ELSD: RT=1.59 minutes. MS(ES):C 40 H 77 For Cl3N4O2, m / z (MH + )753.4. 1 H NMR (300 MHz, CDCl3) δ: ppm 5.42 (m, 1H), 4.46 (br. m, 1H), 3.33 (br. m, 4H), 3.12 (br. m, 5H), 2.95 (m, 4H), 2.40 (d, 3H, J = 9 Hz), 1.75 (br. m, 19H), 1.20 (br. m, 9H), 1.08 (m, 8H), 0.97 (d, 4H, J = 6 Hz ), 0.89 (d, 6H, J = 6 Hz), 0.75 (s, 3H).

[0457] W. Compound SA76: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]heterocyclyl-3-yl(3-aminobutyl)(4-((3-aminobutyl)amino)butyl)carbamate trihydrochloride [ka]

[0458] Step 1: tert-butyl (4-((4-nitrophenyl)sulfonamido)butan-2-yl)carbamate [ka] To a solution of tert-butyl (4-aminobutan-2-yl)carbamate (1.00 g, 5.31 mmol) in dry DCM (15 mL) stirred under nitrogen was added triethylamine (0.89 mL, 6.37 mmol). The solution was cooled to 0° C., and then a solution of 4-nitrobenzenesulfonyl chloride (1.30 g, 5.84 mmol) in 5 mL of dry DCM was added dropwise over 30 minutes. The reaction was allowed to proceed at 0° C. for 1 hour, then at room temperature for an additional 3 hours. The mixture was then diluted with an additional 10 mL of DCM and washed with 1 M aqueous sodium bicarbonate (2 x 15 mL), water (1 x 15 mL), 10% aqueous citric acid (2 x 15 mL), water (1 x 15 mL), and brine (2 x 15 mL), dried over sodium sulfate, filtered, and concentrated to give tert-butyl (4-((4-nitrophenyl)sulfonamido)butan-2-yl)carbamate as a white solid (1.47 g, 3.94 mmol, 74.1%). UPLC / ELSD: RT = 0.61 min. MS (ES): C 15 H 23 For N3O6S, m / z (MH + )374.4. 1H NMR (300 MHz, CDCl3) δ: ppm 8.00 (m, 1H), 7.81 (m, 2H), 7.73 (m, 2H), 6.15 (br. s, 1H), 4.27 (br. s, 1H), 3.64 (br. s, 1H), 3.19 (br. s, 1H), 2.95 (br. s, 1H), 1.64 (m, 1H), 1.30 (s, 10H), 1.00 (d, 3H, J = 6 Hz).

[0459] Step 2: Di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(butane-4,2-diyl)) dicarbamate [ka] To a solution of tert-butyl (4-((4-nitrophenyl)sulfonamido)butan-2-yl)carbamate (1.47 g, 3.94 mmol) in dry DMF (20 mL) stirred under nitrogen, potassium carbonate (1.58 g, 11.44 mmol) and 1,4-diiodobutane (0.25 mL, 1.88 mmol) were added. The solution was heated to 40 °C and allowed to proceed overnight. The next morning, benzyl bromide (0.19 mL, 1.56 mmol) was added, and the reaction was allowed to proceed at room temperature for 24 h. Thiophenol (0.74 mL, 7.22 mmol), potassium carbonate (0.78 g, 5.62 mmol), and an additional 5 mL of dry DMF were then added, and the reaction was again allowed to proceed overnight. The next morning, the salts were removed from the supernatant via multiple rounds of centrifugation and rinsing with DMF. The combined supernatants were concentrated in vacuo to an oil, which was taken up in 40 mL of DCM...

Claims

1. A lipid amine compound of formula A1: 【Chemistry 1】 or a salt thereof (wherein Z is N or CH; R １ is C １－１４ Alkyl, C １－１４ alkenyl, or C １－１４ is hydroxyalkyl; R ２ and R ３ are respectively, C ２－２０ is alkyl, (i) Said C ２－２０ Alkyl is —NR ８ R ９ , OH, and halo, wherein at least one substituent is —NR ８ R ９ and (ii) the C ２－２０ 1, 2, 3, or 4 non-terminal carbons of the alkyl are optionally replaced with O; (iii) the C ２－２０ One, two, three, or four non-terminal carbons of an alkyl may be NR １０ is optionally replaced by; (iv) the C ２－２０ 1, 2, 3, or 4 non-terminal carbons of the alkyl are optionally replaced with C(=O); (v) C above ２－２０ One, two, three, or four non-terminal carbons of the alkyl may be CR ａ R ｂ and optionally replaced by R ａ and R ｂ together with the C atoms to which they are attached, ３－６ Forming a cycloalkyl group; R ２ and R ３ are the same or different; or R ２ and R ３ together with the N atom to which they are attached, form one, two, or three ring-forming NR １０ and forming a 7- to 18-membered heterocycloalkyl group containing a group, said 7- to 18-membered heterocycloalkyl group being １－４ Alkyl, —NR ８ R ９ optionally substituted with 1, 2, or 3 substituents independently selected from , OH, and halo; or R ２ , R ３ , and R ６ together with the atoms to which they are attached and any intervening atoms, represent C １－４ Alkyl, —NR ８ R ９ forming a 7- to 18-membered bridged heterocycloalkyl group optionally substituted with 1, 2, or 3 substituents independently selected from , OH, and halo; R ４ , R ５ , R ６ , and R ７ are H, halo, and C, respectively. １－４ independently selected from alkyl; or R ４ and R ５ together with the carbon atoms to which they are attached, C ３－７ Forming a cycloalkyl group; or R ６ and R ７ together with the carbon atoms to which they are attached, C ３－７ Forming a cycloalkyl group; R ８ , R ９ , and R １０ are H and C, respectively. １－４ independently selected from alkyl; j is 0 or 1; k is 0, 1, 2, 3, 4, 5, or 6; l is 0 or 1; m is 0, 1, 2, 3, 4, 5, or 6; n is 0 or 1; If j is 0, then l is 1; j and l are not both 0, However, the compound is 【Chemistry 2-1】 【Chemistry 2-2】 [Chemistry 2-3] other than).

2. A lipid amine compound of formula A1: 【Transformation 3】 or a salt thereof (wherein Z is N or CH; R １ is C １－１４ Alkyl, C １－１４ alkenyl, or C １－１４ is hydroxyalkyl; R ２ and R ３ are respectively, C ２－２０ is alkyl, (i) Said C ２－２０ Alkyl is —NR ８ R ９ , OH, and halo, wherein at least one substituent is —NR ８ R ９ and (ii) the C ２－２０ 1, 2, 3, or 4 non-terminal carbons of the alkyl are optionally replaced with O; (iii) the C ２－２０ One, two, three, or four non-terminal carbons of an alkyl may be NR １０ is optionally replaced by; R ２ and R ３ are the same or different; or R ２ and R ３ together with the N atom to which they are attached, form one, two, or three ring-forming NR １０ and forming a 7- to 18-membered heterocycloalkyl group containing a group, said 7- to 18-membered heterocycloalkyl group being １－４ Alkyl, —NR ８ R ９ optionally substituted with 1, 2, or 3 substituents independently selected from , OH, and halo; or R ２ , R ３ , and R ６ together with the atoms to which they are attached and any intervening atoms, represent C １－４ Alkyl, —NR ８ R ９ forming a 7- to 18-membered bridged heterocycloalkyl group optionally substituted with 1, 2, or 3 substituents independently selected from , OH, and halo; R ４ , R ５ , R ６ , and R ７ are H, halo, and C, respectively. １－４ independently selected from alkyl; or R ４ and R ５ together with the carbon atoms to which they are attached, C ３－７ Forming a cycloalkyl group; or R ６ and R ７ together with the carbon atoms to which they are attached, C ３－７ Forming a cycloalkyl group; R ８ , R ９ , and R １０ are H and C, respectively. １－４ independently selected from alkyl; j is 0 or 1; k is 0, 1, 2, 3, 4, 5, or 6; l is 0 or 1; m is 0, 1, 2, 3, 4, 5, or 6; n is 0 or 1; If j is 0, then l is 1; j and l are not both 0, However, the compound is 【Chemistry 4-1】 【Chemistry 4-2】 【Chemistry 4-3】 other than).

3. R １ teeth, 【Transformation 5】 3. The compound of claim 1 or 2, wherein

4. (A)R ２ and R ３ are respectively, C ２－１０ independently selected from alkyl, (i) Said C ２－１０ Alkyl is —NR ８ R ９ , OH, and halo, wherein at least one substituent is —NR ８ R ９ and (ii) the C ２－１０ one or two non-terminal carbons of the alkyl are optionally replaced with O; (iii) the C ２－１０ One or two non-terminal carbons of the alkyl may be NR １０ is optionally replaced by; (iv) the C ２－１０ one or two non-terminal carbons of the alkyl are optionally replaced with C(=O); (v) C above ２－１０ One or two non-terminal carbons of the alkyl may be CR ａ R ｂ and optionally replaced by R ａ and R ｂ together with the C atoms to which they are attached, ３－６ forming a cycloalkyl group, or (B) one of R 2 and R 3 is C 2-5 alkyl, said C 2-5 alkyl being substituted with 1, 2, 3, 4, or 5 substituents independently selected from —NR 8 R 9 , OH, and halo, and at least one substituent is —NR 8 R 9 ; the other of R 2 and R 3 is C 7-10 alkyl; (i) said C 7-10 alkyl is substituted with 1, 2, 3, 4, or 5 substituents independently selected from —NR 8 R 9 , OH, and halo, wherein at least one substituent is —NR 8 R 9 ; (ii) 1, 2, 3, or 4 non-terminal carbons of said C 7-10 alkyl are optionally replaced with O; (iii) 1, 2, 3, or 4 non-terminal carbons of said C 7-10 alkyl are optionally replaced with NR 10 ; (iv) 1, 2, 3, or 4 non-terminal carbons of said C 7-10 alkyl are optionally replaced with C(═O); (v) 1, 2, 3, or 4 non-terminal carbons of said C 7-10 alkyl are optionally replaced with CR a R b , and R a and R b together with the C atom to which they are attached form a C 3-6 cycloalkyl group; The compound of claim 1, or a salt thereof.

5. (A)R ２ and R ３ are respectively, C ２－１０ independently selected from alkyl, (i) Said C ２－１０ Alkyl is —NR ８ R ９ , OH, and halo, wherein at least one substituent is —NR ８ R ９ and (ii) the C ２－１０ one or two non-terminal carbons of the alkyl are optionally replaced with O; (iii) the C ２－１０ One or two non-terminal carbons of the alkyl may be NR １０ or (B) one of R 2 and R 3 is C 2-5 alkyl; (i) said C 2-5 alkyl is substituted with 1, 2, 3, 4, or 5 substituents independently selected from —NR 8 R 9 , OH, and halo, wherein at least one substituent is —NR 8 R 9 ; the other of R 2 and R 3 is C 7-10 alkyl; (i) said C 7-10 alkyl is substituted with 1, 2, 3, 4, or 5 substituents independently selected from —NR 8 R 9 , OH, and halo, wherein at least one substituent is —NR 8 R 9 ; (ii) 1, 2, 3, or 4 non-terminal carbons of said C 7-10 alkyl are optionally replaced with O; (iii) 1, 2, 3, or 4 non-terminal carbons of said C 7-10 alkyl are optionally replaced with NR 10 ; or (C) one of R 2 and R 3 is C 3 alkyl substituted with at least one —NR 8 R 9 group and optionally further substituted with one or two groups selected from OH and halo; or (D) R 2 and R 3 together with the N atom to which they are attached form a 7- to 18-membered heterocycloalkyl group containing 1, 2, or 3 ring-forming NR 10 groups, said 7- to 18-membered heterocycloalkyl group being optionally substituted with 1, 2, or 3 substituents independently selected from C 1-4 alkyl, —NR 8 R 9 , OH, and halo; or (E) R 2 and R 3 together with the N atom to which they are attached form an 8- to 10-membered heterocycloalkyl group containing 1, 2, or 3 ring-forming NR 10 groups, said 8- to 10-membered heterocycloalkyl group being optionally substituted with 1, 2, or 3 substituents independently selected from C 1-4 alkyl, —NR 8 R 9 , OH, and halo; 3. The compound according to claim 1 or 2, or a salt thereof.

6. R ２ , R ３ , and R ６ together with the atoms to which they are attached and any intervening atoms, represent C １－４ Alkyl, —NR ８ R ９ 3. The compound according to claim 1 or 2, or a salt thereof, which forms a 7- to 18-membered bridged heterocycloalkyl group optionally substituted with 1, 2, or 3 substituents independently selected from , OH, and halo.

7. R ４ and R ５ are each independently H or C １－４ 3. The compound according to claim 1 or 2, or a salt thereof, wherein the aryl group is alkyl.

8. R ６ and R ７ are each independently H or C １－４ 3. The compound according to claim 1 or 2, or a salt thereof, wherein the aryl group is alkyl.

9. R ８ , R ９ , and R １０ are each independently selected from H and methyl, or a salt thereof.

10. Formula A2: 【Transformation 6】 3. The compound according to claim 1 or 2, wherein the compound has the formula: 【Request Item 11】 【Table 1-1】 Table 1-2 Table 1-3 Table 1-4 Table 1-5 Table 1-6 Table 1-7 Table 1-8 Table 1-9 or a salt thereof. 【Request Item 12】 【Table 2-1】 Table 2-2 Table 2-3 Table 2-4 or a salt thereof. 【Request Item 13】 【Table 3-1】 Table 3-2 Table 3-3 Table 3-4 Table 3-5 Table 3-6 Table 3-7 Table 3-8 Table 3-9 Table 3-10 or a salt thereof.

14. A lipid nanoparticle composition comprising the lipid amine compound according to claim 1 or 2, or a salt thereof.

15. The lipid nanoparticle composition comprises: (i) an ionizable lipid; (ii) phospholipids, (iii) structured lipids, and (iv) optionally a PEG-lipid, and (v) optionally a payload for delivery to the cell The lipid nanoparticle composition of claim 14, further comprising:

16. The ionizable lipid is a compound of formula (I): 【Transformation 7】 or an N-oxide or salt thereof, wherein R １ teeth, 【Transformation 8】 and 【Chemistry 9】 indicates the point of attachment; R ａα , R ａβ , R ａγ , and R ａδ is H, C ２－１２ Alkyl, and C ２－１２ alkenyl; R ２ and R ３ is C １－１４ Alkyl and C ２－１４ alkenyl; R ４ is -(CH ２ ) ｎ OH and 【Chemistry 10】 is selected from n is selected from 1, 2, 3, 4, and 5; 【Chemistry 11】 indicates the point of attachment, R １０ is N(R) ２ and Each R is C １－６ Alkyl, C ２－３ alkenyl, and H; n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; Each R ５ is C １－３ Alkyl, C ２－３ alkenyl, and H; Each R ６ is C １－３ Alkyl, C ２－３ alkenyl, and H; M and M' are each independently selected from -C(O)O- and -OC(O)-; R' is C １－１２ Alkyl or C ２－１２ alkenyl; l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13. The lipid nanoparticle composition of claim 15, wherein

17. The ionizable lipid is 【Chemistry 12】 or an N-oxide or salt thereof.

18. The phospholipid is a compound of formula (IV): 【Chemistry 13】 or a salt thereof (wherein Each R １ are independently H or optionally substituted alkyl; or optionally two R １ are joined together with the intervening atoms to form an optionally substituted monocyclic cycloalkyl or an optionally substituted monocyclic heterocyclyl; or optionally, three R １ are joined together with the intervening atoms to form an optionally substituted bicyclic cycloalkyl or an optionally substituted bicyclic heterocyclyl; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is a compound of the formula: 【Chemistry 14】 of; L ２ Each instance of is independently a bond or an optionally substituted C １－６ alkylene, wherein said optionally substituted C １－６ One methylene unit of alkylene is —O—, —N(R Ｎ )-, -S-, -C(O)-, -C(O)N(R Ｎ ) -, -NR Ｎ C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R Ｎ ) -, -NR Ｎ C(O)O-, or -NR Ｎ C(O)N(R Ｎ )-optionally replaced by; R ２ Each instance of is independently an optionally substituted C １－３０ Alkyl, optionally substituted C １－３０ alkenyl, or optionally substituted C １－３０ alkynyl; optionally R ２ one or more methylene units in each of the groups independently represent an optionally substituted cycloalkylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, —N(R Ｎ )-, -O-, -S-, -C(O)-, -C(O)N(R Ｎ ) -, -NR Ｎ C(O)-, -NR Ｎ C(O)N(R Ｎ )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R Ｎ ) -, -NR Ｎ C(O)O-, -C(O)S-, -SC(O)-, -C(=NR Ｎ ) -, -C(=NR Ｎ ) N (R Ｎ ) -, -NR Ｎ C (=NR Ｎ ) -, -NR Ｎ C (=NR Ｎ ) N (R Ｎ )-, -C(S)-, -C(S)N(R Ｎ ) -, -NR Ｎ C(S)-, -NR Ｎ C(S)N(R Ｎ )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O) ２ -, -S(O) ２ O-, -OS(O) ２ O-, -N(R Ｎ )S(O)-, -S(O)N(R Ｎ ) -, -N(R Ｎ ) S ( O ) N ( R Ｎ )-,-OS(O)N(R Ｎ ) -, -N(R Ｎ )S(O)O-, -S(O) ２ -, -N(R Ｎ ) S (O) ２ -, -S(O) ２ N (R Ｎ ) -, -N(R Ｎ ) S (O) ２ N (R Ｎ ) -, -OS(O) ２ N (R Ｎ ) - or -N(R Ｎ ) S (O) ２ is replaced by O-; R Ｎ each instance of is independently hydrogen, an optionally substituted alkyl, or a nitrogen protecting group; Ring B is an optionally substituted cycloalkyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl; p is 1 or 2; However, the compound has the formula: 【Chemistry 15】 (In the formula, R ２ Each instance of is independently an unsubstituted alkyl, an unsubstituted alkenyl, or an unsubstituted alkynyl. The lipid nanoparticle composition of claim 15, wherein

19. The phospholipids include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether). PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (ME 16.0 PE), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (ME 16.0 PE). PC), 1,2-diphytanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (4ME 16:0 PG), 1,2-diphytanoyl-sn-glycero-3-phospho-L-serine (sodium salt) (4ME 16:0 16. The lipid nanoparticle composition of claim 15, wherein the lipid nanoparticle is selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin.

20. 16. The lipid nanoparticle composition of claim 15, wherein the structural lipid is cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, or mixtures thereof.

21. The PEG-lipid is a compound of formula (V): 【Chemistry 16】 or a salt thereof (wherein R ３ is -OR Ｏ and R Ｏ is hydrogen, an optionally substituted alkyl, or an oxygen protecting group; r is an integer from 1 to 100, inclusive; L １ is an optionally substituted C １－１０ alkylene, wherein said optionally substituted C １－１０ At least one methylene of the alkylene is independently an optionally substituted cycloalkylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, —O—, —N(R Ｎ )-, -S-, -C(O)-, -C(O)N(R Ｎ ) -, -NR Ｎ C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R Ｎ ) -, -NR Ｎ C(O)O-, or -NR Ｎ C(O)N(R Ｎ ) - is replaced by; D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is a compound of the formula: 【Chemistry 17】 of; L ２ Each instance of is independently a bond or an optionally substituted C １－６ alkylene, wherein said optionally substituted C １－６ One methylene unit of alkylene is —O—, —N(R Ｎ )-, -S-, -C(O)-, -C(O)N(R Ｎ ) -, -NR Ｎ C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R Ｎ ) -, -NR Ｎ C(O)O-, or -NR Ｎ C(O)N(R Ｎ )-optionally replaced by; R ２ Each instance of is independently an optionally substituted C １－３０ Alkyl, optionally substituted C １－３０ alkenyl, or optionally substituted C １－３０ alkynyl; optionally R ２ one or more methylene units in each of the groups independently represent an optionally substituted cycloalkylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, —N(R Ｎ )-, -O-, -S-, -C(O)-, -C(O)N(R Ｎ ) -, -NR Ｎ C(O)-, -NR Ｎ C(O)N(R Ｎ )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R Ｎ ) -, -NR Ｎ C(O)O-, -C(O)S-, -SC(O)-, -C(=NR Ｎ ) -, -C(=NR Ｎ ) N (R Ｎ ) -, -NR Ｎ C (=NR Ｎ ) -, -NR Ｎ C (=NR Ｎ ) N (R Ｎ )-, -C(S)-, -C(S)N(R Ｎ ) -, -NR Ｎ C(S)-, -NR Ｎ C(S)N(R Ｎ )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O) ２ -, -S(O) ２ O-, -OS(O) ２ O-, -N(R Ｎ )S(O)-, -S(O)N(R Ｎ ) -, -N(R Ｎ ) S ( O ) N ( R Ｎ )-,-OS(O)N(R Ｎ ) -, -N(R Ｎ )S(O)O-, -S(O) ２ -, -N(R Ｎ ) S (O) ２ -, -S(O) ２ N (R Ｎ ) -, -N(R Ｎ ) S (O) ２ N (R Ｎ ) -, -OS(O) ２ N (R Ｎ ) - or -N(R Ｎ ) S (O) ２ is replaced by O-; R Ｎ each instance of is independently hydrogen, an optionally substituted alkyl, or a nitrogen protecting group; Ring B is an optionally substituted cycloalkyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl; p is 1 or 2. The lipid nanoparticle composition of claim 15, wherein

22. The PEG-lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG DMPE, PEG-DPPC, PEG-DSPE, or PL1: [Chemistry 18] The lipid nanoparticle composition of claim 15, wherein

23. A pharmaceutical composition comprising the lipid nanoparticle composition of claim 14, or a salt thereof, and a pharmaceutically acceptable carrier.

24. 15. A method for delivering a payload to a cell, comprising contacting the cell with the lipid nanoparticle composition of claim 14.

25. A method for delivering a therapeutic or prophylactic payload to a patient, the method comprising administering to the patient the lipid nanoparticle composition of claim 14.

26. 10. A process for preparing a lipid nanoparticle composition, said process comprising contacting a lipid nanoparticle core with a lipid amine compound according to claim 1 or 2, or a salt thereof.

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