CN117658848A

CN117658848A - Lipid compounds for delivery of therapeutic agents and uses thereof

Info

Publication number: CN117658848A
Application number: CN202311408419.6A
Authority: CN
Inventors: 王子君; 桂阳
Original assignee: Yaotang Shanghai Biotechnology Co ltd
Current assignee: Yaotang Shanghai Biotechnology Co ltd
Priority date: 2023-10-26
Filing date: 2023-10-26
Publication date: 2024-03-08

Abstract

The invention provides a lipid compound for delivering a therapeutic agent and application thereof, in particular to a compound of a formula I or pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor thereof.

Description

Lipid compounds for delivery of therapeutic agents and uses thereof

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to a lipid compound for delivering a therapeutic agent and application thereof.

Background

The gene therapy technology is a hotspot of research in the field of modern biological medicine, and can prevent and treat cancers, bacterial and viral infections, diseases with genetic etiology and the like by using nucleic acid medicines. Because nucleic acid drugs are easy to degrade and difficult to enter cells, and the like, the nucleic acid drugs are usually required to be encapsulated by a carrier and delivered to target cells, so that the development of safe and efficient delivery carriers becomes a precondition for clinical application of gene therapy.

Lipid nanoparticles (Lipid nanoparticle, LNP) are currently a research hotspot in the field of non-viral gene vectors. In 2018, the FDA approved LNP delivery patisiran (onpattro) for the treatment of hereditary transthyretin amyloidosis, and studies from the use of LNP technology to deliver nucleic acid drugs have been shown to grow in bursts. In particular, at the end of 2020, the FDA approved new coronavirus vaccines against COVID-19 for Moderna and BioNtech & pyroxene, respectively, both of which delivered mRNA drugs using LNP technology, thus achieving prevention of SARS-CoV-2 virus.

LNP is typically composed of four lipid compounds, namely ionizable lipids, neutral lipids, steroids, and polymer-bound lipids, with the choice of ionizable lipids having the greatest impact on LNP. Current nucleic acid therapeutic agents still face challenges including mainly delivery efficiency, low cell permeability, and high sensitivity to degradation by certain nucleic acid molecules, including RNA.

Thus, there remains a need to develop new lipid compounds that facilitate in vitro or in vivo delivery of nucleic acid molecules for therapeutic and/or prophylactic purposes.

Disclosure of Invention

It is an object of the present invention to provide a novel lipid compound that facilitates in vitro or in vivo delivery of nucleic acid molecules for therapeutic and/or prophylactic purposes.

In a first aspect of the present invention, there is provided a compound of formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor thereof, having the structure shown below:

wherein R is ¹ 、R ² Each independently selected from H, C ₁ -C ₁₅ Alkyl, C ₂ -C ₁₅ Alkenyl, C ₂ -C ₁₅ Alkynyl, C ₁ -C ₁₅ Haloalkyl, -OH, and R _a (R _b )N-，R _a 、R _b Each independently H, C ₁ -C ₁₀ Alkyl, C ₂ -C ₁₀ Alkenyl, C ₂ -C ₁₀ Alkynyl, C ₁ -C ₁₀ A haloalkyl group; or (b)

R ¹ And R is R ² Can form together with the attached N atom a 3-10 membered heterocyclic group containing at least 1 heteroatom, and said heteroatom is selected from N, O, S, preferably N;

G ¹ is- (CH) ₂ ) n-, wherein n is 1-15, preferably 1-10, more preferably 1-6;

R ³ selected from H, C ₁ -C ₂₀ Alkyl, C ₂ -C ₂₀ Alkenyl, C ₂ -C ₂₀ Alkynyl, C ₁ -C ₂₀ Haloalkyl, -OH, or-NH ₂ ；

R ⁴ Is- (CH) ₂ ) m-, wherein m is 1-20, preferably 1-15, more preferably 1-10;

L ¹ 、L ² is free or independent of each otherThe site is selected from the group consisting of:

- (c=o) O-, -O (c=o) -, - (S-S) -, -O (s=o) -, - (c=o) S-, -S (c=o) -, - (c=s) O-, -NH (c=o) -, - (c=s) NH-, -NH (c=s) -, - (c=o) NH-, or a combination thereof, preferably selected from- (c=o) O or-O (c=o) -.

R ⁵ Selected from H, halogen, substituted or unsubstituted C ₁ -C ₂₅ Alkyl, substituted or unsubstituted C ₂ -C ₂₅ Alkenyl or substituted or unsubstituted C ₂ -C ₂₅ Alkynyl, said substitution being with one or more substituents selected from the group consisting of: halogen, C ₁ -C ₁₅ Alkyl, C ₂ -C ₁₅ Alkenyl, C ₂ -C ₁₅ Alkynyl, C ₁ -C ₁₅ Alkoxy, O- (C) ₂ -C ₁₅ Hydrocarbyl, including alkyl, alkenyl, alkynyl), C ₁ -C ₁₅ Haloalkyl, C ₁ -C ₁₅ Haloalkoxy, -OH, -CN, nitro, -NH ₂ ；

R ⁶ Is- (CH) ₂ ) p-, wherein p is 1-20, preferably 1-15, more preferably 1-10;

R ⁷ selected from H, halogen, C ₁ -C ₂₅ Alkyl, C ₂ -C ₂₅ Alkenyl or C ₂ -C ₂₅ Alkynyl, C ₁ -C ₂₅ A haloalkyl group.

In another preferred embodiment, R ¹ 、R ² Each independently selected from H, C ₁ -C ₁₀ Alkyl, C ₂ -C ₁₀ Alkenyl, C ₂ -C ₁₀ Alkynyl, C ₁ -C ₁₀ Haloalkyl, -OH, and R _a (R _b )N-，R _a 、R _b Each independently H, C ₁ -C ₆ Alkyl, C ₂ -C ₆ Alkenyl, C ₂ -C ₆ Alkynyl, C ₁ -C ₆ A haloalkyl group.

In another preferred embodiment, R ¹ 、R ² Each independently selected from H, C ₁ -C ₆ Alkyl, C ₂ -C ₆ Alkenyl, C ₂ -C ₆ Alkynyl, C ₁ -C ₆ HaloalkanesRadicals, -OH, and R _a (R _b )N-，R _a 、R _b Each independently H, C ₁ -C ₆ Alkyl, C ₂ -C ₆ Alkenyl, C ₂ -C ₆ Alkynyl, C ₁ -C ₆ A haloalkyl group.

In another preferred embodiment, R ¹ And R is R ² Together with the attached N atom, a 4-8 membered heterocyclic group containing at least 1 heteroatom selected from N, O, S, preferably N.

In another preferred embodiment, R ¹ And R is R ² Together with the attached N atom, a 4-6 membered heterocyclic group containing at least 1 heteroatom selected from N, O, S, preferably N.

In another preferred embodiment, R ³ Selected from H, C ₁ -C ₁₅ Alkyl, C ₂ -C ₁₅ Alkenyl or C ₂ -C ₁₅ Alkynyl, C ₁ -C ₁₅ Haloalkyl, -OH, or-NH ₂ 。

In another preferred embodiment, R ³ Selected from H, C ₁ -C ₁₀ Alkyl, C ₂ -C ₁₀ Alkenyl or C ₂ -C ₁₀ Alkynyl, C ₁ -C ₁₀ Haloalkyl, -OH, or-NH ₂ 。

In another preferred embodiment, R ⁵ Selected from H, halogen, substituted or unsubstituted C ₁ -C ₂₀ Alkyl, substituted or unsubstituted C ₂ -C ₂₀ Alkenyl or substituted or unsubstituted C ₂ -C ₂₀ Alkynyl, said substitution being with one or more (preferably 1 or 2) substituents selected from the group consisting of: halogen, C ₁ -C ₁₂ Alkyl, C ₂ -C ₁₂ Alkenyl, C ₂ -C ₁₂ Alkynyl, C ₁ -C ₁₂ Alkoxy, O- (C) ₂ -C ₁₂ Hydrocarbyl, including alkyl, alkenyl, alkynyl), C ₁ -C ₁₂ Haloalkyl, C ₁ -C ₁₂ Haloalkoxy, -OH, -CN, nitro, -NH ₂ 。

In another preferred embodiment, R ⁵ Selected from H, halogen, substituted or unsubstituted C ₁ -C ₁₅ Alkyl, substituted or unsubstituted C ₂ -C ₁₅ Alkenyl or substituted or unsubstituted C ₂ -C ₁₅ Alkynyl, said substitution being with one or more (preferably 1 or 2) substituents selected from the group consisting of: halogen, C ₁ -C ₁₀ Alkyl, C ₂ -C ₁₀ Alkenyl, C ₂ -C ₁₀ Alkynyl, C ₁ -C ₁₀ Alkoxy, O- (C) ₂ -C ₁₀ Hydrocarbyl, hydrocarbyl including alkyl, alkenyl, alkynyl), C ₁ -C ₁₀ Haloalkyl, C ₁ -C ₁₀ Haloalkoxy, -OH, -CN, nitro, -NH ₂ 。

In another preferred embodiment, R ⁷ Selected from H, halogen, C ₁ -C ₂₀ Alkyl, C ₂ -C ₂₀ Alkenyl, C ₂ -C ₂₀ Alkynyl or C ₁ -C ₂₀ A haloalkyl group.

In another preferred embodiment, R ⁷ Selected from H, halogen, C ₁ -C ₁₅ Alkyl, C ₂ -C ₁₅ Alkenyl, C ₂ -C ₁₅ Alkynyl or C ₁ -C ₁₅ A haloalkyl group.

In another preferred embodiment, the compound of formula I has the structure of formula I-1:

in the method, in the process of the invention,

R ¹ -R ⁷ 、G ¹ is defined as above.

In another preferred embodiment, the compound of formula I has the structure of formula I-2:

in the method, in the process of the invention,

R ¹ -R ⁷ 、G ¹ is defined as above.

In another preferred embodiment, R ¹ -R ⁷ 、L ¹ -L ² 、G ¹ Specific groups corresponding to the specific compounds in the examples.

In another preferred embodiment, the compound is each specific compound prepared in the examples, the preferred compound being selected from the group consisting of:

TABLE 1

In another preferred example, the compounds of formula I may be used to prepare drug delivery systems including Lipid Nanoparticles (LNP), liposomes, polymer nanoparticles, and the like, preferably for the preparation of lipid nanoparticles.

In a second aspect the present invention provides a lipid carrier comprising a compound of formula I according to the first aspect of the invention or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor thereof.

In another preferred embodiment, the lipid carrier further comprises a helper lipid.

In another preferred embodiment, the compound of formula I is present in the lipid carrier in a molar proportion of 30-65% of the total lipid content.

In another preferred embodiment, the compound of formula I is present in the lipid carrier in a molar proportion of 40-60% of the total lipid content; preferably, 50% molar ratio.

In another preferred embodiment, the helper lipid comprises one or a combination of two or more of an anionic lipid, a neutral lipid, a steroid, and a polymer-bound lipid.

In another preferred embodiment, the lipid carrier further comprises other cationic or ionizable lipid compounds.

In another preferred embodiment, the lipid carrier comprises a first lipid compound comprising a compound of formula I according to the first aspect of the invention or a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug and optionally other ionizable lipids, and a second lipid compound comprising one or a combination of more than two of an anionic lipid, a neutral lipid, a steroid and a polymer-bound lipid.

In another preferred embodiment, the first lipid compound is a compound of formula I or a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug, and other cationic or ionizable lipid in combination.

In another preferred example, the other cationic or ionizable lipid compound comprises one or more of 1, 2-dioleyloxy-N, N-dimethylaminopropane DLinDMA, 1, 2-dioleyloxy-N, N-dimethylaminopropane DODMA, DLin-MC2-MPZ, 2-dioleyl-4- (2-dimethylaminoethyl) - [1,3] -dioxolane DLin-KC2-DMA, 1, 2-dioleoyl-3-trimethylammonium-propane DOTAP, 1'- (2- (4- ((2- (bis (2-hydroxydodecyl) amino) ethyl) (2-hydroxydodecyl) amino) piperazin-1-yl) ethylaminoalkanediyl) di-dodecane-2-ol C12-200, 3β [ N-N' -dimethylaminoethane) -carbamoyl ] cholesterol DC-Chol, and N- [1- (2, 3-dioleoyl chloride) propyl ] -N, N, N-trimethylamine DOTMA.

In another preferred example, the anionic lipid includes one or a combination of two or more of phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, dioleoyl phosphatidylglycerol DOPG, 1, 2-dioleoyl-sn-glycerol-3-phosphatidylserine DOPS, and dimyristoyl phosphatidylglycerol.

In another preferred example, the neutral lipid comprises at least one of 1, 2-dioleoyl-sn-glycero-3-phosphatidylethanolamine DPPC, 1, 2-distearoyl-sn-glycero-3-phosphatidylcholine DPPC, 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine DPPC, 1, 2-dioleoyl-sn-glycero-3-phosphatidylcholine DOPC, dipalmitoyl-phosphatidylglycerol DPPG, oleoyl phosphatidylcholine POPC, 1-palmitoyl-2-oleoyl phosphatidylethanolamine POPE, 1, 2-dipalmitoyl-sn-glycero-3-phosphate ethanolamine DPPE, 1, 2-dimyristoyl-sn-glycero-3-phosphate ethanolamine DMPE, distearoyl phosphatidylethanolamine DSPE, and 1-stearoyl-2-oleoyl phosphatidylethanolamine SOPE or a lipid modified with an anionic or cationic group thereof. The anionic or cationic modifying group is not limited.

In another preferred embodiment, the steroid comprises one or more of cholesterol, non-sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassicasterol, lycosyline, ursolic acid, alpha tocopherol, fecal sterols, and corticosteroids.

In another preferred example, the polymer-bound lipid comprises 1, 2-dimyristoyl-sn-glycerogethoxy-polyethylene glycol PEG-DMG, dimyristoyl-polyethylene glycol PEG-C-DMG, polyethylene glycol-dimyristoyl-glycerol PEG-C14, PEG-1, 2-dimyristoyloxy propyl-3-amine PEG-C-DMA, 1, 2-distearoyl-sn-glycero-3-phosphate ethanolamine-N- [ amino (polyethylene glycol) ] PEG-DSPE, pegylated phosphatidylethanolamine PEG-PE, PEG modified ceramides, PEG modified dialkylamines, PEG modified diacylglycerols, tween-20, tween-80, 1, 2-dipalmitoyl-sn-glycerol-methoxypolyethylene glycol PEG-DPG, 4-O- (2 ',3' -dimyristoyloxy) propyl-1-O- (. Omega. -methoxy (polyethoxy) ethyl) succinate PEG-s-DMG, PEG-dialkoxypropyl PEG-DAA, one or a combination of two or more of mPEG2000-1, 2-di-O-alkyl-sn 3-carbamoyl glyceride PEG-c-DOMG and N-acetylgalactosamine ((R) -2, 3-bis (octadecyloxy) propyl-1- (methoxypoly (ethylene glycol) 2000) propyl carbamate)) GalNAc-PEG-DSG.

In another preferred embodiment, the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid and the polymer-bound lipid in the lipid carrier is (20-65): 0-20): 5-25): 25-55): 0.3-15; illustratively, the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid, and the polymer-bound lipid may be 20:20:5:50:5, 30:5:25:30:10, 20:5:5:55:15, 65:0:9.7:25:0.3, and the like; wherein in the first lipid compound, the molar ratio of the compound of formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug thereof and other cationic or ionizable lipid is (1-10): 0-10; illustratively, the molar ratio may be 1:1, 1:2, 1:5, 1:7.5, 1:10, 2:1, 5:1, 7.5:1, 10:1, etc.

In another preferred embodiment, the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid and the polymer-bound lipid in the lipid carrier is (20-55): 0-13): 5-25): 25-51.5): 0.5-15; wherein in the first lipid compound, the molar ratio of the compound of formula I or a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug, and the other cationic or ionizable lipid is (3-4): 0-5.

In another preferred embodiment, the lipid carrier further comprises a bioactive substance encapsulated in the lipid carrier.

In another preferred embodiment, the bioactive substance is selected from the group consisting of: nucleic acids, proteins, polypeptides, small molecules, or combinations thereof.

In another preferred embodiment, the nucleic acid comprises RNA, DNA, antisense nucleic acid, aptamer, ribozyme, immunostimulatory nucleic acid, or PNA.

In another preferred embodiment, the antisense nucleic acid is an antisense oligonucleotide.

In another preferred embodiment, the RNA is mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomir, a microrna inhibitor, a microrna activator, or a shRNA.

In another preferred embodiment, the DNA comprises a plasmid.

In another preferred embodiment, the mRNA comprises one or more functional nucleotide analogs including pseudouridine, 1-methyl-pseudouridine, and 5-methylcytosine.

In another preferred embodiment, the mRNA encodes at least one antigen.

In another preferred embodiment, the antigen is a pathogenic antigen.

In another preferred embodiment, the antigen is a tumor-associated antigen.

In another preferred embodiment, the mRNA comprises a sequence encoding an RNA-guided DNA binding agent, more specifically, an mRNA encoding a nuclease or a base editor.

In another preferred embodiment, the nucleic acid further comprises a guide RNA, in particular, the guide RNA comprises a gRNA nucleic acid.

In another preferred embodiment, the nucleic acid comprises mRNA and gRNA encoding a nuclease or base editor.

In another preferred embodiment, the gRNA is a modified or unmodified gRNA.

In another preferred embodiment, the lipid carrier has high encapsulation efficiency of the bioactive substance, which greatly improves the in vivo delivery efficiency of the bioactive substance.

In another preferred embodiment, the lipid carrier comprises a first lipid compound comprising a compound of formula I according to the first aspect of the invention and optionally further ionizable lipids and a second lipid compound comprising one or a combination of more than two of an anionic lipid, a neutral lipid, a steroid and a polymer-bound lipid.

In another preferred embodiment, the first lipid compound is any one of the compounds described above or a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug.

In another preferred embodiment, the first lipid compound is any one of the compounds described above or a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug and other cationic or ionizable lipid in combination.

In another preferred embodiment, the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid and the polymer-bound lipid in the lipid carrier is (20-65): 0-20): 5-25): 25-55): 0.3-15; illustratively, the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid, and the polymer-bound lipid may be 20:20:5:50:5, 30:5:25:30:10, 20:5:5:55:15, 65:0:9.7:25:0.3, and the like; wherein in the first lipid compound, the molar ratio of any one of the compounds or pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs and other cationic or ionizable lipids is (1-10): 0-10; illustratively, the molar ratio may be 1:1, 1:2, 1:5, 1:7.5, 1:10, 2:1, 5:1, 7.5:1, 10:1, etc.

In another preferred embodiment, the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid and the polymer-bound lipid in the lipid carrier is (20-55): 0-13): 5-25): 25-51.5): 0.5-15; wherein in the first lipid compound, the molar ratio of any of the above compounds or pharmaceutically acceptable forms thereof, such as salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs and other cationic or ionizable lipids is (3-4): 0-5.

In a third aspect, the present invention provides a Lipid Nanoparticle (LNP) comprising a compound of formula I according to the first aspect of the present invention or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor thereof.

In another preferred embodiment, the lipid nanoparticle further comprises a helper lipid.

In another preferred embodiment, the lipid nanoparticle further comprises a bioactive substance encapsulated in the lipid nanoparticle.

In a fourth aspect, the present invention provides a lipid nanoparticle composition comprising a compound of formula I according to the first aspect of the present invention or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or a precursor thereof according to the second aspect of the present invention or a lipid nanoparticle according to the third aspect of the present invention, and a bioactive substance encapsulated in the lipid carrier or the lipid nanoparticle.

In another preferred embodiment, the mass ratio of bioactive substance to lipid carrier in the composition is 1 (3-40), illustratively 1:3, 1:5, 1:10, 1:15, 1:20, 1:30, 1:40, etc.

In another preferred embodiment, the ratio (w/w) of mRNA to gRNA of the nuclease or base editor in the bioactive substance is about 10:1 to about 1:10.

In another preferred embodiment, the ratio of mRNA to gRNA (w/w) of nuclease or base editor in the bioactive substance is from about 3:1 to about 1:3.

In another preferred embodiment, the ratio (w/w) of mRNA to gRNA of said nuclease or base editor in said biologically active substance is about 3:2.

In another preferred embodiment, the composition comprises a pharmaceutical composition.

In a fifth aspect, the present invention provides a pharmaceutical composition comprising a compound of formula I according to the first aspect of the present invention or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor thereof, or a lipid carrier according to the second aspect of the present invention or a lipid nanoparticle according to the third aspect of the present invention, and a bioactive substance encapsulated in the lipid carrier or the lipid nanoparticle, and a pharmaceutically acceptable excipient, carrier or diluent.

In a sixth aspect, the present invention provides a pharmaceutical formulation comprising a compound of formula I according to the first aspect of the present invention or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor thereof, or a lipid carrier according to the second aspect of the present invention or a lipid nanoparticle according to the third aspect of the present invention, and a biologically active substance encapsulated in the lipid carrier or the lipid nanoparticle, together with a pharmaceutically acceptable excipient, carrier or diluent; or the pharmaceutical formulation comprises the lipid nanoparticle composition of the fourth aspect of the invention, and a pharmaceutically acceptable excipient, carrier or diluent.

In another preferred example, the particle size of the pharmaceutical formulation is 30 to 500nm, and illustratively, the particle size may be 30nm, 50nm, 100nm, 150nm, 250nm, 350nm, 500nm, etc.

In another preferred embodiment, the encapsulation efficiency of the biologically active substance in the pharmaceutical formulation is greater than 50%. Illustratively, the encapsulation efficiency may be 55%, 60%, 65%, 70%, 75%, 79%, 80%, 85%, 89%, 90%, 93%, 95%, etc.

In another preferred embodiment, the hydrated particle size of the drug is 50-200nm, preferably 70-150nm, most preferably 75-110nm.

In another preferred embodiment, the pharmaceutical formulation may be applied for the treatment and/or prevention of diseases.

In another preferred embodiment, the disease is selected from the group consisting of: metabolic disease, genetic disease, cancer, cardiovascular disease, infectious disease, or a combination thereof.

In another preferred embodiment, the metabolic disease comprises Familial Hypercholesterolemia (FH), and the genetic disease preferably comprises transthyretin Amyloidosis (ATTR), primary hyperuricemia (PH 1), and Hereditary Angioedema (HAE).

In another preferred example, the infectious disease comprises hepatitis B (HEPATITIS B).

In another preferred embodiment, the dosage form of the pharmaceutical formulation is selected from the group consisting of: injection, freeze-dried agent, atomized inhalant and smearing type medicament.

In another preferred embodiment, the pharmaceutical formulation is administered by injection, i.e. intravenous, intramuscular, intradermal, subcutaneous, intrathecal, intraduodenal or intraperitoneal injection.

In another preferred embodiment, the pharmaceutical formulation is administered by inhalation, for example intranasal administration.

In another preferred embodiment, the pharmaceutical formulation is administered transdermally, such as by transdermal application or electrode lead-in administration.

The seventh aspect of the present invention provides an application of the compound shown in the formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor thereof in preparing liposome nano-particles.

According to an eighth aspect of the present invention there is provided the use of a compound of formula I according to the first aspect of the present invention or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex thereof, or a lipid carrier according to the second aspect of the present invention, or a lipid nanoparticle according to the third aspect of the present invention, or a lipid nanoparticle composition according to the fourth aspect of the present invention, or a pharmaceutical composition according to the fifth aspect of the present invention, or a pharmaceutical formulation according to the sixth aspect of the present invention, in the preparation of a nucleic acid drug, a genetic vaccine, a small molecule drug, a polypeptide or a protein drug.

According to a ninth aspect of the present invention there is provided a method of preparing a lipid nanoparticle composition according to the fourth aspect of the present invention, the method comprising:

(a) Mixing a compound of formula I according to the first aspect of the invention or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or a precursor thereof, and optionally a co-lipid with an organic solvent, thereby obtaining a lipid organic phase;

(b) Mixing the bioactive substance with an aqueous solvent to obtain an aqueous phase containing the bioactive substance;

(c) Mixing the lipid organic phase of step (a) with the aqueous phase of step (b) to obtain the lipid nanoparticle composition.

In another preferred embodiment, the organic solvent comprises ethanol, methanol, isopropanol, acetonitrile, dimethylformamide, dimethylsulfoxide, dioxane, tetrahydrofuran, or a combination thereof.

In another preferred embodiment, the aqueous solvent is a buffer.

In another preferred embodiment, the aqueous solvent is a buffer having a pH in the range of 3 to 7.

In another preferred embodiment, the acidic buffer is a citrate buffer at pH 4.0.

In another preferred embodiment, the volume ratio of the lipid organic phase to the aqueous phase containing the biologically active substance is 1 (2-5), preferably 1 (3-4).

In another preferred embodiment, in step (c), the lipid organic phase and the aqueous phase are mixed by a microfluidic chip.

In another preferred embodiment, the method further comprises step (d): purifying, concentrating, filtering and sterilizing the nucleic acid lipid nanoparticle composition obtained in the step (c).

In a tenth aspect the present invention provides the use of a compound of formula I according to the first aspect of the present invention or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor thereof for the preparation of a drug delivery system.

In another preferred example, the delivery system comprises Lipid Nanoparticles (LNP), liposomes, polymer nanoparticles, etc., preferably for the preparation of lipid nanoparticles.

In another preferred embodiment, the drug delivery system is for delivering a drug for the treatment and/or prevention of a disease.

The eleventh aspect of the present invention provides the use of a compound of formula I according to the first aspect of the present invention or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or a precursor thereof according to the second aspect of the present invention or a lipid carrier according to the third aspect of the present invention or a lipid nanoparticle composition according to the fourth aspect of the present invention or a pharmaceutical composition according to the fifth aspect of the present invention or a pharmaceutical formulation according to the sixth aspect of the present invention for the preparation of a medicament for the treatment and/or prophylaxis of a disease.

In a twelfth aspect, the invention provides a method of delivering a therapeutic or prophylactic agent to a cell, tissue or organ of a subject, the method comprising contacting the cell, tissue or organ of the subject with a lipid nanoparticle composition according to the fourth aspect of the invention or a pharmaceutical composition according to the fifth aspect of the invention or a pharmaceutical formulation according to the sixth aspect of the invention.

In another preferred embodiment, the therapeutic or prophylactic agent is a biologically active substance, preferably mRNA.

In another preferred embodiment, the cell is selected from the group consisting of: liver cells, lung cells, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells, photoreceptor cells, retinal pigment epithelial cells, secretory cells, heart cells, adipocytes, smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, lymphocytes, B cells, T cells, antigen presenting cells, reticulocytes, leukocytes, granulocytes, and tumor cells.

In another preferred example, the tumor cells include lung cancer cells, colon cancer cells, rectal cancer cells, anal cancer cells, bile duct cancer cells, small intestine cancer cells, stomach cancer cells, esophageal cancer cells, gallbladder cancer cells, liver cancer cells, pancreatic cancer cells, appendiceal cancer cells, breast cancer cells, ovarian cancer cells, cervical cancer cells, prostate cancer cells, kidney cancer cells, cancer cells of the central nervous system, glioblastoma tumor cells, skin cancer cells, lymphoma cells, choriocarcinoma tumor cells, head and neck cancer cells, osteogenic sarcoma tumor cells, and blood cancer cells.

In another preferred embodiment, the tissue or organ is selected from the group consisting of: heart, liver, spleen, lung, kidney, brain, lymph node, muscle, blood, spine or bone.

In a thirteenth aspect, the invention provides a method of producing a protein of interest or a polypeptide of interest in a subject's cells, the method comprising contacting the subject's cells with a lipid nanoparticle composition according to the fourth aspect of the invention or a pharmaceutical composition according to the fifth aspect of the invention or a pharmaceutical formulation according to the sixth aspect of the invention.

In another preferred embodiment, the bioactive substance in the lipid nanoparticle composition, pharmaceutical composition or pharmaceutical formulation is an mRNA, wherein the mRNA encodes a protein or polypeptide of interest, the mRNA being capable of translation in a cell to produce the protein or polypeptide of interest.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 is a schematic representation of the delivery strategy for PCSK9 gene editing efficiency detection in mouse liver cells according to the invention.

Figure 2 shows PCSK9 gene editing efficiency of different lipid compound encapsulated base editors in mouse liver cells.

Detailed Description

The inventors of the present invention have made extensive and intensive studies, and have unexpectedly found for the first time an ionizable lipid compound capable of effectively delivering a drug such as a nucleic acid molecule, a small molecule compound, etc., and by contrast, the lipid nanoparticle of the present invention has a good particle size distribution and a high encapsulation efficiency, and can satisfy the requirements for in vivo delivery. On this basis, the present invention has been completed.

For easier understanding of the present invention, certain technical and scientific terms are defined below in detail. Unless otherwise defined explicitly herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

In the present specification, the numerical range indicated by the term "numerical value a to numerical value B" means a range including the end point numerical value A, B.

In the present specification, the use of "substantially" or "substantially" means that the standard deviation from the theoretical model or theoretical data is within 5%, preferably 3%, more preferably 1%.

In the present specification, the meaning of "can" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

In this specification, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

Reference throughout this specification to "some specific/preferred embodiments," "other specific/preferred embodiments," "an embodiment," and so forth, means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the elements may be combined in any suitable manner in the various embodiments.

Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described herein; it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Unless otherwise indicated, the following terms have the following meanings:

the term "pharmaceutically acceptable salt" refers to salts of the compounds of the invention which are substantially non-toxic to the organism. Pharmaceutically acceptable salts generally include, but are not limited to, salts formed from the compounds of the present invention by reaction with pharmaceutically acceptable inorganic/organic acids or inorganic/organic bases, such salts also being referred to as acid addition salts or base addition salts. Common inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and the like, common organic acids include, but are not limited to, trifluoroacetic acid, citric acid, maleic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, oxalic acid, formic acid, acetic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like, common inorganic bases include, but are not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, barium hydroxide, and the like, and common organic bases include, but are not limited to, diethylamine, triethylamine, ethambutol, and the like.

The term "stereoisomer" (or "optical isomer") refers to a stable isomer that has a perpendicular plane of asymmetry due to at least one chiral factor (including chiral center, chiral axis, chiral plane, etc.), thereby enabling rotation of plane polarized light. The present invention also includes stereoisomers and mixtures thereof, due to the presence of asymmetric centers and other chemical structures in the compounds of the present invention which may lead to stereoisomers. Since the compounds of the present invention and salts thereof include asymmetric carbon atoms, they can exist as single stereoisomers, racemates, mixtures of enantiomers and diastereomers. Typically, these compounds can be prepared in the form of a racemic mixture. However, if desired, such compounds can be prepared or isolated to give pure stereoisomers, i.e., single enantiomers or diastereomers, or mixtures enriched in single stereoisomers (purity. Gtoreq.98%, purity. Gtoreq.95%,. Gtoreq.93%,. Gtoreq.90%,. Gtoreq.88%,. Gtoreq.85% or. Gtoreq.80%). The individual stereoisomers of the compounds are prepared synthetically from optically active starting materials containing the desired chiral centers or by preparation of mixtures of enantiomeric products followed by separation or resolution, e.g., conversion to mixtures of diastereomers followed by separation or recrystallization, chromatography, use of chiral resolving agents, or direct separation of the enantiomers on chiral chromatographic columns. Starting compounds having specific stereochemistry are either commercially available or can be prepared according to the methods described herein and resolved by methods well known in the art.

The term "tautomer" (or "tautomeric form") refers to structural isomers having different energies that can be converted to each other by a low energy barrier. If tautomerism is possible (e.g., in solution), chemical equilibrium of the tautomers can be achieved. For example, proton tautomers (or proton transfer tautomers) include, but are not limited to, interconversions by proton transfer, such as keto-enol isomerisation, imine-enamine isomerisation, amide-imine alcohol isomerisation, and the like. Unless otherwise indicated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

The term "solvate" refers to a substance formed by the association of a compound of the invention, or a pharmaceutically acceptable salt thereof, with at least one solvent molecule by non-covalent intermolecular forces. Common solvates include, but are not limited to, hydrates, ethanolates, acetonates, and the like.

The term "chelate" is a complex having a cyclic structure, obtained by chelation of two or more ligands with the same metal ion to form a chelate ring.

The term "non-covalent complex" is formed by the interaction of a compound with another molecule, wherein no covalent bond is formed between the compound and the molecule. For example, recombination can occur by van der Waals interactions, hydrogen bonding, and electrostatic interactions (also known as ionic bonding).

The term "prodrug" refers to a derivative compound that is capable of providing a compound of the invention directly or indirectly after administration to a patient. Particularly preferred derivative compounds or prodrugs are compounds that, when administered to a patient, may increase the bioavailability of the compounds of the invention (e.g., are more readily absorbed into the blood) or promote delivery of the parent compound to the site of action (e.g., the lymphatic system). All prodrug forms of the compounds of the invention are within the scope of the invention unless otherwise indicated, and the various prodrug forms are well known in the art.

The term "independently" means that at least two groups (or ring systems) present in the structure that are the same or similar in value range may have the same or different meanings in the particular case. For example, substituent X and substituent Y are each independently hydrogen, halogen, hydroxy, cyano, alkyl or aryl, then when substituent X is hydrogen, substituent Y may be either hydrogen or halogen, hydroxy, cyano, alkyl or aryl; similarly, when the substituent Y is hydrogen, the substituent X may be either hydrogen or halogen, hydroxy, cyano, alkyl or aryl.

The terms "comprising" and "including" are used in their open, non-limiting sense.

The term "alkyl" refers to a monovalent, linear or branched alkyl group consisting of only carbon and hydrogen atoms, free of unsaturation, and attached to other moieties by a single bond, including, but not limited to, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, and the like. For example, "C _1-30 Alkyl "refers to a saturated monovalent straight or branched hydrocarbon radical containing 1 to 30 carbon atoms.

The term "alkylene" refers to a divalent straight or branched chain alkane group consisting of only carbon and hydrogen atoms, containing no saturation, and linked to other fragments by two single bonds, respectively, including, but not limited to, methylene, 1-ethylene, 1, 2-ethylene, and the like. For example, "C _1-30 Alkylene "refers to a saturated divalent straight or branched chain alkyl group containing from 1 to 30 carbon atoms.

The term "cycloalkyl" refers to a saturated, monocyclic or polycyclic (e.g., bicyclic, tricyclic or tetracyclic) non-aromatic hydrocarbon group consisting of only carbon and hydrogen atoms. Cycloalkyl groups may include fused, bridged or spiro ring systems. For example, the term "C" as used in the present invention _3-6 Cycloalkyl "refers to cycloalkyl groups having 3 to 6 carbon atoms. For example, cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or bicyclo [2.2.1 ]Heptyl, and the like.

The term "cycloalkylene" refers to a divalent group obtained by removing a hydrogen atom from a cycloalkyl group as defined above, including, but not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and the like. For example, "C _3-30 Cycloalkyl "refers to a divalent group obtained by removing a hydrogen atom from a cycloalkyl group containing 3 to 30 carbon atoms.

The term "branched alkyl" refers to an alkane radical that is attached to the parent molecule and itself forms at least two branched structures. For example

The term "alkenyl" refers to a monovalent, straight or branched chain, alkyl group consisting of only carbon and hydrogen atoms, containing at least one double bond, and attached to other moieties by a single bond, including, but not limited to, ethenyl, propenyl, allyl, isopropenyl, butenyl, and isobutenyl groups, and the like. For example "C _2-30 Alkenyl "means an alkenyl group containing 2 to 30 carbon atoms and having at least 1 carbon-carbon double bond [ ]>C＝C<) A monovalent straight or branched hydrocarbon group.

The term "alkenylene" refers to a divalent straight or branched chain alkane groupWhich consists of only carbon and hydrogen atoms, contains at least one double bond, and is linked to other fragments, including but not limited to vinylidene groups, etc., respectively, by two single bonds. For example, "C _2-30 Alkenylene "means an alkylene group containing 2 to 30 carbon atoms and having at least 1 carbon-carbon double bond>C＝C<) A divalent straight or branched hydrocarbon group.

The term "alkynyl" refers to monovalent, straight or branched chain, alkanyl radicals consisting of only carbon and hydrogen atoms, containing at least one carbon-carbon triple bond, and attached to other moieties by a single bond, including, but not limited to, ethynyl, propynyl, butynyl, pentynyl, and the like. For example "C _2-30 Alkynyl "refers to a monovalent straight or branched hydrocarbon radical containing 2 to 30 carbon atoms and having at least 1 carbon-carbon triple bond.

The term "alkynylene" refers to a divalent straight or branched chain alkane group consisting of only carbon and hydrogen atoms, containing at least one carbon-carbon triple bond, and linked to other fragments, respectively, by two single bonds, including, but not limited to, ethynylene and the like. For example, "C _2-30 Alkynylene "refers to a divalent straight or branched chain hydrocarbon radical containing 2 to 30 carbon atoms and having at least 1 carbon-carbon triple bond.

The term "cycloalkenyl" refers to an unsaturated, monocyclic or polycyclic (e.g., bicyclic, tricyclic or tetracyclic) non-aromatic hydrocarbon group consisting of only carbon and hydrogen atoms. Cycloalkenyl groups may include fused, bridged or spiro ring systems. Such as cyclopropenyl and cyclobutenyl, and the like.

The term "cycloalkenyl" refers to a divalent group obtained by removing a hydrogen atom from a cycloalkenyl group as defined above, including, but not limited to, cyclopropenyl, cyclobutenyl, and the like. For example, "C _3-30 The "cycloalkenylene group" means a divalent group obtained by removing a hydrogen atom from a cycloalkenyl group containing 3 to 30 carbon atoms.

The term "branched alkenyl" is an olefinic radical that is attached to the parent molecule and itself forms at least two branched structures. For example

The term "heterocyclyl" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic, e.g. fused, bridged or spiro) non-aromatic group, the ring atoms of which consist of carbon atoms and at least one heteroatom selected from N, O and S, wherein the S atom is optionally substituted to form S (=o), S (=o) ₂ Or S (=o) (=nr ^x )，R ^x Independently selected from H or C _1-4 An alkyl group. If valence requirements are met, the heterocyclyl may be attached to the remainder of the molecule through any one ring atom. For example, the term "3-8 membered heterocyclyl" as used herein refers to heterocyclyl groups having 3 to 8 ring atoms. For example, the heterocyclic group may be oxiranyl, aziridinyl, azetidinyl, oxetanyl, tetrahydrofuranyl, dioxolyl, pyrrolidinyl, pyrrolidinonyl, imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, dithianyl or trithianyl.

The term "aryl" refers to a monocyclic or fused polycyclic aromatic hydrocarbon group having a conjugated pi-electron system. For example, the term "C" as used in the present invention _6-10 Aryl "refers to aryl groups having 6 to 10 carbon atoms. For example, aryl may be phenyl, naphthyl, anthracenyl, phenanthrenyl, acenaphthylenyl, azulenyl, fluorenyl, indenyl, pyrenyl, and the like.

The term "heteroaryl" refers to a monocyclic or fused polycyclic aromatic group having a conjugated pi-electron system, the ring atoms of which are made up of carbon atoms and at least one heteroatom selected from N, O and S. If valence requirements are met, the heteroaryl group may be attached to the remainder of the molecule through any one of the ring atoms. For example, the term "5-10 membered heteroaryl" as used in the present invention refers to heteroaryl groups having 5 to 10 ring atoms. For example, heteroaryl groups can be thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and its benzo derivatives, pyrrolopyridinyl, pyrrolopyrazinyl, pyrazolopyridinyl, imidazopyridinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, purinyl, and the like.

The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

The term "hydroxy" refers to-OH.

The term "cyano" refers to-CN.

The term "amino" refers to-NH ₂ 。

The term "nitro" refers to-NO ₂ 。

The term "oxo" refers to (=o).

The term "subject" includes both human and non-human animals. Non-human animals include vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, cats, horses, cows, chickens, dogs, mice, rats, goats, rabbits, and pigs. Preferably, the subject is a human. The terms "patient" or "subject" are used interchangeably herein unless indicated.

The term "nuclease" refers to an enzyme that catalyzes the cleavage of phosphodiester bonds between nucleotides in a nucleic acid molecule. In some embodiments, the nuclease is selected from meganucleases, zinc-finger nucleases (ZFNs), TAL-effector DNA binding domain-nuclease fusion proteins (TALENs), and RNA-guided nucleases or variants thereof in which nuclease activity has been reduced or inhibited.

In some embodiments, the RNA-guided nuclease is a naturally-occurring CRISPR-Cas protein or an active variant or fragment thereof. CRISPR-Cas systems are classified as class I or class II systems. Class II systems contain a single effector nuclease and include types II, V, and VI. Each category is further subdivided into types (I, II, III, IV, V, VI), some of which are further classified into subtypes (e.g., type II-A, type II-B, type II-C, type V-A, type V-B).

The term "type II CRISPR-Cas protein", "type II CRISPR-Cas effect protein" or "Cas9" refers to a CRISPR-Cas effect protein that requires transactivating RNA (tracrRNA) and comprises two nuclease domains (RuvC and HNH), each of which is responsible for cleaving a single strand of a double-stranded DNA molecule. In other embodiments, the CRISPR-Cas protein is a naturally occurring V-type CRISPR-Cas protein or an active variant or fragment thereof.

As used herein, the term "V-type CRISPR-Cas protein", "V-type CRISPR-Cas effector protein" or "Cas12" refers to a CRISPR-Cas effector protein that cleaves dsDNA and comprises a single RuvC nuclease domain or a split RuvC nuclease domain and lacks a HNH domain. In other embodiments, the CRISPR-Cas protein is a naturally occurring type VI CRISPR-Cas protein or an active variant or fragment thereof. As used herein, the term "CRISPR-Cas protein type VI", "CRISPR-Cas effector protein type VI" or "Cas13" refers to a CRISPR-Cas effector protein that does not require a tracrRNA and comprises two HEPN domains that cleave RNA.

The term "gRNA" refers to a nucleotide sequence that has sufficient complementarity to a target nucleotide sequence to hybridize to the target sequence and direct the specific binding of a related nuclease to the sequence of the target nucleotide sequence. For a CRISPR-Cas enzyme, the corresponding guide RNAs are one or more RNA molecules (typically one or two) that can bind to the Cas enzyme and direct the Cas enzyme to bind to a particular target nucleotide sequence, and also cleave the target nucleotide sequence in those cases where the Cas enzyme has nickase or nuclease activity.

Definition of groups

As used herein, the term "substituted or unsubstituted" means that the radical may be unsubstituted or that H in the radical is replaced by one or more (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, most preferably 1 to 2) substituents.

As used herein, the term "C ₁ -C ₂₅ Alkyl "or" C ₁ -C ₂₀ Alkyl "or" C ₁ -C ₁₅ Alkyl "or" C ₁ -C ₁₀ Alkyl "or" C ₁ -C ₆ Alkyl "means a straight or branched chain alkyl group having 1 to 25, or 1 to 20, or 1 to 15, or 1 to 10 or 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or the like, and the like.

As used herein, the term "C ₂ -C ₂₅ Alkenyl "or“C ₂ -C ₂₀ Alkenyl "or" C ₂ -C ₁₅ Alkenyl "or" C ₂ -C ₁₀ Alkenyl "or" C ₂ -C ₆ Alkenyl "refers to straight or branched alkenyl groups having 2 to 25 or 2 to 20 or 2 to 15 or 2 to 10 or 2 to 6 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or the like, respectively, and the like.

As used herein, the term C ₂ -C ₂₅ Alkynyl "or" C ₂ -C ₂₀ Alkynyl "or" C ₂ -C ₁₅ Alkynyl "or" C ₂ -C ₁₀ Alkynyl "or" C ₂ -C ₆ Alkynyl "refers to straight or branched chain alkynyl groups having 2 to 25 or 2 to 20 or 2 to 15 or 2 to 10 or 2 to 6 carbon atoms, such as ethynyl, propynyl, or the like.

As used herein, the term "halogenated" refers to groups substituted with the same or different one or more of the above halogen atoms, which may be partially or fully halogenated, such as trifluoromethyl, pentafluoroethyl, heptafluoroisopropyl, or the like.

As used herein, the term "C ₁ -C ₂₅ Haloalkyl "," C ₁ -C ₂₀ Haloalkyl "," C ₁ -C ₁₅ Haloalkyl "or" C ₁ -C ₁₂ Haloalkyl "or" C ₁ -C ₁₀ Haloalkyl "or" C ₁ -C ₆ Haloalkyl "refers to a straight or branched alkyl group having 1 to 25 or 1 to 20 or 1 to 15 or 1 to 12 or 1 to 10 or 1 to 6 carbon atoms, for example, halomethyl, haloethyl, halopropyl, haloisopropyl, or the like, substituted with 1 or more halogens, preferably trifluoromethyl, and so forth.

The term "3-10 membered heterocyclic group" or "4-8 membered heterocyclic group" or "4-6 membered heterocyclic group" as used herein refers to a saturated, partially saturated or unsaturated group (but not aromatic), having a single ring or a fused ring (including bridged and spiro ring systems, having 3 to 10 or 4-8 or 4-6 carbon atoms and 1 to 4 heteroatoms selected from nitrogen, sulfur or oxygen, in which fused ring system one or more rings may be cycloalkyl, aryl or heteroaryl, provided that the point of attachment is through a non-aromatic ring.

The compounds of the invention may contain one or more asymmetric centers and thus occur as racemates, racemic mixtures, single enantiomers, diastereomeric compounds and single diastereomers. Asymmetric centers that may be present depend on the nature of the various substituents on the molecule. Each such asymmetric center will independently produce two optical isomers, and all possible optical isomers and diastereomeric mixtures and pure or partially pure compounds are included within the scope of the invention. The present invention includes all isomeric forms of the compounds.

Ionizable lipids

As used herein, the terms "ionizable lipids of the invention" and "ionizable cationic lipids of the invention" are used interchangeably, and each refer to a compound having formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or precursor thereof.

The ionizable lipids will be protonated to cationic lipids at lower pH values, which in turn will be converted to helper phospholipids at normal physiological pH values. The interaction between the auxiliary phospholipid and the anion cell membrane of blood cells is less, so that the biocompatibility of the lipid nanoparticle is improved. When the lipid nanoparticle is endocytosed by cells, the pH value in the endosome is low, the lipid is protonated and has positive charge, the stability of the membrane structure is poor and even destroyed, and the endosome escape of the lipid nanoparticle is facilitated. In general, it is the pH sensitive nature of lipids that facilitates in vivo delivery of lipid nanoparticles encapsulating bioactive components (e.g., mRNA molecules).

In one aspect of the invention, there is provided an ionizable lipid having a structure as shown in formula I:

in the method, in the process of the invention,

R ¹ 、R ² each independently selected from H, C ₁ -C ₁₅ Alkyl, C ₂ -C ₁₅ Alkenyl, C ₂ -C ₁₅ Alkynyl, C ₁ -C ₁₅ Haloalkyl, -OH, and R _a (R _b )N-，R _a 、R _b Each independently H, C ₁ -C ₁₀ Alkyl, C ₂ -C ₁₀ Alkenyl, C ₂ -C ₁₀ Alkynyl, C ₁ -C ₁₀ A haloalkyl group; or (b)

L ¹ 、L ² is none or each independently selected from the group consisting of:

R ⁷ selected from H, halogen, C ₁ -C ₂₅ Alkyl, C ₂ -C ₂₅ Alkenyl, C ₂ -C ₂₅ Alkynyl or C ₁ -C ₂₅ A haloalkyl group.

In a preferred embodiment of the present invention, the ionizable lipids have the substructure shown in the following formulas I-1, I-2, respectively:

in the structure of the formula I-1 or I-2, R ¹ -R ⁷ 、G ¹ Is defined as above.

In a more preferred embodiment of the present invention, the ionizable lipid has a structure selected from those shown in table 1, wherein the following compounds in table 1 are preferred:

helper lipids

As used herein, the term "helper lipid" refers to other types of lipids in addition to the ionizable lipid in the lipid nanoparticle, including one or a combination of two or more of anionic lipids, neutral lipids, steroids, and polymer-bound lipids.

In some embodiments, helper lipids may be used to improve properties of the lipid nanoparticle, such as enhancing nanoparticle stability, membrane fusion (fusogenicity), and/or flowability, among others.

In another embodiment, the anionic lipid comprises one or a combination of two or more of phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, dioleoyl phosphatidylglycerol DOPG, 1, 2-dioleoyl-sn-glycerol-3-phosphatidylserine DOPS, and dimyristoyl phosphatidylglycerol.

In another embodiment, the neutral lipid comprises at least one of 1, 2-dioleoyl-sn-glycero-3-phosphatidylethanolamine DPPC, 1, 2-distearoyl-sn-glycero-3-phosphatidylcholine DPPC, 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine DPPC, 1, 2-dioleoyl-sn-glycero-3-phosphatidylcholine DOPC, dipalmitoyl-phosphatidylglycerol DPPG, oleoyl phosphatidylcholine POPC, 1-palmitoyl-2-oleoyl phosphatidylethanolamine POPE, 1, 2-dipalmitoyl-sn-glycero-3-phosphate ethanolamine DPPE, 1, 2-dimyristoyl-sn-glycero-3-phosphate ethanolamine DMPE, distearoyl phosphatidylethanolamine DSPE, and 1-stearoyl-2-oleoyl phosphatidylethanolamine SOPE or a lipid modified with an anionic or cationic group thereof. The anionic or cationic modifying group is not limited.

In another embodiment, the steroid comprises one or more of cholesterol, non-sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassicasterol, lycosyline, ursolic acid, alpha tocopherol, fecal sterols, and corticosteroids.

In another embodiment, the polymer-bound lipid comprises 1, 2-dimyristoyl-sn-glycerogethoxy-polyethylene glycol PEG-DMG, dimyristoyl-polyethylene glycol PEG-C-DMG, polyethylene glycol-dimyristoyl-glycerol PEG-C14, PEG-1, 2-dimyristoyloxy propyl-3-amine PEG-C-DMA, 1, 2-distearoyl-sn-glycero-3-phosphate ethanolamine-N- [ amino (polyethylene glycol) ] PEG-DSPE, pegylated phosphatidylethanolamine PEG-PE, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, tween-20, tween-80, 1, 2-dipalmitoyl-sn-glycerol-methoxypolyethylene glycol PEG-DPG, 4-O- (2 ',3' -dimyristoyloxy) propyl-1-O- (ω -methoxy (polyethoxy) ethyl) succinate PEG-s-DMG, PEG-dialkoxypropyl PEG-DAA, one or a combination of two or more of mPEG2000-1, 2-di-O-alkyl-sn 3-carbamoyl glyceride PEG-c-DOMG and N-acetylgalactosamine ((R) -2, 3-bis (octadecyloxy) propyl-1- (methoxypoly (ethylene glycol) 2000) propyl carbamate)) GalNAc-PEG-DSG.

In another embodiment, the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid, and the polymer-bound lipid in the lipid carrier is (20-65): 0-20): 5-25): 25-55): 0.3-15; illustratively, the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid, and the polymer-bound lipid may be 20:20:5:50:5, 30:5:25:30:10, 20:5:5:55:15, 65:0:9.7:25:0.3, and the like; wherein in the first lipid compound, the molar ratio of the compound of formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug thereof and other cationic or ionizable lipid is (1-10): 0-10; illustratively, the molar ratio may be 1:1, 1:2, 1:5, 1:7.5, 1:10, 2:1, 5:1, 7.5:1, 10:1, etc.

In another embodiment, the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid, and the polymer-bound lipid in the lipid carrier is (20-55): 0-13): 5-25): 25-51.5): 0.5-15; wherein in the first lipid compound, the molar ratio of the compound of formula I or a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug, and the other cationic or ionizable lipid is (3-4): 0-5.

In a preferred embodiment of the invention, the helper lipid is selected from DSPC, cholesterol, PEG-DMG.

In another embodiment, the helper lipids include DSPC, cholesterol, and PEG-DMG.

Lipid Nanoparticles (LNP)

As used herein, the term "lipid nanoparticle", "lipid nanoparticle" or "LNP" refers to particles having a diameter of about 5 to 500 nm. In some embodiments, the lipid nanoparticle contains one or more active agents (bioactive substances). In some embodiments, the lipid nanoparticle comprises a nucleic acid therein. In some embodiments, the nucleic acid is condensed inside the nanoparticle with a cationic lipid, polymer, or multivalent small molecule, and an external lipid coating that interacts with the biological environment. Nucleic acids are naturally rigid polymers, more prone to elongate configurations, due to repulsive forces between phosphate groups. In cells, to cope with volume constraints, the DNA can package itself under appropriate solution conditions with the help of ions and other molecules. In general, DNA condensation is defined as the collapse of an extended DNA strand into a compact ordered particle comprising only one or a few molecules. By binding to phosphate groups, cationic lipids can concentrate DNA by neutralizing the phosphate charge and allow it to be tightly packed.

In some embodiments, the bioactive substance is encapsulated into the LNP. In some embodiments, the bioactive substance may be an anionic compound including, but not limited to, DNA (including plasmids), RNA (mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomir, microrna inhibitors, microrna activators or shRNA, etc.), natural and synthetic oligonucleotides (including antisense oligonucleotides, interfering RNAs and small interfering RNAs), nucleoproteins, peptides, nucleic acids, ribozymes, aptamers, immunostimulatory nucleic acids or PNAs, DNA-containing nucleoproteins, such as whole or partially deproteinized viral particles (virions), oligomeric and polymeric anionic compounds other than DNA (e.g., acidic polysaccharides and glycoproteins)). In some embodiments, the bioactive substance may be mixed with an adjuvant. In a preferred embodiment, the mRNA comprises a sequence encoding an RNA-guided DNA binding agent, more specifically, an mRNA encoding a nuclease or a base editor.

In a preferred embodiment, the nucleic acid further comprises a guide RNA, in particular, the guide RNA comprises a gRNA nucleic acid.

In another embodiment, the nucleic acid comprises mRNA and gRNA encoding a nuclease or base editor.

In LNP vaccine products, the biologically active substance is typically contained inside the LNP. In some embodiments, the biologically active substance comprises a nucleic acid. Typically, the water-soluble nucleic acid is condensed with a cationic lipid or polycationic polymer inside the particle, the surface of which is enriched with a helper phospholipid or a PEG lipid derivative. Additional ionizable cationic lipids may also be present on the surface, which upon entry into the cell lysosome are positively charged by ionization in the lysosome's acidic environment, acting with the lysosome membrane, facilitating endosomal escape.

With respect to LNP, ionizable lipids may have different properties or functions. Due to the pKa of the amino group, when the external pH is below the pKa of the lipid molecule, it can be protonated and positively charged. Under these conditions, the lipid molecules can electrostatically bind to the phosphate groups of the nucleic acid, which causes LNP formation and nucleic acid encapsulation, and the surface charge of the LNP in biological fluids (e.g., blood) at physiological pH is substantially neutral. High LNP surface charge is associated with toxicity, immobilization and rapid clearance of circulating, hemolytic toxicity by free macrophages, including immune activation (Fil ion et al Biochim Biophys acta.1997, 10, 23; 1329 (2): 345-56).

In some embodiments, the pKa may be sufficiently high that the ionizable cationic lipid may take a positively charged form at an acidic endosomal pH. In this way, the cationic lipids can bind to endogenous endosomal anionic lipids to facilitate membrane cleavage of non-bilayer structures, such as the hexagonal HI I phase, resulting in more efficient intracellular delivery. In some embodiments, the pKa ranges between 6.2 and 6.5. For example, the pKa may be about 6.2, about 6.3, about 6.4, about 6.5. The unsaturated tail also contributes to the ability of the lipid to adopt a non-bilayer structure. (Jayaraman et al Angew Chem Int Ed Engl.2012, 8, 20; 51 (34): 8529-33).

The release of nucleic acid in the LNP formulation, as well as other characteristics of liposome clearance and circulation half-life, can be altered by the presence of polyethylene glycol and/or sterols (e.g., cholesterol) or other potential additives in the LNP and the overall chemical structure (including the pKa of any ionizable cationic lipid that is part of the formulation).

In one aspect of the present invention there is provided a Lipid Nanoparticle (LNP) comprising a compound of formula I according to the first aspect of the present invention or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor thereof. Further, the lipid nanoparticle also comprises one or more helper lipids including one or a combination of two or more of anionic lipids, neutral lipids, steroids, and polymer-bound lipids.

Pharmaceutical preparation

In a further aspect of the invention there is provided a pharmaceutical formulation (or lipid nanoparticle composition, or LNP composition) comprising a lipid carrier according to the second aspect of the invention, and a bioactive substance encapsulated in the lipid carrier, and a pharmaceutically acceptable carrier. The pharmaceutical formulation is for delivering a biologically active substance to cells in a subject in need thereof.

In some embodiments, the bioactive substance is encapsulated into the LNP. In some embodiments, the bioactive substance may be an anionic compound including, but not limited to, DNA (including plasmids), RNA (mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomir, microrna inhibitors, microrna activators or shRNA, etc.), natural and synthetic oligonucleotides (including antisense oligonucleotides, interfering RNAs and small interfering RNAs), nucleoproteins, peptides, nucleic acids, ribozymes, aptamers, immunostimulatory nucleic acids or PNAs, DNA-containing nucleoproteins, such as whole or partially deproteinized viral particles (virions), oligomeric and polymeric anionic compounds other than DNA (e.g., acidic polysaccharides and glycoproteins). In some embodiments, the bioactive substance may be mixed with an adjuvant. In some embodiments, the LNP composition comprises: nucleic acids, ionizable lipids having a structure represented by formula (I), optionally a helper phospholipid (e.g., distearoyl phosphatidylcholine). In some embodiments, the LNP composition comprises: a nucleic acid; an ionizable lipid having a structure represented by formula I in an amount of 30-65% (molar ratio) of the total lipid of the composition; an optional co-phospholipid (e.g., distearoyl phosphatidylcholine) in an amount of 1-10% of the total lipid of the composition

And, in a preferred embodiment, the molar ratio of the first lipid compound (ionizable lipid of structure of formula (I) and optionally other cationic or ionizable lipids), anionic lipid, neutral lipid, steroid, and polymer-bound lipid in the LNP composition is (20-65): 0-20): 5-25): 25-55): 0.3-15; illustratively, the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid, and the polymer-bound lipid may be 20:20:5:50:5, 30:5:25:30:10, 20:5:5:55:15, 65:0:9.7:25:0.3, and the like.

In a preferred embodiment, the molar ratio of the compound of formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug thereof and the other cationic or ionizable lipid in the first lipid compound is from (1 to 10): from (0 to 10); illustratively, the molar ratio may be 1:1, 1:2, 1:5, 1:7.5, 1:10, 2:1, 5:1, 7.5:1, 10:1, etc.

In some embodiments, the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid, and the polymer-bound lipid in the lipid carrier is (20-55): 0-13): 5-25): 25-51.5): 0.5-15; wherein in the first lipid compound, the molar ratio of any of the above compounds or pharmaceutically acceptable forms thereof, such as salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs and other cationic or ionizable lipids is (3-4): 0-5.

As used herein, the terms "encapsulate" and "encapsulated" refer to mRNA, DNA, siRNA or other nucleic acid drug within or bound to a lipid nanoparticle. As used herein, the term "encapsulating" refers to fully encapsulating or partially encapsulating. For example, the mRNA may be selected to treat and/or prevent a related disease when a lipid nanoparticle composition comprising the mRNA is administered to a subject in need thereof.

As used herein, the term "pharmaceutically acceptable carrier" includes, but is not limited to, any adjuvant, carrier, excipient, scintillator, sweetener, diluter, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifying agent approved by the food and drug administration for use in humans or livestock.

The present invention also provides a method for delivering a nucleic acid drug in vivo, the method comprising administering the lipid nanoparticle composition or the pharmaceutical formulation to a subject in need thereof.

In some embodiments, the nucleic acid lipid nanoparticle composition or the pharmaceutical formulation described above is administered by one of the following routes of administration: oral, intranasal, intravenous, intraperitoneal, intramuscular, intra-articular, intralesional, intratracheal, subcutaneous, and intradermal. In some embodiments, the nucleic acid lipid nanoparticle composition or the pharmaceutical formulation described above is administered, for example, via an enteral or parenteral route of administration. In some embodiments, the nucleic acid lipid nanoparticle composition or pharmaceutical formulation is administered to the subject at a dose of about 0.001mg/kg to about 10 mg/kg.

Process for the preparation of pharmaceutical formulations

In another aspect of the present invention, there is provided a method of preparing a pharmaceutical formulation, the method comprising: (a) Mixing a compound of formula I according to the first aspect of the invention and optionally a co-lipid with an organic solvent, thereby obtaining a lipid organic phase; (b) Mixing the bioactive substance with an aqueous solvent to obtain an aqueous phase containing the bioactive substance; (c) Mixing the lipid organic phase of step (a) with the aqueous phase of step (b) to obtain the lipid nanoparticle composition. Further, the method further comprises step (d): and (3) purifying, concentrating, filtering and sterilizing the lipid nanoparticle medicament obtained in the step (c).

In some embodiments, the organic solvent includes, but is not limited to, ethanol, methanol, isopropanol, acetonitrile, dimethylformamide, dimethylsulfoxide, dioxane, or tetrahydrofuran, or a combination thereof. In some embodiments, the lipid organic phase comprises a small percentage of water or pH buffer. The lipid organic phase may comprise up to 60% by volume of water, for example up to about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60% by volume of water. In one embodiment, the lipid organic phase comprises between about 0.05% and 60% water by volume, for example, between about 0.05% and 50%, between about 0.05% and 40%, or between about 5% and 20% water by volume.

In some embodiments, the aqueous solvent is water. In some embodiments, the aqueous solvent is an aqueous buffer having a pH between 3 and 8 (e.g., a pH of about 3, about 4, about 5, or about 6, etc.). Dissolving a biologically active substance, such as a nucleic acid (e.g., mRNA), in the aqueous solvent, thereby obtaining an aqueous phase containing the biologically active substance. The aqueous phase may comprise a small percentage of a water-miscible organic solvent. The aqueous phase may comprise up to 60% by volume of at least one organic solvent miscible with water, such as up to about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or any% by volume of one organic solvent (e.g., a water miscible organic solvent) between the two. In one embodiment, the aqueous phase comprises between about 0.05% and 60% organic solvent by volume, for example, between about 0.05% and 50%, between about 0.05% and 40%, or between about 5% and 20% organic solution by volume (e.g., water miscible organic solvent). The aqueous buffer may be citrate buffer, tris-HCl buffer, sodium acetate buffer, PBS buffer, or a combination thereof, etc. In some embodiments, the aqueous buffer is a citrate buffer having a pH between 4 and 6 (e.g., a pH of about 4, about 5, or about 6). In one embodiment, the aqueous buffer solution is a citrate buffer having a pH of about 4.

In some embodiments, a solution comprising a mixture of a lipid organic phase and an aqueous phase containing a bioactive substance, the mixture comprising an LNP suspension, may be diluted. In some embodiments, the pH of a solution comprising a mixture of a lipid organic phase of an LNP suspension and an aqueous phase containing a biologically active substance may be adjusted. The pH of the LNP suspension can be diluted or adjusted by the addition of water, acid, base or aqueous buffers. In some embodiments, dilution or adjustment of the pH of the LNP suspension is not performed. In some embodiments, the pH of the LNP suspension is diluted and adjusted.

In some embodiments, excess reagent, solvent, unencapsulated nucleic acids can be removed from the LNP suspension by Tangential Flow Filtration (TFF) (e.g., diafiltration). Organic solvents (e.g., ethanol) and buffers can also be removed from the LNP suspension using TFF. In some embodiments, the LNP suspension is dialyzed. In some embodiments, the LNP suspension is subjected to TFF. In some embodiments, the LNP suspension is subjected to dialysis and TFF.

The main advantages of the invention include:

(1) The invention provides a series of compounds of formula I with novel structure, which can be used as ionizable lipid to prepare lipid carriers together with other lipid compounds, and has controllable particle size, uniform distribution and high encapsulation efficiency.

(2) The lipid compound has the advantages of simple synthesis method, high yield, rapid synthesis and low cost. The compound of the invention can be used for delivering nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs, and enriches the variety of ionizable lipid compounds.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

The reagents and materials used in the examples of the present invention were all commercially available products unless otherwise specified.

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.

In the present invention, "proper amount" means that the amount of the solvent or the amount of the drug to be added is large in adjustable range and less affects the synthesis result, and is not particularly limited.

In the examples described below, both solvents and drugs were used in analytical or chemical purity; redistilling the solvent before use; the anhydrous solvents were treated according to standard methods or literature methods.

The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed. The technical features of the various embodiments of the present invention may be combined with each other as long as they do not collide with each other. All publications and other references mentioned herein are incorporated by reference in their entirety.

Example 1: synthesis of 2-butyloctyl 9- { 7-butyl-20- [3- (diethylamino) propyl ] -10,18,21-trioxymethylene-19, 22-diaza-9-oxatriacontan-19-yl } nonanoate

Step 1 Synthesis of Compounds 1-2

N-octylamine (10.00 g,77.38mmol,1.0 eq) and ethyl formate (30 mL) were added to a 100mL round bottom flask. The temperature was raised to 60℃and the reaction was carried out for 16 hours, and the solvent was distilled off under reduced pressure to give N-octylmethanamide (12.10 g, yield 98.63%) as a compound, which was used directly in the next reaction without further purification.

Step 2 Synthesis of Compounds 1-3

To a 250-mL three-necked flask, compound 1-2 (12.00 g,76.31mmol,1.0 eq), triethylamine (63.47 mL,457.86mmol,6.0 eq) and tetrahydrofuran (100 mL) were successively added, and phosphorus oxychloride (18.72 g,122.10mmol,1.6 eq) was slowly dropped under ice bath. After the completion of the dropwise addition, the reaction was continued for 3 hours at zero ℃. 100ml of saturated sodium bicarbonate solution was added to the reaction mixture, and the mixture was extracted three times with 100ml of ethyl acetate, respectively And the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and subjected to column chromatography to give compound 1-isocyanoooctane (8.00 g, yield 75.3%). MS: M/z [ M+H ]] ⁺ ＝140.1。

Step 3 Synthesis of Compounds 1 to 6

To a 100 mL round bottom flask was added azelaic acid (10.10 g,53.65mmol,5.0 eq), 4-lutidine (0.66 g,5.37mmol,0.5 eq), N, N-diisopropylethylamine (13.87 g,107.30mmol,10.0 eq), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (3.09 g,16.09mmol,1.5 eq) and 50 mL dichloromethane in this order, and after stirring for half an hour at room temperature, 2-butyloct-1-ol (2.00 g,10.73mmol,1.0 eq) was added. After 16 hours at room temperature, the solvent was removed by concentration under reduced pressure, 100 ml of water was added for dilution, and each was extracted three times with 100 ml of ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and column-chromatographed to give the compound 9- [ (2-butyloctyl) oxy ] -9-oxononanoic acid (3.00 g, yield 78.4%).

Step 4 Synthesis of Compounds 1 to 8

Compounds 1-7 (8.45 g,30.93mmol,1.5 eq), 2-hexyldecan-1-ol (5.00 g,20.62mmol,1.0 eq), 4-dimethylpyridine (2.52 g,20.62mmol,1.0 eq), N, N-diisopropylethylamine (7.99 g,61.86mmol,3.0 eq) were added to a round bottom flask containing 60 ml of dichloromethane, and finally 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (5.93 g,30.93mmol,1.5 eq) was added and reacted at room temperature for 16 hours. Concentrating under reduced pressure, and performing column chromatography to obtain compound 9- ({ [ (2-methylpropan-2-yl) oxy) ]Carbonyl } amino) nonanoic acid-2-butyloctyl ester (6.86 g, 75.3% yield). MS: M/z [ M+H ]] ⁺ ＝442.4。

Step 5 Synthesis of Compounds 1 to 9

In a 100ml round bottom flask, adding compound 1-8 (6.86 g,15.54mmol,1.0 eq) and dioxane hydrochloride solution (4M, 100 ml) respectively, stirring at room temperature for 2 hours, concentrating under reduced pressure to obtain compound 4-aminobutyric acid-2-hexyl decyl ester hydrochloride, adding sodium bicarbonate aqueous solution 100ml, stirring for 0.5 hours, extracting with 30 ml ethyl acetate three times respectively, combining organic phases, washing with saturated saline solution, drying with anhydrous sodium sulfate, concentrating under reduced pressure to obtain compound 9-aminononanoic acid-2-butyl octyl ester (4.80 g, product)Rate 90.4%). MS: M/z [ M+H ]] ⁺ ＝342.4。

Step 6 Synthesis of Compounds 1 to 11

In a 25 mL round bottom flask was added 5 mL of methanol, compounds 1-9 (300.0 mg,0.88mmol,1.0 eq), 4-chlorobutyraldehyde (94.0 mg,0.88mmol,1.0 eq). After 0.5 hour of reaction at room temperature, 1-6 (0.31 g,0.88mmol,1.0 eq) was added, after 0.5 hour of stirring at room temperature, 1-3 (120.0 mg,0.88mmol,1.0 eq) was added, the reaction mixture was stirred at room temperature for 16 hours, 50 ml of water was added to dilute the mixture, three times of extraction with 30 ml of n-hexane were respectively carried out, the organic phases were combined, washed with saturated saline solution, dried over anhydrous sodium sulfate, and column chromatography was carried out to obtain the compound 9- [ 7-butyl-20- (3-chloropropyl) -10,18,21-trioxymethylene-19, 22+ -diaza-9-oxatriacontan-19-yl ]2-butyl octyl pelargonate (300.0 mg, 36.9% yield). MS: M/z [ M+H ]] ⁺ ＝925.8。

Step 7 Synthesis of Compound 1

Compounds 1 to 11 (300.0 mg,0.32mmol,1.0 eq), diethylamine (0.12 g,1.6mmol,10.0 eq), potassium carbonate (88.0 mg,0.64mmol,4.0 eq), potassium iodide (27.0 mg,0.16mmol,1.0 eq) were added to 5 ml acetonitrile, respectively, heated and stirred for 16 hours at 80℃and then diluted with 20 ml water, extracted three times with 20 ml ethyl acetate respectively, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and column chromatographed to give compound 9- { 7-butyl-20- [3- (diethylamino) propyl]-10,18,21-trioxymethylene-19, 22-diaza-9-oxatriacontan-19-yl } nonane-2-butyloctyl ester (80.0 mg, 25.7% yield). MS: M/z [ M+H ]] ⁺ ＝962.9。 ¹ H NMR(400MHz,CDCl ₃ )δ6.69(s,1H),4.68(s,1H),3.95(d,J＝4.0Hz,4H),3.55-3.03(m,4H),2.56-2.14(m,16H),2.09-1.58(m,15H),1.53-1.15(m,59H),0.97-0.75(m,15H)。

Example 2: synthesis of 2-butyloctyl 9- { 7-butyl-20- [3- (dimethylamino) propyl ] -10,18,21-trioxymethylene-19, 22-diaza-9-oxatriacontan-19-yl } nonane

Implementation of the embodimentsExample 2 was prepared according to the synthesis method of example 1, substituting diethylamine in step 7 with dimethylamine hydrochloride. The yield was 35.2%. MS: M/z [ M+H ]] ⁺ ＝934.8。 ¹ H NMR(400MHz,CDCl ₃ )δ6.67(s,1H),4.69(s,1H),3.93(d,J＝4.0Hz,4H),3.56-3.04(m,4H),2.60-2.46(m,10H),2.41-2.22(m,8H),2.18-1.61(m,15H),1.53-1.15(m,53H),1.01-0.78(m,15H)。

Example 3: synthesis of 2-butyloctyl 9- { 7-butyl-10,18,21-trioxymethylene-20- [3- (tetrahydro-1H-pyrrol-1-yl) propyl ] -19, 22-diaza-9-oxatriacontan-19-yl } nonanoate

Example 3 was prepared according to the synthetic method of example 1, substituting diethylamine in step 7 with pyrrolidine. The yield was 45.8%. MS: M/z [ M+H ]] ⁺ ＝960.9。 ¹ H NMR(400MHz,CDCl ₃ )δ6.67(s,1H),4.70(s,1H),4.12-3.90(m,4H),3.60-3.05(m,4H),2.58-2.21(m,16H),2.14-1.60(m,15H),1.60-1.22(m,57H),0.99-0.72(m,15H)。

Example 4: synthesis of 2-butyloctyl 9- { 7-butyl-20- [2- (diethylamino) ethyl ] -10,18,21-trioxymethylene-19, 22-diaza-9-oxatriacontan-19-yl } nonanoate

Example 4 was prepared according to the synthesis method of example 1, substituting chloroacetaldehyde in step 6 with chloroacetaldehyde. The yield was 28.4%. MS: M/z [ M+H ]] ⁺ ＝948.9。 ¹ H NMR(400MHz,CDCl ₃ )δ6.74(s,1H),4.76(s,1H),4.03(d,J＝4.0Hz,4H),3.58-3.06(m,4H),2.68-2.15(m,16H),2.11-1.57(m,15H),1.56-1.14(m,57H),0.95-0.78(m,15H)。

Example 5: synthesis of 4, 4-bis { [ (5Z) -oct-5-enyl ] oxy } butanoic acid-7-butyl-19- (3-ethyl-8-oxolan-3, 9-diazaheptadec-7-yl) -10, 18-dioxolan-19-aza-9-oxadocan-22-yl ester

Example 5 was prepared following the synthetic procedure of example 1 substituting 1-9 in step 6 with 3-aminopropan-1-ol.

Step 1 Synthesis of Compound 5

5-1 (500.0 mg,0.72mmol,1.0 eq), 5-2 (0.37 g,1.08mmol,1.5 eq), DMAP (88.0 mg,0.072mmol,0.1 eq), EDCI (0.21 g,1.08mmol,1.5 eq), DIEA (0.19 g,1.44mmol,2.0 eq) were added to 3 ml of dichloromethane in this order, stirred at room temperature for 16 hours, diluted with 20 ml of water, extracted three times with 10 ml of dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and chromatographed to give the compound 4, 4-bis { [ (5Z) -oct-5-enyl ]Oxy } butanoic acid-7-butyl-19- (3-ethyl-8-oxy-3, 9-diazaheptadec-7-yl) -10, 18-dioxo-19-aza-9-oxa-docosan-22-yl ester (0.25 g, 34.2% yield). MS: M/z [ M+H ]] ⁺ ＝1018.9。 ¹ H NMR(400MHz,CDCl ₃ )δ6.75(s,1H),5.48-5.20(m,4H),4.84(s,1H),4.49(s,1H),4.10-3.91(m,4H),3.61-3.25(m,6H),3.20-2.80(m,6H),2.48-2.12(m,6H),2.05-1.52(m,26H),1.48-1.04(m,45H),0.98-0.75(m,15H)。

Example 6

Preparation, characterization and in vivo editing experimental evaluation of lipid nanoparticles

1. Design of animal experiment

mRNA and sgRNA delivery experiments targeting the base editor ABE8e of PCSK9

Cholesterol in blood is mainly synthesized by the liver, which is also a main organ for decomposing excessive cholesterol, and a low-density lipoprotein LDL receptor (LDLR) exists on the surface of the liver, which can be combined with cholesterol circulating back to the liver, so that the cholesterol is decomposed into bile acid, and is excreted outside the body through the intestinal tract; PCSK9 is a liver-synthesized protease capable of binding to LDL receptors, promoting their entry into hepatocytes, leading to degradation of LDL receptors by lysosomes, and a reduction in the number of LDL receptors; thus, inhibiting the activity of PSCK9, the amount of LDLR can be increased, and thus the uptake and decomposition ability of cholesterol can be enhanced. Basic and clinical studies show that the PCSK9 gene is an effective target for treating hyperlipidemia and atherosclerosis. FIG. 1 shows the changes in the number of LDL receptors and ultimately the changes in cholesterol metabolism caused before and after editing of a specific site of the PCSK9 gene.

The strategy of PCSK9 gene editing and delivery of the mouse liver cells is shown in figure 1, and the main flow is as follows: the prepared lipid nanoparticle is utilized to target and deliver mRNA and sgRNA encoding ABE8e to mouse liver cells by intravenous injection, mutation is introduced into PCSK9 genes under the action of the ABE8e and the sgRNA, the mutation of bases A to G is realized at a specific site, and the editing efficiency is calculated by sequencing.

The specific experimental design is as follows:

1.1 selecting appropriate mutation sites and editing designs

The single base editor ABE8e realizes accurate base substitution from a to G without a donor template and without DSB, based on which the first exon of the PCSK9 gene is selected as the screening mutation site. mRNA encoding a single-base editing tool ABE8e and sgRNA are delivered into an animal body together through lipid nanoparticles, the mRNA encoding the base editor ABE8e is translated into protein in cytoplasm, the protein forms a complex with the sgRNA and enters a cell nucleus, the base editor ABE8e targets a first exon splice donor site of a PCSK9 gene under the guidance of the sgRNA, adenine (A) on a first exon template strand is deaminated into inosine (I), I can be regarded as G at DNA level for reading and copying, and finally, replacement from A to G is realized, so that the splice donor site is destroyed so that a PCSK9 gene reading frame is terminated in advance.

1.2 preparation of mRNA and sgRNA of base editor ABE8e

The sequence of the first exon and the first intron of the mouse PCSK9 Gene (NCBI Gene ID: 100102) is selected as a targeting region, and a target sequence PCSK9-sgRNA for single-base editing of the PCSK9 Gene is determined.

By analyzing the sequence of the PCSK9 gene across the first exon and the intron, the sgrnas of the targeting region were designed: PCSK9-sgRNA (synthesized by auresli, south kyo) having the sequence:

PCSK9-sgRNA：5’-CCCATACCTTGGAGCAACGG-3’(SEQ ID NO:1)；

the sgRNA is designed according to the target sequence and oligonucleotide (oligo) is synthesized, and the sequence of the used sgRNA is shown as SEQ ID NO. 1. CACC sequences were added to the 5 'end of the upstream sequence and AAAC sequences to the 5' end of the downstream sequence of each sgRNA. After synthesis, the upstream and downstream sequences were annealed by a preset procedure (95 ℃,5min;95 ℃ to 85 ℃ at-2 ℃/S;85 ℃ to 25 ℃ at-0.1 ℃/S; kept at 4 ℃) and the annealed product was ligated to the lenti U6-sgRNA/EF1a-mCherry vector (Addgene, plasmid, # 114199) linearized by BbsI (NEB, R3539S).

Wherein, the system used in the construction of the sgRNA plasmid is as follows:

the linearization system of the lenti U6-sgRNA/EF1a-mCherry vector is as follows: 3 μg of carrier; 6. Mu.L of buffer (NEB: R0539L); bbsI 2. Mu.L; ddH ₂ O was added to 60. Mu.L and digested overnight at 37 ℃.

The linkage system of the sgRNA annealing product and the linearization carrier is as follows: t4 ligase buffer (NEB: M0202L) 1. Mu.L, linearized vector 20ng, annealed oligo fragment (10. Mu.M) 5. Mu.L, T4 ligase (NEB: M0202L) 0.5. Mu.L, ddH ₂ O was made up to 10. Mu.L and ligated overnight at 16 ℃.

The ligated vector was transformed into E.coli DH5a competent cells (Geotex only, DL 1001). The specific flow is as follows: DH5 alpha competent cells were taken out from-80℃and rapidly inserted into ice for 5 min, after which the pellet was thawed, the ligation product was added and gently mixed by hand-pulling the bottom of the centrifuge tube, and left to stand in ice for 25 min. The mixture was heat-shocked in a 42℃water bath for 45 seconds, quickly returned to ice and allowed to stand for 2 minutes. To the centrifuge tube, 700. Mu.l of sterile LB medium without antibiotics was added, and after mixing, resuscitated at 37℃for 60 minutes at 200 rpm. The bacteria were harvested by centrifugation at 5000rpm for one minute, and about 100. Mu.l of the supernatant was gently swirled to resuspension the pellet and spread on LB medium of Amp antibiotics. The plates were placed in an incubator at 37℃overnight. Single colonies were picked, sequenced and confirmed, positive clones were shaken and plasmids (TIANGEN: DP 120-01) were extracted, and the concentration was determined and stored in a-20deg.C refrigerator for use.

The base editor ABE8E used in this experiment was the high efficiency base editor ABE8E evolved by David R.Liu team (Richter MF, zhao KT, eton E, lapinaite A, newby GA, thuronyi BW, wilson C, koblan LW, zeng J, bauer DE, doudna JA, liu DR.Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity.Nat Biotechnol.2020Jul;38 (7): 883-891.doi:10.1038/s41587-020-0453-z.Epub 2020Mar 16.Erratumin:Nat Biotechnol.2020May 20;PMID:32433547;PMCID:PMC7357821). Plasmid ABE8e (Plasmid # 138489) was purchased from adedge and mRNA of ABE8e was expressed and purified for use by the laboratory.

2. The ionizable lipid or the compound of the invention/DSPC/cholesterol/PEG-lipid was prepared in a molar ratio of 50:10:38.5:1.5.

2.1 Di-oleylmethylene-4-dimethylaminobutyrate (DLin-MC 3-DMA, commonly abbreviated as MC 3) and Compound 1-Compound 5 of the present invention were dissolved in absolute ethanol at the above molar ratios with DSPC, cholesterol, PEG-DMG, respectively.

2.2 ethanol solutions of different lipid carriers were mixed with buffer of mRNA at 1:3 (volume/volume) where the mass ratio of total lipid to mRNA was 40:1,sgRNA:ABE8e mRNA (w/w) was 1:1, and nucleic acid lipid nanoparticles 1-10 were obtained by a microfluidic nano-drug manufacturing system (NanoAssemblr Ignite, canada) at a flow rate of 12 ml/min. The obtained nucleic acid lipid nanoparticles were immediately diluted 40-fold in 1 x DPBS buffer. The diluted nucleic acid lipid nanoparticle solution passes through an overspeed centrifuge tube and is concentrated to reach the target volume. After dilution, the particles were used for DLS particle size measurement and encapsulation efficiency detection.

2.3 determination of the particle size and polydispersity index of lipid nanoparticles by dynamic light scattering in 173 ° back scattering detection mode using Malvern Zetasizer Nano ZS (Malvern UK). The encapsulation efficiency of the lipid nanoparticles was determined using the Quant-it Ribogreen RNA quantification kit (ThermoFisher Scientific, UK) according to the manufacturer's instructions and the test results are shown in table 2.

Table 2 nano lipid particle characterization

3 in vivo editing experiment evaluation

3.1 lipid nanoparticles comprising a compound of the invention (see Table 2) encapsulating mRNA and sgRNA encoding base editor ABE8e were systemically administered to 6-7 week old C57BL/6 female mice (purchased from Jiangsu Jiujiaka) by tail vein injection at a dose of 0.2 mg/kg. Lipid nanoparticles comprising dioleylmethylene-4-dimethylaminobutyrate (DLin-MC 3-DMA, abbreviated as MC 3) encapsulating mRNA and sgRNA of base editor ABE8e were applied in a similar manner to a comparable group of mice of both week-old and sex-old as positive controls. In addition, PBS buffer was also used as a negative control for mice of comparable age and sex in a similar manner to tail vein injection.

3.2 edit efficiency detection

Editing efficiency detection was performed one week after mice were dosed, liver tissue was taken after mice were sacrificed, genome was extracted after lysis, and efficiency analysis was performed by deep sequencing.

The deep sequencing procedure was as follows:

(1) Primers were designed based on the target gene location, see in particular table 3.

TABLE 3 design of primers for targeting PCSK9 Gene

Sequence object	Sequence(s)
		PCSK9-F2	5’-ACCAGACGGCTAGATGAGCA-3’(SEQ ID NO:2)
PCSK9-R2	5’-CCCAGGACGAGGATGGAGATTA-3’(SEQ ID NO:3)

(2) Editing efficiency detection.

The PCR procedure was as follows: 94 ℃ for 2min;98℃10s,60℃30s,68℃20s,34 cycles; 68 ℃ for 5min. After the PCR is finished, gel electrophoresis is used for verification, a single band with proper size is selected, the amplified product is determined to be correct, and the obtained PCR product is sent to Nanjing gold Style company for sequencing.

(3) And analyzing the reading of the deep sequencing result through the crispress software, analyzing the specific site, and calculating the editing efficiency, wherein the calculated result is shown in table 4, and the editing efficiency corresponding to each lipid nanoparticle can be shown in fig. 2.

Table 4 in vivo edit efficiency assessment

As shown in table 4, the ionizable lipid compounds employed in the present invention are capable of effectively delivering nucleic acid molecules, small molecule compounds, and the like; by contrast, the lipid nanoparticle has the advantages of better particle size distribution, high encapsulation efficiency and obviously better delivery effect than the lipid nanoparticle contrast, and can meet the in-vivo delivery requirement.

The description of the exemplary embodiments presented above is merely illustrative of the technical solution of the present invention and is not intended to be exhaustive or to limit the invention to the precise form described. Obviously, many modifications and variations are possible in light of the above teaching to those of ordinary skill in the art. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable others skilled in the art to understand, make and utilize the invention in various exemplary embodiments and with various alternatives and modifications. The scope of the invention is intended to be defined by the claims and equivalents thereof.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor thereof, having the structure shown below:

2. A lipid carrier comprising a compound of formula I as defined in claim 1 or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor thereof.

3. A Lipid Nanoparticle (LNP) comprising a compound of formula I according to claim 1 or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor thereof.

4. A lipid nanoparticle composition comprising a compound of formula I according to claim 1 or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or a precursor thereof, or a lipid carrier according to claim 2 or a lipid nanoparticle according to claim 3, and a bioactive substance encapsulated in the lipid carrier or the lipid nanoparticle.

5. A pharmaceutical composition comprising a compound of formula I according to claim 1 or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor thereof, or a lipid carrier according to claim 2 or a lipid nanoparticle according to claim 3, and a biologically active substance encapsulated in the lipid carrier or the lipid nanoparticle, together with a pharmaceutically acceptable excipient, carrier or diluent.

6. A pharmaceutical formulation comprising a compound of formula I according to claim 1 or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor thereof, or a lipid carrier according to claim 2 or a lipid nanoparticle according to claim 3, and a biologically active substance encapsulated in the lipid carrier or the lipid nanoparticle, together with a pharmaceutically acceptable excipient, carrier or diluent; or the pharmaceutical formulation comprises the lipid nanoparticle composition of claim 4, and a pharmaceutically acceptable excipient, carrier or diluent.

7. Use of a compound of formula I as defined in claim 1 or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor thereof for the preparation of a liposomal nanoparticle.

8. Use of a compound of formula I according to claim 1 or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or lipid carrier according to claim 2 or lipid nanoparticle according to claim 3 or lipid nanoparticle composition according to claim 4 or pharmaceutical composition according to claim 5 or pharmaceutical formulation according to claim 6 for the preparation of a nucleic acid drug, genetic vaccine, small molecule drug, polypeptide or protein drug.

9. A method of preparing the lipid nanoparticle composition of claim 4, comprising:

(a) Mixing a compound of formula I as defined in claim 1 or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or a precursor thereof, and optionally a co-lipid with an organic solvent, thereby obtaining a lipid organic phase;

10. Use of a compound of formula I as defined in claim 1 or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or precursor thereof for the preparation of a drug delivery system.

11. Use of a compound of formula I according to claim 1 or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or a precursor thereof, or a lipid carrier according to claim 2, or a lipid nanoparticle according to claim 3, or a lipid nanoparticle composition according to claim 4, or a pharmaceutical composition according to claim 5, or a pharmaceutical formulation according to claim 6, for the manufacture of a medicament for the treatment and/or prophylaxis of a disease.

12. A method of delivering a therapeutic or prophylactic agent to a cell, tissue or organ of a subject, comprising contacting the cell, tissue or organ of the subject with the lipid nanoparticle composition of claim 4 or the pharmaceutical composition of claim 5 or the pharmaceutical formulation of claim 6.

13. A method of producing a protein or polypeptide of interest in a subject cell, comprising contacting the subject cell with the lipid nanoparticle composition of claim 4 or the pharmaceutical composition of claim 5 or the pharmaceutical formulation of claim 6.