CN117820149B - Ionizable lipid compounds - Google Patents

Abstract

The present invention provides a novel ionizable lipid compound, or a pharmaceutically acceptable salt, isotope variant, tautomer or stereoisomer thereof, a pharmaceutical composition thereof, and use thereof in preparing a preventive or therapeutic nucleic acid vaccine.

Description

Ionizable lipid compounds

Technical Field

The invention relates to an ionizable cationic lipid compound, or pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, and a preparation method and application thereof.

Background

The nucleic acid vaccine is prepared through introducing exogenous gene (DNA or RNA) encoding specific antigen protein directly into host cell, expressing the antigen protein in the host cell and inducing the host to produce immune response to the antigen protein for the purpose of preventing and treating diseases.

Moderna and BioNTech use Lipid Nanoparticles (LNP) as delivery systems, the LNP major components including Cationic Lipid molecules (Cationic Lipid), cholesterol, neutral lipids and polyethylene glycol conjugated lipids. The cationic lipid molecule is the core of the LNP delivery system, and the molecular structure plays a decisive role in the aspects of the delivery efficiency, targeting, preparation stability and the like of the whole liposome nano-particle.

Since specific delivery to achieve different kinds of nucleic acid substance delivery and different targets has different requirements for the delivery system, further development of new lipid molecules is required in order to meet different demands of gene therapy.

Disclosure of Invention

BioNTech the cationic lipid molecule ALC0315 used in nucleic acid vaccines is currently one of the well-accepted cationic lipid compounds with optimal nucleic acid delivery effects, which can generate an efficient immune response in the body. The present inventors have developed a class of cationic lipid compounds containing gem-dimethyl (see CN115850104 a) that have a high efficiency of nucleic acid delivery, mainly to the liver, when injected into the body by intravenous administration. However, nucleic acid vaccines can be used for generating immune responses in the lymphoid organs of the human body by intramuscular injection, and whether the lipid compounds of the invention generate good immune responses in the human body is a key problem in the research of the invention due to different applications. Unexpectedly, the compounds of the present invention are useful for preparing nucleic acid vaccines, and have better immune response effects than ALC 0315.

Specifically, in one aspect of the present invention, there is provided a compound of formula (I), or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof:

Wherein,

R ₁ and R ₂ are independently selected from C _4-20 alkyl, C _4-20 alkenyl, and C _4-20 alkynyl, said C _4-20 alkyl, C _4-20 alkenyl, or C _4-20 alkynyl being optionally substituted with one or more R, one or more methylene units in said C _4-20 alkyl, C _4-20 alkenyl, or C _4-20 alkynyl being optionally and independently replaced with-NR ";

R is independently selected from H, C _1-14 alkyl, -L _a-OR_a, and-L _a-NR_aR'_a;

r' is independently selected from H and C _1-20 alkyl;

r ₃ and R ₄ are independently selected from H, C _1-6 alkyl, C _1-6 haloalkyl, 3 to 10 membered cycloalkyl, and 3 to 10 membered heterocyclyl, said C _1-6 alkyl, C _1-6 haloalkyl, 3 to 10 membered cycloalkyl, or 3 to 10 membered heterocyclyl optionally substituted with 1,2, 3, 4, or 5R;

or R ₃、R₄ together with the N atom to which they are attached form a3 to 10 membered heterocyclyl, said 3 to 10 membered heterocyclyl optionally substituted with 1,2, 3, 4 or 5R;

a is independently selected from 0,1, 2, 3, 4 and 5;

b and d are independently selected from 3,4, 5, 6, 7, 8 and 9;

c and e are independently selected from 0,1, 2, 3 and 4;

b+c=3, 4, 5, 6, 7, 8 or 9, d+e=3, 4, 5, 6, 7, 8 or 9;

optionally and independently substituted with 1,2,3,4 or 5C _1-6 alkyl groups;

r ₅、R₆、R₇ and R ₈ are independently selected from C _1-6 alkyl, said C _1-6 alkyl optionally substituted with 1,2, 3, 4 or 5R;

R is independently at each occurrence selected from H, halogen, cyano, C _1-6 alkyl, C _1-6 haloalkyl, -L _b-OR_b, or-L _b-NR_bR'_b;

L _a is independently selected from a bond and C _1-6 alkylene;

L _b is independently selected from a bond and C _1-6 alkylene;

R _a and R' _a are independently selected from H, C _1-6 alkyl, 3 to 10 membered cycloalkyl and 3 to 10 membered heterocyclyl;

R _b and R' _b are independently selected from H, C _1-6 alkyl, 3 to 10 membered cycloalkyl and 3 to 10 membered heterocyclyl.

The compounds of the present invention are ionizable lipid compounds.

In one embodiment, a is selected from 2,3 and 4, more preferably 2 or 3, more preferably 2.

In one embodiment, b and d are independently 4 or 5.

In one embodiment, c and e are independently 0 or 1.

In one embodiment, b+c=4, 5 or 6.

In one embodiment, d+e=4, 5 or 6.

In one embodiment, b+c=5 and d+e=5.

In one embodiment, R ₁ or R ₂ are independently selected from linear alkyl groups having a total length of 6, 7, 8, 9 or 10 carbon atoms, 1, 2 or 3 methylene groups of which are optionally and each independently substituted with C _1-8 alkyl.

In one embodiment, R ₁ and R ₂ are each independently a straight chain alkyl group having a total length of 9 carbon atoms, and one of R ₁ or R ₂ is substituted with a C _4-6 alkyl group.

In one embodiment, R ₁ and R ₂ are each independently a straight chain alkyl group having a total length of 9 carbon atoms, and one of R ₁ or R ₂ is substituted with n-butyl, n-pentyl or n-hexyl.

In one embodiment, R ₃ and R ₄ are independently selected from C _1-6 alkyl, said C _1-6 alkyl optionally substituted with 1,2 or 3R.

In one embodiment, R is independently selected from H, C _1-6 alkyl, C _1-6 haloalkyl, and-OR _a, wherein R _a is independently selected from H and C _1-6 alkyl.

In one embodiment, R ₃ and R ₄ are independently selected from methyl.

In one embodiment, R ₅、R₆、R₇ and R ₈ are independently selected from C _1-6 alkyl, preferably methyl or ethyl, more preferably methyl.

In another aspect of the invention, there is provided the following compounds, or pharmaceutically acceptable salts, isotopic variants, tautomers or stereoisomers thereof:

in one embodiment, the above compounds are used for the preparation of nucleic acid vaccines, preferably for the preparation of RNA vaccines, more preferably for the preparation of mRNA vaccines.

In another aspect of the invention, a pharmaceutical composition is provided comprising a compound as described above, or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, and optionally a pharmaceutically acceptable adjuvant, such as a carrier, adjuvant, or vehicle.

In one embodiment, the pharmaceutical composition is a nanoparticle composition comprising a compound of the invention.

In one embodiment, the nanoparticle composition comprises a lipid component, and optionally an active ingredient.

In one embodiment, the active ingredient is a nucleic acid.

In one embodiment, the lipid component comprises the following components in mole percent:

20mol% to 85mol% of ionizable cationic lipid;

10mol% to 75mol% of structural lipids;

Neutral lipid 1.0mol% -30mol%;

0.25mol% to 10mol% of polymer lipid.

Wherein the ionizable cationic lipid is a compound described above, or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof.

In one embodiment, the mole percent content of the ionizable cationic lipid is 42.5 mole% to 50 mole%, 32.5 mole% to 50 mole%, 25 mole% to 65 mole%, 30 mole% to 60 mole%, 30 mole% to 50 mole%, 30 mole% to 55 mole%, or 40 mole% to 52.5 mole%.

In one embodiment, the mole percent content of the structural lipid is 30.6 mole% to 61 mole%, 30.6 mole% to 51 mole%, 25 mole% to 70 mole%, 27.5 mole% to 66 mole%, 30.5 mole% to 66 mole%, 28 mole% to 64 mole%, or 28 mole% to 54 mole%.

In one embodiment, the mole percent content of neutral lipids is 1 mole% to 25 mole%, 1.5 mole% to 20 mole%, 5 mole% to 20 mole%, or 5 mole% to 20 mole%.

In one embodiment, the molar percentage content of the polymer lipid is 1mol% to 5mol%, 1mol% to 2mol%, 0.5mol% to 8mol%, 1mol% to 5mol%, 1mol% to 3mol%, or 1mol% to 3mol%.

In one embodiment, the molar ratio of N atoms in the ionizable cationic lipid to N to P atoms in the loading molecule is 1-15:1, e.g., can be 3-12:1, 3-7:1, 1-10:1, 3-6:1, or 3-5:1.

In one embodiment, the particles have a particle size of 65-200nm, for example 65-180nm, 70-170nm, 70-130nm, 70-180nm, 80-180nm, 90-180nm or 100-135nm.

In one embodiment, the neutral lipid is selected from one or more of DSPC, DMPC, DOPC, DPPC, POPC, DOPE, DMPE, POPE or DPPE.

In one embodiment, the structural lipid is selected from one or more of cholesterol, sitosterol, stigmasterol, rock-soap sterol, brassicasterol, ergosterol, lycorine, ursolic acid, alpha-tocopherol, stigmasterol, aveosterol, ergocalcitol, or campesterol.

In one embodiment, the polymeric lipid is a pegylated lipid.

In one embodiment, the pegylated lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol.

In one embodiment, the pegylated lipid is selected from one or more of DMPE-PEG1000、DPPE-PEG1000、DSPE-PEG1000、DOPE-PEG1000、DMG-PEG2000、Ceramide-PEG2000、DMPE-PEG2000、DPPE-PEG2000、DSPE-PEG2000、Azido-PEG2000、DSPE-PEG2000-Mannose、Ceramide-PEG5000、DSPE-PEG5000.

In another aspect of the invention, there is provided a compound as described above, or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, or a pharmaceutical composition as described above, for use in the treatment, diagnosis and/or prevention of a disease.

In another aspect of the invention, there is provided the use of a compound as described above, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, or a pharmaceutical composition as described above, in the manufacture of a medicament for the treatment, diagnosis or prophylaxis of a disease.

In one embodiment, the medicament for treating, diagnosing or preventing a disease is selected from a therapeutic vaccine or a prophylactic vaccine.

In one embodiment, the vaccine is used for preventing/treating diseases caused by viral infection.

In one embodiment, the vaccine is for the prevention/treatment of a disease caused by coronavirus infection, preferably a disease caused by SARS-CoV-2;

In one embodiment, the vaccine is used for preventing/treating diseases caused by influenza virus infection.

In one embodiment, the vaccine is used for preventing/treating diseases caused by herpes zoster virus infection.

In another aspect of the invention, there is provided a method of treating, diagnosing or preventing a disease in a subject comprising administering to the subject a pharmaceutical composition comprising a compound as described above, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof.

In one embodiment, the pharmaceutical composition is administered in an effective amount.

In one embodiment, the disease is a disease caused by a viral infection.

In one embodiment, the disease is a disease caused by a coronavirus infection, preferably a disease caused by SARS-CoV-2.

In one embodiment, the disease is a disease caused by influenza virus infection.

In one embodiment, the disease is a disease caused by an infection with a herpes zoster virus.

In another aspect of the invention there is provided the use of a compound as described above, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, or a pharmaceutical composition as described above, in the manufacture of a medicament for delivering a load selected from one or more of a therapeutic agent, a prophylactic agent or a diagnostic agent.

In another aspect of the invention, there is provided a method of delivering a load in a subject comprising administering to the subject a pharmaceutical composition comprising a compound as described above, or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, said load being selected from one or more of a therapeutic agent, a prophylactic agent, or a diagnostic agent.

In one embodiment, the therapeutic, prophylactic or diagnostic agent is a nucleic acid.

In one embodiment, the nucleic acid is selected from the group consisting of antisense oligonucleotides (ASOs).

In one embodiment, the nucleic acid is selected from one or more of RNA or DNA.

In one embodiment, the RNA is selected from one or more of small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (lncRNA), microrna (miRNA), small activating RNA (saRNA), poly-coding nucleic acid (MCNA), poly-coding nucleic acid (PCNA), guide RNA (gRNA), CRISPRRNA (CRRNA), or ribozyme.

In one embodiment, the RNA is mRNA, more preferably modified mRNA.

Definition of the definition

Chemical definition

The definition of specific functional groups and chemical terms is described in more detail below.

The term "substituted" or "substituted" means that any one or more hydrogen atoms on a particular atom is replaced with a substituent.

The term "chemical bond" refers to a single, double or triple bond, preferably a single bond.

When numerical ranges are listed, it is intended to include each and every value and subrange within the range. For example, "C _1-6 alkyl" includes C₁、C₂、C₃、C₄、C₅、C₆、C_1-6、C_1-5、C_1-4、C_1-3、C_1-2、C_2-6、C_2-5、C_2-4、C_2-3、C_3-6、C_3-5、C_3-4、C_4-6、C_4-5 and C _5-6 alkyl.

"C _4-20 alkyl" refers to a straight or branched chain saturated hydrocarbon group having 4 to 20 carbon atoms, further including C _9-15 alkyl. Examples of C _1-6 alkyl groups include methyl (C ₁), ethyl (C ₂), n-propyl (C ₃), Isopropyl (C ₃), n-butyl (C ₄), tert-butyl (C ₄), sec-butyl (C ₄), Isobutyl (C ₄), n-pentyl (C ₅), 3-pentyl (C ₅), pentyl (C ₅), Neopentyl (C ₅), 3-methyl-2-butyl (C ₅), tertiary pentyl (C ₅) and n-hexyl (C ₆), and the like. The term "C _1-6 alkyl" also includes heteroalkyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced with a heteroatom (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkyl group may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Conventional alkyl abbreviations include ：Me(-CH₃)、Et(-CH₂CH₃)、iPr(-CH(CH₃)₂)、nPr(-CH₂CH₂CH₃)、n-Bu(-CH₂CH₂CH₂CH₃) or i-Bu (-CH ₂CH(CH₃)₂).

"C _4-20 alkenyl" refers to a straight or branched hydrocarbon group having 4 to 20 carbon atoms and at least one carbon-carbon double bond. Examples of C _2-6 alkenyl groups include vinyl (C ₂), 1-propenyl (C ₃), 2-propenyl (C ₃), 1-butenyl (C ₄), 2-butenyl (C ₄), butadienyl (C ₄), pentenyl (C ₅), pentadienyl (C ₅), hexenyl (C ₆), and the like. The term "C _2-6 alkenyl" also includes heteroalkenyl groups in which one or more (e.g., 1,2,3, or 4) carbon atoms are replaced with heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkenyl group may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or1 substituent.

"C _4-20 alkynyl" refers to a straight or branched hydrocarbon group having 4 to 20 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds. The term "C _2-6 alkynyl" also includes heteroalkynyl groups in which one or more (e.g., 1,2,3, or 4) carbon atoms are replaced with heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Alkynyl groups may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

The term "the total length of the variables a and B is x carbon atoms" means that the sum of the number of carbon atoms in the backbone of the group represented by the variable a and the number of carbon atoms in the backbone of the group represented by the variable B is x.

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

Thus, "C _1-6 haloalkyl" refers to the "C _1-6 alkyl" described above, which is substituted with one or more halo groups. The haloalkyl group may be substituted at any available point of attachment, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

"C _3-10 cycloalkyl" or "3-to 10-membered cycloalkyl" refers to a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms and zero heteroatoms, optionally containing 1,2 or 3 double or triple bonds. Cycloalkyl also includes ring systems in which the cycloalkyl ring is fused to one or more aryl or heteroaryl groups, where the point of attachment is on the cycloalkyl ring, and in such cases the number of carbons continues to represent the number of carbons in the cycloalkyl system. Cycloalkyl also includes wherein the cycloalkyl rings described above, wherein substituents on any non-adjacent carbon atoms are joined to form a bridged ring, taken together to form a multicyclic alkane sharing two or more carbon atoms. cycloalkyl also includes the cycloalkyl rings described above, wherein substituents on the same carbon atom are joined to form a ring, together forming a multicycloalkane sharing one carbon atom. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl (C ₃), cyclopropenyl (C ₃), cyclobutyl (C ₄), cyclobutenyl (C ₄), Cyclopentyl (C ₅), cyclopentenyl (C ₅), cyclohexyl (C ₆), cyclohexenyl (C ₆), Cyclohexadienyl (C ₆), cycloheptyl (C ₇), cycloheptenyl (C ₇), cycloheptadienyl (C ₇), cycloheptatriene (C ₇), and the like. Cycloalkyl groups may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

"3-10 Membered heterocyclyl" or "3-10 membered heterocyclyl" refers to a saturated or unsaturated group of a 3-10 membered non-aromatic ring system having ring carbon atoms and 1 to 5 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus and silicon, optionally containing 1,2 or 3 double or triple bonds. In a heterocyclic group containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom as the valence permits. Heterocyclyl also includes ring systems in which the above heterocyclyl ring is fused to one or more cycloalkyl groups, wherein the point of attachment is on the heterocyclyl ring, or ring systems in which the above heterocyclyl ring is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such cases the number of ring members continues to represent the number of ring members in the heterocyclyl ring system. Heterocyclyl also includes the heterocyclic rings described above in which substituents on any non-adjacent carbon or nitrogen atom are joined to form a bridged ring, taken together to form a polycyclic heteroalkane sharing two or more carbon or nitrogen atoms. Heterocyclyl groups also include those wherein the above-mentioned heterocyclyl rings are wherein the substituents on the same carbon atom are joined to form a ring, together forming a polycyclic heteroalkane sharing one carbon atom. Exemplary 3-membered heterocyclic groups containing one heteroatom include, but are not limited to, aziridine, oxetane, thiirane (thiorenyl). Exemplary 4-membered heterocyclic groups containing one heteroatom include, but are not limited to, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclic groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2, 5-dione. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to, pyrazolidinyl, dioxolanyl, oxathiolanyl (oxasulfuranyl), dithiolanyl (disulfuranyl), and oxazolidin-2-one. Exemplary 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl (thianyl). Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithiocyclohexenyl, dioxanyl. Exemplary 6-membered heterocyclic groups containing three heteroatoms include, but are not limited to, hexahydrotriazinyl (triazinanyl). Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to, azepanyl, oxepinyl, and thiepanyl. Exemplary 5-membered heterocyclic groups fused to a C ₆ aryl ring (also referred to herein as 5, 6-bicyclic heterocyclic groups) include, but are not limited to, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinone groups, and the like. exemplary 6-membered heterocyclyl groups fused to a C ₆ aryl ring (also referred to herein as 6, 6-bicyclic heterocyclyl) include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like. Heterocyclyl also includes those wherein the heterocyclyl shares one or two atoms with a cycloalkyl, heterocyclyl, aryl or heteroaryl group to form a bridged or spiro ring, where the shared atoms may be carbon or nitrogen atoms as the valency permits. Heterocyclyl also includes the above heterocyclyl and heterocyclyl groups may be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

"C _6-10 aryl" refers to a group of a monocyclic or polycyclic (e.g., bicyclic) 4n+2 aromatic ring system (e.g., having 6 or 10 pi electrons shared in a cyclic arrangement) having 6 to 10 ring carbon atoms and zero heteroatoms. In some embodiments, aryl groups have six ring carbon atoms ("C ₆ aryl"; e.g., phenyl). In some embodiments, aryl groups have ten ring carbon atoms ("C ₁₀ aryl"; e.g., naphthyl, e.g., 1-naphthyl and 2-naphthyl). Aryl also includes ring systems in which the above aryl ring is fused to one or more cycloalkyl or heterocyclyl groups, and the point of attachment is on the aryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the aryl ring system. The aryl group may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

"5-14 Membered heteroaryl" or "5-14 membered heteroaryl" refers to a group of a 5-14 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6, 10, or 14 pi electrons shared in a cyclic arrangement) having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. In heteroaryl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, as the valency permits. The heteroaryl bicyclic ring system may include one or more heteroatoms in one or both rings. Heteroaryl also includes ring systems in which the above heteroaryl ring is fused to one or more cycloalkyl or heterocyclyl groups, and the point of attachment is on the heteroaryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the heteroaryl ring system. In some embodiments, a 5-10 membered heteroaryl group is preferred, which is a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms. In other embodiments, 5-6 membered heteroaryl groups are particularly preferred, which are 5-6 membered monocyclic or bicyclic 4n+2 aromatic ring systems having ring carbon atoms and 1-4 ring heteroatoms. Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thienyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary three heteroatom containing 5 membered heteroaryl groups include, but are not limited to, triazolyl, oxadiazolyl (e.g., 1,2, 4-oxadiazolyl), and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl or pyridonyl. Exemplary 6 membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azetidinyl, oxepinyl, and thietaneyl. Exemplary 5, 6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazole, benzothienyl, isobenzothienyl, benzofuranyl, benzisotofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzisothiazolyl, benzothiadiazolyl, indenazinyl, and purinyl. Exemplary 6, 6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Heteroaryl groups may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

"Optionally substituted" means that the substituent may be designated, or may be unsubstituted.

The divalent groups formed by removing another hydrogen from the above-defined alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl groups and the like are collectively referred to as "subunits". Cyclic groups such as cycloalkyl, heterocyclyl, aryl, and heteroaryl are collectively referred to as "cyclic groups".

Alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and the like as defined herein are optionally substituted groups.

"Nucleic acid" refers to a single-or double-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecule, and hybrid molecules thereof. Examples of nucleic acid molecules include, but are not limited to, messenger RNAs (mrnas), micrornas (mirnas), small interfering RNAs (sirnas), self-amplifying RNAs (sarnas), antisense oligonucleotides (ASOs), and the like. The nucleic acid may be further chemically modified, the chemical modification being selected from one of pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 5-methylcytosine, or a combination thereof. The mRNA molecules contain protein coding regions and may further contain expression control sequences, typical expression control sequences include, but are not limited to, 5 'caps (5' caps), 5 'untranslated regions (5' utrs), 3 'untranslated regions (3' utrs), polyadenylation sequences (polyas), miRNA binding sites.

"Cationic lipid" refers to a lipid molecule capable of being positively charged under physiological pH conditions. In some embodiments, the cationic lipid is an amino lipid.

"Neutral lipids" refers to lipid molecules that are uncharged at specific pH conditions, such as physiological pH conditions. Examples of neutral lipids include, but are not limited to, 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylethanolamine (DOPE), 1, 2-dimyristoyl-sn-glycero-3-phosphorylethanolamine (DMPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylethanolamine (DPPE), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylethanolamine (DPPE).

"Structural lipids" refers to lipids, such as steroids, that are commonly used to enhance nanoparticle stability by filling in the interstices between the lipids. The steroid is a compound having a cyclopenta-polyhydrophenanthrene carbon skeleton, and in a preferred embodiment, the steroid is selected from cholesterol, sitosterol, stigmasterol, soapsterol, brassicasterol, ergosterol, lycorine, ursolic acid, alpha-tocopherol, stigmasterol, oat sterol, ergocalcitol, or campesterol.

"Polymer lipid" refers to a molecule that contains a polymer moiety and a lipid moiety. In some embodiments, the polymer lipid is a polyethylene glycol (PEG) lipid. Other lipids capable of reducing aggregation, such as products of coupling compounds having uncharged, hydrophilic, steric-blocking moieties to lipids, may also be used.

"Lipid nanoparticle" refers to particles having a nanoscale size that contain a lipid component.

"Biodegradable group" refers to a functional group containing a biodegradable linkage, such as an ester, disulfide, amide, and the like. Biodegradation can affect the process of scavenging compounds from the body. The biodegradable groups of the present invention are oriented from the head to the tail of the ionizable lipid molecules.

Other definitions

The term "treating" as used herein relates to reversing, alleviating, inhibiting the progression or prevention of a disorder or condition to which the term applies, or one or more symptoms of such disorder or condition. The term "treatment" as used herein relates to the action of a verb treatment, the latter as just defined.

Compounds are named herein using standard nomenclature. Compounds having asymmetric centers, it is to be understood (unless otherwise indicated) that all optical isomers and mixtures thereof are encompassed. Furthermore, unless otherwise specified, all isomeric compounds encompassed by the present invention may occur with carbon-carbon double bonds in the form of Z and E. Compounds that exist in different tautomeric forms, one of the compounds is not limited to any particular tautomer, but is intended to encompass all tautomeric forms.

The term "pharmaceutically acceptable salts" as used herein means those carboxylate salts, amino acid addition salts of the compounds of the invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without undue toxicity, irritation, allergic response and the like commensurate with a reasonable benefit/risk ratio, and effective for their intended use, including (if possible) zwitterionic forms of the compounds of the invention.

Pharmaceutically acceptable base addition salts are formed with metals or amines, for example alkali metal and alkaline earth metal hydroxides or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N, N' -dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine and procaine.

The base addition salts of the acidic compounds may be prepared by contacting the free acid form with a sufficient amount of the desired base to form the salt, in a conventional manner. The free acid can be regenerated by contacting the salt form with the acid in a conventional manner, isolating the free acid. The free acid forms differ somewhat in certain physical properties from their respective salt forms, such as solubility in polar solvents, but for the purposes of the present invention, the salts are also equivalent to their respective free acids.

The salt may be a sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide prepared from an inorganic acid, an acid such as hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, and the like. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthate, mesylate, glucoheptonate, lactobionate, laurylsulfonate, isethionate, and the like. Salts may also be prepared from organic acids, such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Representative salts include acetates, propionates, octanoates, isobutyrates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, mandelates, benzoates, chlorobenzoates, methylbenzates, dinitrobenzoates, naphthoates, benzenesulfonates, toluenesulfonates, phenylacetates, citrates, lactates, maleates, tartrates, methanesulfonates, and the like. Pharmaceutically acceptable salts may include cations based on alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Salts of amino acids, such as arginine salts, gluconate salts, galacturonate salts, and the like are also contemplated (see, e.g., berge s.m. et al, "Pharmaceutical Salts," j.pharm.sci.,1977;66:1-19, incorporated herein by reference).

The "subject" administered includes, but is not limited to, a human (i.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle aged adult, or senior adult)) and/or a non-human animal, e.g., a mammal, e.g., primate (e.g., cynomolgus monkey, rhesus monkey), cow, pig, horse, sheep, goat, rodent, cat, and/or dog. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The terms "human", "patient" and "subject" are used interchangeably herein.

"Disease," "disorder," and "condition" are used interchangeably herein.

As used herein, unless otherwise indicated, the term "treating" includes an effect that occurs when a subject has a particular disease, disorder, or condition, which reduces the severity of the disease, disorder, or condition, or delays or slows the progression of the disease, disorder, or condition ("therapeutic treatment"), as well as an effect that occurs before the subject begins to have the particular disease, disorder, or condition ("prophylactic treatment").

As used herein, unless otherwise indicated, a "prophylactically effective amount" of a pharmaceutical composition is an amount sufficient to prevent a disease, disorder, or condition, or to prevent one or more symptoms associated with a disease, disorder, or condition, or to prevent recurrence of a disease, disorder, or condition. A prophylactically effective amount of a pharmaceutical composition refers to an amount of a therapeutic agent, alone or in combination with other agents, that provides a prophylactic benefit in preventing a disease, disorder, or condition. The term "prophylactically effective amount" may include an amount that improves overall prophylaxis, or an amount that enhances the prophylactic effect of other prophylactic agents.

"Combination" and related terms refer to the simultaneous or sequential administration of a pharmaceutical composition of the invention and another therapeutic agent. For example, the pharmaceutical compositions of the invention may be administered simultaneously or sequentially with the other therapeutic agent in separate unit dosage forms, or simultaneously with the other therapeutic agent in a single unit dosage form.

The term "vaccine" as used herein refers to a composition that can provide an active acquired immune and/or therapeutic effect (e.g., treatment) against a particular disease or pathogen. Vaccines typically contain one or more agents that can induce an immune response against a pathogen or disease (i.e., a target pathogen or disease) in an individual. The immunogenic formulation stimulates the immune system of the body to recognize the agent as a threat or indication of the presence of the target pathogen or disease, thereby inducing immune memory so that the immune system more readily recognizes and eliminates any pathogen upon subsequent contact. Vaccines may be prophylactic (e.g., to prevent or ameliorate the effects of any natural or pathogen future infection or the effects of a cancer that is expected to occur in a susceptible individual) or therapeutic (e.g., to treat cancer in an individual who has been diagnosed with cancer)). The administration of a vaccine is called vaccination. In some examples, the vaccine composition may provide nucleic acid, e.g., mRNA encoding an antigenic molecule (e.g., peptide), to an individual. Nucleic acids delivered into an individual by a vaccine composition can be expressed as antigenic molecules and allow the individual to gain immunity against the antigenic molecules. In the case of vaccination against infectious diseases, the vaccine composition may provide mRNA encoding antigenic molecules associated with a particular pathogen, such as one or more peptides known to be expressed in the pathogen (e.g., pathogenic bacteria or viruses). In the case of viral vaccines, the vaccine composition may provide mRNA encoding certain viral peptides that are characteristic of the virus for which immunity is sought, such as peptides (e.g., capsid proteins) that are substantially exclusively or highly expressed on the surface of the virus. The individual may have immunity against the viral peptide after inoculation with the viral vaccine composition, specifically killing the cells expressing it.

The term "nucleic acid vaccine", also called gene vaccine, as used herein, is a vaccine that is obtained by introducing genetic material (DNA or RNA) that determines a specific antigen of a pathogen directly into human cells, allowing the human cells to produce these antigens themselves and stimulating the body to mount an immune response to the antigen, thereby allowing the vaccinator to obtain a corresponding immune protection, and which further comprises an adjuvant (e.g., lipid nanoparticles).

The term "delivery system" as used herein refers to a class of substances that is capable of carrying antigenic substances to the immune system of the body and storing and exerting their antigenic effects therein for a prolonged period of time. The vaccine delivery systems described herein may be liposome adjuvant vaccine delivery systems or nanoadjuvant vaccine delivery systems.

The term "adjuvant" as used herein refers to pharmaceutically acceptable substances that enhance the immune response to an antigen when co-administered with the antigen or when administered before, during or after administration of the antigen to a subject, including, but not limited to, nucleic acid adjuvants (e.g., nucleic acid vectors), plant adjuvants (e.g., alkylamines, phenolic components, quinines, sapogenins, sesquiterpenes, proteins, polypeptides, polysaccharides, glycolipids, phytohemagglutinin, etc.), bacterial adjuvants (e.g., cholera toxin, escherichia coli heat labile toxin, bacterial lipopolysaccharide, etc.), aluminum adjuvants and other inorganic component adjuvants (e.g., calcium adjuvants), emulsion adjuvants (e.g., freund's adjuvant).

The term "virus" is used in accordance with its ordinary meaning in the field of biology and is meant to include viral genomes (e.g. DNA, RNA, single strand, double strand), protein protective capsids (e.g. capsid proteins) and related proteins, and in the case of enveloped viruses (e.g. herpes viruses), the envelope comprises lipids and optionally host cell membrane components, and/or viral proteins.

The term "viral infection" or "viral disease" refers to a disease or condition caused by a virus, including symptomatic and asymptomatic infections. Non-limiting examples of viral infections include liver viral diseases (e.g., a, b, c, d, e), herpes viral infections (e.g., HSV-1, HSV-2, shingles), flaviviral infections, zika (Zika) viral infections, cytomegalovirus infections, respiratory viral infections (e.g., adenovirus infections, influenza, severe acute respiratory syndrome, coronavirus infections (e.g., SARS-CoV-1, SARS-CoV-2, MERS-CoV, covd-19, MERS), gastrointestinal viral infections (e.g., norovirus infections, rotavirus infections, astrovirus infections), eruptive viral infections (e.g., measles, shingles, smallpox, rubella), viral hemorrhagic diseases (e.g., ebola virus, lassa fever, dengue fever, yellow fever), nervous system viral infections (e.g., west nile virus infections, polio, meningitis, viral encephalitis, japanese encephalitis, rabies), and human papilloma virus infections.

SARS-CoV-2 belongs to the family of beta coronaviruses, and its members include two other human and animal co-viruses that cause severe disease outbreaks in the new thousand years, severe acute respiratory syndrome coronavirus (SARS-CoV) and middle east respiratory syndrome coronavirus (MERS-CoV). The term "SARS-CoV" refers to SARS coronavirus. The term "SARS-CoV" includes any coronavirus, such as SARS-CoV-2, SARS-CoV-1 and MERS-CoV.

In the context of a disease, the term "preventing" and its various grammatical variations are used in the sense that the clinical symptoms of the disease do not occur in individuals who have not experienced or developed symptoms of the disease.

As used herein, unless otherwise indicated, the term "treating" includes an effect that occurs when a subject has a particular disease, disorder, or condition, which reduces the severity of the disease, disorder, or condition, or delays or slows the progression of the disease, disorder, or condition ("therapeutic treatment"), as well as an effect that occurs before the subject begins to have the particular disease, disorder, or condition ("prophylactic treatment").

The term "treating" as used herein relates to reversing, alleviating, inhibiting the progression or prevention of a disorder or condition to which the term applies, or one or more symptoms of such disorder or condition. The term "treatment" as used herein relates to the action of a verb treatment, the latter as just defined.

The "subject" administered includes, but is not limited to, a human (i.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle aged adult, or senior adult)) and/or a non-human animal, e.g., a mammal, e.g., primate (e.g., cynomolgus monkey, rhesus monkey), cow, pig, horse, sheep, goat, rodent, cat, and/or dog. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The terms "human", "patient" and "subject" are used interchangeably herein.

"Disease," "disorder," and "condition" are used interchangeably herein.

The term "effective amount" refers to an amount of a formulation according to the invention that is sufficient to effect such treatment when administered to a patient for treating a state, disorder or condition or to generate an immune response when administered to a patient for generating such immune response. The "effective amount" will vary depending on the active ingredient, the state, disorder or condition to be treated and its severity, as well as the age, weight, physical condition and responsiveness of the subject to be treated.

Unless otherwise indicated, a "prophylactically effective amount" is an amount sufficient to prevent a disease, disorder, or condition, or an amount sufficient to prevent one or more symptoms associated with a disease, disorder, or condition, or to prevent recurrence of a disease, disorder, or condition. A prophylactically effective amount of a drug refers to an amount of a therapeutic agent, alone or in combination with other agents, that provides a prophylactic benefit in preventing a disease, disorder, or condition. The term "prophylactically effective amount" may include an amount that improves overall prophylaxis, or an amount that enhances the prophylactic effect of other prophylactic agents.

Examples

The present invention will be further described in detail with reference to the following examples in order to make the technical solution of the present invention clearer and more specific. The following examples are presented only to illustrate specific embodiments of the invention so that those skilled in the art can understand the invention and are not intended to limit the scope of the invention. In the specific embodiment of the present invention, technical means, methods, and the like not specifically described are conventional technical means, methods, and the like in the art. Materials, reagents and the like used in the examples are commercially available unless otherwise specified.

TABLE 1

EXAMPLE 1 Synthesis of Compound 1

THF (160 mL), compound 1-1 (80 g,688.7 mmol) and LDA (344.4 mL,688.7 mmol) were added to a 2L four-necked round bottom flask at room temperature, the system was cooled to-40℃and then stirred at-40℃for 1h. Dibromobutane (206.70 g,957.3 mmol) was then added dropwise at-40℃for 30 minutes, and finally DMPU (12.7 g,99.2 mmol) was added dropwise at-40℃for 2h and incubated at-40℃for 1h. And (5) freely heating and stirring for 15h. The reaction was monitored by TLC and the starting material was consumed. The reaction was quenched with saturated NH ₄ Cl (200 mL) at 0deg.C, diluted with water (160 mL) and ethyl acetate (160 mL), extracted, and separated. The aqueous phase was back extracted once more with ethyl acetate (200 mL). The organic phases were combined and washed twice with saturated NaCl (300 mL), dried over anhydrous Na ₂SO₄, filtered and concentrated. The crude product was purified by silica gel column to give compound 1-2 (100 g) as pale yellow oil.

THF (1L) and compound 1-2 (98 g,391.91 mmol) were added to a 2L four-necked round bottom flask at room temperature, the system was cooled to-10℃and LiAlH ₄ (313.5 mL,313.5 mmol) was added dropwise over 30 minutes, the system was stirred at-10℃for 30 minutes, and then TLC was monitored for reaction, and the conversion of the starting material was complete. The reaction was quenched with Na ₂SO₄.10H₂ O (125 g) at-4℃and the system was diluted with water (500 mL) and ethyl acetate (5L), extracted and separated. The aqueous phase was back extracted once more with ethyl acetate (300 mL). The organic phases were combined and washed twice with saturated NaCl (500 mL), dried over anhydrous Na ₂SO₄, filtered and concentrated. The crude product was passed through a silica gel column to give 1-3 (73 g) as a pale yellow oil.

DMSO (40 mL), naH (3.83 g,95.7 mmol) and TosMIC (4.67 g,23.9 mmol) were added in portions under 25℃nitrogen, followed by TBAI (883.5 mg,2.39 mmol) and the system stirred at 25℃for 15 min. Finally, compounds 1-3 (10 g,47.8 mmol) were dissolved in DMSO (100 mL) and the above system was added dropwise at 25℃and the system was stirred for an additional 1h at 25 ℃. TLC monitored the reaction and the conversion of starting material was complete. The reaction was quenched with ice water (400 mL) at 20 ℃, the system extracted with MTBE (2×500 mL), the organic phases combined and washed once with saturated NaCl (500 mL), the organic phase dried over anhydrous Na ₂SO₄, filtered and concentrated. Crude product (10 g) was obtained and taken directly to the next step.

In a 500mL three-necked flask, crude compound 1-4 (10 g), CH ₃ OH (100 mL) and 12N HCl (5.3 mL) were added at room temperature. The system was reacted at 80℃for 3h. LCMS monitored reaction, complete conversion of starting material. The system was cooled to room temperature, ph=7 was adjusted with saturated sodium carbonate, methanol was removed by concentration under reduced pressure, the system was diluted with water (100 mL) and ethyl acetate (100 mL), extracted, and the solution was separated. The aqueous phase was back-extracted once with ethyl acetate (100 mL), the organic phases combined and washed once with saturated NaCl (100 mL), and the organic phase dried over anhydrous Na ₂SO₄, filtered and concentrated. The crude product was passed through a silica gel column to give compounds 1-5 (4.6 g).

In a 100mL three-necked flask, compounds 1-5 (2.5 g,8.73 mmol), n-decanoic acid (1.50 g,8.73 mmol), DMAP (53.3 mg,0.431 mmol) and DCM (25 mL) were added at room temperature. EDCI (1.67 g,8.73 mmol) was added at 0deg.C and the system was reacted for 18h at 20deg.C. TLC and LCMS together monitor the reaction with product signal. The reaction was quenched with saturated NH ₄ Cl (25 mL), extracted and separated. The aqueous phase was back-extracted once with DCM (25 mL), the organic phases combined and washed once with saturated NaCl (50 mL), and the organic phase dried over anhydrous Na ₂SO₄, filtered and concentrated. The crude product was passed through a silica gel column to give pale yellow compounds 1-6 (1.71 g).

Compounds 1-6 (240 mg,0.55 mmol), compounds 1-7 (145.2 mg,0.60 mmol), EDCI (156.6 mg,0.82 mmol), DMAP (13.3 mg,0.11 mmol) and DCM (3 mL) were added at room temperature in an 8mL lock tube. The system was reacted at 20℃for 18h. TLC monitored the reaction and the conversion of starting material was complete. The reaction was quenched with saturated NH ₄ Cl (10 mL), diluted with DCM (10 mL), extracted, and separated. The aqueous phase was back-extracted once with DCM (15 mL), the organic phases combined and washed once with saturated NaCl (20 mL), and the organic phase dried over anhydrous Na ₂SO₄, filtered and concentrated. The crude product was purified by silica gel column to give compounds 1-8 (301.8 mg) as pale yellow oil.

In a 25mL three-necked flask, compounds 1-8 (301.8 mg,0.45 mmol), methanol (4 mL) and THF (1 mL) were added at room temperature, and NaBH ₄ (17.2 mg,0.45 mmol) was added at 0 ℃. The reaction was monitored by TLC at 20℃for 1h, starting material was complete. The reaction was quenched with water (20 mL), diluted with ethyl acetate (20 mL), extracted, and separated. The aqueous phase was back-extracted once with ethyl acetate (20 mL), the organic phases combined and washed once with saturated NaCl (20 mL), and the organic phase was dried over anhydrous Na ₂SO₄, filtered and concentrated. The crude product (273.8 mg) was taken directly to the next step.

Compounds 1-9 (273.8 mg,0.41 mmol), 4-dimethylaminobutyric acid (137.6 mg,0.82 mmol), EDCI (236.0 mg,1.23 mmol), DMAP (50.1 mg,0.41 mmol) and DCM (3 mL) were added at room temperature in an 8mL tube seal. The system was reacted at 20℃for 2h. TLC monitored the reaction and the conversion of starting material was complete. The reaction was quenched with saturated NH ₄ Cl (10 mL), diluted with DCM (10 mL), extracted, and separated. The aqueous phase was back-extracted once with DCM (10 mL), the organic phases combined and washed once with saturated NaCl (25 mL), and the organic phase dried over anhydrous Na ₂SO₄, filtered and concentrated. The crude product was purified by silica gel column to give compound 1 (239.6 mg) as a pale yellow oil.

¹H NMR(300MHz,CDCl₃)δ:4.85(p,J＝6.2Hz,1H),3.77(s,4H),2.31(ddd,J＝14.8,7.4,4.3Hz,12H),1.65-1.49(m,11H),1.36-1.20(m,52H),0.89(s,15H),0.88(t,J＝6.8Hz,7H);ESI-MS m/z:780.65[M+H]⁺.

EXAMPLE 2 Synthesis of Compound 2

To an 8mL vial was added starting compound 1-6 (150 mg,0.34 mmol), compound 2-1 (96.01 mg,0.374 mmol), EDCI (97.87 mg,0.51 mmol), DMAP (8.32 mg,0.068 mmol) and DCM (3 mL). Stirring at room temperature for 18 hours, TLC monitoring no starting material. Adding silica gel, stirring, and purifying by column chromatography to obtain 196mg of pale yellow oily substance 2-2.

Into a 25mL three-necked flask were charged starting compound 2-2 (196 mg,0.29 mmol), meOH (4 mL) and THF (1 mL). Cooled to 0 ℃, naBH ₄ (10.92 mg,0.29 mmol) was added. Stir at room temperature for 1 hour, TLC monitored no starting material. Saturated aqueous ammonium chloride (10 mL) was added and MTBE extracted (1X 20 mL). The organic phase was washed with saturated brine (1X 20 mL), dried over anhydrous Na ₂SO₄, and concentrated under reduced pressure to give 140mg of product 2-3 which was used directly in the next reaction.

To an 8 mL tube sealer was added raw material compound 2-3 (140 mg,0.21 mmol), 4-dimethylaminobutyric acid (41.35 mg,0.25 mmol), EDCI (59.10 mg,0.31 mmol), DMAP (5.02 mg,0.041 mmol) and DCM (2 mL). The reaction was carried out at room temperature for 5 hours, and TLC monitored for absence of starting material. Adding silica gel, mixing, and purifying by column chromatography to obtain 105.9mg of compound 2.

¹H NMR(300MHz,CDCl₃)δ:4.85(q,J＝6.3Hz,1H),3.77(s,4H),2.45-2.22(m,13H),1.84(p,J＝7.4Hz,2H),1.61(d,J＝7.2Hz,4H),1.52(d,J＝6.1Hz,5H),1.27(qd,J＝6.8,4.5,3.4Hz,46H),0.89(s,17H),0.88(d,J＝13.3Hz,3H);ESI-MS m/z:794.70[M+H]⁺.

EXAMPLE 3 Synthesis of Compound 3

In an 8mL vial, compound 1-6 (150 mg,0.34 mmol), compound 3-1 (85.50 mg, 0.514 mmol), EDCI (97.87 mg,0.510 mmol), DMAP (8.32 mg,0.068 mmol) and DCM (2 mL) were added. Stir at room temperature for 18 hours, monitored by TLC. Directly mixing, and purifying by column chromatography to obtain 165.6mg yellow oily substance 3-2.

In a 25mL three-necked flask, compound 3-2 (165.6 mg,0.25 mmol), meOH (4 mL) and THF (1 mL) were added. NaBH ₄ (9.62 mg,0.25 mmol) was added after cooling to 0℃and stirring was continued for 1 hour at room temperature, monitored by TLC. After the reaction of the starting materials was completed, 10mL of saturated aqueous NH ₄ Cl solution was added, EA was extracted (1X 20 mL), and the organic phase was washed with saturated brine (1X 10 mL). Dried over anhydrous Na ₂SO₄ and concentrated in vacuo to give 166mg of crude compound 3-3 which is used directly in the next step.

In an 8mL vial, compound 3-3 (166 mg,0.25 mmol), 4-dimethylaminobutyric acid (51.13 mg,0.31 mmol), EDCI (73.09 mg,0.38 mmol), DMAP (6.21 mg,0.051 mmol) and DCM (2 mL) were added. Stir at room temperature for 5 hours, monitored by TLC. Directly stirring, column chromatography of pure DCM/MeOH (50:1) gave 142.4mg of Compound 3 as a pale yellow oil.

¹H NMR(300MHz,CDCl₃)δ:4.87(q,J＝6.2Hz,1H),3.77(s,4H),2.39-2.26(m,12H),1.84(q,J＝7.4Hz,2H),1.68-1.54(m,5H),1.52(d,J＝6.2Hz,5H),1.34-1.19(m,47H),0.91-0.86(m,21H);ESI-MS m/z:766.55[M+H]⁺.

EXAMPLE 4 Synthesis of Compound 4

A solution of Compound 4-1 (100 g,979 mmol) in tetrahydrofuran (800 mL) was cooled to-40℃and LDA (2M, 490 mL) was slowly added dropwise to the solution, and stirring was continued for 1 hour after the completion of the dropwise addition, and a solution of 4-2 (315 g,1.37 mol) in tetrahydrofuran (100 mL) was added dropwise to the reaction system at the same temperature, and the reaction system was stirred overnight. The reaction system was quenched with saturated aqueous ammonium chloride, extracted with ethyl acetate, and the organic phases were combined and dried over anhydrous sodium sulfate. Filtering, concentrating the filtrate to obtain crude product. The crude product was purified by silica gel column separation to give compound 4-3 (115 g).

A solution of compound 4-3 (100 g,398 mmol), tosMIC (38.9 g, 199mmol) and TBAI (14.7 g,39.8 mmol) in tetrahydrofuran (800 mL) was cooled to 0deg.C, sodium hydride (20.7 g,517 mmol) was added slowly in portions and reacted overnight at ambient temperature. The reaction system was quenched with saturated aqueous sodium chloride, extracted with ethyl acetate, and the organic phases were combined and dried over anhydrous sodium sulfate. Filtering, concentrating the filtrate until the filtrate is dried to obtain 115g of 4-4 crude product, and directly using the crude product in the next reaction without separation and purification.

To a solution of compound 4-4 (110 g,205 mmol) in dichloromethane (880 mL) was added 330mL of concentrated hydrochloric acid and the reaction was continued at room temperature for 2 hours, and TLC monitored the completion of the substrate reaction. The reaction system was quenched with saturated aqueous ammonium chloride, extracted with ethyl acetate, and the organic phases were combined and dried over anhydrous sodium sulfate. Filtering, concentrating the filtrate to obtain crude product. Compound 4-5 (30.0 g,80.9mmol, 39.4% yield) was obtained by column chromatography on silica gel.

TMSOK (11.0 g,86.4 mmol) was added to a solution of compounds 4-5 (8.0 g,21.6 mmol) in tetrahydrofuran (35.0 mL) at ambient temperature, the reaction was heated to 70℃and stirred, and TLC monitored complete consumption of starting material. The reaction solution was cooled to room temperature, the organic solvent was removed by rotary evaporation, 20mL of water was added to the crude product and extracted with dichloromethane, the aqueous phase was collected, the pH of the solution was adjusted to less than 5 with 1M hydrochloric acid, extraction was performed with dichloromethane, the organic phases were combined and dried over anhydrous sodium sulfate, the filtrate was collected by filtration, and concentrated to give compound 4-6 (7.0 g).

Potassium carbonate (1.55 g,11.2 mmol) was added to a solution of compound 4-6 (959 mg,2.8 mmol) and 1-bromononane (428 mg,3.08 mmol) in DMF, the reaction was then warmed to 60℃and allowed to react for 4 hours, cooled to room temperature, the reaction system quenched with saturated aqueous sodium chloride solution, extracted with ethyl acetate, the organic phases combined and dried over anhydrous sodium sulfate. Filtering, concentrating the filtrate to dry to obtain crude product, and purifying with silica gel column to obtain compound 4-8 (682 mg).

To a20 mL vial was added compound 4-8 (600 mg,1.28 mmol), compound 4-9 (329.3 mg,1.54 mmol), EDCI (294.5 mg,1.54 mmol), DMAP (156.4 mg,1.28 mmol), DCM (5 mL). The reaction was carried out at room temperature overnight. LCMS monitored for product signal. Quenched with 10mL of water, extracted with CH ₂Cl₂ (1X 10 mL), washed with saturated brine (1X 10 mL), dried over Na ₂SO₄, filtered, spun-dried and purified by column to give compound 4-10 (543 mg).

To a 50mL three-necked flask, compound 4-10 (543 mg,0.83 mmol) and MeOH (10 mL) were added under nitrogen, naBH ₄ (157.8 mg,4.17 mmol) was added in portions at 0℃and reacted at room temperature for 2 hours. Dropwise add 20mL of water at 0deg.C and extract with EtOAc (1X 20 mL). The organic phase was washed with saturated brine (1X 20 mL), dried over Na ₂SO₄, and filtered and spun-dried to give Compound 4-11 (480 mg).

To an 8mL vial was added 4-11 (480 mg,0.74 mmol), SM4 (289.23 mg,2.21 mmol), EDCI (422.68 mg,2.21 mmol), DMAP (89.8 mg,0.74 mmol), DCM (3 mL), and the mixture was reacted overnight at room temperature. LCMS monitored for product signal. Quenched with 10mL of water, extracted with DCM (2X 20 mL), the organic phase washed with brine (1X 20 mL), dried over Na ₂SO₄, filtered off and purified by Prep-TLC (DCM/MeOH 10:1) to give 4 (118.3 mg).

¹H NMR(300MHz,CDCl₃)δ:4.86(t,J＝6.2Hz,1H),4.06(td,J＝6.6,2.5Hz,4H),2.40(t,J＝7.0Hz,2H),2.00(s,2H),1.66-1.56(m,5H),1.50(s,9H),1.27(d,J＝12.8Hz,45H),1.17(s,13H),0.96-0.86(m,9H);ESI-MS m/z:766.80[M+H]⁺.

EXAMPLE 5 Synthesis of Compound 5

Compound 5 was prepared according to the synthesis method of CN115850104a example 70.

Pharmacological experiments

Experimental example 1 nanoparticle preparation

Materials used for assembly of lipid nanoparticles are (1) ionizable lipid compounds such as example compounds 1-5 or ALC0315 (available from AVT), (2) structural lipids such as Cholesterol Cholesterol (available from Sigma-Aldrich), (3) phospholipids such as DSPC 1, 2-distearoyl-SN-glycerol-3-phosphorylcholine (Distearoylphosphatidylcholine available from AVT) or DOPE 1,2 dioleoyl-SN-glycerol-3-phosphoethanolamine (Dioleoyl Phosphoethanolamine available from AVT), (4) pegylated lipid compounds such as DMG-PEG2000 (1, 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 available from AVT) or ALC0159 (available from Sapinoban) and (5) nucleic acid fragments effective components of novel coronavirus spike mRNA (SARS encoding sequence identical to WO2021154763A1 (Table 1-2S Protein Variant 1-4) or varicella-3-methoxypolyethylene glycol-2000) or varicella-UniJ virus mRNA encoding sequence (UniJ 8). The names and structural formulas of the lipid nanoparticle assembly materials are shown in Table 2.

The preparation method of the lipid nanoparticle comprises (1) dissolving and mixing an ionizable lipid compound, cholesterol, phospholipid and polyethylene glycol lipid in ethanol according to a preferred formula, wherein the optimized formula of the compound 1-5 is shown in table 3, the preferred formula of the ALC0315 is shown in document (mRNA-lipid nanoparticle COVID-19vaccines:Structure and stability,International Journal of Pharmaceutics,2021),LNP and table 3, (2) dissolving mRNA active ingredient in 25mM sodium acetate solution (pH=4.5), (3) mixing an organic phase dissolved with the lipid mixture and an aqueous phase dissolved with the mRNA ingredient by using an automatic high-throughput microfluidic system according to a flow rate ratio ranging from 1:1 to 1:4, wherein the mixing speed is 10mL/min to 18mL/min, (4) diluting the prepared lipid nanoparticle with a phosphate buffer salt solution, ultrafiltering the nanoparticle solution to an original preparation volume by using an ultrafiltration tube (purchased from Millipore) with a molecular weight cutoff of 30kDa, (5) filtering and sterilizing the obtained nanoparticle by using a 0.2 μm sterile filter membrane, and preserving the nanoparticle in a sealed glass bottle at low temperature.

The preparation method of the lipid nanoparticle comprises a microfluidic mixing system, but is not limited to the method, and also comprises a T-type mixer, an ethanol injection method and the like.

TABLE 2

TABLE 3 Table 3

Experimental example 2 characterization of physical Properties of lipid nanoparticles

Particle size and particle size dispersion coefficient (PDI) of the prepared lipid nanoparticles were measured using a Zetasizer Pro (available from Malvern Instruments Ltd) and DynaPro NanoStar (available from Wyatt) dynamic light scattering instrument. The extent of entrapment of the lipid nanoparticle to the RNA was characterized by the encapsulation efficiency (Encapsulation Efficiency%), a coefficient reflecting the extent of binding of the lipid nanoparticle to the RNA fragment. The coefficients were measured by the method of Quant-it ^TM RiboGreen RNA Assay (available from Invitrogen). Lipid nanoparticle samples were diluted in TE buffer (10 mM Tris-HCl,1mM EDTA,pH =7.5), part of the sample solution was taken out, and 0.5% Triton (Triton X-100) was added thereto and allowed to stand at 37℃for 30 minutes. Adding inImmediately after the reaction, the fluorescence value was read by a Varioskan LUX multifunctional microplate reader (commercially available from Thermofisher) under the conditions of an absorption light band of 485nm and an emission light band of 528nm to obtain the entrapment rate value.

Experimental example 3 muscle immunization of mice

The adaptive immune effect of lipid nanoparticles entrapped with mRNA encoding viral antigen proteins in mice was evaluated. The test mice were SPF-grade BALB/c mice, female, 6-8 weeks old, weighing 18-22g, purchased from Beijing Bei Fu Biotechnology Co. All animals were fed with water for more than 7 days before the test, and the test period was free to eat water, light and shade were alternated for 12/12h, the indoor temperature was 20-26 ℃ and the humidity was 40-70%. Mice were randomly divided into immune and negative control groups (DPBS, ph=7.4), each group of 5 mice. The prepared lipid nanoparticles encapsulating mRNA encoding viral antigen protein were injected into immunized mice in muscle administration according to the immunization dose of Table 4. Primary and booster immunizations were performed, with several days between them.

TABLE 4 Table 4

Sequence number	Group of	Dosage of	Immune interval
				1	Novel coronavirus spike protein group	0.025mg/kg mRNA	14 Days
2	Varicella-zoster virus envelope glycoprotein E group	0.05mg/kg mRNA	For 28 days

Experimental example 4 evaluation of humoral immune Effect

The humoral immune response in mice was assessed by taking blood through the mouse orbit on day 10 after each immunization to obtain sufficient serum, and encapsulating lipid nanoparticles encoding viral antigen protein mRNA.

1. Novel coronavirus specific binding antibody detection

Diluting novel coronavirus spike protein (purchased from Soy) to 2ng/μl with carbonate buffer (50 mM, pH 9.6,0.22 μm filter membrane filtration) to form protein coating solution, adding 96-well plate seal, adding 4 ℃ overnight, pouring 96-well plate to remove protein coating solution after coating overnight, adding washing solution (1 XTBS containing 0.2% Tween-20 purchased from Soy) into each well, washing plate 6 times, adding blocking solution (1 XTBS containing 2% BSA purchased from Soy), incubating in 37 ℃ incubator for blocking, repeating washing plate operation after blocking for 2 hours, diluting immune mouse serum with antibody diluent (washing solution containing 0.5% BSA) for 10 times gradient to obtain serum of 10 ^-1 -10 ^-6 different dilutions, adding well plate for 37 ℃ incubator incubation, repeating washing plate operation after incubation for 2 hours, adding horseradish peroxidase labeled anti-mouse (purchased from Biday) with antibody for 0.5% BSA, stopping incubation for 20 minutes after waiting for incubation for 50 ℃ for incubation, repeating time, adding dye-buffer enzyme assay for 20 minutes after incubation for 50 ℃ for incubation, and stopping incubation for detection of the substrate.

And judging the IgG titer of the serum binding antibody, namely, the OD of one diluted serum is more than or equal to 2.1 with the OD of the negative control, the OD of the next dilution is less than 2.1 with the OD of the negative control, and the dilution is the corresponding antibody titer of the serum sample (calculated as 0.05 if the OD of the negative control is less than 0.05). The novel coronavirus spike protein binding antibody titers of the lipid nanoparticle-immunized mouse serum loaded with the novel coronavirus spike protein mRNA are shown in Table 5.

2. Evaluation of neutralization by novel coronavirus-specific antibody

The neutralizing effect of the antibodies to mouse serum was evaluated using a novel coronavirus spike protein pseudovirus.

Mouse immune serum was serially diluted 3-fold with DMEM complete medium (available from Gibco) to give 6 different dilutions of serum, which was then incubated with 650TCID ₅₀ pseudovirus (available from mitsubishi biotechnology limited) at 37 ℃. A pseudovirus-free cell control group and a serum sample-free pseudovirus control group are simultaneously provided. After one hour of incubation, 2×10 ⁴ Huh 7 cells (hepatoma cells) were each added and cell culture was performed at 37 ℃ with 5% CO ₂. Since pseudoviruses enter cells, firefly luciferase is expressed, and after 24 hours, the pseudoviruses react with a luminescent substrate and carry out luminescence detection. The percentage of pseudovirus inhibition was calculated by comparison with the luminescence value of the pseudovirus control group. The dilution factor of serum, half-neutralization dilution, was calculated by the Reed-Muench method when pseudovirus was 50% inhibited (NT ₅₀).NT₅₀ illustrates the neutralizing activity of serum antibodies to pseudovirus, NT ₅₀, the neutralizing activity of lipid nanoparticles immune mouse serum loaded with novel coronavirus spike protein mRNA, is detailed in table 5.

As can be seen from the data in table 5, the novel coronavirus vaccine prepared from the compound of the present invention has significantly better humoral immune effect than the control ALC0315, both in serum IgG titer and in serum neutralization ability. When the degradable group of the compounds of the invention (corresponding to the groups M1 and M2) is-OC (O) -the neutralizing capacity in serum is better than that of compounds containing the group-C (O) O-.

TABLE 5

3. Varicella-zoster virus envelope glycoprotein E specific binding antibody detection

Recombinant varicella-zoster virus (Oka strain vaccine) envelope glycoprotein E (purchased from offshore protein, DRA 224) was diluted to 1 ng/. Mu.l with carbonate buffer (50 mM, pH 9.6,0.22 μm filter membrane filtration) to form a protein coating solution, sealed in 96-well plates, and subjected to 4 ℃ overnight; after coating overnight, the protein coating solution was removed by pouring 96-well plates, washing the plates with washing solution (1 XTBS containing 0.2% Tween-20, purchased from Soy pal) each well, repeating 6 times of washing the plates, adding blocking solution (1 XTBS containing 2% BSA, purchased from Soy pal) each well, incubating with 37℃incubator for blocking, repeating the plate washing operation after blocking for 2 hours, diluting immune mouse serum with antibody dilution (washing solution containing 0.5% BSA) 10 times of gradient to obtain serum with different dilutions of 10 ^-1 to 10 ^-6 and adding the serum into the well plates for incubation with 37℃incubator, repeating the plate washing operation after incubation for 2 hours, diluting horseradish peroxidase-labeled goat anti-mouse IgG (H+L) (purchased from Biyun days, A0216) 250 times with antibody dilution (washing solution containing 0.5% BSA) to obtain secondary antibody dilution, incubating with 37℃incubator, adding TMB substrate (purchased from root incubation, room temperature, 20min, and finally stopping the reaction when the enzyme-labeled on the apparatus is stopped for detection of light-proof, and the enzyme-labeled on the Lai pal (OD) is stopped). And judging the IgG titer of the serum binding antibody, namely, the OD of one diluted serum is more than or equal to 2.1 with the OD of the negative control, the OD of the next dilution is less than 2.1 with the OD of the negative control, and the dilution is the corresponding antibody titer of the serum sample (calculated as 0.05 if the OD of the negative control is less than 0.05). The antibody titers of varicella-zoster virus envelope glycoprotein E binding to mouse serum immunized by lipid nanoparticles loaded with varicella-zoster virus envelope glycoprotein E mRNA are shown in Table 6.

The data in Table 6 shows that the compounds of the invention produce a strong humoral immune response in the preparation of nucleic acid vaccines encoding varicella zoster virus.

TABLE 6

Experimental example 5 evaluation of cellular immune Effect

1. Novel coronavirus specific cellular immunity

On day 14 after booster immunization, spleen dissection was performed on immunized mice and negative control mice, and spleen cells were isolated to prepare single cell suspensions. The mouse spleen cells were added with a novel coronavirus spike protein peptide (from Jinsri, 2. Mu.g/ml/peptide) and specifically stimulated at 37℃under 5% CO ₂ cell culture conditions, and after 18h stimulation, the cytokines IFN-. Gamma.and IL-2 secretion were detected using the enzyme-linked immunospot detection kit (from MABTECH). And naturally airing the spot plate to be developed after the experiment is finished, counting the spots by using a full-automatic AID iSpot ELISPOT plate reader, and recording various parameters of the spots. The specific IFN-gamma and IL-2 secretion of novel coronavirus spike proteins are detailed in Table 7.

As can be seen from the data in table 7, the compounds of the present invention produced novel coronavirus vaccines with significantly better cellular immune effects than the control compound ALC0315.

TABLE 7

2. Varicella zoster virus specific cellular immunity

On day 14 after booster immunization, spleen dissection was performed on immunized mice and negative control mice, and spleen cells were isolated to prepare single cell suspensions. Mouse spleen cells were specifically stimulated with a varicella-zoster virus (Oka strain vaccine) envelope glycoprotein E white mix overlapping peptide library (purchased from Kirsrui, 2 μg/ml/peptide) under 37 ℃ and 5% CO2 cell culture conditions, and secretion of cytokines IFN- γ and IL-2 was detected by an enzyme-linked immunospot (ELISPot) method after 18 hours of stimulation. ELISPot detection kit is purchased from MABTECH, the specific implementation method refers to the kit instruction book, after the experiment is finished, after the color development spot plate is naturally dried, the spots are counted by using a full-automatic AID iSpot ELISPOT plate reader, and various parameters of the spots are recorded. Specific IFN-. Gamma.and IL-2 secretion of varicella zoster virus envelope glycoprotein E are shown in Table 8.

The data in Table 8 shows that the compounds of the present invention produce a strong cellular immune response in the preparation of varicella-zoster virus nucleic acid vaccine.

TABLE 8

Experimental example 6 in vitro safety assessment

HERG test

The potential inhibition of human hERG channels by ionizable lipid compounds was evaluated using SyncroPatch 384i/384 automated patch clamp system. Evaluation was performed using CHO cells stably overexpressing the hERG gene with cisapride as a positive control. The ionizable lipid compounds were set at 5 concentrations of 30, 10, 3.33, 1.11, 0.37 μm and 50% inhibitory concentrations (IC ₅₀) were calculated. The evaluation was done by kanglong formation. The test results are shown in Table 9.

2. Salmonella typhimurium and escherichia coli Mini-Ames test

The ability of ionizable lipid compounds to induce back mutations in histidine-auxotrophs, salmonella typhimurium (TA 98, TA100, TA1535 and TA 1537) and tryptophan-auxotrophs, E.coli WP2 uvrA (pKM 101), was tested and the potential mutagenetics of the test subjects were assessed. The test doses of ionizable lipid compounds were 1.5, 4, 10, 25, 64, 160, 400, and 1000 μg/well. Both negative/solvent control (methanol) and positive control were tested. The test was completed by kanglong formation. The test results are shown in Table 9.

The data in Table 9 show that the compounds of the invention have substantially no potassium channel inhibition and toxicity and are safe.

TABLE 9

While the present invention has been fully described by way of embodiments thereof, it is to be noted that various changes and modifications will become apparent to those skilled in the art. Such variations and modifications are intended to be included within the scope of the appended claims.

Claims (23)

1. A compound of formula (I), or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof:

(I)

Wherein,

R ₁ and R ₂ are independently selected from C _4-20 alkyl, said C _4-20 alkyl optionally substituted with one or more R;

R is independently selected from H and C _1-14 alkyl;

R ₃ and R ₄ are independently selected from H and C _1-6 alkyl;

a is selected from 2,3 or 4;

b and d are independently 4 or 5;

c and e are independently 1;

b+c=5 or 6, d+e=5 or 6;

Optionally and independently substituted with 1 or 2C _1-6 alkyl groups;

R ₅、R₆、R₇ and R ₈ are independently selected from methyl or ethyl.

2. The compound of claim 1, wherein a is 2 or 3.

3. The compound of claim 2, wherein a is 2.

4. A compound according to any one of claims 1-3, wherein b and d are independently 4, b+c=5, d+e=5.

5. A compound according to any one of claims 1 to 3, wherein R ₁ and R ₂ are independently selected from linear alkyl groups of total length 6, 7, 8, 9 or 10 carbon atoms, 1,2 or 3 methylene groups of the linear alkyl groups being optionally and each independently substituted by C _1-8 alkyl.

6. The compound of claim 5, wherein R ₁ or R ₂ are each independently a straight chain alkyl group having a total length of 9 carbon atoms, and one of R ₁ or R ₂ is substituted with a C _4-6 alkyl group.

7. A compound according to any one of claims 1-3, wherein R ₃ and R ₄ are methyl.

8. A compound according to any one of claims 1-3, wherein R ₅、R₆、R₇ and R ₈ are methyl.

9. A compound, or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, of:

。

10. A pharmaceutical composition comprising a compound of any one of claims 1-9, or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, and optionally a pharmaceutically acceptable adjuvant.

11. The pharmaceutical composition according to claim 10, wherein the pharmaceutical composition is a nanoparticle composition comprising a lipid component and optionally an active ingredient;

the lipid component comprises 20 mol% -85 mol% of ionizable cationic lipid, 10 mol% -75 mol% of structural lipid, 1.0 mol% -30 mol% of neutral lipid and 0.25 mol% -10 mol% of polymer lipid;

wherein the ionizable cationic lipid is a compound of formula (I), or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof.

12. The pharmaceutical composition of claim 11, wherein the lipid component comprises ionizable cationic lipid 30 mol% -50 mol%, structural lipid 30 mol% -60 mol%, neutral lipid 10mol% -30 mol%, and polymeric lipid 0.25% -mol% -5 mol%.

13. The pharmaceutical composition of claim 11, wherein the lipid component comprises an ionizable cationic lipid 35 mol% -45 mol%, a structural lipid 40 mol% -50 mol%, a neutral lipid 10 mol% -20 mol%, and a polymeric lipid 0.5 mol% -2 mol%.

14. The pharmaceutical composition of claim 11, wherein the neutral lipid is selected from one or more of DSPC, DMPC, DOPC, DPPC, POPC, DOPE, DMPE, POPE or DPPE.

15. The pharmaceutical composition of claim 11, wherein the structural lipid is selected from one or more of cholesterol, sitosterol, stigmasterol, sapogenol, brassicasterol, ergosterol, lycorine, ursolic acid, alpha-tocopherol, stigmasterol, aveosterol, ergocalcitol, or campesterol.

16. The pharmaceutical composition of claim 11, wherein the polymeric lipid is a pegylated lipid selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol.

17. Use of a compound of any one of claims 1-9, or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, or a pharmaceutical composition of any one of claims 10-16, for the manufacture of a medicament for delivering a load selected from one or more of a therapeutic agent, a prophylactic agent, or a diagnostic agent.

18. The use of claim 17, wherein the therapeutic, prophylactic or diagnostic agent is a nucleic acid.

19. The use of claim 18, wherein the nucleic acid is selected from ASO.

20. The use of claim 18, wherein the nucleic acid is selected from one or more of RNA or DNA.

21. The use of claim 20, wherein the RNA is selected from one or more of small interfering RNA, short hairpin RNA, antisense RNA, messenger RNA, modified messenger RNA, long non-coding RNA, microrna, small activating RNA, poly-coding nucleic acid, guide RNA, CRISPRRNA, or ribozyme.

22. The use of claim 21, wherein the RNA is mRNA.

23. The use of claim 22, wherein the RNA is a modified mRNA.