TWI870048B - Lipid compound or a derivative thereof and pharmaceutical composition employing the same - Google Patents

Abstract

A lipid compound or a derivative thereof and a pharmaceutical composition employing the same are provided. The lipid compound has a structure represented by Formula (I) ，wherein Z ¹，Z ²，Z ³and Z ⁴are disclosed in the specification.

Description

Lipid compound or its derivative and pharmaceutical composition containing the same

The present disclosure relates to a lipid compound or its derivative and a pharmaceutical composition containing the same.

The success of messenger RNA (mRNA) vaccines has prompted the biotech industry to actively invest in the field of nucleic acid drugs, among which the key lipid nanoparticle (LNP) delivery technology of nucleic acid drugs is the focus of development. However, RNA drugs are easily degraded in the human body and have negative charges and cannot easily pass through the cell membrane, so they need the assistance of a carrier to be encapsulated in the carrier for delivery.

Ionizable cationic lipids are currently the most commonly used carriers for lipid nanoparticles. However, traditional ionizable cationic lipids may cause significant side effects when administered in vivo. Problems observed include low percentages of effective delivery to the target, which results in relatively low therapeutic effects or low efficacy. In addition, the ionizable cationic lipids used in lipid nanoparticles as carriers need to have a specifically tuned pH value so that the carrier can be formulated with the active agent and protect the active agent from degradation during administration, and can release the active agent immediately once the carrier reaches its target.

Therefore, the industry needs to develop new lipids that can meet the lipid-nucleic acid delivery system.

，其中Z¹係O或S；Z²係-Q¹-A¹-Q²-A²-X¹；Z³與Z²相同；Z⁴係-Q³-A³-Q⁴-A⁴-X²；Q¹以及Q³係獨立為單鍵、-O-、或-NH-；Q²以及Q⁴係獨立為單鍵、-O-、-NH-、-S-S-、

、

、

、或

；A¹係C1-C6伸烷基(alkylene group)；A³係單鍵、或C1-C6伸烷基(alkylene group)；A²以及A⁴係獨立為單鍵、C1-C12伸烷基(alkylene group)、C2-C12伸烯基(alkenylene group)、或C2-C12伸炔基(alkynylene group)；X¹係氫、C1-C24烷基(alkyl group)、C2-C24烯基(alkenyl group)、C2-C24炔基(alkynyl group)、C1-C24烷醇基(alkanol group)、C2-C24烯醇基(alkenol group)、C2-C24炔醇基(alkynol group)、-NR¹R²、

、

、

、或

；X²係氫、C6-C12芳香基(aryl group)、C1-C24烷基(alkyl group)、C2-C24烯基(alkenyl group)、C2-C24炔基(alkynyl group)、C1-C24烷醇基(alkanol group)、C2-C24烯醇基(alkenol group)、C2-C24炔醇基(alkynol group)、-NR⁷R⁸、

、

、

、或

；R¹係氫、C1-C24烷基(alkyl group)、C2-C24烯基(alkenyl group)、C2-C24炔基(alkynyl group)、C1-C24烷醇基(alkanol group)、C2-C24烯醇基(alkenol group)、C2-C24炔醇基(alkynol group)、或-A⁹-Q⁷-X⁵；R²係氫、C1-C24烷基(alkyl group)、C2、C24烯基(alkenyl group)、C2-C24炔基(alkynyl group)、C1-C24烷醇基(alkanol group)、C2-C24烯醇基(alkenol group)、C2-C24 炔醇基(alkynol group)、或-A¹⁰-Q⁸-X⁶；R³、R⁴、R⁵、R⁶、R⁹、R¹⁰、R¹¹、與R¹²係獨立為氫、或C1-C6烷基(alkyl group)；R⁷係氫、C1-C24烷基(alkyl group)、C2-C24烯基(alkenyl group)、C2-C24炔基(alkynyl group)、C1-C24烷醇基(alkanol group)、C2-C24烯醇基(alkenol group)、C2-C24炔醇基(alkynol group)、或-A¹¹-Q⁹-X⁷；R⁸係氫、C1-C24烷基(alkyl group)、C2-C24烯基(alkenyl group)、C2-C24炔基(alkynyl group)、C1-C24烷醇基(alkanol group)、C2-C24烯醇基(alkenol group)、C2-C24炔醇基(alkynol group)、或-A¹²-Q¹⁰-X⁸；A⁵、A⁶、A⁷、A⁸、A⁹、A¹⁰、A¹¹、與A¹²係獨立為C1-C12伸烷基(alkylene group)；Q⁵、Q⁶、Q⁷、Q⁸、Q⁹、與Q¹⁰係獨立為-O-、-NH-、-S-S-、

、

、

、

、

、或

；以及，X³、X⁴、X⁵、X⁶、X⁷、與X⁸係獨立為C1-C24烷基(alkyl group)。根據本揭露某些實施例，本揭露提供一種醫藥組合物，其中該醫藥組合物包含本揭露所述脂質化合物或其衍生物、以及一輔助脂質。 According to the embodiments of the present disclosure, the present disclosure provides a lipid compound or a derivative thereof, wherein the derivative of the lipid compound can be a pharmaceutically acceptable salt of the lipid compound or a solvate thereof. According to the embodiments of the present disclosure, the lipid compound has a structure shown in formula (I):

, wherein Z ¹ is O or S; Z ² is -Q ¹ -A ¹ -Q ² -A ² -X ¹ ; Z ³ is the same as Z ² ; Z ⁴ is -Q ³ -A ³ -Q ⁴ -A ⁴ -X ² ; Q ¹ and Q ³ are independently a single bond, -O-, or -NH-; Q ² and Q ⁴ are independently a single bond, -O-, -NH-, -SS-,

,

,

,or

^A1 is a C1-C6 alkylene group; ^A3 is a single bond or a C1-C6 alkylene group; ^A2 and ^A4 are independently a single bond, a C1-C12 alkylene group, a C2-C12 alkenylene group, or a C2-C12 alkynylene group; ^X1 is hydrogen, a C1-C24 alkyl group, a C2-C24 alkenyl group, a C2-C24 alkynyl group, a C1-C24 alkanol group, ^a C2-C24 alkenol group, a C2-C24 alkynol group, ^-NR1R2 ,

,

,

,or

; X ² is hydrogen, C6-C12 aryl group, C1-C24 alkyl group, C2-C24 alkenyl group, C2-C24 alkynyl group, C1-C24 alkanol group, C2-C24 alkenol group, C2-C24 alkynol group, -NR ⁷ R ⁸ ,

,

,

,or

; R ¹ is hydrogen, C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C1-C24 alkanol, C2-C24 alkenol, C2-C24 alkynol, or -A ⁹ -Q ⁷ -X ⁵ ; R ² is hydrogen, C1-C24 alkyl, C2, C24 alkenyl, C2-C24 alkynyl, C1-C24 alkanol, C2-C24 alkenol, C2-C24 alkynol, or -A ¹⁰ -Q ⁸ -X ⁶ ; R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁹ , R ¹⁰ , ^R11 , and ^R12 are independently hydrogen or C1-C6 alkyl; ^R7 is hydrogen, C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C1-C24 alkanol, C2-C24 alkenol, C2-C24 alkynol, or ^-A11 - ^Q9 - ^X7 ; ^R8 is hydrogen, C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C1-C24 alkanol, C2-C24 alkenol, C2-C24 alkynol, or -A12- ^Q9 -X7. ^A5 , ^A6 , ^A7 , ^A8 , ^A9 , ^A10 , ^A11 , and ^A12 are independently C1-C12 alkylene groups; ^Q5 , ^Q6 , ^Q7 , ^Q8 , ^Q9 , and ^Q10 are independently -O-, ^-NH- , ^-SS- ,

,

,

,

,

,or

and X ³ , X ⁴ , X ⁵ , X ⁶ , X ⁷ , and X ⁸ are independently C1-C24 alkyl groups. According to certain embodiments of the present disclosure, the present disclosure provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the lipid compound or its derivative described in the present disclosure and an auxiliary lipid.

The following is directed to the lipid compounds or their derivatives and pharmaceutical compositions containing the same described in the present disclosure. It should be understood that the following description provides many different embodiments or examples for implementing different aspects of the present disclosure. The specific elements and arrangements described below are only for the purpose of simply describing the present disclosure. Of course, these are only used as examples and are not intended to limit the present disclosure. In the present disclosure, the term "about" means that the specified amount can be increased or decreased by an amount that can be recognized by a person skilled in the art as a general and reasonable size.

The present disclosure provides a lipid compound or a derivative thereof. According to an embodiment of the present disclosure, the lipid compound described in the present disclosure is an ionizable cationic lipid with a specific structure, the main core of which is pentavalent phosphorus, and may include 1 to 3 ionizable tertiary amine groups. The lipid compound described in the present disclosure is easier to metabolize in the body and has lower toxicity. The lipid compound or a derivative thereof described in the present disclosure can be positively charged in an acidic environment, and then adsorb negatively charged nucleic acids to form lipid nanoparticles (LNP). In addition, the present disclosure also provides a pharmaceutical composition comprising the lipid compound or a derivative thereof described in the present disclosure. By using the lipid compound or its derivatives disclosed herein, the pharmaceutical composition disclosed herein can form lipid nanoparticles (LNPs) that encapsulate ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA), thereby achieving the purpose of protecting ribonucleic acid and/or deoxyribonucleic acid, slowing down their degradation rate, and improving cell transfection efficiency.

，其中Z¹可為O或S；Z²可為-Q¹-A¹-Q²-A²-X¹；Z³與Z²相同；Z⁴可為-Q³-A³-Q⁴-A⁴-X²；Q¹以及Q³可獨立為單鍵、-O-、或-NH-；Q²以及Q⁴可獨立為單鍵、-O-、-NH-、-S-S-、

、

、

、或

；A¹可為C1-C6伸烷基(alkylene group)；A³係單鍵、或C1-C6伸烷基(alkylene group)；A²以及A⁴係獨立為單鍵、C1-C12伸烷基(alkylene group)、C2-C12伸烯基(alkenylene group)、或C2-C12伸炔基(alkynylene group)；X¹係氫、C1-C24烷基(alkyl group)、C2-C24烯基(alkenyl group)、C2-C24炔基(alkynyl group)、C1-C24烷醇基(alkanol group)、C2-C24烯醇基(alkenol group)、C2-C24炔醇基(alkynol group)、-NR¹R²、

、或

；X²可為氫、C6-C12芳香基(aryl group)、C1-C24烷基(alkyl group)、C2-C24烯 基(alkenyl group)、C2-C24炔基(alkynyl group)、C1-C24烷醇基(alkanol group)、C2-C24烯醇基(alkenol group)、C2-C24炔醇基(alkynol group)、-NR⁷R⁸、

、

、

、或

； R¹可為氫、C1-C24烷基(alkyl group)、C2-C24烯基(alkenyl group)、C2-C24炔基(alkynyl group)、C1-C24烷醇基(alkanol group)、C2-C24烯醇基(alkenol group)、C2-C24炔醇基(alkynol group)、或-A⁹-Q⁷-X⁵；R²可為氫、C1-C24烷基(alkyl group)、C2-C24烯基(alkenyl group)、C2-C24炔基(alkynyl group)、C1-C24烷醇基(alkanol group)、C2-C24烯醇基(alkenol group)、C2-C24炔醇基(alkynol group)、或-A¹⁰-Q⁸-X⁶；R³、R⁴、R⁵、R⁶、R⁹、R¹⁰、R¹¹、與R¹²可獨立為氫、或C1-C6烷基(alkyl group)；R⁷可為氫、C1-C24烷基(alkyl group)、C2-C24烯基(alkenyl group)、C2-C24炔基(alkynyl group)、C1-C24烷醇基(alkanol group)、C2-C24烯醇基(alkenol group)、C2-C24炔醇基(alkynol group)、或-A¹¹-Q⁹-X⁷；R⁸可為氫、C1-C24烷基(alkyl group)、C2-C24烯基(alkenyl group)、C2-C24炔基(alkynyl group)、C1-C24烷醇基(alkanol group)、C2-C24烯醇基(alkenol group)、C2-C24炔醇基(alkynol group)、或-A¹²-Q¹⁰-X⁸；A⁵、A⁶、A⁷、A⁸、A⁹、A¹⁰、A¹¹、與A¹²可獨立為C1-C12伸烷基(alkylene group)；Q⁵、Q⁶、Q⁷、Q⁸、Q⁹、與Q¹⁰可獨立為-O-、-NH-、-S-S-、

、

、

、

、

、 或

；以及X³、X⁴、X⁵、X⁶、X⁷、與X⁸可獨立為C1-C24烷基(alkyl group)。 According to the embodiments of the present disclosure, the lipid compound provided in the present disclosure has a structure shown in formula (I):

, wherein Z ¹ can be O or S; Z ² can be -Q ¹ -A ¹ -Q ² -A ² -X ¹ ; Z ³ is the same as Z ² ; Z ⁴ can be -Q ³ -A ³ -Q ⁴ -A ⁴ -X ² ; Q ¹ and Q ³ can independently be a single bond, -O-, or -NH-; Q ² and Q ⁴ can independently be a single bond, -O-, -NH-, -SS-,

,

,

,or

^A1 can be a C1-C6 alkylene group; ^A3 is a single bond or a C1-C6 alkylene group; ^A2 and ^A4 are independently a single bond, a C1-C12 alkylene group, a C2-C12 alkenylene group, or a C2-C12 alkynylene group; ^X1 is hydrogen, a C1-C24 alkyl group, a C2-C24 alkenyl group, a C2-C24 alkynyl group, ^a C1-C24 alkanol group, a C2-C24 alkenol group, a C2-C24 alkynol group, ^-NR1R2 ,

,or

; X ² can be hydrogen, C6-C12 aryl group, C1-C24 alkyl group, C2-C24 alkenyl group, C2-C24 alkynyl group, C1-C24 alkanol group, C2-C24 alkenol group, C2-C24 alkynol group, -NR ⁷ R ⁸ ,

,

,

,or

; R ¹ may be hydrogen, C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C1-C24 alkanol, C2-C24 alkenol, C2-C24 alkynol, or -A ⁹ -Q ⁷ -X ⁵ ; R ² may be hydrogen, C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C1-C24 alkanol, C2-C24 alkenol, C2-C24 alkynol, or -A ¹⁰ -Q ⁸ -X ⁶ ; R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁹ , R ^{R 10} , R ¹¹ , and R ¹² may independently be hydrogen or a C1-C6 alkyl group; R ⁷ may be hydrogen, a C1-C24 alkyl group, a C2-C24 alkenyl group, a C2-C24 alkynyl group, a C1-C24 alkanol group, a C2-C24 alkenol group, a C2-C24 alkynol group, or -A ¹¹ -Q ⁹ -X ⁷ ; R ⁸ may be hydrogen, a C1-C24 alkyl group, a C2-C24 alkenyl group, a C2-C24 alkynyl group, a C1-C24 alkanol group, a C2-C24 alkenol group, a C2-C24 alkynol group, or -A ¹² -Q 9 -X 7 ^A5 , ^A6 , ^A7 , ^{A8 , A9} , ^A10 , ^A11 , and ^A12 can independently be a C1-C12 alkylene group; ^Q5 , ^Q6 , ^Q7 , ^Q8 , ^Q9 , and ^Q10 can independently be -O-, -NH-, ^-SS- ,

,

,

,

,

, or

; and X ³ , X ⁴ , X ⁵ , X ⁶ , X ⁷ , and X ⁸ may independently be a C1-C24 alkyl group.

According to the embodiments of the present disclosure, the derivative of the lipid compound described in the present disclosure may be a pharmaceutically acceptable salt of the lipid compound, or a solvate thereof.

According to the disclosed embodiments, the pharmaceutically acceptable salt of the lipid compound can be formed by reacting the lipid compound with a pharmaceutically acceptable acid. Here, the pharmaceutically acceptable salt includes a salt formed by the lipid compound and an inorganic acid, such as a hydrochloride, phosphate, diphosphate, hydrobromide, sulfate, sulfinate, or nitrate. According to the disclosed embodiments, the pharmaceutically acceptable salt includes a salt formed by the lipid compound and an organic acid, such as lactate, oxalate, appletate, maleate, fumarate, tartrate, succinate, citrate, lactate, sulfonate, p-toluenesulfonate, 2-hydroxyethylsulfonate, benzoate, salicylate, stearate, trifluoroacetic acid, amino acid salt, or acetate. In addition, pharmaceutically acceptable cations include but are not limited to sodium, potassium, calcium, aluminum, lithium, and ammonium.

According to the disclosed embodiments, the solvate of the lipid compound includes a hydrate of the lipid compound or an alcoholate of the lipid compound.

According to the embodiments of the present disclosure, the single bond described in the present disclosure refers to the situation where there is no single atom at the relevant position. For example, in the structure where ^Z2 is ^-Q1 - ^A1 - ^Q2 - ^A2 - ^X1 , when ^Q2 is a single bond, there is no single atom at the position represented by ^Q2 , and ^A1 and ^A2 are directly connected, so that ^Z2 is the structure of ^-Q1 - ^A1 - ^A2 - ^X1 .

According to the embodiments of the present disclosure, the alkyl group described in the present disclosure may be a linear or branched alkyl group. According to the embodiments of the present disclosure, the alkenyl group described in the present disclosure may be a linear or branched alkenyl group, and contain at least one carbon-carbon double bond. According to the embodiments of the present disclosure, the alkynyl group described in the present disclosure may be a linear or branched alkynyl group, and contain at least one carbon-carbon triple bond.

For example, the C1-C24 alkyl group may be methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, or isomers thereof.

According to the embodiments of the present disclosure, the alkanol group described in the present disclosure may be a linear or branched alkanol group. According to the embodiments of the present disclosure, the alkenol group described in the present disclosure may be a linear or branched alkenol group and contain at least one carbon-carbon double bond. According to the embodiments of the present disclosure, the alkynol group described in the present disclosure may be a linear or branched alkynyl group and contain at least one carbon-carbon triple bond. Here, an alkanol group means that at least one of the carbon atoms of an alkyl group is replaced by a hydroxyl group; an alkenol group means that at least one of the carbon atoms of an alkenyl group is replaced by a hydroxyl group; and an alkynol group means that at least one of the carbon atoms of an alkynyl group is replaced by a hydroxyl group. In addition, according to the embodiments of the present disclosure, the C6-C12 aromatic group described in the present disclosure may be a phenyl group, a biphenyl group, or a naphthyl group.

According to the disclosed embodiments, the alkylene group described in the disclosed embodiments may be a linear or branched alkylene group. According to the disclosed embodiments, the alkenylene group described in the disclosed embodiments may be a linear or branched alkenylene group. According to the disclosed embodiments, the alkynylene group described in the disclosed embodiments may be a linear or branched alkynylene group.

For example, the C1-C24 alkylene group may be a methylene group, an ethyl group, a propylene group, a butylene group, a pentylene group, a hexylene group, or an isomer thereof.

According to the embodiments of the present disclosure, in the lipid compound having the structure shown in formula (I) described in the present disclosure, ^Z4 is different from ^Z2 (ie, ^Z4 is different from ^Z3 ).

According to the embodiments of the present disclosure, in the lipid compound having the structure shown in formula (I) described in the present disclosure, Z ² , Z ³ and Z ⁴ are the same.

According to the embodiments of the present disclosure, in the lipid compound having the structure shown in formula (I) described in the present disclosure, ^Q1 and ^Q2 are not single bonds at the same time. In other words, when ^Q1 is a single bond, ^Q2 is not a single bond; and when ^Q2 is a single bond, ^Q1 is not a single bond.

、

、

、

、

、

、

、

、或

。 According to the disclosed embodiments, ^Q1 is a single bond, and ^Q2 can be -O-, -NH-, -SS-,

,

,

,

,

,

,

,

,or

.

According to the disclosed embodiments, ^Q2 is a single bond, and ^Q1 can be -O-, or -NH-.

、

、

、

、或

。 According to the disclosed embodiments, ^Q1 can be -O-, or -NH-, and ^Q2 can be -O-, -NH-, -SS-,

,

,

,

,or

.

According to the embodiments of the present disclosure, in the lipid compound having the structure shown in formula (I) described in the present disclosure, ^Q3 and ^Q4 are not single bonds at the same time. In other words, when ^Q3 is a single bond, ^Q4 is not a single bond; and when ^Q4 is a single bond, ^Q3 is not a single bond.

、

、

、

、

、

、

、

、或

。 According to the disclosed embodiment, ^Q3 is a single bond, and ^Q4 can be -O-, NH-, -SS-,

,

,

,

,

,

,

,

,or

.

According to the disclosed embodiments, Q ⁴ is a single bond, and Q ³ may be -O-, or -NH-.

、

、

、

、

、

、

、

、或

、且A3非為單鍵。 According to the disclosed embodiments, Q ³ can be -O-, or -NH-, Q ⁴ can be -O-, -NH-, -SS-,

,

,

,

,

,

,

,

,or

, and A3 is not a single key.

According to the embodiments of the present disclosure, when A ² is a C1-C12 alkylene group, a C2-C12 alkenylene group, or a C2-C12 alkynylene group, Q ² is not a single bond.

According to the embodiments of the present disclosure, when A ² is a C1-C12 alkylene group, a C2-C12 alkenylene group, or a C2-C12 alkynylene group, Q ² is not a single bond.

According to the embodiments of the present disclosure, when A ³ is a single bond, A ⁴ is a C1-C12 alkylene group, a C2-C12 alkenylene group, or a C2-C12 alkynylene group.

According to the embodiments of the present disclosure, when A ⁴ is a single bond, A ³ is a C1-C6 alkylene group.

According to embodiments of the present disclosure, when A ³ is a single bond and A ⁴ is a single bond, Q ⁴ is a single bond, and Q ³ is -O- or -NH-.

、或

，其中Q¹、Q²、Q³、Q⁴、A¹、A²、A³、A⁴、X¹、以及X²的定義上述相同。 According to the embodiments of the present disclosure, the lipid compound having the structure shown in formula (I) described in the present disclosure can be

,or

, wherein Q ¹ , Q ² , Q ³ , Q ⁴ , A ¹ , A ² , A ³ , A ⁴ , X ¹ , and X ² have the same meanings as above.

According to the embodiments of the present disclosure, the present disclosure also provides a pharmaceutical composition, such as a lipid nanoparticle composition. According to the embodiments of the present disclosure, the pharmaceutical composition comprises the lipid compound or its derivative described in the present disclosure, and a sterol.

According to the disclosed embodiment, in the pharmaceutical composition, the lipid compound (and/or its derivative) is 100 mol, and the content of the sterol can be 50 to 300 mol, such as 60 mol, 80 mol, 100 mol, 120 mol, 150 mol, 180 mol, 200 mol, 230 mol, 250 mol, or 280 mol. According to the disclosed embodiment, the sterol can be cholesterol, cholesterol hexasuccinate, ergosterol, lanosterol, or a combination thereof.

According to the disclosed embodiments, the pharmaceutical composition may further comprise an auxiliary lipid, wherein the auxiliary lipid may be 5 to 200 moles, such as 6 moles, 8 moles, 10 moles, 20 moles, 30 moles, 50 moles, 75 moles, 100 moles, 120 moles, 150 moles, 170 moles, or 190 moles.

According to the embodiments of the present disclosure, the auxiliary lipid can be 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3 -1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine ( 2-oleoyl-sn-glycero-3-phosphatidylcholine, POPC), 1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC, hydrogenated soy phospholipid acylcholine, phosphatidylcholine (HSPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-tac-glycerol) (sodium salt) (DMPG), Sulfoquinovosyl diglycerol (SDG) Diacylglycerol, SQDG), monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine ( 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dioleoyl-sn-glycero-3-phospho-1-serine (DOPS), L-a-phosphatidylcholine (EPC), phosphatidylcholine (PC), or a combination thereof.

According to the disclosed embodiments, the pharmaceutical composition may further comprise a PEG-ylated lipid, wherein the content of the PEG-ylated lipid may be 1 to 30 moles, such as 2 moles, 3 moles, 4 moles, 5 moles, 6 moles, 7 moles, 8 moles, 9 moles, 10 moles, 11 moles, 12 moles, 13 moles, 14 moles, 15 moles, 16 moles, 17 moles, 18 moles, 19 moles, 20 moles, 21 moles, 22 moles, 23 moles, 24 moles, 25 moles, 26 moles, 27 moles, 28 moles, or 29 moles.

According to the disclosed embodiments, the PEG-ylated lipid can be phosphatidylethanolamine modified with polyethylene glycol, phosphatidic acid modified with polyethylene glycol, ceramide modified with polyethylene glycol, dialkylamine modified with polyethylene glycol, diacylglycerol modified with polyethylene glycol, dialkylglycerol modified with polyethylene glycol, sterol modified with polyethylene glycol, phospholipid modified with polyethylene glycol, or a combination thereof.

According to the embodiments of the present disclosure, the PEG-ylated lipid may be 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DMPE-PEG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG), 1,2-dimyristo ... glycol, DMPE-PEG), 1,2-dipalmitoyl-sn-glycerol-3-succinate-polyethylene glycol (DPGS-PEG), cholesterol polyethylene glycol (cholesteryl-polyethylene glycol), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DPPE-PEG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DOPE-PEG), 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (1,2-dimyristoyl-sn-glycerol-methoxypolyethylene glycol glycol, DMG-PEG), 1,2-distearoyl-sn-glycerol-3-methoxy-polyethylene glycol (DSG-PEG), 1,2-dipalmitoyl-sn-glycerol-methoxy-polyethylene glycol (DPG-PEG), α- [2-(ditetradecylamino)-2-oxoethyl] -ω -methoxy-poly(oxy-1,2-ethanediyl) (ALC-0159), or a combination thereof.

According to the disclosed embodiments, the pharmaceutical composition may further comprise a nucleic acid, wherein the content of the nucleic acid may be 0.1 to 30 moles, such as 0.2 moles, 0.5 moles, 1 mole, 1.5 moles, 2 moles, 3 moles, 4 moles, 5 moles, 6 moles, 7 moles, 8 moles, 9 moles, 10 moles, 11 moles, 12 moles, 13 moles, 14 moles, 15 moles, 16 moles, 17 moles, 18 moles, 19 moles, 20 moles, 21 moles, 22 moles, 23 moles, 24 moles, 25 moles, 26 moles, 27 moles, 28 moles, or 29 moles.

According to the disclosed embodiments, the nucleic acid may be deoxyribonucleic acid (DNA), plasmid DNA, messenger RNA (mRNA), small interfering RNA (siRNA), self-amplifying RNA (saRNA), circular RNA, or a combination thereof.

In order to make the above contents and other purposes, features, and advantages of this disclosure more clearly understood, the following specifically lists a preferred embodiment and describes it in detail with the accompanying drawings.

Lipid compounds

Table 1 lists the lipid compounds described in the embodiments of this disclosure.

To further illustrate the preparation method of the lipid compound disclosed herein, the following examples are used to illustrate the preparation process of the lipid compound described in Examples 1-3, 6-15, 18, and 19.

Example 1

Compound (1) (2 equivalents) and tetrahydrofuran (THF) were added to a reaction bottle under nitrogen to obtain a solution (concentration of 0.1 M). Then, lithium bis(trimethylsilyl)amide (LiHMDS) solution (dissolved in tetrahydrofuran, concentration of 1.0 M, LiHMDS of 3 equivalents) was added to the reaction bottle. After stirring the reaction at room temperature for 1 hour, compound (2) (1 equivalent) was added dropwise to the reaction bottle. After the reaction was complete, the obtained product was concentrated and extracted with n-hexane and brine. After collecting the organic phase, dehydrating with sodium sulfate (Na ₂ SO ₄ ) and removing the solvent, the obtained product was purified by high performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (concentration of 0.1%) as eluents) to obtain lipid compound (1). The reaction formula of the above reaction is shown below:

Next, the lipid compound (1) described in Example 1 was analyzed by nuclear magnetic resonance spectroscopy, and the obtained spectral information was as follows: ¹ H-NMR (500 MHz, CDCl ₃ ): δ 4.48 (s, 4H), 4.18 (t, J = 7.0, 7.5 Hz, 2H), 3.40 (s, 4H), 3.09 (m, 9H), 1.68 (s, 9H), 1.36-1.26 (m, 55H), 0.87 (t, J = 7.0, 7.0 Hz, 12H). Next, the lipid compound (1) was analyzed by liquid chromatography-mass spectrometry (LC-MS), and M/Z was measured as [M+H] ⁺ = 718.7.

Example 2

Compound (1) (2.5 equivalents) and tetrahydrofuran (THF) were added to a reaction bottle under nitrogen to obtain a solution (concentration of 0.1 M). Then, lithium bis(trimethylsilyl)amide (LiHMDS) solution (dissolved in tetrahydrofuran, concentration of 1.0 M, LiHMDS of 3 equivalents) was added to the reaction bottle. After stirring the reaction at room temperature for 1 hour, compound (3) (1 equivalent) was added dropwise to the reaction bottle. After the reaction was complete, the obtained product was concentrated and extracted with n-hexane and brine. After collecting the organic phase, dehydrating with sodium sulfate (Na ₂ SO ₄ ) and removing the solvent, the obtained product was purified by high performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (concentration of 0.1%) as eluents) to obtain lipid compound (2). The reaction formula of the above reaction is shown below:

Next, the lipid compound (2) described in Example 2 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is as follows: ¹ H-NMR (500 MHz, CDCl ₃ ): δ 4.42 (s, 2H), 4.29 (s, 2H), 4.10-4.09 (m, 2H), 3.73 (s, 4H), 3.47-3.43 (m, 2H), 3.32-3.30 (m, 4H), 3.11-3.04 (m, 8H), 1.67 (s, 12H), 1.32-1.25 (m, 77H), 0.88 (t, J =7.5, 7.5 Hz, 18H). Then, the lipid compound (3) was analyzed by liquid chromatography-mass spectrometry (LC-MS), and the M/Z was measured as: [M+H] ⁺ =986.1.

Example 3

Compound (4) (2 equivalents) and tetrahydrofuran (THF) were added to a reaction bottle under nitrogen to obtain a solution (concentration of 0.1 M). Then, lithium bis(trimethylsilyl)amide (LiHMDS) solution (dissolved in tetrahydrofuran, concentration of 1.0 M, LiHMDS of 3 equivalents) was added to the reaction bottle. After stirring the reaction at room temperature for 1 hour, compound (2) (1 equivalent) was added dropwise to the reaction bottle. After the reaction was complete, the obtained product was concentrated and extracted with n-hexane and brine. After collecting the organic phase, dehydrating with sodium sulfate (Na ₂ SO ₄ ) and removing the solvent, the obtained product was purified by high performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (concentration of 0.1%) as eluents) to obtain lipid compound (3). The reaction formula of the above reaction is shown below:

Next, the lipid compound (3) described in Example 3 was analyzed by nuclear magnetic resonance spectroscopy, and the obtained spectral information was as follows: ¹ H-NMR (500 MHz, CDCl ₃ ): 4.13 (t, J = 6.5, 7.5 Hz, 6H), 3.19 (s, 4H), 3.03-3.02 (m, 8H), 2.17-2.14 (m, 11H), 1.67 (s, 8H), 1.35-1.26 (m, 52H), 0.87 (t, J = 7.0, 6.5 Hz, 12H). Next, the lipid compound (3) was analyzed by liquid chromatography-mass spectrometry (LC-MS), and the measured M/Z was: [M+H] ⁺ = 746.5.

Example 6

Compound (5) (1 equivalent) and dichloromethane were added to a reaction bottle under nitrogen to obtain a solution (concentration of 0.04 M). Then, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction bottle. After stirring the reaction at room temperature for 10 minutes, compound (6) (4 equivalents) was added dropwise to the reaction bottle. After 8 hours of reaction, the obtained product was purified by high performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (concentration of 0.1%) as eluents) to obtain lipid compound (6). The reaction formula of the above reaction is shown below:

Next, the lipid compound (6) described in Example 6 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is as follows: ¹ H-NMR (500 MHz, CDCl ₃ ): δ 10.62 (s, 1H), 8.73 (s, 1H), 3.60 (s, 3H), 3.22 (s, 3H), 3.09-3.06 (m, 4H), 2.54-2.56 (m, 2H), 2.11-2.13 (m, 4H), 1.63 (s, 12H), 1.32-1.26 (m, 77H), 0.88 (t, J=7.5, 7.5 Hz, 18H). Then, the lipid compound (6) was analyzed by liquid chromatography-mass spectrometry (LC-MS), and the M/Z was measured as: [M+H] ⁺ =1151.3.

Example 7

Compound (5) (1 equivalent) and dichloromethane were added to a reaction bottle under nitrogen to obtain a solution (concentration of 0.04 M). Then, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction bottle. After stirring the reaction at room temperature for 10 minutes, compound (7) (4 equivalents) was added dropwise to the reaction bottle. After 8 hours of reaction, the obtained product was purified by high performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (concentration of 0.1%) as eluents) to obtain lipid compound (7). The reaction formula of the above reaction is shown below:

Next, the lipid compound (7) described in Example 7 was analyzed by nuclear magnetic resonance spectroscopy, and the obtained spectral information was as follows: ¹ H-NMR (500 MHz, CDCl ₃ ): δ 7.98 (s, 3H), 4.49 (s, 7H), 3.21 (s, 6H), 3.10-2.8 (m, 18H), 2.48 (s, 5H), 2.07 (s, 5H), 1.80-1.40 (m, 26H), 1.40-1.10 (m, 78H), 0.84 (t, J = 6.0, 7.0 Hz, 18H). Next, the lipid compound (7) was analyzed by liquid chromatography-mass spectrometry (LC-MS), and M/Z was measured as [M+H] ⁺ = 1235.5.

Example 8

Compound (5) (1 equivalent) and dichloromethane were added to a reaction bottle under nitrogen to obtain a solution (concentration of 0.04 M). Then, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction bottle. After stirring the reaction at room temperature for 10 minutes, compound (8) (4 equivalents) was added dropwise to the reaction bottle. After 8 hours of reaction, the obtained product was purified by high performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (concentration of 0.1%) as eluents) to obtain lipid compound (8). The reaction formula of the above reaction is shown below:

Next, the lipid compound (8) described in Example 8 was analyzed by nuclear magnetic resonance spectroscopy, and the obtained spectral information was as follows: ¹ H-NMR (500 MHz, CDCl ₃ ): δ 11.52 (s, 1H), 5.57 (s, 1H), 3.18 (s, 7H), 2.30 (s, 10H), 2.50 (s, 3H), 2.06 (s, 4H), 1.67-1.65 (m, 11H), 1.34-1.25 (m, 51H), 0.87 (t, J = 2.0, 5.5 Hz, 9H). Next, the lipid compound (8) was analyzed by liquid chromatography-mass spectrometry (LC-MS), and the measured M/Z was: [M+H] ⁺ = 1319.6.

Example 9

Under nitrogen, compound (5) (1 equivalent) and dichloromethane were added to a reaction bottle to obtain a solution (concentration of 0.05 M). Then, diisopropylethylamine (DIPEA) (3 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (3 equivalents) were added to the reaction bottle. After stirring the reaction at room temperature for 10 minutes, compound (7) (4 equivalents) and compound (9) (1 equivalent) were added dropwise to the reaction bottle. After 8 hours of reaction, the obtained product was purified by high performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (concentration of 0.1%) as eluents) to obtain lipid compound (9). The reaction formula of the above reaction is shown below:

Next, the lipid compound (9) described in Example 9 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is as follows: ¹ H-NMR (500 MHz, CDCl ₃ ): ¹ H-NMR (500 MHz, CDCl ₃ ): δ 10.84 (s, 1H), 8.03 (s, 2H), 3.66 (s, 2H), 3.33-3.24 (m, 6H), 3.04-2.99 (m, 16H), 2.81 (s, 1H), 2.53 (s, 4H), 2.11 (s, 4H), 1.76-1.56 (m, 12H), 1.33-1.27 (m, 38H), 0.88 (t, J =6.5, 7.5 Hz, 9H). Then, the lipid compound (9) was analyzed by liquid chromatography-mass spectrometry (LC-MS), and the M/Z was measured to be: [M+H] ⁺ =955.8.

Example 10

Compound (5) (1 equivalent) and dichloromethane were added to a reaction bottle under nitrogen to obtain a solution (concentration of 0.04 M). Then, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction bottle. After stirring the reaction at room temperature for 10 minutes, compound (10) (4 equivalents) was added dropwise to the reaction bottle. After 8 hours of reaction, the obtained product was purified by high performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (concentration of 0.1%) as eluents) to obtain lipid compound (10). The reaction formula of the above reaction is shown below:

Next, the lipid compound (10) described in Example 10 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is as follows: ¹ H-NMR (500 MHz, CDCl ₃ ): δ 8.79 (s, 1H), 3.70-3.68 (m, 3H), 3.54 (s, 2H), 3.38 (s, 2H), 3.23-3.03 (m, 9H), 2.60-2.57 (m, 16H), 2.16-2.02 (m, 5H), 1.63 (s, 6H), 1.50 (t, J =7.0, 7.5 Hz, 1H), 1.33-1.27 (m, 39H), 0.89 (t, J =6.5, 7.0 Hz, 9H). Then, the lipid compound (10) was analyzed by liquid chromatography-mass spectrometry (LC-MS), and the M/Z was measured to be: [M+H] ⁺ =1024.8.

Example 11

Under nitrogen, compound (5) (1 equivalent) and dichloromethane were added to a reaction bottle to obtain a solution (concentration of 0.05 M). Then, diisopropylethylamine (DIPEA) (3 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (3 equivalents) were added to the reaction bottle. After stirring the reaction at room temperature for 10 minutes, compound (6) (4 equivalents) and compound (9) (1 equivalent) were added dropwise to the reaction bottle. After 8 hours of reaction, the obtained product was purified by high performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (concentration of 0.1%) as eluents) to obtain lipid compound (11). The reaction formula of the above reaction is shown below:

Next, the lipid compound (11) described in Example 11 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is as follows: ¹ H-NMR (500 MHz, CDCl ₃ ): ¹ H-NMR (500 MHz, CDCl ₃ ): δ 8.79 (s, 1H), 3.70-3.68 (m, 3H), 3.54 (s, 2H), 3.38 (s, 2H), 3.23-3.03 (m, 9H), 2.60-2.57 (m, 16H), 2.16-2.02 (m, 5H), 1.63 (s, 6H), 1.50 (t, J =7.0, 7.5 Hz, 1H), 1.33-1.27 (m, 39H), 0.89 (t, J =6.5, 7.0 Hz, 9H). Then, the lipid compound (11) was analyzed by liquid chromatography-mass spectrometry (LC-MS), and the M/Z was measured to be: [M+H] ⁺ =899.7.

Example 12

Compound (5) (1 equivalent) and dichloromethane were added to a reaction bottle under nitrogen to obtain a solution (concentration 0.04 M). Then, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction bottle. After stirring the reaction at room temperature for 10 minutes, compound (11) (4 equivalents) was added dropwise to the reaction bottle. After 8 hours of reaction, the obtained product was purified by high performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (concentration of 0.1%) as eluents) to obtain lipid compound (12). The reaction formula of the above reaction is shown below:

Next, the lipid compound (12) described in Example 12 was analyzed by nuclear magnetic resonance spectroscopy, and the obtained spectral information was as follows: ¹ H-NMR (500 MHz, CDCl ₃ ): δ 8.50 (s, 2H), 6.46 (s, 5H), 4.10-3.90 (m, 3H), 3.90-3.0 (m, 13H), 2.55 (s, 3H), 2.15 (s, 3H), 1.60-1.20 (m, 61H), 0.88 (t, J = 7.0, 7.5 Hz, 10H). Next, the lipid compound (12) was analyzed by liquid chromatography-mass spectrometry (LC-MS), and M/Z was measured as [M+H] ⁺ = 1499.4.

Example 13

Compound (5) (1 equivalent) and dichloromethane were added to a reaction bottle under nitrogen to obtain a solution (concentration 0.04 M). Then, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction bottle. After stirring the reaction at room temperature for 10 minutes, compound (12) (4 equivalents) was added dropwise to the reaction bottle. After 8 hours of reaction, the obtained product was purified by high performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (concentration of 0.1%) as eluents) to obtain lipid compound (13). The reaction formula of the above reaction is shown below:

Next, the lipid compound (13) described in Example 13 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is as follows: ¹ H-NMR (500 MHz, CDCl ₃ ): δ 10.68 (s, 2H), 8.78 (s, 3H), 3.59 (d, J = 4.0 Hz, 5H), 3.45-3.20 (m, 12H), 3.20-3.0 (m, 11H), 2.60-2.40 (m, 5H), 2.20-2.0 (m, 5H), 1.80-1.45 (m, 10H), 1.40-1.20 (m, 32H), 0.88 (t, J = 6.5 Hz, 15H). Then, the lipid compound (13) was analyzed by liquid chromatography-mass spectrometry (LC-MS), and the M/Z was measured to be: [M+H] ⁺ =898.8.

Example 14

Compound (5) (1 equivalent) and dichloromethane were added to a reaction bottle under nitrogen to obtain a solution (concentration of 0.04 M). Then, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction bottle. After stirring the reaction at room temperature for 10 minutes, compound (13) (4 equivalents) was added dropwise to the reaction bottle. After 8 hours of reaction, the obtained product was purified by high performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (concentration of 0.1%) as eluents) to obtain lipid compound (14). The reaction formula of the above reaction is shown below:

Next, the lipid compound (14) described in Example 14 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is as follows: ¹ H-NMR (500 MHz, CDCl ₃ ): δ 10.67 (s, 2H), 8.75 (s, 2H), 4.25-4.0 (m, 5H), 3.62 (d, J = 4.5 Hz, 4H), 3.25-3.20 (m, 4H), 3.20-3.0 (m, 9H), 2.65-2.45 (m, 4H), 2.25-2.15 (m, 4H), 1.80-1.60 (m, 9H), 1.45-1.20 (m, 44H), 0.89 (t, J = 6.0, 7.5 Hz, 12H). Then, the lipid compound (14) was analyzed by liquid chromatography-mass spectrometry (LC-MS), and the M/Z was measured to be: [M+H] ⁺ =1067.

Example 15

Compound (5) (1 equivalent) and dichloromethane were added to a reaction bottle under nitrogen to obtain a solution (concentration: 0.04 M). Then, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction bottle. After stirring the reaction at room temperature for 10 minutes, compound (14) (4 equivalents) was added dropwise to the reaction bottle. After 8 hours of reaction, the obtained product was purified by high performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (concentration of 0.1%) as eluents) to obtain lipid compound (15). The reaction formula of the above reaction is shown below:

Next, the lipid compound (15) described in Example 15 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is as follows: ¹ H-NMR (500 MHz, CDCl ₃ ): δ 10.77 (s, 2H), 8.80 (s, 2H), 3.62-3.50 (m, 7H), 3.30-3.20 (m, 5H), 3.20-3.0 (m, 11H), 2.60-2.50 (m, 5H), 2.20-2.0 (m, 5H), 1.80-1.50 (m, 11H), 1.50-1.20 (m, 40H), 0.89 (t, J =5.0, 7.0 Hz, 14H). Then, the lipid compound (15) was analyzed by liquid chromatography-mass spectrometry (LC-MS), and the M/Z was measured to be: [M+H] ⁺ =982.8.

Example 18

Compound (5) (1 equivalent) and dichloromethane were added to a reaction bottle under nitrogen to obtain a solution (concentration 0.04 M). Then, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction bottle. After stirring the reaction at room temperature for 10 minutes, compound (15) (4 equivalents) was added dropwise to the reaction bottle. After 8 hours of reaction, the obtained product was purified by high performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (concentration of 0.1%) as eluents) to obtain lipid compound (18). The reaction formula of the above reaction is shown below:

Next, the lipid compound (18) described in Example 18 was analyzed by nuclear magnetic resonance spectroscopy, and the obtained spectral information is as follows: ¹ H-NMR (500 MHz, CDCl ₃ ): δ 8.66 (s, 2H), 4.60-3.90 (m, 13H), 3.90-3.0 (m, 9H), 2.70-2.40 (m, 4H), 2.30-2.0 (m, 4H), 1.70-1.20 (m, 45H), 0.89 (t, J = 6.0, 7.0 Hz, 13H). Next, the lipid compound (18) was analyzed by liquid chromatography-mass spectrometry (LC-MS), and M/Z was measured as [M+H] ⁺ = 864.3.

Example 19

Compound (5) (1 equivalent) and dichloromethane were added to a reaction bottle under nitrogen to obtain a solution (concentration of 0.04 M). Then, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction bottle. After stirring the reaction at room temperature for 10 minutes, compound (16) (4 equivalents) was added dropwise to the reaction bottle. After 8 hours of reaction, the obtained product was purified by high performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (concentration of 0.1%) as eluents) to obtain lipid compound (19). The reaction formula of the above reaction is shown below:

Next, the lipid compound (19) described in Example 19 was analyzed by nuclear magnetic resonance spectroscopy, and the obtained spectral information was as follows: ¹ H-NMR (500 MHz, CDCl ₃ ): δ 8.63 (s, 2H), 4.70-3.90 (m, 12H), 3.90-3.0 (m, 17H), 2.70-2.50 (m, 4H), 2.30-2.10 (m, 4H), 1.60-1.20 (m, 48H), 0.88 (t, J = 6.5, 7.5 Hz, 11H). Next, the lipid compound (19) was analyzed by liquid chromatography-mass spectrometry (LC-MS), and M/Z was measured as [M+H] ⁺ = 1247.1.

Preparation of the composition

Example 35

Luciferase mRNA (product number R1018, purchased from APExBIO) was dissolved in an acidic buffer (product number J63669, purchased from Alfa Aesar) (pH 4.5) to obtain a nucleic acid aqueous solution (concentration 0.1 mg/mL).

The lipid compound (1), cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and DMG-PEG lipid (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol) (product number DMG-PEG-2K, purchased from Nippon Fine Chemical) prepared in Example 1 were dissolved in ethanol to obtain a lipid solution (concentration of 10-15 mg/mL), wherein the contents of lipid compound (1), cholesterol, DSPC, and DMG-PEG lipid are shown in Table 2.

Next, the nucleic acid aqueous solution and the lipid solution are mixed through a microfluidic system (NanoAssemblr Ignite® system, purchased from Precision NanoSystem Inc.), and the volumes of the acid aqueous solution and the lipid solution are adjusted so that the ratio (N/P) of the nitrogen atom (N) of the lipid compound (1) to the phosphorus atom (P) from the luciferase mRNA is 12. Next, after ultrafiltration or dialysis, the resulting composition is self-assembled to form a nucleic acid lipid nanoparticle carrier (1).

Implementation Examples 36-43

The same method as Example 35 is followed, but the components and/or content in the lipid solution and the N/P value (adjusted by the volume ratio of the nucleic acid aqueous solution and the lipid solution) are changed according to Table 1 to obtain nucleic acid lipid nanoparticle carriers (2)-(9).

Comparison Example 1

The same method as in Example 35 was followed, except that the lipid compound (1) was replaced with (6Z,9Z,28Z,31Z)-triacontriacontria-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (DLin-MC3-DMA), and the N/P value was adjusted (by adjusting the volume ratio of the nucleic acid aqueous solution and the lipid solution) to obtain the nucleic acid lipid nanoparticle carrier (10).

Comparison Example 2

The same method as in Example 35 was followed, except that 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoic acid heptadecan-9-yl ester (SM-102) was used to replace the lipid compound (1), and the N/P value was adjusted (by adjusting the volume ratio of the nucleic acid aqueous solution and the lipid solution) to obtain the nucleic acid lipid nanoparticle carrier (11).

Implementation Examples 44-52

The same method as Example 35 is followed, but the components and/or content in the lipid solution are changed according to Table 3, and the N/P value is adjusted (adjusted by the volume ratio of the nucleic acid aqueous solution and the lipid solution) to obtain nucleic acid lipid nanoparticle carriers (12)-(20).

Table 3

Implementation Examples 53-61

The same method as Example 35 is followed, but the components and/or content in the lipid solution are changed according to Table 4, and the N/P value is adjusted (adjusted by the volume ratio of the nucleic acid aqueous solution and the lipid solution) to obtain nucleic acid lipid nanoparticle carriers (21)-(29).

Implementation Examples 62-70

The same method as Example 35 is followed, but the components and/or content in the lipid solution are changed according to Table 5, and the N/P value is adjusted (adjusted by the volume ratio of the nucleic acid aqueous solution and the lipid solution) to obtain nucleic acid lipid nanoparticle carriers (30)-(38).

Evaluation of the properties of pharmaceutical compositions

The particle size, polydispersity coefficient (PdI), zeta potential, mRNA recovery, mRNA encapsulation efficiency and acid dissociation constant (pKa) of the nucleic acid lipid nanoparticle carriers (1)-(9) obtained in Examples 35-43 and the nucleic acid lipid nanoparticle carriers (10) and (11) obtained in Comparative Examples 1 and 2 were evaluated. The results are shown in Table 6.

The particle size, polydispersity coefficient (PdI), and Zeta potential of nucleic acid lipid nanoparticle carriers are evaluated as follows. Take 20μL of sample and add it to 330μL of Dulbecco's Phosphate-Buffered Saline (DPBS), shake it evenly, and measure the particle size and polydispersity coefficient of liposomes with a dynamic light scattering instrument (Zetasizer Nano-ZS, Malvern Panalytical). In addition, take 20μL of sample and add it to 680μL of sodium chloride aqueous solution (concentration of 10mM), shake it evenly, and measure the Zeta potential of liposomes with a dynamic light scattering instrument (Zetasizer Nano-ZS, Malvern Panalytical).

The evaluation method of mRNA recovery is as follows. After sterile filtration of the nucleic acid lipid nanoparticle carrier, take 100 μL of the sample and add 25 μL of 5% Triton X-100. Then, after incubation at 37°C for 10 minutes, add 100 μL of chloroform. After high-speed shaking (speed of 2000 rpm) for 10 minutes and centrifugation for 30 minutes (centrifugal force of 14000×g), take 80 μL of the supernatant and analyze its absorbance using a UV-visible spectrophotometer (UV7, Mettler Toledo). Then, calculate the concentration of mRNA using the following formula (1), and calculate the recovery rate using the following formula (2). Wherein, C is the concentration of RNA, A _260nm is the absorbance of RNA at 260 wavelength, Vf is the volume of supernatant (μL), and W is the initial weight of RNA (ng).

C=(A _260nm x 40)/(100/125) Formula (1)

Recovery rate (%) = [(C x Vf)/W] x 100% Formula (2)

The RNA encapsulation efficiency (EE) was measured using the Quant-iT RiboGreen RNA Assay kit (purchased from Invitrogen). That is, the mRNA concentration in the dispersion containing the nucleic acid lipid particles was quantified in the presence and absence of 0.2% Triton X-100 surfactant, and the RNA encapsulation efficiency was calculated using the following formula (3). EE is the RNA encapsulation efficiency, C _Free is the unencapsulated RNA concentration, and C _A11 is the total RNA concentration.

EE(%)=(1-C _Free /C _A11 )x 100% formula (3)

The acid dissociation constant (pKa) was calculated by using a titration method based on 2-(p-toluidine)-6-naphthalenesulfonic acid (TNS) in combination with a microplate fluorescence analyzer (Infinite F200 Pro, Tecan, excitation wavelength 320nm, emission wavelength 448nm) to analyze the fluorescence intensity titration curve of the sample at 18 points in the buffer solution with a pH value of 2.5~11.0. The titration curve was sigmoidal fit to calculate the acid dissociation constant (pKa), which is the pH value at which the nucleic acid lipid nanoparticle carrier reaches half of the maximum fluorescence intensity.

The particle size, polydispersity coefficient (PdI), zeta potential, mRNA recovery, mRNA encapsulation efficiency, and acid dissociation constant (pKa) of the nucleic acid lipid nanoparticle carriers (12)-(20) obtained in Examples 44-52 were evaluated. The results are shown in Table 7.

The particle size, polydispersity coefficient (PdI), zeta potential, mRNA recovery, mRNA encapsulation efficiency and acid dissociation constant (pKa) of the nucleic acid lipid nanoparticle carriers (21)-(29) obtained in Examples 53-61 were evaluated. The results are shown in Table 8.

The particle size, polydispersity coefficient (PdI), zeta potential, mRNA recovery, mRNA encapsulation efficiency and acid dissociation constant (pKa) of the nucleic acid lipid nanoparticle carriers (30)-(38) obtained in Examples 62-70 were evaluated. The results are shown in Table 9.

From Tables 6 to 9, it can be seen that the particle size of the nucleic acid lipid nanoparticle carrier formed by self-assembly of the pharmaceutical composition disclosed herein can be about 50nm to 250nm, and the polydispersity coefficient can be between 0.03 and 0.33. In certain embodiments, the RNA recovery rate (%) of the nucleic acid lipid nanoparticle carrier formed by self-assembly of some pharmaceutical compositions disclosed herein can be higher than about 80%, and the acid dissociation constant (pKa) can be between 5 and 8.5.

Transfection efficiency and cell activity assessment

HEK293 cells were seeded at 5×10 ⁴ cells/well in a 96-well culture plate and cultured for 24 hours. Then, 97 μL of Opti-MEM ^TM medium was mixed evenly with 3 μL of lipofectamine reagent (purchased from Invitrogen) and reacted at room temperature for 10 minutes to obtain a first solution. 1 μL of Luciferase mRNA (product number R1018, purchased from APExBIO) (concentration of 1 mg/mL) was mixed evenly with 99 μL of Opti-MEM ^TM medium to obtain a second solution. Then, the second solution was added to the first solution, mixed evenly, and reacted at room temperature for 5 minutes to obtain a transfection solution. Next, the lipid nanoparticle (LNP) solutions prepared in Examples 35-43 were diluted with 1xPBS to obtain diluted lipid nanoparticle solutions (volume 200 μL, concentration 5 μg/mL). Next, 800 μL of cell culture solution (product number MT-10-009-CVS, purchased from Corning) was added to the diluted lipid nanoparticle solution and the transfection solution to obtain a cell co-culture solution (the concentration of luciferase mRNA was 0.1 μg/100 μL). Next, 100 μL of different cell co-culture solutions were added to 96-well culture plates and co-cultured with cells (0.1 μg mRNA/per well). After 24 hours of incubation, 20 μL of supernatant was taken out and 20 μL of cell viability assay reagent (GF-AFC) was added. After reacting at 37°C for 35-40 minutes, the fluorescence value (Ex: 380nm; Em: 540nm) of the obtained product was measured using a fluorometer. By dividing the fluorescence detection reading and the fluorescence value of the untreated negative control group, the cell viability in the presence of different lipid nanoparticle (LNP) solutions was calculated. The results are shown in Table 10.

From the above, it can be seen that the lipid compounds disclosed in this disclosure have almost no effect on cell activity when conducting in vitro cell experimental analysis.

Then, 100 μL of ONE-Glo ^™ Reagent was added to each well of the culture plate, and the luminescence value was measured within 3 minutes to evaluate the transfection ability of different lipid nanoparticle (LNP) solutions. The results are shown in Table 11.

Table 11

As shown in Table 11, the lipid compound disclosed in this disclosure can indeed improve the transfection efficiency of HEK293 cells.

Although the present disclosure has been disclosed as above with several embodiments, they are not intended to limit the present disclosure. Anyone with ordinary knowledge in the technical field can make any changes and modifications without departing from the spirit and scope of the present disclosure. Therefore, the protection scope of the present disclosure shall be determined by the definition of the attached claims.

without .

Claims (17)

A lipid compound or a derivative thereof, wherein the lipid compound has a structure represented by formula (I):

, wherein Z ¹ is O; Z ² is -Q ¹ -A ¹ -Q ² -A ² -X ¹ ; Z ³ is the same as Z ² ; Z ⁴ is -Q ³ -A ³ -Q ⁴ -A ⁴ -X ² ; Q ¹ and Q ³ are independently a single bond, or -O-; Q ² and Q ⁴ are independently a single bond, -O-,

,

,or

^A1 is C1-C6 alkylene; ^A3 is a single bond, or C1-C6 alkylene; ^A2 and ^A4 are independently a single bond, C1-C12 alkylene, C2-C12 alkenylene, or C2-C12 ^alkynylene ; ^X1 is C1-C24 alkanol, ^-NR1R2 , or

; X ² is hydrogen, C6-C12 aromatic group, C1-C24 alkyl group, C2-C24 alkenyl group, C2-C24 alkynyl group, C1-C24 alkanol group, C2-C24 alkenol group, C2-C24 alkynol group, -NR ⁷ R ⁸ ,

,

,

,or

; R ¹ is hydrogen, C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C1-C24 alkyl alcohol, C2-C24 enol, C2-C24 alkynol, or -A ⁹ -Q ⁷ -X ⁵ ; R ² is hydrogen, C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C1-C24 alkyl alcohol, C2-C24 enol, C2-C24 alkynol, or -A ¹⁰ -Q ⁸ -X ⁶ ; R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁹ , R ¹⁰ , R ¹¹ , and R ¹² are independently hydrogen or C1-C6 alkyl; R ^R7 is hydrogen, C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C1-C24 alkyl alcohol, C2-C24 enol, C2-C24 alkynol, or ^-A11 - ^Q9 - ^X7 ; ^R8 is hydrogen, C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C1-C24 alkyl alcohol, C2-C24 enol, C2-C24 alkynol, or ^-A12 - ^Q10 - ^X8 ; ^A5 , ^A6 , ^A7 , ^A8 , ^A9 , ^A10 , ^A11 , and ^A12 are independently C1-C12 alkylene; ^Q5 , ^Q6 , ^Q7 , ^Q8 , ^Q9 , and Q10 are each C1-C12 alkylene; ^{10 series} are independent of -O-, -NH-, -SS-,

,

,

,

,

,or

; and X ³ , X ⁴ , X ⁵ , X ⁶ , X ⁷ , and X ⁸ are independently C1-C24 alkyl; when Q1 and Q3 are -O-, and X ² is -NR ⁷ R ⁸ , X ¹ is -NR ¹ R ² ; the derivative of the lipid compound is a pharmaceutically acceptable salt of the lipid compound, or a solvate of the lipid compound, and the solvate of the lipid compound is a hydrate of the lipid compound, or an alcoholate of the lipid compound. The lipid compound or its derivative as described in claim 1, wherein when ^Q1 is a single bond, ^Q2 is -O-,

,

,or

. The lipid compound or its derivative as described in claim 1, wherein when ^Q2 is a single bond, ^Q1 is -O-. The lipid compound or its derivative as described in claim 1, wherein when Q ³ is a single bond, Q ⁴ is -O-,

,

,or

. The lipid compound or its derivative as described in claim 1, wherein when Q ⁴ is a single bond, Q ³ is -O-. The lipid compound or its derivative as described in claim 1, wherein when A ³ is a single bond, A ⁴ is C1-C12 alkylene, C2-C12 alkenylene, or C2-C12 alkynylene. The lipid compound or its derivative as described in claim 1, wherein when A ⁴ is a single bond, A ³ is a C1-C6 alkylene group. The lipid compound or its derivative as described in claim 1, wherein when ^A3 is a single bond and ^A4 is a single bond, ^Q4 is a single bond, and ^Q3 is -O-. The lipid compound or its derivative as described in claim 1, wherein ^Z4 is different from ^Z2 . The lipid compound or its derivative as described in claim 1, wherein Z ² , Z ³ and Z ⁴ are the same. The lipid compound or its derivative as described in claim 1, wherein the lipid compound is

A pharmaceutical composition comprising: a lipid compound or a derivative thereof as described in any one of claims 1 to 11; and a sterol. The pharmaceutical composition as described in claim 12, wherein the sterol is cholesterol, cholesterol hexasuccinate, ergosterol, lanosterol, or a combination thereof. The pharmaceutical composition as described in claim 12 further comprises: an auxiliary lipid, wherein the auxiliary lipid is 1,2-lauryl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-stearyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine, 1,2-distearyl-sn-glycero-3-phosphocholine, Choline, 1,2-dioleyl-sn-glycero-3-phosphocholine, hydrogenated soybean phospholipid acylcholine, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-racemic-glycerol) (sodium salt), sulfo-6-deoxyglucosyl diglycerol, monogalactosyl diglyceride, digalactosyl diglyceride, 1,2-dioleyl-sn-glycero-3-phosphoethanolamine, L-a-phosphatidylcholine, lecithin, or a combination thereof. The pharmaceutical composition as described in claim 12 further comprises: a PEGylated lipid, wherein the PEGylated lipid is phosphatidylethanolamine modified with polyethylene glycol, phosphatidic acid modified with polyethylene glycol, ceramide modified with polyethylene glycol, dialkylamine modified with polyethylene glycol, diacylglycerol modified with polyethylene glycol, dialkylglycerol modified with polyethylene glycol, sterol modified with polyethylene glycol, phospholipid modified with polyethylene glycol, or a combination thereof. The pharmaceutical composition as claimed in claim 15, wherein the PEGylated lipid is 1,2-dimyristyl-sn-glycero-3-phosphoethanolamine polyethylene glycol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine polyethylene glycol, 1,2-dimyristyl-sn-glycero-3-phosphoethanolamine polyethylene glycol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine polyethylene glycol, 1,2-distearoyl-sn-glycero-succinic acid polyethylene glycol, cholesterol polyethylene glycol, 1,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine polyethylene glycol, 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine polyethylene glycol, 1,2-dimyristyl-sn-glycerol, methoxy polyethylene glycol, 1,2-distearyl-sn-glycerol-methoxy polyethylene glycol, 1,2-dipalmitoyl-sn-glycerol-methoxy polyethylene glycol, α-[2-(di-tetradecylamino)-2-oxoethyl]-ω-methoxy-poly(oxy-1,2-ethanediyl), or a combination thereof. The pharmaceutical composition as described in claim 12 further comprises: a nucleic acid, wherein the nucleic acid is deoxyribonucleic acid, plasmid DNA, messenger RNA, small molecule interfering RNA, self-amplifying RNA, circular RNA, or a combination thereof.