WO2021170034A1 - 胺基脂质化合物、其制备方法和应用 - Google Patents

胺基脂质化合物、其制备方法和应用 Download PDF

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WO2021170034A1
WO2021170034A1 PCT/CN2021/077852 CN2021077852W WO2021170034A1 WO 2021170034 A1 WO2021170034 A1 WO 2021170034A1 CN 2021077852 W CN2021077852 W CN 2021077852W WO 2021170034 A1 WO2021170034 A1 WO 2021170034A1
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lipid
group
amino
alkyl
compound
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PCT/CN2021/077852
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French (fr)
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李林鲜
查高峰
龙韵馨
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深圳深信生物科技有限公司
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Priority to JP2022552177A priority Critical patent/JP7411824B2/ja
Priority to AU2021225909A priority patent/AU2021225909B2/en
Priority to CN202180017433.6A priority patent/CN115190876B/zh
Priority to EP21759547.9A priority patent/EP4112607A4/en
Priority to CA3173259A priority patent/CA3173259A1/en
Priority to KR1020227033411A priority patent/KR20220146590A/ko
Priority to US17/907,951 priority patent/US20230123334A1/en
Publication of WO2021170034A1 publication Critical patent/WO2021170034A1/zh

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    • C07D251/12—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D251/26—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with only hetero atoms directly attached to ring carbon atoms
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Definitions

  • the present disclosure relates to an amino lipid compound, a preparation method thereof, and lipid particles containing the amino lipid compound.
  • the lipid particles can be used to deliver bioactive agents into cells.
  • the invention also relates to the use of lipid particles containing amino lipids as medicines.
  • Gene therapy refers to the introduction of the target genetic material into specific tissue cells through appropriate vectors for proper expression, replacing or correcting the disorder in the structure or function of their own genes, killing diseased cells or enhancing the body's ability to clear diseased cells, etc., so as to achieve Purpose of treatment.
  • nucleic acid substances are easily hydrolyzed by nucleases and have a large amount of negative charges which are not conducive to cell phagocytosis. Therefore, the research and development of high-efficiency, safe and tissue-specific gene delivery systems is particularly important.
  • the gene vectors currently studied can be divided into viral vectors and non-viral vectors. Although the transfection efficiency of viral vectors is high, problems such as toxicity, immunogenicity, and serious safety limit their application in clinical treatment. However, non-viral vectors have attracted the attention of more and more experts and researchers due to their advantages of easy preparation, transportation, storage, safety, effectiveness, and non-immunogenicity.
  • Non-viral vectors are mainly divided into cationic polymers and cationic liposomes.
  • Cationic polymers refer to polymers containing cations that are artificially synthesized or naturally formed.
  • Polyethyleneimine (PEI) is one of the most common cationic polymer non-viral gene carriers. It can encapsulate negatively charged genes and form nanocomplexes, which can enter cells through endocytosis and escape through lysosomes, showing better transfection efficiency.
  • PEI is highly cytotoxic, and its toxicity is directly proportional to the transfection efficiency.
  • chitosan has good biological safety, its transfection efficiency is low.
  • Cationic lipid particles are artificially prepared phospholipid vesicles with a double-layer membrane.
  • lipid particles have properties similar to biological membranes, when the lipid particle DNA complex contacts the cell membrane, it enters the cell through endocytosis. In addition to high transfection efficiency, lipid particles are not immunogenic, have low cytotoxicity, and are easy to prepare. Therefore, cationic lipid particles have become one of the most commonly used vectors for gene transfection.
  • amino lipid compound which is a compound represented by the following formula I:
  • R 1 and R 2 are the same or different from each other, and are each independently selected from C 6 -C 24 alkyl, C 6 -C 24 alkenyl, C 6 -C 24 alkynyl, and C 4 -C 24 acyl, wherein The C 6 -C 24 alkyl group, the C 6 -C 24 alkenyl group, the C 6 -C 24 alkynyl group and the C 4 -C 24 acyl group are optionally substituted with a C 1 -C 6 hydrocarbyl group;
  • R 3 and R 4 are the same or different from each other, and are each independently selected from C 1 -C 12 alkyl, C 2 -C 12 alkenyl, and C 2 -C 12 alkynyl, wherein the C 1 -C 12 alkyl ,
  • the C 2 -C 12 alkenyl group and the C 2 -C 12 alkynyl group are optionally substituted with a C 1 -C 6 hydrocarbon group, or R 3 and R 4 are combined with each other to form a compound selected from nitrogen, sulfur and oxygen
  • R 5 is not present, or R 5 is hydrogen or C 1 -C 12 alkyl to provide a quaternary amine
  • R 6 , R 7 , and R 8 are hydrogen or C 1 -C 12 alkyl
  • L is a C 1 -C 12 alkylene group, a C 2 -C 12 alkenylene group or a C 2 -C 12 alkynylene group, wherein the C 1 -C 12 alkylene group, the C 2 -C 12 alkenylene group And the C 2 -C 12 alkynylene group are optionally substituted with one or more substituents selected from the group consisting of hydrocarbon group, carboxyl group, acyl group and alkoxy group, or L contains a group selected from nitrogen, sulfur and oxygen Heteroatom optionally substituted 4 to 10 membered heterocyclic ring.
  • Another aspect of the present invention provides a method for preparing the amino lipid compound.
  • Another aspect of the present invention provides the use of the amino lipid compound for preparing lipid particles.
  • lipid particles which comprise the amino-based lipid compound.
  • Another aspect of the present invention provides the use of the lipid particles in the preparation of medicines
  • Figure 1 shows the titers of humoral antibodies produced by subcutaneous administration of representative amino lipid compounds to deliver ovalbumin mRNA (OVA mRNA).
  • OVA mRNA ovalbumin mRNA
  • Figure 2 shows a microscopic image of HEK293 cells treated with a reference reagent (Lipofectamine2000) and S8N12D6 according to Example 4.
  • Figure 2(a) shows the brightness of HEK293 cells treated with a reference reagent (Lipofectamine2000) Visual field image
  • Figure 2(b) shows a bright field image of HEK293 cells treated with S8N12D6 according to Example 4
  • Figure 2(c) shows a Hoechst stained image of HEK293 cell nuclei treated with a reference reagent (Lipofectamine2000)
  • 2(d) shows the Hoechst stained image of HEK293 cell nuclei treated with S8N12D6 according to Example 4.
  • Figure 2(e) shows the GFP image of HEK293 cells treated with the reference reagent (Lipofectamine2000), and Figure 2(f) shows The GFP image of HEK293 cells treated with S8N12D6 according to Example 4 is shown.
  • the term "optionally substituted” means that one or more hydrogen atoms attached to an atom or group are independently unsubstituted or substituted by one or more such as one, two, three or four substituents ,
  • the substituents are independently selected from: deuterium (D), halogen, -OH, mercapto, cyano, -CD 3 , C 1 -C 6 alkyl (preferably C 1 -C 3 alkyl), C 2- C 6 alkenyl, C 2 -C 6 alkynyl, cycloalkyl (preferably C 3 -C 8 cycloalkyl), aryl, heterocyclyl (preferably 3-8 membered heterocyclyl), heteroaryl, aryl Group C 1 -C 6 alkyl-, heteroaryl C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, -OC 1 -C 6 alkyl (preferably -OC 1 -C 3 alkyl),- OC 2 - -
  • hydrocarbyl group refers to the group remaining after aliphatic hydrocarbon has lost one hydrogen atom, including linear or branched, saturated or unsaturated hydrocarbyl groups.
  • the hydrocarbyl groups include alkyl, alkenyl and alkynyl groups.
  • the hydrocarbyl group is a C 1 -C 10 hydrocarbyl group, a C 1 -C 6 hydrocarbyl group, or a C 1 -C 3 hydrocarbyl group.
  • alkyl refers to C 1 -C 24 alkyl, C 1 -C 20 alkyl, C 1 -C 18 alkyl, C 1 -C 12 alkyl, C 1 -C 6 alkyl, C 3 -C 24 alkyl, C 3 -C 20 alkyl, C 3 -C 18 alkyl, C 3 -C 12 alkyl, C 3 -C 6 alkyl, C 6 -C 24 alkyl, C 6 -C 20 alkyl, C 6 -C 18 alkyl or C 6 -C 12 alkyl.
  • alkenyl refers to C 2 -C 24 alkenyl, C 2 -C 20 alkenyl, C 2 -C 18 alkenyl, C 2 -C 12 alkenyl, C 2 -C 6 alkenyl, C 3 -C 20 alkenyl, C 3 -C 18 alkenyl, C 3 -C 12 alkenyl, C 3 -C 6 alkenyl, C 6 -C 24 alkenyl, C 6 -C 20 alkenyl, C 6 -C 18 alkenyl or C 6 -C 12 alkenyl.
  • alkynyl refers to C 2 -C 24 alkynyl, C 2 -C 20 alkynyl, C 2 -C 18 alkynyl, C 2 -C 12 alkynyl, C 2 -C 6 alkynyl, C 3 -C 20 alkynyl, C 3 -C 18 alkynyl, C 3 -C 12 alkynyl, C 3 -C 6 alkynyl, C 6 -C 24 alkynyl, C 6 -C 20 alkynyl, C 6 -C 18 alkynyl or C 6 -C 12 alkynyl.
  • acyl as used herein means a hydrocarbyl-carbonyl group, preferably the acyl group is C 4 -C 24 acyl, C 6 -C 18 acyl, C 6 -C 12 acyl, C 6 -C 10 acyl, C 4- C 6 acyl group.
  • alkoxy as used herein means an alkyl-oxy group, preferably the alkoxy group is a C 1 -C 10 alkoxy group, more preferably, the alkoxy group is a C 1 -C 6 alkoxy group Group, most preferably, the alkoxy group is a C 1 -C 3 alkoxy group.
  • heterocyclic ring means a saturated or unsaturated cyclic group containing heteroatoms selected from N, O, S, etc., preferably, the heterocyclic ring includes a heterocyclic group selected from N, O, S An optionally substituted 4- to 10-membered heterocyclic ring with 1 to 6 heteroatoms, or an optionally substituted 4 to 6 containing 1, 2, or 3 heteroatoms selected from N, O, S Saturated heterocyclic ring.
  • heterocycles include, but are not limited to: azetidine, oxetanyl, tetrahydrofuran, pyrrolidine, imidazolidine, pyrazolidine, tetrahydropyran, piperidine, morpholine, thiomorpholine, piperazine , Pyrrolidine, piperidine, piperazine and morpholine are preferred.
  • the heterocyclic ring may be optionally substituted by one or more substituents. For the types of substituents, please refer to the above definition of "optionally substituted”.
  • amino lipid compound which is a compound represented by the following formula I:
  • R 1 and R 2 are the same or different from each other, and are each independently selected from C 6 -C 24 alkyl, C 6 -C 24 alkenyl, C 6 -C 24 alkynyl, and C 4 -C 24 acyl, wherein The C 6 -C 24 alkyl group, the C 6 -C 24 alkenyl group, the C 6 -C 24 alkynyl group and the C 4 -C 24 acyl group are optionally substituted with a C 1 -C 6 hydrocarbyl group;
  • R 1 and R 2 are the same or different from each other, and are each independently a C 6 -C 18 alkyl group or a C 6 -C 18 alkenyl group.
  • R 3 and R 4 are the same or different from each other, and are each independently selected from C 1 -C 12 alkyl, C 2 -C 12 alkenyl and C 2 -C 12 alkynyl, wherein the C 1 -C 12 alkane Group, the C 2 -C 12 alkenyl group and the C 2 -C 12 alkynyl group are optionally substituted with a C 1 -C 6 hydrocarbyl group, or R 3 and R 4 are combined with each other to form a compound selected from nitrogen, sulfur and An optionally substituted 4 to 10 membered heterocyclic ring of 1 to 6 heteroatoms in oxygen;
  • R 5 is not present, or R 5 is hydrogen or C 1 -C 12 alkyl to provide a quaternary amine
  • R 6 , R 7 , and R 8 are hydrogen or C 1 -C 12 alkyl
  • L is a C 1 -C 12 alkylene group, a C 2 -C 12 alkenylene group, or a C 2 -C 12 alkynylene group, wherein the C 1 -C 12 alkylene group, the C 2 -C 12 alkylene group
  • the alkenyl group and the C 2 -C 12 alkynylene group are optionally substituted with one or more substituents selected from the group consisting of a hydrocarbyl group, a carboxyl group, an acyl group and an alkoxy group, or L is a group selected from nitrogen, sulfur and oxygen.
  • L is a C 1 -C 8 alkylene group, a C 2 -C 6 alkylene group, a C 2 -C 4 alkylene group or a C 3 -C 4 alkylene group, wherein the C 1 -C 8 alkylene group
  • the alkyl group, the C 2 -C 6 alkylene group, the C 2 -C 4 alkylene group or the C 3 -C 4 alkylene group is optionally substituted with a C 1 -C 6 hydrocarbyl group.
  • the present invention provides the amino lipid compound of formula I, wherein: R 1 and R 2 are the same or different from each other, and are each independently C 6 -C 24 alkyl or C 6 -C 24 alkenyl (e.g.
  • R 3 and R 4 are the same or different from each other, and are each independently a C 1- C 12 alkyl group , Wherein the C 1- C 12 alkyl group is optionally substituted with a C 1 -C 6 hydrocarbyl group, or R 3 and R 4 are combined with each other to form a compound containing 1 to 6 heteroatoms selected from nitrogen, sulfur and oxygen An optionally substituted 4- to 10-membered heterocyclic ring; and R 5 is absent.
  • the present invention provides an amino lipid compound of formula I as described above, wherein L is a C 1 -C 4 alkylene group, wherein the C 1 -C 4 alkylene group is optionally C 1 -C 6 hydrocarbyl substitution.
  • L is a C 2 -C 4 alkylene group, such as (CH 2 ) q , where q is 1, 2, 3, or 4.
  • the present invention provides the amino lipid compound of formula I as described above, wherein R 1 and R 2 are the same or different from each other, and are each independently a C 6 -C 18 alkyl group or a C 6- C 18 alkenyl. In some embodiments, R 1 and R 2 are the same or different from each other, and are each independently a C 6 -C 18 alkyl group. In some embodiments, one of R 1 and R 2 is a C 6 -C 18 alkyl group and the other is a C 6 -C 18 alkenyl group
  • the present invention provides the amino lipid compound of formula I as described above, wherein:
  • n 0 or 1
  • each independently is selected from the following S6, S7, S8, S9, S10, S11, S12, S14, S15, S16, S18, S19, S20, N6, N7, N8, N9, N11, One of N12, N13, N15, N16, N18 and 2 N 12 :
  • S6 CH 3 (CH 2 ) 5 S-;
  • S7 CH 3 (CH 2 ) 6 S-;
  • S8 CH 3 (CH 2 ) 7 S-;
  • S12 CH 3 (CH 2 ) 11 S-;
  • S14 CH 3 (CH 2 ) 13 S-;
  • S15 CH 3 (CH 2 ) 14 S-;
  • N6 CH 3 (CH 2 ) 5 NH-; N7: CH 3 (CH 2 ) 6 NH-; N8: CH 3 (CH 2 ) 7 NH-;
  • N9 CH 3 (CH 2 ) 8 NH-; N11: CH 3 (CH 2 ) 10 NH-; N12: CH 3 (CH 2 ) 11 NH-;
  • N13 CH 3 (CH 2 ) 12 NH-; N15: CH 3 (CH 2 ) 14 NH-; N16: CH 3 (CH 2 ) 15 NH-;
  • N18 CH 3 (CH 2 ) 17 NH-; 2 N 12 : (CH 3 (CH 2 ) 11 ) 2 N-.
  • the present invention provides the amino lipid compound of formula I as described above, wherein:
  • the present invention provides the amino lipid compound of formula I as described above, wherein:
  • X 3 is O
  • R 5 does not exist
  • O1, O2, O3, O4, O5, O6, O7, O8, O9 and O10 selected from the following:
  • the present invention provides the amino lipid compound of formula I as described above, wherein the amino lipid compound is a compound represented by the following formula I':
  • X 1 'and X 2' are the same or different and are each independently NH, O or S, preferably NH, or S;
  • R 1 , R 2 , R 3 , R 4 and q are the same as those in the description of the amino lipid compound of formula I above.
  • the present invention provides the amino lipid compound of formula I as described above, wherein the amino lipid compound is a compound represented by the following formula I":
  • R 1 , R 2 , R 3 , R 4 and q are the same as those in the description of the amino lipid compound of formula I above.
  • Another aspect of the present invention provides a method for preparing the amino lipid compound of formula I as described above, the method comprising the following steps:
  • the first intermediate is combined with the one represented by R 2 (R 7 ) n -X 2 H
  • the compound undergoes a second reaction in the presence of a base as an acid binding agent to obtain a second intermediate of formula I-2;
  • the second intermediate is combined with a diamine represented by HX 3 (R 8 ) p -L-NR 3 R 4 R 5 Carry out the third reaction under heating to obtain the amino lipid compound of formula I;
  • X 1 , X 2 , X 3 , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , L, m, n and p are the same as those in the above In the description of the amino lipid compound of formula I, they have the same definition.
  • X 1 and X 2 are the same or different from each other, and are each independently N or S.
  • R 5 is absent.
  • L is C 1 -C 12 alkylene group, a C 1 -C 12 alkylene group optionally substituted with C 1 -C 6 hydrocarbon group, for example, L is (CH 2) q, wherein q Is an integer from 1 to 12, for example, an integer from 1 to 6.
  • R 6 is hydrogen.
  • R 7 is hydrogen.
  • R 8 is hydrogen.
  • the third reaction is carried out in the presence of a base as an acid binding agent.
  • the present invention provides a method for preparing the amino lipid compound of formula I', the method comprising the following steps:
  • the third reaction is carried out in the presence of a base as an acid binding agent.
  • the present invention provides a method of preparing the above-mentioned amino lipid compound of formula I",
  • the method includes the following steps:
  • the first intermediate and the compound represented by R 2 -SH are used as an acid binding agent at room temperature or under heating conditions.
  • the second reaction is carried out in the presence of a base to obtain a second intermediate of formula I"-2;
  • R 1 , R 2 , R 3 , R 4 and q are the same as those in the above description of the amino lipid compound of formula I".
  • the first reaction is performed at less than 20°C (for example, less than 15°C, less than 10°C, less than 5°C, less than 0°C, less than It is carried out at a temperature of -10°C or lower than -20°C, and the lower limit of the reaction temperature is not specifically limited) and a temperature higher than -40°C (for example, higher than -35°C, or preferably equal to or higher than -30°C).
  • room temperature is the usual temperature under atmospheric pressure, and can refer to 0°C to 30°C, 0°C to 25°C, 0°C to 20°C, 5°C to 30°C, 5°C to 20°C, 10 Temperature range from °C to 30 °C, 10 °C to 25 °C, 15 °C to 30 °C, or 15 °C to 25 °C.
  • heating conditions specifically refers to heating to 50°C to 120°C, 50°C to 110°C, 50°C to 80°C, 60°C to 120°C, 60°C to 110°C, 60°C to 100°C, 70°C to 120°C, 70°C to 100°C, 70°C to 90°C temperature range.
  • base may particularly refer to basic compounds commonly used in the art, such as but not limited to: organic bases such as triethylamine, DIPEA, pyridine, DMAP; or inorganic bases such as sodium hydroxide, hydrogen Potassium oxide, sodium carbonate, potassium carbonate.
  • organic bases such as triethylamine, DIPEA, pyridine, DMAP
  • inorganic bases such as sodium hydroxide, hydrogen Potassium oxide, sodium carbonate, potassium carbonate.
  • q is an integer from 1 to 8, preferably an integer from 1 to 6, and more preferably, q is an integer from 1 to 4, such as 1, 2, 3, or 4.
  • the present invention provides a method for preparing an amino lipid compound of formula II, and the method can be carried out by the following reaction formula 1:
  • R 1 , R 2 , R 3 , R 4 and X 2 are the same as those in the above description of the preparation method of the amino lipid compound of formula I.
  • the present invention provides a method for preparing an amino lipid compound of formula III, and the method can be carried out by the following reaction formula 2:
  • R 1 , R 2 , R 3 , R 4 and X 2 are the same as those in the above description of the preparation method of the amino lipid compound of formula I.
  • the present invention provides a method for preparing an amino lipid compound of formula IV, which can be carried out by the following reaction formula 3:
  • R 1 , R 2 , R 3 , R 4 , X 2 and q are the same as those in the above description of the amino lipid compound of formula I;
  • the present invention provides a method for preparing an amino lipid compound of formula V, which can be carried out by the following reaction formula 4:
  • R 1 , R 2 , R 3 , R 4 , X 2 and q are the same as those in the above description of the amino lipid compound of formula I;
  • the preparation method of the amino lipid compound according to the present invention is very versatile and can be used for the rapid synthesis of an amino lipid compound library, and can be used in a very inexpensive manner for rapid cell-based screening experiments.
  • Another aspect of the present invention provides the use of the amino lipid compound according to any one of the formulas I, I', II, III, IV and V for preparing lipid particles.
  • Another aspect of the present invention provides lipid particles comprising the amino lipid compound according to the present invention as described above.
  • the amine-based lipid compound of the present invention contains long non-polar residues, all the obtained compounds have hydrophobic characteristics, and because they have amine groups, they also have hydrophilic characteristics.
  • Such amphoteric characteristics can be used to form lipid particles, for example, lipid nanoparticles, lipid bilayers, micelles, liposomes, and the like.
  • lipid particle means a nano-sized substance prepared by putting an amino lipid compound in an aqueous solution. These particles are in particular lipid nanoparticles, lipid bilayer vesicles (liposomes), multilamellar vesicles or micelles.
  • the lipid particle is a liposome containing the amino lipid compound according to the present invention as described above.
  • liposomes are microvesicles composed of a bilayer of lipid amphiphilic (amphiphilic) molecules enclosing a water-containing compartment.
  • Liposome formation is not a spontaneous process.
  • lipid vesicles When lipids are placed in water, lipid vesicles are first formed, thus forming a bilayer or a series of bilayers, each separated by a water molecule.
  • Liposomes can be formed by treating lipid vesicles with ultrasound in water.
  • lipid bilayer means a film formed by two layers of lipid molecules.
  • micelle means an aggregate of surfactant molecules dispersed in a liquid colloid. Typical micelles in aqueous solutions form aggregates with the hydrophilic head region when they contact water, chelating the hydrophobic single tail region in the center of the micelle.
  • the term "cell” means a general term and includes single cells, tissues, organs, insect cells, avian cells, fish cells, amphibian cells, mammalian cells, primary cells, continuous cell lines, stem cells And/or the cultivation of genetically engineered cells (such as recombinant cells expressing heterologous polypeptides or proteins).
  • Recombinant cells include, for example, cells expressing heterologous polypeptides or proteins (such as growth factors or blood factors).
  • the lipid particles or liposomes further contain one or more of auxiliary lipids, sterols and bioactive agents.
  • the lipid particles or liposomes contain helper lipids.
  • the auxiliary lipid is a non-cationic lipid.
  • the auxiliary lipid is a non-cationic phospholipid.
  • non-cationic lipids may contain cationic functional groups (e.g., ammonium groups), but should contain anionic functional groups to at least neutralize molecules. The totality of all functional groups in the lipid molecule should be non-cationic.
  • Liposomes composed of a mixture of amine-based lipids and non-cationic (neutral) phospholipids according to the present invention are the most effective for delivering nucleic acids into cells.
  • the non-cationic lipid is DOPE, DSPC, or a combination thereof.
  • the molar ratio of the amino lipid compound to the auxiliary lipid is about (2 to 10):1, preferably about (3 to 8): 1, more preferably about (4 to 6):1, for example about 4:1, about 4.5:1 or about 5:1.
  • the lipid particles or liposomes comprise sterols.
  • Sterols such as cholesterol, are natural components in cell membranes. It can be used to stabilize particles and help integrate with cell membranes.
  • the sterol may be one or more selected from cholesterol, sitosterol, stigmasterol and ergosterol, preferably cholesterol.
  • the molar ratio of the amino lipid compound to the sterol is about (1 to 1.5):1, preferably about (1 to 1.4):1 , For example, about (1 to 1.3):1.
  • the lipid particles or liposomes contain a biologically active agent.
  • a biologically active agent is a substance that has a biological effect when introduced into a cell or host, for example, it acts by stimulating an immune response or an inflammatory response, by exerting an enzyme activity, or by supplementing mutations.
  • the biologically active agents are especially nucleic acids, peptides, proteins, antibodies and small molecules.
  • the biologically active agent is a nucleic acid, including but not limited to messenger RNA (mRNA), antisense oligonucleotide, DNA, plasmid, ribosomal RNA (rRNA), microRNA (miRNA) , Transfer RNA (tRNA), small inhibitory RNA (siRNA) and small nuclear RNA (snRNA).
  • mRNA messenger RNA
  • rRNA ribosomal RNA
  • miRNA microRNA
  • tRNA Transfer RNA
  • siRNA small inhibitory RNA
  • small nuclear RNA small nuclear RNA
  • the biologically active agent is selected from anti-tumor agents, antibiotics, immunomodulators, anti-inflammatory agents, agents that act on the central nervous system, antigens or fragments thereof, proteins, peptides, polypeptides, and polypeptides. Polypeptoids, vaccines and small molecules, and mixtures thereof.
  • the lipid particles or liposomes further contain at least one polyethylene glycol (PEG)-lipid.
  • PEG lipids help protect the particles and their contents from degradation in vitro or in vivo.
  • PEG forms a protective layer on the surface of liposomes and increases the circulation time in the body. It can be used in liposomal drug delivery (PEG-liposomes).
  • the PEG-lipid may be one or more selected from PEG1000-DMG, PEG5000-DMG, PEG2000-DMG and PEG2000-DSPE, preferably PEG2000-DMG.
  • the molar ratio of the amino lipid compound to the PEG-lipid is about (9 to 42):1, preferably about (12 to 38) :1, more preferably about (16 to 36):1, for example about (18 to 34):1.
  • the lipid particles or liposomes according to the present invention have excellent properties of encapsulating bioactive agents.
  • Lipid particles or liposomes containing biologically active agents can be used to deliver any of a variety of therapeutic agents into cells.
  • the present invention includes the use of lipid particles (especially liposomes) as described above for the delivery of biologically active agents into cells.
  • the present invention also provides a method for delivering a biologically active agent into a cell, the method comprising contacting the lipid particle or liposome of the present invention containing the biologically active agent with the cell.
  • the lipid particles or liposomes containing the amino lipid compounds of the present invention are suitable for delivering biologically active agents into cells.
  • a variety of different amine-based lipid compounds synthesized by the general synthesis method can be screened for the specific characteristics of liposomes. Important characteristics are, for example, transfection efficiency, cytotoxicity, adhesion of the drug to be delivered to the cell, stability of liposomes, size of liposomes, and the like.
  • the method of the present invention can form specifically adapted liposomes for specific applications.
  • lipid particles or liposomes can be used to transfect multicellular tissues or organs. Therefore, the present invention also provides a method for transfecting cells, multicellular tissues or organs, the method comprising contacting the lipid particles or liposomes of the present invention containing the nucleic acid with the cells. This provides patients with the possibility of new treatments.
  • the patient can be any mammal, preferably selected from mice, rats, pigs, cats, dogs, horses, goats, cows and monkeys and/or others. In other preferred embodiments, the patient is a human.
  • Another aspect of the present invention also provides the use of the lipid particles or liposomes comprising any one of formula I, I', I", II, III, IV and V as a medicament
  • Another aspect of the present invention also provides the preparation of the lipid particles or liposomes containing the amino lipid compound of any one of formula I, I', I", II, III, IV, and V In the use.
  • the lipid particles are used as carriers for encapsulating bioactive agents.
  • lipid particles or liposomes, or the drugs can be administered to patients for gene therapy, gene vaccination, antisense therapy or treatment by interfering RNA.
  • Specific applications include but are not limited to:
  • the lipid particles of the present invention can deliver nucleic acids for gene therapy.
  • foreign genes can be introduced into target cells to correct or compensate diseases caused by defects and abnormal genes, so as to achieve the purpose of treatment. It also includes the application of transgene technology, that is, inserting foreign genes into the appropriate recipient cells of the patient through gene transfer technology, so that the products produced by the foreign genes can treat certain diseases, such as common lung cancer, Gastric cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer, prostate cancer, etc. It can also introduce gene-edited nucleic acid materials for the treatment of a variety of genetic diseases, such as hemophilia, thalassemia, Gaucher's disease and so on.
  • the lipid particles of the present invention can be used for vaccination.
  • the lipid particles or liposomes of the present invention can be used to deliver antigens or nucleic acids encoding antigens.
  • the lipid particles of the present invention can also be used to elicit an immune response against various antigens, which are used to treat and/or prevent a variety of diseases, such as cancer, allergies, toxicity and pathogens (e.g., viruses, bacteria, fungi and Other pathogenic organisms) infection.
  • diseases such as cancer, allergies, toxicity and pathogens (e.g., viruses, bacteria, fungi and Other pathogenic organisms) infection.
  • the present invention also provides methods for gene therapy, gene vaccination, antisense therapy or treatment by interfering RNA, the method comprising administering to a patient in need the bioactive agent (for example, the desired nucleic acid or antigen) of the present invention Lipid particles or liposomes.
  • bioactive agent for example, the desired nucleic acid or antigen
  • the lipid particles of the present invention can be used to prepare drugs for nucleic acid transfer.
  • the nucleic acid is RNA, messenger RNA (mRNA), antisense oligonucleotide, DNA, plasmid, ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small inhibitory RNA ( siRNA) and small nuclear RNA (snRNA).
  • mRNA messenger RNA
  • rRNA ribosomal RNA
  • miRNA microRNA
  • tRNA transfer RNA
  • siRNA small inhibitory RNA
  • snRNA small nuclear RNA
  • Encapsulation of non-biologically active agents is also an object of the lipid particles or liposomes of the present invention. Therefore, the present invention also provides such lipid particles or liposomes, which contain a non-biologically active agent.
  • non-biologically active agents include, but are not limited to: antioxidants, colors, pigments, fragrances and flavors, etc., such as those used in cosmetics. It is expected that the lipid particles or liposomes according to the present invention also have excellent properties of encapsulating non-biologically active agents.
  • An amino lipid compound which is a compound represented by the following formula I:
  • R 1 and R 2 are the same or different from each other, and are each independently selected from C 6 -C 24 alkyl, C 6 -C 24 alkenyl, C 6 -C 24 alkynyl, and C 4 -C 24 acyl, wherein , The C 6 -C 24 alkyl group, the C 6 -C 24 alkenyl group, the C 6 -C 24 alkynyl group and the C 4 -C 24 acyl group are optionally substituted with a C 1 -C 6 hydrocarbyl group ;
  • R 3 and R 4 are the same or different from each other, and are each independently selected from C 1 -C 12 alkyl, C 2 -C 12 alkenyl, and C 2 -C 12 alkynyl, wherein the C 1 -C 12 alkane Group, the C 2 -C 12 alkenyl group and the C 2 -C 12 alkynyl group are optionally substituted with a C 1 -C 6 hydrocarbyl group, or R 3 and R 4 are combined with each other to form a compound selected from nitrogen, sulfur and An optionally substituted 4 to 10 membered heterocyclic ring of 1 to 6 heteroatoms in oxygen;
  • R 5 is not present, or R 5 is hydrogen or C 1 -C 12 alkyl to provide a quaternary amine
  • R 6 , R 7 , and R 8 are hydrogen or C 1 -C 12 alkyl
  • L is a C 1 -C 12 alkylene group, a C 2 -C 12 alkenylene group, or a C 2 -C 12 alkynylene group, wherein the C 1 -C 12 alkylene group, the C 2 -C 12 alkylene group
  • the alkenyl group and the C 2 -C 12 alkynylene group are optionally substituted with one or more substituents selected from the group consisting of a hydrocarbyl group, a carboxyl group, an acyl group and an alkoxy group, or L is a group selected from nitrogen, sulfur and oxygen.
  • R 1 and R 2 are the same or different from each other, and are each independently a C 6 -C 24 alkyl group;
  • R 3 and R 4 are the same or different from each other, and are each independently a C 1 -C 12 alkyl group, wherein the C 1 -C 12 alkyl group is optionally substituted with a C 1 -C 6 hydrocarbon group, or R 3 and R 4 combine with each other to form an optionally substituted 4- to 10-membered heterocyclic ring containing 1 to 6 heteroatoms selected from nitrogen, sulfur and oxygen;
  • R 5 does not exist.
  • R 1 -X 1 -and R 2 -X 2 - are the same as or different from each other, and are each independently selected from S6, S7, S8, S9, S10, S11, S12, S14, S15, S16, One of S18, N6, N7, N8, N9, N11, N12, N13, N15, N16, N18, 2 N 12 ;
  • -X 3 -LN(R 3 )(R 4 )(R 5 ) is one selected from the group consisting of D1, D2, D3, D4, D5, D6, D7, D8, D9, and D10 as described above.
  • q is an integer from 1 to 12, and the definitions of R 1 , R 2 , R 3 and R 4 are the same as those in any one of items 1 to 6.
  • R 1 , R 2 , R 3 , R 4 , and X 2 are the same as those defined in the foregoing description of the preparation method of the amino lipid compound.
  • reaction formula 2 The method for preparing the amino lipid compound according to the present invention, the method can be carried out by the following reaction formula 2:
  • R 1 , R 2 , R 3 , R 4 , and X 2 are the same as those defined in the foregoing description of the preparation method of the amino lipid compound.
  • lipid particles are lipid nanoparticles, liposomes, multilamellar vesicles or Micelles; more preferably, the lipid particles are lipid nanoparticles.
  • Lipid particles comprising the amino lipid compound according to any one of items 1 to 6; preferably, the lipid particles are lipid nanoparticles, liposomes, multilamellar vesicles or gels Bundle; More preferably, the lipid particles are lipid nanoparticles.
  • the lipid particle according to item 11 which further contains one or more of auxiliary lipids, sterols and bioactive agents;
  • the auxiliary lipid is a non-cationic lipid, more preferably, the auxiliary lipid is a non-cationic phospholipid, and further preferably, the non-cationic lipid is DOPE;
  • the sterol is cholesterol
  • the biologically active agent is a nucleic acid, an anti-tumor agent, an antibiotic, an immunomodulator, an anti-inflammatory agent, an agent that acts on the central nervous system, an antigen or fragments thereof, peptides, proteins, antibodies, vaccines, and small molecules.
  • the nucleic acid is RNA, messenger RNA (mRNA), antisense oligonucleotide, DNA, plasmid, ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA) ), small inhibitory RNA (siRNA) and small nuclear RNA (snRNA).
  • lipid particles according to item 11 or 12 in the preparation of a medicine, which is a medicine used for gene therapy, gene vaccination, antisense therapy or treatment by interfering RNA;
  • the gene therapy can be used for the treatment of cancer and genetic diseases; more preferably, the cancer is lung cancer, gastric cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma cancer, blood cancer or prostate cancer.
  • the genetic disease is one or more of hemophilia, thalassemia and Gaucher’s disease;
  • the genetic vaccination is used to treat cancer, allergies, toxicity, and pathogen infection; more preferably, the pathogen is one or more of viruses, bacteria or fungi.
  • the nucleic acid is RNA, messenger RNA (mRNA), antisense oligonucleotide, DNA, Plasmids, ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small inhibitory RNA (siRNA) and small nuclear RNA (snRNA).
  • mRNA messenger RNA
  • rRNA ribosomal RNA
  • miRNA microRNA
  • tRNA transfer RNA
  • siRNA small inhibitory RNA
  • snRNA small nuclear RNA
  • the amino lipid compound of the present invention has the characteristics of strong functional group diversity and large number.
  • the preparation method of the amino lipid compound has the advantages of easy availability of raw materials, mild reaction conditions, good reaction selectivity, high reaction yield, and equipment With the advantages of low requirements and simple operation, the preparation method of the present invention is very versatile, can be used for the rapid synthesis of ionizable amino lipid compound libraries, and can be used in a very inexpensive manner for rapid cell-based screening experiments .
  • the types and combinations of functional groups of amine-based lipid compounds provided by the present invention are diverse. Through conventional methods for transfection efficiency, cytotoxicity, adhesion of drugs to be delivered to cells, stability of lipid particles, and The screening of features such as size can be conveniently applied to deliver different nucleic acid substances to different cells, which improves the pertinence and effectiveness of the delivery system.
  • the amino lipid compound of the present invention can be synthesized by combinatorial chemistry methods with high throughput and high efficiency, with simple synthesis method and high yield.
  • Lipid particles or liposomes containing the amine-based lipid compound of the present invention have excellent properties of encapsulating biologically active agents, and can be used for biologically active agents, especially poorly water-soluble agents or active agents that are easily decomposed or degraded (such as (Nucleic acid) delivery to improve its bioavailability and effectiveness.
  • the chemical reagents used in the examples are all analytically pure, purchased from TCI, Aladdin or J&K.
  • the synthesized compound has one of the structures shown in Table I below:
  • Sz each independently represents a group selected from S6 to S12, S14 to S16 and S18 listed in Table II below;
  • Nx each independently represents a group selected from N6 to N9, N11 to N13, N15, N16, N18, and 2 N 12 listed in Table II below;
  • Dy each independently represents a group selected from D1 to D10 listed in Table II below:
  • Example 1.1 Parallel synthesis and characterization of S8NxDy series amino lipid compound library
  • n3, -NRR, R 1 and R 2 are as defined above.
  • Example 1.1.1 One-pot synthesis and characterization of a representative amino lipid compound S8N11D6.
  • Example 1.2 Parallel synthesis and characterization of S6SxDy series amino lipid compound library
  • n3, -NRR, R 1 and R 2 are as defined above.
  • Step I Use a pipette to transfer the reaction solution of step I to 11 1.5mL reaction flasks (each 1.15mL, 0.15mmol), and add linear alkyl mercaptan (0.15mmol) and DIPEA THF to the corresponding reaction flasks.
  • the solution (0.35mL, 0.5M) was stirred and reacted at room temperature for 2h, and there was no Step I raw material detected by TLC.
  • Example 1.2.1 One-pot synthesis and characterization of a representative amino lipid compound S6S11D6
  • n3, -NRR, R 1 and R 2 are as defined above.
  • Example 1.4 Parallel synthesis and characterization of S6SxOy series amino lipid compound library
  • n4 1, 2, 3 or 4
  • -NR'R' represents the dialkylamino or cyclic amino moiety in O1 to O10 above
  • R 1 and R 2 are as defined above.
  • Step I Use a pipette to transfer the reaction solution of step I to 11 1.5mL reaction flasks (each 1.15mL, 0.15mmol), and add linear alkyl mercaptan (0.15mmol) and DIPEA in THF to the corresponding reaction flasks. (0.35mL, 0.5M), the reaction was stirred at room temperature for 2h, and there was no Step I raw material detected by TLC.
  • Example 1.4.1 One-pot synthesis and characterization of a representative amino lipid compound S19S9O8
  • Example 2 In vivo delivery performance evaluation of luciferase mRNA of lipid nanoparticles prepared from amino-based lipid compounds
  • the amino lipid compound of the present invention is mixed with DOPE, cholesterol, and PEG2000-DMG at a molar ratio of 45:10:42.5:2.5 and dissolved in absolute ethanol so that the molar concentration of the amino lipid compound is 0.001-0.01 mmol/L.
  • lipid nanoparticles were obtained, and then it was dialyzed with a dialysis cassette (Fisher, MWCO 20,000) under 1X PBS, temperature controlled at 4°C for 6 hours, and filtered through a 0.22 ⁇ m microporous membrane before use.
  • the mass ratio of amino lipid compound to Fluc mRNA is about 10:1.
  • the resulting lipid nanoparticle (LNP) solution was administered to the test animal by subcutaneous administration.
  • the particle size and PDI of the prepared lipid nanoparticles were measured by Nano-ZSZEN3600 (Malvern). Take 40uL of LNP solution for particle size measurement, cycle three times, each cycle is 30s.
  • RNA HS Assay kit detects the concentration of LNP RNA. Theoretical RNA concentration is the total RNA input divided by the total volume of the final solution.
  • the preparation method is the same as the preparation method 1, except that the molar ratio of the amino lipid compound, DSPC, cholesterol and PEG2000-DMG is 50:10:38.5:1.5.
  • the resulting lipid nanoparticle (LNP) solution was administered to the test animal via tail vein and intramuscular injection.
  • Animal preparation 6-week-old female BALB/c mice weighing about 20g were selected and raised in an SPF-class breeding room. Animal experiments are carried out in strict accordance with the guidelines of the national health agency and animal ethics requirements.
  • In vivo delivery 9 mice were randomly selected from each group, and the lipid nanoparticle solution was injected into the lipid nanoparticle solution by subcutaneous, intramuscular, and tail vein injection at a dosage of 0.5 mg/kg Fluc mRNA (3 mice per administration method) Mice). After 12 hours, each mouse was injected with 200 ⁇ L of 10 mg/mL D-luciferin potassium salt (PerkinElmer) through the tail vein. After 10 minutes, the mouse was placed under the in vivo imaging system (IVIS-200, Xenogen). Observe the total fluorescence intensity of each mouse and take a photo to record it. The expression intensities of Fluc mRNA delivered by representative amino lipid compounds through the three administration methods are shown in Table 5-7. DLin-MC3 served as a control.
  • Table 5 Expression intensity of Fluc mRNA delivered by subcutaneous administration of representative amino lipid compounds.
  • Table 6 Fluc mRNA expression intensity of representative amino lipid compounds delivered by intramuscular injection.
  • Table 7 Expression intensity of Fluc mRNA delivered by tail vein administration of representative amino lipid compounds.
  • Example 3 In vivo delivery of ovalbumin mRNA and immune performance evaluation of lipid nanoparticles prepared from amino-based lipid compounds
  • the amino lipid compound of the present invention is mixed with DOPE, cholesterol, and PEG2000-DMG at a molar ratio of 50:10:38.5:1.5 and dissolved in absolute ethanol so that the molar concentration of the amino lipid compound is 0.001-0.01 mmol/L.
  • the mass ratio of amino lipid compound to ovalbumin mRNA (OVA mRNA) is about 10:1.
  • Animal preparation 6-week-old female BALB/c mice weighing about 20g were selected and raised in an SPF-class breeding room. Animal experiments are carried out in strict accordance with the guidelines of the national health agency and animal ethics requirements.
  • mice were randomly selected from each group, and a lipid nanoparticle solution was injected into the leg muscles at a dose of 0.5 mg/kg mRNA (Day 0). After 7 days, use the same amount to reinforce it again (Day 7). Blood was collected from the tail vein on the 21st day for serological analysis.
  • DLin-MC3 as a control ( Figure 1)
  • Enzyme-linked immunosorbent assay pre-coated a flat-bottomed 96-well plate (Nunc) in 50mM carbonate buffer, the OVA protein concentration is 0.5 ⁇ g protein per well (pH 9.6) at 4°C overnight, and then used 5% glycine blocked. Obtained from animals receiving the mRNA containing lipid nanoparticles with a solution of serum in pH PBS-0.05 7.4% Tween (PBS-T) of from 10 to 106 fold diluted, and added to the wells and incubated at room temperature Place it at 37°C for 1 hour.
  • PBS-T pH PBS-0.05 7.4% Tween
  • HRP horseradish peroxidase conjugated goat anti-mouse IgG was labeled in PBS-T-1% BSA at a dilution of 1:10,000. After adding the HRP substrate, the absorbance at 450 nm was measured in an ELISA microplate reader (Bio-Rad). As shown in Figure 1, the IgG antibody titration of S7S18D3, S16N7D9, S19S9O8 and S19S9O9 was significantly better than that of the control group.
  • HEK293 cells ATCC CRL-1573 TM
  • DMEM Human fetal bovine serum
  • Detection An example of the percentage of the number of GFP fluorescent cells relative to the total number of cells (using the nuclear dye Hoechst to determine the total number of cells-see Figure 2). According to the manufacturer's instructions, Lipofectamine 2000 (Invitrogen) was used as a positive control group.
  • lipid/DNA transfection complex After incubating 10 ⁇ L of lipid/DNA transfection complex at room temperature for 30 minutes, add 90 ⁇ L of freshly resuspended cells (3-5 ⁇ 10 4 cells) and mix with a pipette. 100 ⁇ L of cell + lipid/DNA complexes were immediately transferred to separate wells of a 96-well culture plate, and placed in an incubator containing 5% CO 2 at 37°C.
  • Hoechst33258 (Invitrogen) was added to the cells at a final concentration of 0.2 ⁇ g/ml, and incubated at 37°C in the dark for 15 minutes. Then the cells were washed once with PBS solution, and culture medium was added for 20 to 24 hours.
  • Table 8 Absolute transfection efficiency of 2530 compounds on DNA of HEK293 cells.
  • Example 5 Preliminary screening of amino lipid compounds as mRNA carriers
  • HEK293 cells ATCC CRL-1573 TM
  • DMEM Human fetal bovine serum
  • Detection An example of the percentage of the number of GFP fluorescent cells relative to the total number of cells (the total number of cells is measured using the nuclear dye Hoechst). Lipofectamine 2000 (Invitrogen) was used as a positive control group.
  • the experimental method is basically the same as that in Example 4.
  • the mass of transfected EGFP mRNA (TriLink) is 50ng per well.
  • Table 9 Absolute mRNA transfection efficiency of 2530 compounds on HEK293 cells.
  • DMEM Human fetal bovine serum
  • Detection an example of the percentage of the number of GFP fluorescent cells relative to the total number of cells (the total number of cells is measured using the nuclear dye Hoechst). According to the manufacturer's instructions, Lipofectamine 2000 (Invitrogen) was used as a positive control group.
  • Table 10 Absolute DNA transfection efficiency of 170 compounds on Hela cells.
  • DMEM Human fetal bovine serum
  • Detection an example of the percentage of the number of GFP fluorescent cells relative to the total number of cells (the total number of cells is measured using the nuclear dye Hoechst). According to the manufacturer's instructions, the commercially available lipofection reagent Lipofectamine 2000 (Invitrogen) was used as a positive control group.
  • the experimental method is basically the same as that in Example 4.
  • the mass of transfected EGFP mRNA is 50ng per well.
  • Table 11 Absolute mRNA transfection efficiency of 170 compounds to Hela cells.
  • Example 8 Transfection of MCF7 cell line with amino lipid compounds as DNA carriers
  • DMEM fetal bovine serum
  • Detection an example of the percentage of the number of GFP fluorescent cells relative to the total number of cells (the total number of cells is measured using the nuclear dye Hoechst). According to the manufacturer's instructions, Lipofectamine 2000 was used as a positive control group.
  • Example 9 Transfection of MCF7 cell line with amino lipid compounds as mRNA carriers
  • DMEM Human fetal bovine serum
  • Detection an example of the percentage of the number of GFP fluorescent cells relative to the total number of cells (the total number of cells is measured using the nuclear dye Hoechst). Lipofectamine 2000 (Invitrogen) was used as a positive control group.
  • the experimental method is basically the same as that in Example 4.
  • the mass of transfected EGFP mRNA (TriLink) is 50ng per well.
  • Table 13 Absolute mRNA transfection efficiency of 102 compounds to MCF7 cells.
  • Example 10 Screening of amino lipid compounds as DNA carriers in difficult-to-transfect cell lines (embryonic stem cells)
  • Embryonic stem cells hESC2, culture Stem Cells according to the reported method, 2005, 23, 544-549
  • Detection An example of the percentage of the number of GFP fluorescent cells relative to the total number of cells. Lipofectamine Stem was used as a positive control group.
  • Table 14 Absolute DNA transfection efficiency of 60 compounds on embryonic stem cells.
  • Example 11 Screening of amino lipid compounds as mRNA carriers in difficult-to-transfect cell lines (embryonic stem cells)
  • Embryonic stem cells hESC2, culture Stem Cells according to the reported method, 2005, 23, 544-549
  • Detection An example of the percentage of the number of GFP fluorescent cells relative to the total number of cells. Lipofectamine Stem was used as a positive control group.
  • the experimental method is basically the same as that in Example 10.
  • the mass of transfected EGFP mRNA is 50ng per well.
  • Table 15 Absolute transfection efficiency of 60 compounds to mRNA of embryonic stem cells.
  • Example 12 Screening of amino lipid compounds as DNA carriers in difficult-to-transfect cell lines (cardiomyocytes)
  • Cardiomyocytes (derived from hESC-induced differentiation according to the reported method, J.Mol.Cell.Cardiol.2011,51,288-298)
  • DMEM Human fetal bovine serum
  • Detection an example of the percentage of the number of GFP fluorescent cells relative to the total number of cells (the total number of cells is measured using the nuclear dye Hoechst). According to the manufacturer's instructions, Lipofectamine Stem (Invitrogen) was used as a positive control group.
  • Table 16 Absolute DNA transfection efficiency of 60 compounds on cardiomyocytes.
  • Example 13 Screening of amino lipid compounds as mRNA carriers in difficult-to-transfect cell lines (cardiomyocytes)
  • Cardiomyocytes (derived from hESC-induced differentiation according to the reported method, J.Mol.Cell.Cardiol.2011,51,288-298)
  • DMEM Human fetal bovine serum
  • Detection an example of the percentage of the number of GFP fluorescent cells relative to the total number of cells (the total number of cells is measured using the nuclear dye Hoechst). According to the manufacturer's instructions, Lipofectamine Stem (Invitrogen) was used as a positive control group.
  • the experimental method is basically the same as that in Example 4.
  • the mass of transfected EGFP mRNA is 50ng per well.
  • Table 17 Absolute mRNA transfection efficiency of 60 compounds on cardiomyocytes.
  • PEG2000-DMG (1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000(1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol-2000));

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Abstract

本发明公开了胺基脂质化合物、其制备方法和应用。本发明还提供含有所述胺基脂质的脂质颗粒及其用于将生物活性剂传送至细胞中的用途。本发明还提供含有所述胺基脂质的脂质颗粒作为药物的用途。

Description

胺基脂质化合物、其制备方法和应用 技术领域
本公开涉及胺基脂质化合物、其制备方法和包含所述胺基脂质化合物的脂质颗粒。所述脂质颗粒可用于递送生物活性剂至细胞中。本发明还涉及含有胺基脂质的脂质颗粒作为药物的用途。
背景技术
基因治疗是指通过适当的载体将目的遗传物质导入特定组织细胞内进行适当的表达，替代或纠正自身基因结构或功能上的错乱，杀死病变细胞或增强机体清除病变细胞的能力等，从而达到治疗目的。但核酸物质易被核酸酶水解且带大量负电荷而不利于细胞吞噬，因此，研究和开发具有高效安全和组织特异性的基因传递体系成就显得尤为重要。
目前研究的基因载体可以分为病毒载体和非病毒载体。虽然病毒载体的转染效率高，但毒性、免疫原性及严重的安全性等问题，限制了其在临床治疗中的应用。而非病毒载体由于容易制备、运输、储藏，且安全、有效、无免疫原性等优点，已引起越来越多专家学者的注意。
非病毒类载体主要分为阳离子聚合物和阳离子脂质体。阳离子聚合物是指人工合成或者天然形成的含有阳离子的聚合物。聚乙烯亚胺(Polyethyleneimine,PEI)是一种最常见的阳离子聚合物非病毒基因载体。它可以将带负电的基因包裹住并形成纳米复合物，此复合物能够通过细胞内吞的方式进入细胞经过溶酶体逃逸展现出较好的转染效率。然而，PEI的细胞毒性大，且毒性与转染效率成正比关系。壳聚糖生物安全性虽好，但转染效率较低。阳离子脂质颗粒是一种人工制备的携有双层膜的磷脂质小囊，这些脂质颗粒主要区别在于它们所带电荷及细微结构的不同。当把目的DNA与这种脂质颗粒混合后，DNA就会浓缩并与之形成较稳定的脂质颗粒DNA复合物(lipoplex)。由于脂质颗粒具有类似生物膜的性质，因此当脂质颗粒DNA复合物与细胞膜接触后，通过胞吞作用而进入胞内。除了较高的转染效率，脂质颗粒没有免疫原性，细胞毒性小，也易于制备，因此阳离子脂质颗粒已成为现今用于基因转染的最常用的载体之一。
虽然阳离子脂质颗粒已有较多报道，且有商业化转染试剂可供选择。待递送基因结构的细微差异需要有脂质颗粒电荷及细微结构与之相匹配，难有一种或几种转染试剂能够适用于所有基因物质的递送。而待转入基因物质的细胞结构的细微差异，也同样需要不同性质的脂质颗粒来与之匹配。因此，相对于基因疗法的快速发展和庞大的市场需求，迫切需要开发一种小型平台式的递送系统来适应不同结构的核酸物质的递送需要。
发明内容
本发明的一个方面提供胺基脂质化合物，所述胺基脂质化合物是由以下式I表示的化合物：
Figure PCTCN2021077852-appb-000001
其中：
R 1和R 2彼此相同或不同，并且各自独立地选自C 6-C 24烷基、C 6-C 24烯基、C 6-C 24炔基和C 4-C 24酰基，其中所述C 6-C 24烷基、所述C 6-C 24烯基、所述C 6-C 24炔基和所述C 4-C 24酰基任选地被C 1-C 6烃基取代；
X 1、X 2和X 3彼此相同或不同，并且各自独立地选自C、N、O、S、S＝O、S(＝O) 2和S-S；
当X 1是C时，m＝2，并且两个R 6彼此相同或不同；当X 1是N时，m＝1；当X 1是O、S、S＝O、S(＝O) 2或S-S时，m＝0；
当X 2是C时，n＝2，并且两个R 7彼此相同或不同；当X 2是N时，n＝1；当X 2是O、S、S＝O、S(＝O) 2或S-S时，n＝0；
当X 3为C时，p＝2，并且两个R 8彼此相同或不同；当X 3是N时，p＝1；当X 3是O、S、S＝O、S(＝O) 2或S-S时，p＝0；
R 3和R 4彼此相同或不同，并且各自独立地选自C 1-C 12烷基、C 2-C 12烯基和C 2-C 12炔基，其中所述C 1-C 12烷基、所述C 2-C 12烯基和所述C 2-C 12炔基任选地被C 1-C 6烃基取代，或者R 3和R 4彼此结合以形成包含选自氮、硫和氧中的1至6个杂原子的任选地取代的4至10元杂环；
R 5不存在，或者R 5是氢或C 1-C 12烷基以提供季胺；
R 6、R 7、R 8是氢或C 1-C 12烷基；
L是C 1-C 12亚烷基、C 2-C 12亚烯基或C 2-C 12亚炔基，其中所述C 1-C 12亚烷基、所述C 2-C 12亚烯基和所述C 2-C 12亚炔基任选地被选自烃基、羧基、酰基和烷氧基中的一个或多个取代基取代，或者L是包含选自氮、硫和氧中的杂原子的任选地取代的4至10元杂环。
本发明的另一方面提供制备所述胺基脂质化合物的方法。
本发明的另一方面提供所述胺基脂质化合物用于制备脂质颗粒的用途。
本发明的另一方面提供脂质颗粒，其包含所述胺基脂质化合物。
本发明的另一方面提供所述脂质颗粒在药物的制备中的用途
附图说明
图1显示代表性胺基脂质化合物皮下给药递送卵清蛋白mRNA(OVA mRNA)所产生的体液抗体滴度。
图2显示了采用参照试剂(Lipofectamine2000)和根据实施例4的用S8N12D6处理后的HEK293细胞的显微图像，其中，图2(a)显示了采用参照试剂(Lipofectamine2000)处理后的HEK293细胞的亮视野图像，图2(b)显示了根据实施例4采用S8N12D6处理后的HEK293细胞的亮视野图像；图2(c)显示了采用参照试剂(Lipofectamine2000)处理后的HEK293细胞核的Hoechst染色图像；图2(d)显示了根据实施例4采用S8N12D6处理后的HEK293细胞核的Hoechst染色图像，图2(e)显示了采用参照试剂(Lipofectamine2000)处理后的HEK293细胞的GFP图像，图2(f)显示了根据实施例4采用S8N12D6处理后的HEK293细胞的GFP图像。
具体实施方式
下文中，将更详细地描述本发明。
本文所用的术语“任选地取代的”意指与原子或基团连接的一个或多个氢原子独立地未被取代，或被一个或多个例如一、二、三或四个取代基取代，所述取代基独立地选自：氘(D)、卤素、-OH、巯基、氰基、-CD 3、C 1-C 6烷基(优选C 1-C 3烷基)、C 2-C 6烯基、C 2-C 6炔基、环烷基(优选C 3-C 8环烷基)、芳基、杂环基(优选3-8元杂环基)、杂芳基、芳基C 1-C 6烷基-、杂芳基C 1-C 6烷基、C 1-C 6卤代烷基、-OC 1-C 6烷基(优选-OC 1-C 3烷基)、-OC 2-C 6烯基、OC 1-C 6烷基苯基、C 1-C 6烷基-OH(优选C 1-C 4烷基-OH)、C 1-C 6烷基-SH、C 1-C 6烷基-O-C 1-C 6烷基、OC 1-C 6卤代烷基、NH 2、C 1-C 6烷基-NH 2(优选C 1-C 3烷基-NH 2)、-N(C 1-C 6烷基) 2(优选-N(C 1-C 3烷基) 2)、-NH(C 1-C 6烷基)(优选-NH(C 1-C 3烷基))、-N(C 1-C 6烷基)(C 1-C 6烷基苯基)、-NH(C 1-C 6烷基苯基)、硝基、-C(O)-OH、-C(O)OC 1-C 6烷基(优选-C(O)OC 1-C 3烷基)、-CONRiRii(其中Ri和Rii是H、D和C 1-C 6烷基，优选C 1-C 3烷基)、-NHC(O)(C 1-C 6烷基)、-NHC(O)(苯基)、-N(C 1-C 6烷基)C(O)(C 1-C 6烷基)、-N(C 1-C 6烷基)C(O)(苯基)、-C(O)C 1-C 6烷基、-C(O)杂芳基(优选-C(O)-5-7元杂芳基)、-C(O)C 1-C 6烷基苯基、-C(O)C 1-C 6卤代烷基、-OC(O)C 1-C 6烷基(优选-OC(O)C 1-C 3烷基)、-S(O) 2-C 1-C 6烷基、-S(O)-C 1-C 6烷基、-S(O) 2-苯基、-S(O) 2-C 1-C 6卤代烷基、-S(O) 2NH 2、-S(O) 2NH(C 1-C 6烷基)、-S(O) 2NH(苯基)、-NHS(O) 2(C 1-C 6烷基)、-NHS(O) 2(苯基)和-NHS(O) 2(C 1-C 6卤代烷基)，其中所述的烷基、环烷基、苯基、芳基、杂环基和杂芳基中的每一个任选地被选自以下中的一个或多个取代基进一步取代：卤素、-OH、-NH 2、环烷基、3-8元杂环基、C 1-C 4烷基、C 1-C 4卤代烷基-、-OC 1-C 4烷基、-C 1-C 4烷基-OH、-C 1-C 4烷基-O-C 1-C 4烷基、-OC 1-C 4卤代烷基、氰基、硝基、-C(O)-OH、-C(O)OC 1-C 6烷基、-CON(C 1-C 6烷基) 2、-CONH(C 1-C 6烷基)、-CONH 2、 -NHC(O)(C 1-C 6烷基)、-NH(C 1-C 6烷基)C(O)(C 1-C 6烷基)、-SO 2(C 1-C 6烷基)、-SO 2(苯基)、-SO 2(C 1-C 6卤代烷基)、-SO 2NH 2、-SO 2NH(C 1-C 6烷基)、-SO 2NH(苯基)、-NHSO 2(C 1-C 6烷基)、-NHSO 2(苯基)和-NHSO 2(C 1-C 6卤代烷基)。当一个原子或基团被多个取代基取代时，所述多个取代基可以相同或不同。
本文所用的术语“烃基”意指脂肪烃失去一个氢原子后剩余的基团，包括直链的或支链的、饱和的或不饱和的烃基，所述烃基包括烷基、烯基和炔基，优选地所述烃基为C 1-C 10的烃基，C 1-C 6烃基，或C 1-C 3的烃基。
本文所用的术语“烷基”是指C 1-C 24烷基，C 1-C 20烷基，C 1-C 18烷基，C 1-C 12烷基，C 1-C 6烷基，C 3-C 24烷基，C 3-C 20烷基，C 3-C 18烷基，C 3-C 12烷基，C 3-C 6烷基，C 6-C 24烷基，C 6-C 20烷基，C 6-C 18烷基或C 6-C 12烷基。
本文所用的术语“烯基”是指C 2-C 24烯基，C 2-C 20烯基，C 2-C 18烯基，C 2-C 12烯基，C 2-C 6烯基，C 3-C 20烯基，C 3-C 18烯基，C 3-C 12烯基，C 3-C 6烯基，C 6-C 24烯基，C 6-C 20烯基，C 6-C 18烯基或C 6-C 12烯基。烯基可以包含一个或多个(例如2、3或4个)C＝C双键。
本文所用的术语“炔基”是指C 2-C 24炔基，C 2-C 20炔基，C 2-C 18炔基，C 2-C 12炔基，C 2-C 6炔基，C 3-C 20炔基，C 3-C 18炔基，C 3-C 12炔基，C 3-C 6炔基，C 6-C 24炔基，C 6-C 20炔基，C 6-C 18炔基或C 6-C 12炔基。
本文所用的术语“酰基”意指烃基-羰基，优选地所述酰基是C 4-C 24酰基、C 6-C 18酰基、C 6-C 12酰基、C 6-C 10酰基、C 4-C 6酰基。
本文所用的术语“烷氧基”意指烷基-氧基，优选所述烷氧基是C 1-C 10烷氧基，更优选地，所述烷氧基为C 1-C 6烷氧基，最优选地，所述烷氧基为C 1-C 3烷氧基。
本文所用的术语“杂环”意指包含选自N、O、S等杂原子的饱和的或不饱和的环状基团，优选地，所述杂环为包含选自N、O、S中的1至6个杂原子的任选地取代的4至10元杂环，或者为包含选自N、O、S中的1，2，或3个杂原子的任选地取代的4至6元饱和杂环。杂环的实例包括但不限于：氮杂环丁烷、氧杂环丁基、四氢呋喃、吡咯烷、咪唑烷、吡唑烷、四氢吡喃、哌啶、吗啉、硫吗啉、哌嗪，优选吡咯烷、哌啶、哌嗪和吗啉。所述杂环可以任选被一个或多个取代基取代，取代基的种类参见上述有关“任选地取代的”的定义。
本发明的一个方面提供胺基脂质化合物，所述所述胺基脂质化合物是由以下式I表示的化合物：
Figure PCTCN2021077852-appb-000002
其中：
R 1和R 2彼此相同或不同，并且各自独立地选自C 6-C 24烷基、C 6-C 24烯基、C 6-C 24炔基和C 4-C 24酰基，其中，所述C 6-C 24烷基、所述C 6-C 24烯基、所述C 6-C 24炔基和所述C 4-C 24酰基任选地被C 1-C 6烃基取代；
优选地，R 1和R 2彼此相同或不同，并且各自独立地是C 6-C 18烷基或C 6-C 18烯基。
X 1、X 2和X 3彼此相同或不同，并且各自独立地选自C、N、O、S、S＝O、S(＝O) 2和S-S；
当X 1是C时，m＝2，并且两个R 6彼此相同或不同；当X 1是N时，m＝1；当X 1是O、S、S＝O、S(＝O) 2或S-S时，m＝0；
当X 2是C时，n＝2，并且两个R 7彼此相同或不同；当X 2是N时，n＝1；当X 2是O、S、S＝O、S(＝O) 2或S-S时，n＝0；
当X 3为C时，p＝2，并且两个R 8彼此相同或不同；当X 3是N时，p＝1；当X 3是O、S、S＝O、S(＝O) 2或S-S时，p＝0；
R 3和R 4彼此相同或不同，并且各自独立地选自C 1-C 12烷基、C 2-C 12烯基和C 2-C 12炔基，其中，所述C 1-C 12烷基、所述C 2-C 12烯基和所述C 2-C 12炔基任选地被C 1-C 6烃基取代，或者R 3和R 4彼此结合以形成包含选自氮、硫和氧中的1至6个杂原子的任选地取代的4至10元杂环；
R 5不存在，或者R 5是氢或C 1-C 12烷基以提供季胺；
R 6、R 7、R 8是氢或C 1-C 12烷基；
L是C 1-C 12亚烷基、C 2-C 12亚烯基或C 2-C 12亚炔基，其中，所述C 1-C 12亚烷基、所述C 2-C 12亚烯基和所述C 2-C 12亚炔基任选地被选自烃基、羧基、酰基和烷氧基中的一个或多个取代基取代，或者L是包含选自氮、硫和氧中的杂原子的任选地取代的4至10元杂环。
优选地，L是C 1-C 8亚烷基、C 2-C 6亚烷基、C 2-C 4亚烷基或C 3-C 4亚烷基，其中所述C 1-C 8亚烷基、所述C 2-C 6亚烷基、所述C 2-C 4亚烷基或所述C 3-C 4亚烷基任选地被C 1-C 6烃基取代。
在一些实施方案中，本发明提供所述式I的胺基脂质化合物，其中X 1为C，R 6为H，m＝2；或者X 1为N，R 6为H，m＝1；或者X 1为O、S、S＝O、S(＝O) 2或S-S，m＝0；替代地或更进一步地，X 2为C，R 7为H，n＝2；或者X 2为N，R 7为H，n＝1；或者X 2为O、S、S＝O、S(＝O) 2或S-S，n＝0；替代地或更进一步地，X 3为C，R 8为H，p＝2；或者X 3为N，R 8为H，p＝1；或者X 3为O、S、S＝O、S(＝O) 2或S-S，p＝0。
在另一些实施方案中，本发明提供所述式I的胺基脂质化合物，其中X 1和X 2彼此相同或不同，并且各自独立地为N或S；当X 1是N时，m＝1；当X 1为S时，m＝0；当X 2是N时，n＝1；当X 2为S时，n＝0；替代地或更进一步地，X 3为N且p＝1，或者X 3为O且p＝0；替代地或更进一步地，R 5不存在；替代地或更进一步地，L是C 1-C 12亚烷基，所述C 1-C 12亚烷基任选地被C 1-C 6烃基取代，例如L是(CH 2) q，其中q是1至12的整数，例如1至8或1至6的整数。在一些此类实施方案中，当m＝1时，R 6是氢；替代地或更进一步地，当n＝1时，R 7是氢；替代地或更进一步地，R 8是氢。在另一些此类实施方案中，本发明提供所述式I的胺基脂质化合物，其中：R 1和R 2彼此相同或不同，并且各自独立地是C 6-C 24烷基或C 6-C 24烯基(例如C 12-C 18烯基)；X 1为N，R 6为H，m＝1；或者X 1为S，m＝0；X 2为N，R 7为H，n＝1；或者X 2为S，n＝0；X 3为N，R 8为H，p＝1；R 3和R 4彼此相同或不同，并且各自独立地是C 1-C 12烷基，其中所述C 1-C 12烷基任选地被C 1-C 6烃基取代，或者R 3和R 4彼此结合以形成包含选自氮、硫和氧中的1至6个杂原子的任选地取代的4至10元杂环；并且R 5不存在。
在一些实施方案中，本发明提供上文所述的式I的胺基脂质化合物，其中L是C 1-C 4亚烷基，其中所述C 1-C 4亚烷基任选地被C 1-C 6烃基取代。例如，L是C 2-C 4亚烷基，如(CH 2) q，其中q是1、2、3或4。
在一些实施方案中，本发明提供上文所述的式I的胺基脂质化合物，其中R 1和R 2彼此相同或不同，并且各自独立地是C 6-C 18烷基或C 6-C 18烯基。在一些实施方案中，R 1和R 2彼此相同或不同，并且各自独立地是C 6-C 18烷基。在一些实施方案中，R 1和R 2中的一者是C 6-C 18烷基，而另一者是C 6-C 18烯基
在一些实施方案中，本发明提供上文所述的式I的胺基脂质化合物，其中：
m＝0或1，n＝0或1，并且
Figure PCTCN2021077852-appb-000003
彼此相同或不同，并且各自独立地是选自以下的S6、S7、S8、S9、S10、S11、S12、S14、S15、S16、S18、S19、S20、N6、N7、N8、N9、N11、N12、N13、N15、N16、N18和 2N 12中的一种：
S6:CH 3(CH 2) 5S-；S7:CH 3(CH 2) 6S-；S8:CH 3(CH 2) 7S-；
S9:CH 3(CH 2) 8S-；S10:CH 3(CH 2) 9S-；S11:CH 3(CH 2) 10S-；
S12:CH 3(CH 2) 11S-；S14:CH 3(CH 2) 13S-；S15:CH 3(CH 2) 14S-；
S16:CH 3(CH 2) 15S-；S18:CH 3(CH 2) 17S-；
S19：
Figure PCTCN2021077852-appb-000004
S20：
Figure PCTCN2021077852-appb-000005
N6:CH 3(CH 2) 5NH-；N7:CH 3(CH 2) 6NH-；N8:CH 3(CH 2) 7NH-；
N9:CH 3(CH 2) 8NH-；N11:CH 3(CH 2) 10NH-；N12:CH 3(CH 2) 11NH-；
N13:CH 3(CH 2) 12NH-；N15:CH 3(CH 2) 14NH-；N16:CH 3(CH 2) 15NH-；
N18:CH 3(CH 2) 17NH-； 2N 12:(CH 3(CH 2) 11) 2N-。
在一些实施方案中，本发明提供上文所述的式I的胺基脂质化合物，其中：
p＝1，并且
Figure PCTCN2021077852-appb-000006
是选自以下的D1、D2、D3、D4、D5、D6、D7、D8、D9和D10中的一种：
D1：
Figure PCTCN2021077852-appb-000007
D2：
Figure PCTCN2021077852-appb-000008
D3：
Figure PCTCN2021077852-appb-000009
D4：
Figure PCTCN2021077852-appb-000010
D5：
Figure PCTCN2021077852-appb-000011
D6：
Figure PCTCN2021077852-appb-000012
D7：
Figure PCTCN2021077852-appb-000013
D8：
Figure PCTCN2021077852-appb-000014
D9：
Figure PCTCN2021077852-appb-000015
D10：
Figure PCTCN2021077852-appb-000016
在一些实施方案中，本发明提供上文所述的式I的胺基脂质化合物，其中：
X 3是O；
p是0；
R 5不存在；并且
Figure PCTCN2021077852-appb-000017
是选自以下的O1、O2、O3、O4、O5、O6、O7、O8、O9和O10中的一种：
O1：
Figure PCTCN2021077852-appb-000018
O2：
Figure PCTCN2021077852-appb-000019
O3：
Figure PCTCN2021077852-appb-000020
O4：
Figure PCTCN2021077852-appb-000021
O5：
Figure PCTCN2021077852-appb-000022
O6：
Figure PCTCN2021077852-appb-000023
O7：
Figure PCTCN2021077852-appb-000024
O8：
Figure PCTCN2021077852-appb-000025
O9：
Figure PCTCN2021077852-appb-000026
O10：
Figure PCTCN2021077852-appb-000027
在另一些实施方案中，本发明提供上文所述的式I的胺基脂质化合物，其中所述胺基脂质化合物是由以下式I’表示的化合物：
Figure PCTCN2021077852-appb-000028
其中：
X 1’和X 2’彼此相同或不同，并且各自独立地为NH、O或S，优选NH或S；
R 1、R 2、R 3、R 4和q的定义与在上文关于所述式I的胺基脂质化合物的描述中它们的定义相同。
在另一些实施方案中，本发明提供上文所述的式I的胺基脂质化合物，其中所述胺基脂质化合物是由以下式I”表示的化合物：
Figure PCTCN2021077852-appb-000029
其中：
R 1、R 2、R 3、R 4和q的定义与在上文关于所述式I的胺基脂质化合物的描述中它们的定义相同。
本发明的另一方面提供制备上文所述的式I的胺基脂质化合物的方法，所述方法包括以下步骤：
(1)在-40℃至30℃的温度下，在作为缚酸剂的碱的存在下，使三聚氯氰与由R 1(R 6) m-X 1H表示的化合物进行第一反应，得到式I-1的第一中间体；
Figure PCTCN2021077852-appb-000030
(2)在分离或(优选)不分离所述第一中间体的情况下，在室温或加热条件下，使所述第一中间体与由R 2(R 7) n-X 2H表示的化合物在作为缚酸剂的碱的存在下进行第二反应，得到式I-2的第二中间体；
Figure PCTCN2021077852-appb-000031
(3)在分离或(优选)不分离所述第二中间体的情况下，使所述第二中间体与由HX 3(R 8) p-L-NR 3R 4R 5表示的二胺在加热条件下进行第三反应，得到所述式I的胺基脂质化合物；
其中，X 1、X 2、X 3、R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、L、m、n和p的定义与在上文关于所述式I的胺基脂质化合物的描述中它们的定义相同。
例如，在一些实施方案中，X 1和X 2彼此相同或不同，并且各自独立地为N或S。当X 1是N时，m＝1；当X 1为S时，m＝0。当X 2是N时，n＝1；当X 2为S时，n＝0。在一些实施方案中，X 3为N，且p＝1。在一些实施方案中年，R 5不存在。在一些实施方案中，L是C 1-C 12亚烷基，所述C 1-C 12亚烷基任选地被C 1-C 6烃基取代，例如L是(CH 2) q，其中q是1至12的整数，例如1至6的整数。在一些实施方案中，当m＝1时，R 6是氢。在一些实施方案中，当n＝1时，R 7是氢。在一些实施方案中，R 8是氢。
优选地，在该步骤(3)中，所述第三反应是在作为缚酸剂的碱的存在下进行的。
在一些实施方案中，本发明提供制备所述式I’的胺基脂质化合物的方法，所述方法包括以下步骤：
(1)在-40℃至30℃的温度下，在作为缚酸剂的碱的存在下，使三聚氯氰与由R 1-X 1’H表示的 化合物进行第一反应，得到式I’-1的第一中间体；
Figure PCTCN2021077852-appb-000032
(2)在分离或(优选)不分离所述第一中间体的情况下，在室温或加热条件下，使所述第一中间体与由R 2-X 2’H表示的化合物在作为缚酸剂的碱的存在下进行第二反应，得到式I’-2的第二中间体；
Figure PCTCN2021077852-appb-000033
(3)在分离或(优选)不分离所述第二中间体的情况下，使所述第二中间体与由H 2N-(CH 2) q-NR 3R 4表示的二胺在加热条件下进行第三反应，得到所述式I’的胺基脂质化合物；
其中，X 1’、X 2’、R 1、R 2、R 3、R 4和q的定义与在上文关于所述式I’的胺基脂质化合物的描述中它们的定义相同。
优选地，在该步骤(3)中，所述第三反应是在作为缚酸剂的碱的存在下进行的。
在一些实施方案中，本发明提供制备上文所述的式I”的胺基脂质化合物的方法，
Figure PCTCN2021077852-appb-000034
所述方法包括以下步骤：
(1)在-40℃至30℃的温度下，在作为缚酸剂的碱的存在下，使三聚氯氰与由R 1-SH表示的化合物进行第一反应，得到式I”-1的第一中间体；
(2)在分离或(优选)不分离所述第一中间体的情况下，在室温或加热条件下，使所述第一中间体与由R 2-SH表示的化合物在作为缚酸剂的碱的存在下进行第二反应，得到式I”-2的第二中间体；
Figure PCTCN2021077852-appb-000035
(3)在分离或(优选)不分离所述第二中间体的情况下，在作为缚酸剂的碱的存在下，使所述第二中间体与由HO-(CH 2) q-NR 3R 4表示的醇胺在在室温或加热条件进行第三反应，得到所述式I”的胺基脂质化合物；
其中，R 1、R 2、R 3、R 4和q的定义与在上文关于所述式I”的胺基脂质化合物的描述中它们的定义相同。
在上文所述的任一方法的优选实施方案中，所述第一反应在低于20℃(例如，低于15℃、低于10℃、低于5℃、低于0℃、低于-10℃或低于-20℃，并且对反应温度的下限没有具体限制)和高于-40℃(例如高于-35℃，或者优选等于或高于-30℃)的温度下进行。
如本文所用的，术语“室温”是大气压力下的通常温度，可以指0℃至30℃，0℃至25℃，0℃至20℃，5℃至30℃，5℃至20℃，10℃至30℃，10℃至25℃，15℃至30℃，或15℃至25℃的温度范围。
如本文所用的，术语“加热条件”具体指加热至50℃至120℃，50℃至110℃，50℃至80℃，60℃至120℃，60℃至110℃，60℃至100℃，70℃至120℃，70℃至100℃，70℃至90℃的温度范围。
如本文所用的，术语“碱”可以特别地是本领域通常使用的碱化合物，例如但不限于：有机碱，如 三乙胺、DIPEA、吡啶、DMAP；或无机碱，如氢氧化钠、氢氧化钾、碳酸钠、碳酸钾。
优选地，q是1至8的整数，优选是1至6的整数，更优选地，q是1至4的整数，例如1、2、3或4。
在一些实施方案中，本发明提供制备式II的胺基脂质化合物的方法，所述方法可以通过如下的反应式1来进行：
[反应式1]
Figure PCTCN2021077852-appb-000036
其中，R 1、R 2、R 3、R 4和X 2的定义与在上文关于所述式I的胺基脂质化合物的制备方法的描述中它们的定义相同。
在一些实施方案中，本发明提供制备式III的胺基脂质化合物的方法，所述方法可以通过如下的反应式2来进行：
[反应式2]
Figure PCTCN2021077852-appb-000037
其中，R 1、R 2、R 3、R 4和X 2的定义与在上文关于所述式I的胺基脂质化合物的制备方法的描述中它们的定义相同。
在一些实施方案中，本发明提供制备式IV的胺基脂质化合物的方法，所述方法可以通过如下的反应式3来进行：
[反应式3]
Figure PCTCN2021077852-appb-000038
其中，R 1、R 2、R 3、R 4、X 2和q的定义与在上文关于所述式I的胺基脂质化合物的描述中它们的定义相同；
优选的，X 2选自O、S、S＝O、S(＝O) 2和S-S，优选为O或S，更优选为S。
在一些实施方案中，本发明提供制备式V的胺基脂质化合物的方法，所述方法可以通过如下的反应式4来进行：
[反应式4]
Figure PCTCN2021077852-appb-000039
其中，R 1、R 2、R 3、R 4、X 2和q的定义与在上文关于所述式I的胺基脂质化合物的描述中它们的定义相同；
优选的，X 2选自O、S、S＝O、S(＝O) 2和S-S，优选为O或S，更优选为S。
可以看出，根据本发明的胺基脂质化合物的制备方法是非常通用的，可以用于胺基脂质化合物 库的快速合成，以非常廉价的方式用于快速的基于细胞的筛选实验。
本发明的另一方面提供根据所述式I、I’、II、III、IV和V中任一者的胺基脂质化合物用于制备脂质颗粒的用途。
本发明的另一方面提供脂质颗粒，其包含如上文所述的根据本发明的胺基脂质化合物。
由于本发明的胺基脂质化合物含有长的非极性残基，所得到的化合物全部具有疏水性特征，并且由于具有胺基，同时又具有亲水性特征。这种两性特征可以用于形成脂质颗粒，例如，脂质纳米粒、脂质双层、胶束、脂质体等。
在本发明的范围内，术语“脂质颗粒”意思是将胺基脂质化合物放入水溶液中制得的纳米大小的物质。这些颗粒特别地是脂质纳米粒、脂质双层泡囊(脂质体)、多层泡囊或胶束。
在本发明的优选实施方案中，所述脂质颗粒是含有如上文所述的根据本发明的胺基脂质化合物的脂质体。在本发明的范围内，脂质体是由包裹含水隔室的脂质两性(两亲(amphiphilic)分子的双层组成的微泡囊。
脂质体形成不是一个自发的过程。当脂质放入水中时首先形成脂质泡囊，因此形成一个双层或一系列双层，每个通过水分子分开。可以通过在水中用超声波处理脂质泡囊来形成脂质体。
在本发明的范围内，术语“脂质双层”意思是由两层脂质分子形成的薄膜。术语“胶束”意思是分散在液体胶体中的表面活性剂分子的聚集物。水溶液中的典型胶束接触水时与亲水性头部区域形成聚集物，螯合胶束中心的疏水性单尾区。
在本发明的范围内，术语“细胞”意思是一般术语，并且包括单个细胞、组织、器官、昆虫细胞、禽类细胞、鱼细胞、两栖类细胞、哺乳动物细胞、初级细胞、连续细胞系、干细胞和/或遗传工程化细胞(如表达异源多肽或蛋白的重组细胞)的培养。重组细胞包括，例如，表达异源多肽或蛋白(如生长因子或血液因子)的细胞。
在一些优选的实施方案中，所述脂质颗粒或脂质体进一步含有辅助脂质、固醇和生物活性剂中的一种或多种。
例如，在一些实施方案中，所述脂质颗粒或脂质体含有辅助脂质。在优选的实施方案中，所述辅助脂质是非阳离子脂质。在更优选的实施方案中，所述辅助脂质是非阳离子磷脂。在本发明的范围内，非阳离子脂质可以含有阳离子官能团(例如，铵基)，但应当含有阴离子官能团，以至少中和分子。脂质分子中的所有官能团的总体应当是非阳离子的。由根据本发明的胺基脂质和非阳离子(中性)磷脂的混合物组成的脂质体对于将核酸传送至细胞中是最有效的。在甚至更优选的实施方案中，所述非阳离子脂质是DOPE、DSPC或其组合。在一些优选的实施方案中，在所述脂质颗粒中，所述胺基脂质化合物与所述辅助脂质的摩尔比为约(2至10):1，优选约(3至8):1，更优选约(4至6):1，例如约4:1、约4.5:1或约5:1。
在一些替代的或进一步的实施方案中，所述脂质颗粒或脂质体包含固醇。固醇，如胆固醇，是细胞膜中的天然成分。其可以用于稳定颗粒，并且帮助与细胞膜的整合。所述固醇可以是选自胆固醇、谷甾醇、豆甾醇和麦角固醇中的一种或多种，优选为胆固醇。在一些优选的实施方案中，在所述脂质颗粒中，所述胺基脂质化合物与所述固醇的摩尔比为约(1至1.5):1，优选约(1至1.4):1，例如约(1至1.3):1。
在一些替代的或进一步的实施方案中，所述脂质颗粒或脂质体含有生物活性剂。在本发明的范围内，生物活性剂是在引入细胞或宿主中时具有生物作用的物质，例如，通过刺激免疫应答或炎性应答、通过发挥酶活性或通过补充突变等来起作用。所述生物活性剂特别是核酸、肽、蛋白、抗体和小分子。将脂质体用于将药物包裹在脂质双层内或脂质体的内部含水空间中时，都可以使用术语“脂质颗粒药物”。
在最优选的实施方案中，所述生物活性剂是核酸，包括但不限于，信使RNA(mRNA)、反义寡核苷酸、DNA、质粒、核糖体RNA(rRNA)、微RNA(miRNA)、转移RNA(tRNA)、小的抑制RNA(siRNA)和小的核RNA(snRNA)。在另一些优选的实施方案中，所述生物活性剂选自抗肿瘤剂、抗生素、免疫调节剂、抗炎剂、作用于中枢神经系统的药剂、抗原或其片段、蛋白、肽、多肽、多肽类(polypeptoid)、疫苗和小分子，及其混合物。
在另一些实施方案中，所述脂质颗粒或脂质体进一步含有至少一种聚乙二醇(PEG)-脂质。PEG 脂质有助于保护颗粒及其内含物免受体外或体内降解。此外，PEG在脂质体表面上形成保护层，并且提高了体内循环时间。其可以用于脂质体药物传送中(PEG-脂质体)。优选地，所述PEG-脂质可以是选自PEG1000-DMG、PEG5000-DMG、PEG2000-DMG和PEG2000-DSPE中的一种或多种，优选为PEG2000-DMG。在一些优选的实施方案中，在所述脂质颗粒中，所述胺基脂质化合物与所述PEG-脂质的摩尔比为约(9至42):1，优选约(12至38):1，更优选约(16至36):1，例如约(18至34):1。
根据本发明的脂质颗粒或脂质体具有优良的包封生物活性剂的性能。包含生物活性剂的脂质颗粒或脂质体可以用于将多种治疗剂中的任何一种传送至细胞中。本发明包括如上所述的脂质颗粒(尤其是脂质体)用于将生物活性剂传送至细胞中的用途。本发明还提供将生物活性剂传送至细胞中的方法，所述方法包括使包含所述生物活性剂的本发明的脂质颗粒或脂质体与所述细胞接触。
如上所示，含有本发明的胺基脂质化合物的脂质颗粒或脂质体适用于将生物活性剂传送至细胞中。可以对通过所述通用合成方法合成的多种不同胺基脂质化合物给予脂质体的特定特征进行筛选。重要的特征例如是转染效率、细胞毒性、待传送至细胞中的药剂的粘附、脂质体的稳定性、脂质体的大小等。本发明的方法可以形成特定适应的脂质体，以用于特定的应用。
例如，脂质颗粒或脂质体可以用于转染多细胞组织或器官。因此，本发明还提供转染细胞、多细胞组织或器官的方法，所述方法包括使包含所述核酸的本发明的脂质颗粒或脂质体与所述细胞接触。这给患者提供了新的治疗处理的可能性。
根据本发明，患者可以是任何哺乳动物，优选选自小鼠、大鼠、猪、猫、狗、马、山羊、牛和猴子和/或其他。在另一些优选的实施方案中，患者是人。
本发明的另一方面还提供所述包含式I、I’、I”、II、III、IV和V中任一者的胺基脂质化合物的脂质颗粒或脂质体用作药物的用途。本发明的另一方面还提供所述包含式I、I’、I”、II、III、IV和V中任一者的胺基脂质化合物的脂质颗粒或脂质体在药物的制备中的用途。例如，在一些实施方案中，所述脂质颗粒用作包封生物活性剂的载体。
特别地，所述脂质颗粒或脂质体、或者所述药物可以给予患者，用于基因治疗、基因疫苗接种、反义治疗或通过干扰RNA的治疗中。具体的应用包括但不限于：
(1)本发明的脂质颗粒可以传递核酸以用于基因治疗。通过本发明的胺基脂质将外源基因导入靶细胞，以纠正或补偿缺陷和异常基因引起的疾病，以达到治疗目的。其中也包括转基因等方面的技术应用，也就是将外源基因通过基因转移技术将其插入患者的适当的受体细胞中，使外源基因制造的产物能治疗某种疾病，如常见的肺癌、胃癌、肝癌、食管癌、结肠癌、胰腺癌、脑癌、淋巴癌、血癌、前列腺癌等。还可导入经过基因编辑的核酸物质以用于多种遗传疾病的治疗，如血友病，地中海贫血、高雪氏病等。
(2)本发明的脂质颗粒可以用于疫苗接种中。本发明的脂质颗粒或脂质体可以用于传送抗原或编码抗原的核酸。本发明的脂质颗粒还可以用于引发对抗各种抗原的免疫应答，所述抗原用于治疗和/或预防多种病症，如癌症、过敏、毒性和病原体(如，病毒、细菌、真菌和其他致病生物体)感染。
本发明还提供基因治疗、基因疫苗接种、反义治疗或通过干扰RNA治疗的方法，所述方法包括向有需要的患者施用包含生物活性剂(例如，所需要的核酸或抗原)的本发明的脂质颗粒或脂质体。
本发明的脂质颗粒可用于制备用于核酸转移的药物。优选地，所述核酸为RNA、信使RNA(mRNA)、反义寡核苷酸、DNA、质粒、核糖体RNA(rRNA)、微RNA(miRNA)、转移RNA(tRNA)、小的抑制RNA(siRNA)和小的核RNA(snRNA)。
包封非生物活性剂也是本发明的脂质颗粒或脂质体的一个目的。因此，本发明还提供这样的脂质颗粒或脂质体，其包含非生物活性剂。非生物活性剂的实例包括但不限于：抗氧化剂、色素、颜料、香料和调味剂等，例如用于化妆品中的那些。预期根据本发明的脂质颗粒或脂质体也具有优良的包封非生物活性剂的性能。
本发明还包括以下实施方案：
1.胺基脂质化合物，所述胺基脂质化合物是由以下式I表示的化合物：
Figure PCTCN2021077852-appb-000040
其中，R 1和R 2彼此相同或不同，并且各自独立地选自C 6-C 24烷基、C 6-C 24烯基、C 6-C 24炔基和C 4-C 24酰基，其中，所述C 6-C 24烷基、所述C 6-C 24烯基、所述C 6-C 24炔基和所述C 4-C 24酰基任选地被C 1-C 6烃基取代；
X 1、X 2和X 3彼此相同或不同，并且各自独立地选自C、N、O、S、S＝O、S(＝O) 2和S-S；
当X 1是C时，m＝2，并且两个R 6彼此相同或不同；当X 1是N时，m＝1；当X 1是O、S、S＝O、S(＝O) 2或S-S时，m＝0；
当X 2是C时，n＝2，并且两个R 7彼此相同或不同；当X 2是N时，n＝1；当X 2为O、S、S＝O、S(＝O) 2或S-S时，n＝0；
当X 3是C时，p＝2，并且两个R 8彼此相同或不同；当X 3是N时，p＝1；当X 3为O、S、S＝O、S(＝O) 2或S-S时，p＝0；
R 3和R 4彼此相同或不同，并且各自独立地选自C 1-C 12烷基、C 2-C 12烯基和C 2-C 12炔基，其中，所述C 1-C 12烷基、所述C 2-C 12烯基和所述C 2-C 12炔基任选地被C 1-C 6烃基取代，或者R 3和R 4彼此结合以形成包含选自氮、硫和氧中的1至6个杂原子的任选地取代的4至10元杂环；
R 5不存在，或者R 5是氢或C 1-C 12烷基以提供季胺；
R 6、R 7、R 8是氢或C 1-C 12烷基；
L是C 1-C 12亚烷基、C 2-C 12亚烯基或C 2-C 12亚炔基，其中，所述C 1-C 12亚烷基、所述C 2-C 12亚烯基和所述C 2-C 12亚炔基任选地被选自烃基、羧基、酰基和烷氧基中的一个或多个取代基取代，或者L是包含选自氮、硫和氧中的杂原子的任选地取代的4至10元杂环。
2.根据第1项所述的胺基脂质化合物，其中：
X 1为C，R 6为H，m＝2；或者X 1为N，R 6为H，m＝1；或者X 1为O、S、S＝O、S(＝O) 2或S-S，m＝0；
X 2为C，R 7为H，n＝2；或者X 2为N，R 7为H，n＝1；或者X 2为O、S、S＝O、S(＝O) 2或S-S，n＝0；
X 3为C，R 8为H，p＝2；或者X 3为N，R 8为H，p＝1；或者X 3为O、S、S＝O、S(＝O) 2或S-S，p＝0。
3.根据第1项所述的胺基脂质化合物，其中：
R 1和R 2彼此相同或不同，并且各自独立地是C 6-C 24烷基；
X 1为N，R 6为H，m＝1；或者X 1为S，m＝0；
X 2为N，R 7为H，n＝1；或者X 2为S，n＝0；
X 3为N，R 8为H，p＝1；
R 3和R 4彼此相同或不同，并且各自独立地是C 1-C 12烷基，其中，所述C 1-C 12烷基任选地被C 1-C 6烃基取代，或者R 3和R 4彼此结合以形成包含选自氮、硫和氧中的1至6个杂原子的任选地取代的4至10元杂环；
R 5不存在。
4.根据第1至3项中任意一项所述的胺基脂质化合物，其中L是C 1-C 4亚烷基，其中所述C 1-C 4亚烷基任选地被C 1-C 6烃基取代。
5.根据第1至3项中任意一项所述的胺基脂质化合物，其中R 1和R 2彼此相同或不同，并且各自独立地是C 6-C 18烷基。
6.根据第1项所述的胺基脂质化合物，其中：
m＝0，n＝0，p＝0，
R 1-X 1-和R 2-X 2-彼此相同或不同，并且各自独立地是选自如上文所述的S6、S7、S8、S9、S10、S11、S12、S14、S15、S16、S18、N6、N7、N8、N9、N11、N12、N13、N15、N16、N18、 2N 12中的一种；
-X 3-L-N(R 3)(R 4)(R 5)是选自如上文所述的D1、D2、D3、D4、D5、D6、D7、D8、D9、D10中的一种。
7.制备根据第1至6项中任意一项所述的胺基脂质化合物的方法，所述方法包括以下步骤：
(1)在-30℃至30℃的温度下，在作为缚酸剂的碱的存在下，使三聚氯氰与由R 1-X 1H表示的化合物进行第一反应，得到第一中间体，其中X 1是NH或S；
(2)在不分离所述第一中间体的情况下，在室温或加热条件下，加入由R 2-X 2H表示的化合物，在作为缚酸剂的碱的存在下，进行第二反应，得到第二中间体，其中X 2是NH或S；
(3)在不分离所述第二中间体的情况下，加入由H 2N-(CH 2) q-NR 3R 4表示的二胺，在加热条件下进行第三反应，得到胺基脂质化合物；
其中，q是1至12的整数，R 1、R 2、R 3和R 4的定义与第1至6项中任意一项中它们的定义相同。
8.制备根据本发明的胺基脂质化合物的方法，所述方法可以通过如下的反应式1来进行：
[反应式1]
Figure PCTCN2021077852-appb-000041
其中，R 1，R 2，R 3，R 4，X 2的定义与前面关于胺基脂质化合物的制备方法的描述中定义的相同。
9.制备根据本发明的胺基脂质化合物的方法，所述方法可以通过如下的反应式2来进行：
[反应式2]
Figure PCTCN2021077852-appb-000042
其中，R 1，R 2，R 3，R 4，X 2的定义与前面关于胺基脂质化合物的制备方法的描述中定义的相同。
10.根据第1至6项中任意一项所述的胺基脂质化合物用于制备脂质颗粒的用途，其中所述脂质颗粒为脂质纳米粒、脂质体、多层囊泡或胶束；更优选地，所述脂质颗粒为脂质纳米粒。
11.脂质颗粒，其包含第1至6项中任意一项所述的胺基脂质化合物；优选地，所述脂质颗粒为脂质纳米粒、脂质体、多层囊泡或胶束；更优选地，所述脂质颗粒为脂质纳米粒。
12.根据第11项所述的脂质颗粒，所述脂质颗粒进一步含有辅助脂质、固醇和生物活性剂中的一种或多种；
优选地，所述辅助脂质为非阳离子脂质，更优选地，所述辅助脂质是非阳离子磷脂，进一步优选地，所述非阳离子脂质是DOPE；
优选地，所述固醇为胆固醇；
优选地，所述生物活性剂为核酸、抗肿瘤剂、抗生素、免疫调节剂、抗炎剂、作用于中枢神经系统的药剂、抗原或其片段、肽、蛋白、抗体、疫苗和小分子中的一种或多种；更优选地，所述核酸为RNA、信使RNA(mRNA)、反义寡核苷酸、DNA、质粒、核糖体RNA(rRNA)、微RNA(miRNA)、转移RNA(tRNA)、小的抑制RNA(siRNA)和小的核RNA(snRNA)。
13.根据第11或12项所述的脂质颗粒在药物的制备中的用途，所述药物是用于基因治疗、基因疫苗接种、反义治疗或通过干扰RNA的治疗的药物；
优选地，所述基因治疗可用于癌症和遗传疾病的治疗；更优选地，所述癌症为肺癌、胃癌、肝 癌、食管癌、结肠癌、胰腺癌、脑癌、淋巴癌、血癌或前列腺癌中的一种或多种，所述遗传疾病为血友病，地中海贫血、高雪氏病中的一种或多种；
优选地，所述基因疫苗接种用于治疗癌症、过敏、毒性和病原体感染；更优选地，所述病原体为病毒、细菌或真菌中的一种或多种。
14.根据第11或12项所述的脂质颗粒在制备用于核酸转移的药物中的用途，优选地，所述核酸为RNA、信使RNA(mRNA)、反义寡核苷酸、DNA、质粒、核糖体RNA(rRNA)、微RNA(miRNA)、转移RNA(tRNA)、小的抑制RNA(siRNA)和小的核RNA(snRNA)。
有益效果
本发明的胺基脂质化合物具有官能团多样性强、数量众多的特点，所述胺基脂质化合物的制备方法具有原料易得、反应条件温和、反应选择性好、反应产率高、仪器设备要求低和操作简单的优点，本发明的制备方法是非常通用的，可以用于可离子化的胺基脂质化合物库的快速合成，可以以非常廉价的方式用于快速的基于细胞的筛选实验。
本发明所提供的胺基脂质化合物官能团种类及组合方式多样，通过常规的针对转染效率、细胞毒性、待传送至细胞中的药剂的粘附、脂质颗粒的稳定性、脂质颗粒的大小等特征的筛选，可方便地适用于将不同核酸物质递送至不同细胞，提高了递送系统的针对性和有效性。
本发明的胺基脂质化合物可采用组合化学的方法高通量地、高效率地合成，合成方法简单、收率高。
含有本发明的胺基脂质化合物的脂质颗粒或脂质体具有优良的包封生物活性剂的性能，可以用于生物活性剂、特别是水难溶性药剂或者易分解或降解活性剂(例如核酸)的递送，提高其生物利用度和效力。
为了使本发明的目的、技术方案和优点更加清楚，下面结合具体实施例对本发明进行描述。所描述的实施例是本发明一部分实施方案，而不是全部的实施方案。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动的情况下所获得的所有其他实施方案，都属于本发明保护的范围。
实施例
如无特别说明，实施例中所用的化学试剂均为分析纯的，购自TCI、Aladdin或J&K等。
实施例1：胺基脂质化合物的合成
为系统研究烷基链长短、烷基链组合方式、可离子化的头部基团等结构与转染能力的关系，选取了11种6-18个碳链的直链烷基硫醇(S6-S18)、11种6-24个碳链的烷基胺(N6- 2N12)、结构各异的10种二胺(D1-D6)。应用组合化学的方法，高通量地合成了2530个同时含有两个烷基链以及可离子化的头部基团的化合物库，合成路径如下：
(a)三嗪作为连接基团
Figure PCTCN2021077852-appb-000043
(b)典型例子
Figure PCTCN2021077852-appb-000044
(c)合成砌块
Figure PCTCN2021077852-appb-000045
(d)化合物库
Figure PCTCN2021077852-appb-000046
其中n3＝1、2或3；-NRR表示上文中的二胺D1至D10中的二烷基氨基或环氨基部分；并且R 1和R 2如上文所定义。
所合成的化合物具有下表I所示的结构之一：
表I：所合成的化合物
Figure PCTCN2021077852-appb-000047
其中Sz各自独立地表示选自下表II所列的S6至S12、S14至S16和S18的基团；
Nx各自独立地表示选自下表II所列的N6至N9、N11至N13、N15、N16、N18和 2N 12的基团；以及
Dy各自独立地表示选自下表II所列的D1至D10的基团：
表II
Figure PCTCN2021077852-appb-000048
Figure PCTCN2021077852-appb-000049
对于所合成的化合物，所测定的质谱([M+H] +)数据示于下表III中：
表III
  D1 D2 D3 D4 D5 D6 D7 D8 D9 D10
S6N6 383.4 425.3 409.3 423.4 438.4 397.3 411.4 439.3 452.4 423.4
S6N7 397.3 439.3 423.3 437.4 452.4 411.3 425.4 453.3 466.4 437.5
S6N8 411.4 453.3 437.4 451.4 466.6 425.4 439.4 467.3 480.6 451.4
S6N9 425.3 467.4 451.3 465.4 480.4 439.3 453.4 481.3 494.4 465.4
S6N11 453.3 495.3 479.3 493.4 508.4 467.3 481.5 509.4 522.4 493.4
S6N12 467.4 509.3 493.4 507.4 522.4 481.3 495.4 523.3 536.4 507.6
S6N13 481.3 523.5 507.3 521.5 536.4 495.3 509.4 537.5 550.6 521.4
S6N15 509.6 551.3 535.5 549.4 564.4 523.3 537.5 565.3 578.4 549.4
S6N16 523.3 565.3 549.3 563.4 578.6 537.3 551.4 579.5 592.4 563.4
S6N18 551.3 593.3 577.3 591.4 606.4 565.3 579.4 607.3 620.4 591.6
S6 2N12 635.6 677.4 661.4 675.5 690.5 649.6 663.5 691.4 704.5 675.5
S7N6 397.3 439.6 423.7 437.4 452.4 411.3 425.4 453.3 466.4 437.6
S7N7 411.3 453.3 437.3 451.4 466.4 425.3 439.4 467.3 480.6 451.4
S7N8 425.5 467.3 451.3 465.4 480.6 439.3 453.5 481.3 494.4 465.4
S7N9 439.3 481.3 465.3 479.4 494.4 453.5 467.4 495.5 508.4 479.4
S7N11 467.5 509.3 493.7 507.7 522.4 481.3 495.4 523.3 536.4 507.4
S7N12 481.3 523.3 507.6 521.4 536.7 495.3 509.6 537.3 550.6 521.4
S7N13 495.6 537.6 521.3 535.4 550.4 50963 523.4 551.3 564.4 535.4
S7N15 523.3 565.3 549.3 563.4 578.4 537.3 551.4 579.3 592.4 563.4
S7N16 537.3 579.3 563.3 577.4 592.4 551.3 565.4 593.3 606.4 577.4
S7N18 565.3 607.3 591.3 605.4 620.4 579.3 593.4 621.3 634.4 605.4
S7 2N12 649.4 691.4 675.4 689.5 704.5 663.4 677.5 705.4 718.5 689.5
S8N6 411.3 453.3 437.3 451.4 466.4 425.3 439.4 467.3 480.4 451.4
S8N7 425.3 467.3 451.3 465.4 480.6 439.3 453.4 481.3 494.4 465.4
S8N8 439.8 481.3 465.3 479.4 494.4 453.6 467.4 495.3 508.6 479.4
S8N9 453.3 495.8 479.8 493.6 508.4 467.3 481.5 509.5 522.4 493.6
S8N12 495.7 537.3 521.3 535.4 550.6 509.3 523.4 551.3 564.4 535.4
  D1 D2 D3 D4 D5 D6 D7 D8 D9 D10
S8N13 509.3 551.3 535.6 549.4 564.4 523.3 537.6 565.3 578.5 549.6
S8N15 537.3 579.3 563.3 577.4 592.4 551.3 565.4 593.3 606.4 577.4
S8N16 551.3 593.3 577.3 591.4 606.4 565.3 579.4 607.3 620.4 591.4
S8N18 579.6 621.3 605.3 619.4 634.4 593.3 607.4 635.3 648.4 619.4
S8 2N12 663.4 705.4 689.4 703.5 718.5 677.4 691.5 719.4 732.5 703.5
S9N6 425.3 467.3 451.3 465.4 480.7 439.3 453.4 481.3 494.4 465.6
S9N7 439.3 481.3 465.3 479.6 494.4 453.3 467.4 495.3 508.4 479.4
S9N8 453.6 495.3 479.3 493.4 508.4 467.3 481.4 509.6 522.4 493.4
S9N9 467.3 509.3 493.3 507.4 522.4 481.3 495.4 523.3 536.4 507.4
S9N11 495.3 537.3 521.6 535.4 550.4 509.3 523.4 551.3 564.4 535.6
S9N12 509.3 551.6 535.3 549.4 564.4 523.3 537.4 565.3 578.4 549.4
S9N13 523.6 565.6 549.3 563.4 578.4 537.3 551.4 579.3 592.4 563.4
S9N15 551.3 593.3 577.3 591.4 606.4 565.3 579.4 607.3 620.4 591.4
S9N16 565.3 607.3 591.3 605.4 620.4 579.3 593.4 621.3 634.4 605.4
S9N18 593.3 635.3 619.6 633.4 648.4 607.3 621.4 649.3 662.4 633.4
S9 2N12 677.4 719.4 703.4 717.5 732.5 691.4 705.5 733.4 746.5 717.5
S10N6 439.5 481.6 465.6 479.4 494.4 453.5 467.4 495.3 508.4 479.4
S10N7 453.3 495.3 479.3 493.5 508.4 467.3 481.4 509.3 522.4 493.4
S10N8 467.3 509.3 493.3 507.7 522.4 481.3 495.4 523.3 536.4 507.4
S10N9 481.3 523.3 507.3 521.4 536.4 495.5 509.4 537.3 550.4 521.4
S10N11 509.3 551.3 535.3 549.4 564.4 523.3 537.4 565.3 578.4 549.4
S10N12 523.3 565.3 549.3 563.4 578.4 537.3 551.4 579.3 592.4 563.4
S10N13 537.3 579.3 563.3 577.4 592.4 551.3 565.4 593.3 606.7 577.7
S10N15 565.3 607.3 591.3 605.4 620.4 579.3 593.4 621.3 634.4 605.4
S10N16 579.3 621.3 605.3 619.4 634.4 593.3 607.4 635.6 648.4 619.4
S10N18 607.3 649.3 633.3 647.4 662.4 621.3 635.4 663.3 676.4 647.4
S10 2N12 691.4 733.4 717.4 731.5 746.5 705.4 719.7 747.4 760.5 731.5
S11N6 453.3 495.3 479.3 493.4 508.4 467.3 481.4 509.3 522.4 493.4
S11N7 467.3 509.3 493.3 507.4 522.4 481.3 495.4 523.3 536.4 507.4
S11N8 481.3 523.3 507.3 521.4 536.4 495.3 509.4 537.3 550.4 521.4
S11N9 495.3 537.3 521.6 535.4 550.4 509.3 523.4 551.3 564.4 535.4
S11N11 523.3 565.3 549.3 563.4 578.6 537.3 551.4 579.3 592.4 563.4
S11N12 537.6 579.3 563.3 577.4 592.4 551.3 565.4 593.3 606.4 577.6
S11N13 551.3 593.3 577.3 591.4 606.4 565.3 579.4 607.3 620.4 591.4
S11N15 579.6 621.3 605.6 619.4 634.4 593.3 607.4 635.3 648.4 619.4
S11N16 593.3 635.3 619.3 633.4 648.4 607.3 621.4 649.3 662.4 633.4
S11N18 621.3 663.3 647.3 661.4 676.4 635.3 649.4 677.3 690.4 661.4
S11 2N12 705.4 747.4 731.6 745.7 760.5 719.4 733.5 761.4 774.5 745.5
S12N6 467.3 509.3 493.3 507.4 522.4 481.3 495.4 523.3 536.4 507.4
S12N7 481.3 523.3 507.3 521.4 536.4 495.3 509.4 537.3 550.4 521.4
S12N8 495.3 537.3 521.3 535.4 550.4 509.3 523.4 551.3 564.4 535.4
S12N9 509.3 551.3 535.3 549.4 564.4 523.3 537.4 565.3 578.4 549.4
S12N11 537.3 579.3 563.3 577.5 592.7 551.3 565.4 593.3 606.4 577.4
S12N12 551.3 593.3 577.3 591.4 606.4 565.3 579.7 607.3 620.4 591.4
S12N13 565.3 607.3 591.3 605.4 620.4 579.3 593.4 621.3 634.4 605.4
S12N15 593.3 635.3 619.3 633.4 648.4 607.3 621.4 649.3 662.4 633.4
  D1 D2 D3 D4 D5 D6 D7 D8 D9 D10
S12N16 607.3 649.3 633.3 647.4 662.4 621.3 635.4 663.3 676.4 647.4
S12N18 635.3 677.3 661.3 675.4 690.4 649.3 663.4 691.5 704.4 675.4
S12 2N12 719.5 761.4 745.4 759.5 774.5 733.4 747.5 775.4 788.5 759.5
S14N6 495.3 537.3 521.3 535.4 550.4 509.3 523.4 551.3 564.5 535.5
S14N7 509.3 551.5 535.3 549.4 564.4 523.3 537.4 565.3 578.4 549.4
S14N8 523.5 565.3 549.3 563.4 578.4 537.5 551.4 579.3 592.4 563.4
S14N9 537.3 579.6 563.3 577.4 592.5 5516 565.4 593.6 606.6 577.4
S14N11 565.3 607.3 591.6 605.4 620.4 579.3 593.4 621.3 634.4 605.4
S14N12 579.5 621.3 605.3 619.4 634.4 593.3 607.4 635.3 648.4 619.4
S14N13 593.3 635.6 619.3 633.4 648.4 607.3 621.4 649.3 662.4 633.4
S14N15 621.3 663.3 647.3 661.4 676.4 635.3 649.4 677.3 690.4 661.4
S14N16 635.6 677.3 661.3 675.4 690.4 649.3 663.4 691.3 704.4 675.4
S14N18 663.3 705.3 689.3 703.4 718.4 677.3 691.4 719.3 732.4 703.6
S14 2N12 747.4 789.4 773.4 787.5 802.5 761.4 775.6 803.4 816.5 787.5
S15N6 509.3 551.7 535.3 549.4 564.4 523.6 537.4 565.3 578.6 549.4
S15N7 523.7 565.3 549.3 563.4 578.4 537.3 551.4 5796 592.4 563.4
S15N8 537.3 579.3 563.3 577.4 592.4 551.3 565.4 593.3 606.4 577.4
S15N9 551.3 593.3 577.7 591.4 606.4 565.3 579.4 607.3 620.4 591.4
S15N11 579.7 621.3 605.3 619.7 634.6 593.3 607.7 635.3 648.8 619.9
S15N12 593.3 635.3 619.3 633.4 648.4 607.3 621.4 649.3 662.4 633.4
S15N13 607.5 649.3 633.3 647.7 662.4 621.3 635.4 663.3 676.4 647.4
S15N15 635.3 677.3 6615 675.4 690.4 649.3 663.4 691.3 704.4 675.4
S15N16 649.3 691.5 675.3 689.4 704.7 663.3 677.4 705.3 718.4 689.4
S15N18 677.3 719.3 703.3 717.4 732.4 691.3 705.4 733.3 746.4 717.4
S15 2N12 761.4 803.4 787.4 801.5 816.5 775.4 789.5 817.4 830.5 801.5
S16N6 523.3 565.3 549.3 563.4 578.4 537.3 551.4 579.3 592.4 563.4
S16N7 537.3 579.3 563.7 577.4 592.4 551.3 565.4 593.3 606.4 577.4
S16N8 551.6 593.6 577.3 591.4 606.4 565.3 579.4 607.3 620.4 591.4
S16N9 565.3 607.3 591.5 605.6 620.4 579.3 593.4 621.3 634.4 605.4
S16N11 593.3 635.3 619.3 633.4 648.7 607.3 621.4 649.3 662.4 633.4
S16N12 607.3 649.3 633.3 647.4 662.4 621.3 635.4 663.3 676.4 647.4
S16N13 621.3 663.6 647.3 661.4 676.4 635.3 649.4 677.3 690.4 661.4
S16N15 649.3 691.3 675.6 689.4 704.4 663.7 677.4 705.3 718.4 689.4
S16N16 663.3 705.3 689.3 703.4 718.4 677.3 691.4 719.3 732.4 703.4
S16N18 691.3 733.3 717.3 731.4 746.4 705.3 719.4 747.3 760.4 731.4
S16 2N12 775.4 817.4 801.4 815.5 830.5 789.4 803.5 831.7 844.7 815.5
S18N6 551.3 593.3 577.3 591.4 606.4 565.3 579.4 607.3 620.4 591.4
S18N7 565.3 607.3 591.3 605.4 620.4 579.3 593.4 621.3 634.4 605.8
S18N8 579.3 621.3 605.6 619.4 634.7 593.3 607.4 635.3 648.4 619.4
S18N9 593.3 635.3 619.3 633.4 648.4 607.5 621.6 649.7 662.8 633.8
S18N11 621.3 663.3 647.3 661.4 676.4 635.3 649.4 677.3 690.4 661.4
S18N12 635.3 677.3 661.3 675.4 690.4 649.3 663.4 691.3 704.4 675.4
S18N13 649.5 691.3 675.3 689.4 704.4 663.3 677.4 705.3 718.4 689.4
S18N15 677.3 719.3 703.3 717.4 732.4 691.3 705.4 733.3 746.4 717.4
S18N16 691.3 733.3 717.3 731.4 746.4 705.3 719.4 747.3 760.4 731.4
S18N18 719.3 761.6 745.3 759.4 774.4 733.3 747.4 775.3 788.4 759.4
  D1 D2 D3 D4 D5 D6 D7 D8 D9 D10
S18 2N12 803.4 845.4 829.7 843.7 858.5 817.4 831.5 859.4 872.5 843.5
S6S6 400.3 442.3 426.3 440.4 455.7 414.3 428.4 456.3 469.4 440.4
S6S7 414.3 456.3 440.3 454.4 469.4 428.3 442.4 470.3 483.4 454.4
S6S8 428.3 470.3 454.3 468.4 483.4 442.3 456.4 484.3 497.4 468.4
S6S9 442.3 484.3 468.3 482.4 497.4 456.3 470.4 498.3 511.4 482.4
S6S10 456.3 498.3 482.3 496.4 511.4 470.3 484.4 512.3 525.4 496.4
S6S11 470.3 512.3 496.3 521.4 525.4 484.3 484.3 526.5 539.4 510.4
S6S12 484.6 526.6 510.3 524.4 539.4 498.3 512.4 540.3 553.4 524.4
S6S14 512.3 554.3 538.5 552.4 567.4 526.3 540.4 568.3 581.4 552.4
S6S15 526.5 568.3 552.3 566.4 581.4 540.4 554.4 582.3 595.4 566.4
S6S16 540.3 582.3 566.3 580.4 595.4 554.3 568.4 596.3 609.4 580.4
S6S18 568.3 610.3 594.3 608.4 623.4 582.3 596.4 624.3 637.4 608.4
S7S7 428.5 470.3 454.3 468.4 483.4 442.3 456.4 484.3 497.4 468.4
S7S8 442.3 484.3 468.3 482.4 497.4 456.3 470.4 498.3 511.4 482.4
S7S9 456.3 498.3 482.3 496.4 511.4 470.3 484.4 512.3 525.4 496.4
S7S10 470.3 512.5 496.3 510.4 525.4 484.3 498.4 526.3 539.4 510.4
S7S11 484.3 526.3 510.3 524.4 539.4 498.3 512.4 540.3 553.4 524.4
S7S12 498.7 540.3 524.3 538.4 553.4 512.3 526.4 554.3 567.4 538.4
S7S14 526.3 568.3 552.3 566.4 581.4 540.3 554.4 582.3 595.4 566.4
S7S15 540.3 582.3 566.3 580.4 595.4 554.3 568.4 596.3 609.4 580.4
S7S16 554.6 596.3 580.3 594.4 609.4 568.3 582.4 610.3 623.4 594.4
S7S18 582.3 624.3 608.3 622.4 637.4 596.3 610.4 638.3 651.4 622.4
S8S8 456.8 498.3 482.3 496.4 511.4 470.3 484.4 512.3 525.4 496.4
S8S9 470.3 512.3 496.3 510.6 525.6 484.3 498.6 526.3 539.4 510.4
S8S10 484.7 526.3 510.3 524.4 539.4 498.3 512.4 540.3 553.8 524.7
S8S11 498.3 540.3 524.3 538.4 553.4 512.3 526.4 554.3 567.4 538.4
S8S12 512.3 554.3 538.3 552.4 567.4 526.3 540.4 568.3 581.4 552.4
S8S14 540.3 582.3 566.3 580.4 595.4 554.3 568.4 596.3 609.4 580.4
S8S15 554.3 596.3 580.3 594.4 609.4 568.3 582.4 610.3 623.4 594.4
S8S16 568.3 610.3 594.3 608.4 623.4 582.3 596.4 624.3 637.4 608.4
S8S18 596.3 638.3 622.3 636.4 651.4 610.3 624.4 652.3 665.4 636.4
S9S9 484.3 526.3 510.3 524.4 539.4 498.3 512.4 540.3 553.4 524.4
S9S10 498.3 540.7 524.6 538.4 553.4 512.3 526.4 554.3 567.4 538.4
S9S11 512.3 554.3 538.3 552.4 567.4 526.3 540.4 568.3 581.4 552.4
S9S12 526.3 568.3 552.3 566.4 581.4 540.3 554.4 582.3 595.4 566.4
S9S14 554.3 596.3 580.3 594.4 609.4 568.3 582.4 610.3 623.4 594.4
S9S15 568.3 610.6 594.3 608.5 623.4 582.5 596.6 624.3 637.4 608.4
S9S16 582.3 624.3 608.3 622.4 637.4 596.3 610.4 638.7 651.4 622.5
S9S18 610.3 652.3 636.3 650.4 665.4 624.3 638.4 666.3 679.4 650.4
S10S10 512.3 554.3 538.3 552.4 567.4 526.3 540.4 568.3 581.4 552.4
S10S11 526.3 568.3 552.3 566.4 581.4 540.3 554.4 582.3 595.4 566.4
S10S12 540.3 582.3 566.3 580.4 595.4 554.3 568.4 596.3 609.4 580.4
S10S14 568.3 610.6 594.3 608.4 623.4 582.3 596.4 624.3 637.4 608.4
S10S15 582.3 624.3 608.3 622.4 637.4 596.3 610.4 638.3 651.4 622.4
S10S16 596.3 638.3 622.3 636.4 651.4 610.3 624.4 652.3 665.4 636.4
S10S18 624.3 666.3 650.3 664.4 679.4 638.3 652.4 680.3 693.4 664.4
S11S11 540.5 582.3 566.5 580.4 595.4 554.3 568.4 596.3 609.4 580.4
  D1 D2 D3 D4 D5 D6 D7 D8 D9 D10
S11S12 554.3 596.3 580.3 594.4 609.4 568.3 582.4 610.3 623.4 594.4
S11S14 582.3 624.5 608.3 622.4 637.4 596.3 610.4 638.3 651.4 622.4
S11S15 596.3 638.3 622.6 636.4 651.4 610.3 624.4 652.3 665.4 636.4
S11S16 610.3 652.3 636.3 650.7 665.4 624.3 638.4 666.3 679.4 650.4
S11S18 638.3 680.3 664.3 678.4 693.4 652.3 666.4 694.3 707.4 678.4
S12S12 568.3 610.5 594.3 608.4 623.6 582.7 596.4 624.3 637.4 608.4
S12S14 596.3 638.3 622.3 636.4 651.4 610.3 624.4 652.3 665.4 636.4
S12S15 610.3 652.3 636.3 650.4 665.4 624.3 638.4 666.3 679.4 650.4
S12S16 624.3 666.3 650.3 664.4 679.4 638.3 652.4 680.3 693.4 664.4
S12S18 652.3 694.3 678.3 692.4 707.4 666.3 680.8 708.9 721.4 692.4
S14S14 624.3 666.3 650.3 664.4 679.4 638.3 652.4 680.3 693.4 664.4
S14S15 638.3 680.6 664.3 678.4 693.4 652.5 666.4 694.3 707.4 678.4
S14S16 652.3 694.3 678.3 692.4 707.4 666.3 680.4 708.3 721.4 692.4
S14S18 680.4 722.3 706.3 720.8 735.7 694.3 708.4 736.3 749.4 720.4
S15S15 652.3 694.5 678.3 692.4 707.4 666.3 680.4 708.3 721.4 692.4
S15S16 666.3 708.3 692.3 706.4 721.4 680.3 694.4 722.3 735.4 706.4
S15S18 694.3 736.3 720.3 734.4 749.4 708.3 722.4 750.3 763.4 734.4
S16S16 680.3 722.3 706.3 720.4 735.4 694.3 708.4 736.3 749.4 720.4
S16S18 708.3 750.3 734.3 748.4 763.4 722.3 736.4 764.3 777.4 748.4
S18S18 736.3 778.3 762.3 776.4 791.4 750.3 764.4 792.3 805.4 776.4
N6N6 366.4 408.4 392.4 406.8 421.5 380.4 394.5 422.4 435.5 406.5
N6N7 380.4 422.4 406.4 420.5 435.5 394.4 408.5 436.4 449.5 420.5
N6N8 394.4 436.4 420.4 434.5 449.6 408.4 422.5 450.4 463.5 434.5
N6N9 408.4 450.4 434.4 448.5 463.5 422.4 436.5 464.4 477.5 448.5
N6N11 436.4 478.4 462.4 476.4 491.4 450.4 464.4 492.4 492.4 476.4
N6N12 450.4 492.4 476.4 490.5 505.5 464.4 478.5 506.4 519.5 490.5
N6N13 464.4 506.4 490.4 504.5 519.5 478.4 492.5 520.4 533.5 504.5
N6N15 492.4 534.4 518.4 532.5 547.5 506.4 520.5 548.4 561.5 532.5
N6N16 506.4 548.4 532.4 546.5 561.5 520.4 534.5 562.4 575.5 546.5
N6N18 534.4 576.4 560.4 574.5 589.5 548.4 562.5 590.4 603.5 574.5
N6 2N12 618.5 660.5 644.5 658.6 673.6 632.5 646.6 674.5 687.6 658.6
N7N7 394.4 436.4 420.4 434.5 449.5 408.4 422.5 450.4 463.5 434.5
N7N8 408.4 450.4 434.4 448.5 463.5 422.4 436.5 464.4 477.5 448.5
N7N9 422.4 464.4 448.7 462.5 477.5 436.4 450.5 478.4 491.5 462.5
N7N11 450.4 492.4 476.4 490.5 505.5 464.4 478.5 506.4 519.5 490.5
N7N12 464.4 506.4 490.4 504.5 519.5 478.4 492.5 520.4 533.5 504.5
N7N13 478.4 520.4 504.4 518.5 533.5 492.4 506.5 534.4 547.5 518.5
N7N15 506.8 548.4 532.8 546.5 561.5 520.5 534.5 562.4 575.5 546.5
N7N16 520.4 562.4 546.4 560.5 575.5 534.4 548.5 576.4 589.5 560.5
N7N18 548.4 590.4 574.4 588.5 603.5 562.4 576.5 604.4 617.5 588.5
N7 2N12 632.7 674.7 658.5 672.6 687.6 646.5 660.6 688.5 701.6 672.6
N8N8 422.4 464.8 448.4 462.5 477.5 436.4 450.5 478.4 491.5 462.5
N8N9 436.4 478.4 462.4 476.5 491.5 450.4 464.5 492.4 505.5 476.5
N8N11 464.7 506.4 490.4 504.5 519.5 478.4 492.5 520.4 533.5 504.5
N8N12 478.5 520.4 504.4 518.5 533.8 492.4 506.5 534.4 547.5 518.5
N8N13 492.4 534.7 518.4 532.5 547.5 506.4 520.5 548.4 561.5 532.5
N8N15 520.4 562.6 546.4 560.5 575.5 534.4 548.5 576.4 589.5 560.5
  D1 D2 D3 D4 D5 D6 D7 D8 D9 D10
N8N16 534.4 576.8 560.4 574.5 589.5 548.4 562.5 590.4 603.5 574.5
N8N18 562.4 604.4 588.4 602.5 617.5 576.4 590.5 618.4 631.5 602.5
N8 2N12 646.5 688.5 672.5 686.6 701.6 660.5 674.6 702.5 715.6 686.6
N9N9 450.4 492.4 476.4 490.5 505.5 464.4 478.5 506.4 519.5 490.5
N9N11 478.4 520.4 504.4 518.5 533.5 492.4 506.5 534.4 547.5 518.5
N9N12 492.4 534.4 518.7 532.5 547.5 506.4 520.5 548.4 561.5 532.5
N9N13 506.4 548.4 532.4 546.5 561.5 520.4 534.5 562.4 575.5 546.5
N9N15 534.4 576.4 560.4 574.9 589.5 548.4 562.5 590.4 603.5 574.5
N9N16 548.4 590.4 574.4 588.5 603.5 562.9 576.5 604.4 617.5 588.5
N9N18 576.8 618.4 602.4 616.5 631.5 590.4 604.5 632.4 645.9 616.5
N9 2N12 660.5 702.5 686.9 700.6 715.6 674.5 688.6 716.5 729.6 700.6
N11N11 506.4 548.4 532.4 546.5 561.5 520.4 534.6 562.4 575.5 546.5
N11N12 520.4 562.4 546.4 560.5 575.5 534.4 548.5 576.4 589.5 560.5
N11N13 534.4 576.4 560.4 574.5 589.5 548.4 562.5 590.4 603.5 574.5
N11N15 562.4 604.4 588.4 602.5 617.5 576.4 590.5 618.4 631.5 602.5
N11N16 576.4 618.4 602.4 616.5 631.5 590.4 604.5 632.4 645.5 616.5
N11N18 604.4 646.4 630.4 644.5 659.5 618.4 632.5 660.4 673.5 644.5
N11 2N12 688.5 730.5 714.5 728.6 743.6 702.5 716.6 744.5 757.6 728.6
N12N12 534.4 576.4 560.4 574.5 589.5 548.4 562.5 590.4 603.5 574.5
N12N13 548.4 590.4 574.4 588.5 603.5 562.4 576.5 604.4 617.8 588.5
N12N15 576.4 618.4 602.4 616.5 631.5 590.4 604.5 632.8 645.5 616.5
N12N16 590.4 632.4 616.4 630.5 645.5 604.4 618.8 646.4 659.5 630.8
N12N18 618.4 660.4 644.4 658.5 673.5 632.4 646.5 674.4 687.5 658.5
N12 2N12 702.5 744.5 728.8 742.6 757.6 716.5 730.8 758.5 771.6 742.6
N13N13 562.4 604.4 588.4 602.8 617.5 576.4 590.5 618.4 631.5 602.5
N13N15 590.4 632.4 616.4 630.5 645.5 604.4 618.5 646.4 659.5 630.5
N13N16 604.4 646.4 630.4 644.5 659.8 618.4 632.5 660.4 673.5 644.5
N13N18 632.4 674.4 658.4 672.5 687.5 646.4 660.5 688.4 701.5 672.5
N13 2N12 716.5 758.5 742.5 756.6 771.6 730.6 744.6 772.5 785.6 756.6
N15N15 618.4 660.4 644.4 658.5 673.5 632.4 646.5 674.4 687.5 658.5
N15N16 632.4 674.4 658.4 672.5 687.9 646.4 660.5 688.4 701.5 672.5
N15N18 660.4 702.4 686.4 700.5 715.5 674.4 688.5 716.4 729.5 700.5
N15 2N12 744.5 786.5 770.5 784.6 799.6 758.5 772.6 800.5 813.6 784.6
N16N16 646.9 688.4 672.4 686.9 701.5 660.4 674.5 702.4 715.5 686.5
N16N18 674.4 716.9 700.4 714.7 729.5 688.4 702.5 730.4 743.5 714.5
N16 2N12 758.5 800.5 784.5 798.6 813.6 772.5 786.6 814.5 827.6 798.6
N18N18 702.9 744.4 728.4 742.5 757.5 716.8 730.5 758.4 771.5 742.5
N18 2N12 786.5 828.5 812.5 826.6 841.6 800.5 814.6 842.5 855.6 826.6
2N12 2N12 870.6 912.6 896.9 910.7 926.1 884.6 898.9 926.6 939.7 911.2
实施例1.1：S8NxDy系列胺基脂质化合物库的平行合成与表征
Figure PCTCN2021077852-appb-000050
其中n3、-NRR、R 1和R 2如上文所定义。
Figure PCTCN2021077852-appb-000051
在50mL干燥的反应瓶中加入三聚氯氰(2mmol)、THF(10mL)，冷浴搅拌降温至-20℃后，依次加入1-辛硫醇(2mmol)、DIPEA(2.4mmol)，反应液立即析出大量沉淀，反应5min后TLC检测，反应结束。将反应液转移至15mL离心管中，定容至15mL后于离心机离心(2000r/min,1min)。得Step I反应液(15mL,0.13M)。
用移液枪将Step I反应液转移至11个1.5mL的反应瓶中(每个1.15mL,0.15mmol)，相应的反应瓶中各加入直链烷基胺(0.15mmol)、DIPEA的THF溶液(0.35mL,0.5M)，于室温下搅拌反应2h，TLC检测无Step I原料。
用移液枪将每个Step II反应液分别转移至10个1.5mL的EP管中(每个0.1mL,0.01mmol)，相应的EP管中各加入二胺的THF溶液(0.12mL,0.012mmol，0.1M)、DIPEA的THF溶液(0.02mL,0.1M)，于加热型振摇反应器(Thermo-Shaker)中于78℃反应1h，TLC检测无Step II原料。反应结束后，将反应管内的溶剂常温挥干，即得到110个胺基脂质化合物S8NxDy。选取其中10个代表性化合物进行质谱检测，结果见下面的表1。
表1：代表性化合物的MW/z值
Figure PCTCN2021077852-appb-000052
Figure PCTCN2021077852-appb-000053
实施例1.1.1：代表性胺基脂质化合物S8N11D6的一锅法合成和表征.
Figure PCTCN2021077852-appb-000054
在20mL干燥的反应管中加入三聚氯氰(1mmol)、THF(10mL)，冷浴搅拌降温至-20℃后，依次加入1-辛硫醇(1mmol)、DIPEA(1.2mmol)，反应液立即析出大量沉淀，反应5min后TLC检测，反应结束。将反应液升至室温后，加入十一链烷基胺(1mmol)、DIPEA(1.2mol)，于室温下搅拌反应2h，TLC检测无Step I原料。再加入N,N-二甲基丙二胺(1.2mmol)、DIPEA(2mol)，搅拌下升温至78℃反应1h，TLC检测无Step II原料。使用自动纯化系统纯化(正向硅胶柱，DCM:MeOH从0至10％梯度洗脱)得白色固体440.4mg，三步反应的产率89％，纯度>99％。 1H NMR(400MHz,CDCl 3)δ3.54(m,2H),3.34(m,2H),3.01(m,4H),2.80-2.73(m,6H),2.07(m,2H),1.67(m,2H),1.53(m,2H),1.38(m,2H),1.28(m,2H),1.24(m,24H),0.88-0.84(m,6H).ESI-MS calculated for C27H55N6S+[M+H]+495.4,found 495.6.
实施例1.2：S6SxDy系列胺基脂质化合物库的平行合成与表征
Figure PCTCN2021077852-appb-000055
其中n3、-NRR、R 1和R 2如上文所定义。
Figure PCTCN2021077852-appb-000056
在50mL干燥的反应瓶中加入三聚氯氰(2mmol)、THF(10mL)，冷浴搅拌降温至-20℃后，依次加入1-己硫醇(2mmol)、DIPEA(2.4mmol)，反应液立即析出大量沉淀，反应5min后TLC检测，反应结束。将反应液转移至15mL离心管中，定容至15mL后于离心机离心(2000r/min,1min)。得Step I反应液(15mL,0.13M)。
用移液枪将步骤I反应液转移至11个1.5mL的反应瓶中(每个1.15mL,0.15mmol)，相应的反应瓶中各加入直链烷基硫醇(0.15mmol)、DIPEA的THF溶液(0.35mL,0.5M)，于室温下搅拌反应2h，TLC检测无Step I原料。
用移液枪将每个Step II反应液分别转移至10个1.5mL的EP管中(每个0.1mL,0.01mmol)，相应的EP管中各加入二胺的THF溶液(0.12mL,0.012mmol，0.1M)、DIPEA的THF溶液(0.02mL,0.1M)，于加热型振摇反应器(Thermo-Shaker)中于78℃反应1h，TLC检测无Step II原料。反应结束后，将反应管内的溶剂常温挥干，即得到110个胺基脂质化合物S6SxDy。选取其中10个代表性化合物进行质谱检测，结果见下面的表2。
表2：代表性化合物MW/z值
Figure PCTCN2021077852-appb-000057
Figure PCTCN2021077852-appb-000058
实施例1.2.1：代表性胺基脂质化合物S6S11D6的一锅法合成和表征
Figure PCTCN2021077852-appb-000059
在20mL干燥的反应管中加入三聚氯氰(1mmol)、THF(10mL)，冷浴搅拌降温至-20℃后，依次加入1-己硫醇(1mmol)、DIPEA(1.2mmol)，反应液立即析出大量沉淀，反应5min后TLC检测，反应结束。将反应液升至室温后，加入十一链烷基硫醇(1mmol)、DIPEA(1.2mol)，于室温下搅拌反应2h，TLC检测无Step I原料。再加入N,N-二甲基丙二胺(1.2mmol)、DIPEA(2mol)搅拌下升温至78℃反应1h，TLC检测无Step II原料。使用自动纯化系统纯化(正向硅胶柱，DCM:MeOH从0至10％梯度洗脱)得白色固体445.1mg,三步反应的产率92％，纯度99％。 1H NMR(400MHz,CDCl 3)δ3.51(m,2H),3.04-3.01(m,4H),2.81-2.74(m,6H),2.09(m,2H),1.68(m,2H),1.55(m,2H),1.33(m,2H),1.29(m,2H),1.23(m,22H),0.88-0.84(m,6H).ESI-MS calculated for C 25H 50N 5S 2 +[M+H] +484.4,found 484.4.
实施例1.3：N6NxDy系列胺基脂质化合物库的平行合成与表征
Figure PCTCN2021077852-appb-000060
其中n3、-NRR、R 1和R 2如上文所定义。
Figure PCTCN2021077852-appb-000061
在50mL干燥的反应瓶中加入三聚氯氰(2mmol)、THF(10mL)，冷浴搅拌降温至-20℃后， 依次加入1-己胺(2mmol)、DIPEA(2.4mmol)，反应液立即析出大量沉淀，反应5min后TLC检测，反应结束。将反应液转移至15mL离心管中，定容至15mL后于离心机离心(2000r/min,1min)。得Step I反应液(15mL,0.13M)。
用移液枪将Step I反应液转移至11个1.5mL的反应瓶中(每个1.15mL,0.15mmol)，相应的反应瓶中各加入直链烷基胺(0.15mmol)，DIPEA的THF溶液(0.35mL,0.5M)，于室温下搅拌反应2h，TLC检测无Step I原料。
用移液枪将每个Step II反应液分别转移至10个1.5mL的EP管中(每个0.1mL,0.01mmol)，相应的EP管中各加入二胺的THF溶液(0.12mL,0.012mmol，0.1M)、DIPEA的THF溶液(0.02mL,0.1M)，于加热型振摇反应器(Thermo-Shaker)中于78℃反应1h，TLC检测无Step II原料。反应结束后，将反应管内的溶剂常温挥干，即得到110个胺基脂质化合物N6NxDy。选取其中10个代表性化合物进行质谱检测，结果见表3。
表3：代表性化合物MW/z值
Figure PCTCN2021077852-appb-000062
Figure PCTCN2021077852-appb-000063
实施例1.3.1：代表性胺基脂质化合物N6N11D6的一锅法合成和表征
Figure PCTCN2021077852-appb-000064
在20mL干燥的反应管中加入三聚氯氰(1mmol)、THF(10mL)，冷浴搅拌降温至-20℃后，依次加入1-己胺(1mmol)、DIPEA(1.2mmol)，反应液立即析出大量沉淀，反应5min后TLC检测，反应结束。将反应液升至室温后，加入十一链烷基胺(1mmol)、DIPEA(1.2mol)，于室温下搅拌反应2h，TLC检测无Step I原料。再加入N,N-二甲基丙二胺(1.2mmol)、DIPEA(2mol)搅拌下升温至78℃反应1h，TLC检测无Step II原料额。使用自动纯化系统纯化(正向硅胶柱，DCM:MeOH从0至10％梯度洗脱)得白色固体395.7mg,三步反应的产率88％，纯度99％。 1H NMR(400MHz,CDCl 3)δ3.54(m,2H),3.05-3.02(m,4H),2.82-2.73(m,6H),2.08(m,2H),1.69(m,2H),1.54(m,2H),1.34(m,2H),1.30(m,2H),1.24(m,22H),0.89-0.85(m,6H).ESI-MS calculated for C 25H 52N 7 +[M+H] +450.4,found 450.4.
实施例1.4：S6SxOy系列胺基脂质化合物库的平行合成与表征
Figure PCTCN2021077852-appb-000065
其中n4＝1、2、3或4；-NR’R’表示上文中的O1至O10中的二烷基氨基或环氨基部分；并且R 1和R 2如上文所定义。
Figure PCTCN2021077852-appb-000066
在50mL干燥的反应瓶中加入三聚氯氰(2mmol)、THF(10mL)，冷浴搅拌降温至-20℃后，依次加入1-己硫醇(2mmol)、DIPEA(2.4mmol)，反应液立即析出大量沉淀，反应5min后TLC检测，反应结束。将反应液转移至15mL离心管中，定容至15mL后于离心机离心(2000r/min,1min)。得Step I反应液(15mL,0.13M)。
用移液枪将步骤I反应液转移至11个1.5mL的反应瓶中(每个1.15mL,0.15mmol)，相应的反应瓶中各加入直链烃基硫醇(0.15mmol)、DIPEA的THF溶液(0.35mL,0.5M)，于室温下搅拌反应2h，TLC检测无Step I原料。
用移液枪将每个Step II反应液分别转移至10个1.5mL的EP管中(每个0.1mL,0.01mmol)，相应的EP管中各加入羟胺的THF溶液(0.12mL,0.012mmol，0.1M)、DIPEA的THF溶液(0.02mL,0.1M)，于振摇反应器(Thermo-Shaker)中室温反应2h，TLC检测无Step II原料。反应结束后，将反应管内的溶剂常温挥干，即得到110个胺基脂质化合物S6SxOy。选取其中10个代表性化合物进行质谱检测，结果见下面的表1.4。
表1.4：代表性化合物MW/z值
Figure PCTCN2021077852-appb-000067
Figure PCTCN2021077852-appb-000068
实施例1.4.1：代表性胺基脂质化合物S19S9O8的一锅法合成和表征
Figure PCTCN2021077852-appb-000069
在20mL干燥的反应管中加入三聚氯氰(1mmol)、THF(10mL)，冷浴搅拌降温至-20℃后，依次加入1-油硫醇(1mmol)、DIPEA(1.2mmol)，反应液立即析出大量沉淀，反应5min后TLC检测，反应结束。将反应液升至室温后，加入1-壬硫醇(1mmol)、DIPEA(1.2mol)，于室温下搅拌反应2h，TLC检测无Step I原料。再加入N-(2-羟乙基)-吡咯烷(1.2mmol)、DIPEA(2mol)，于室温下反应2h，TLC检测无Step II原料。使用自动纯化系统纯化(正向硅胶柱，DCM:MeOH从0至10％梯度洗脱)得白色固体546.2mg,三步反应的产率86％，纯度99％。 1H NMR(400MHz,CDCl 3)δ5.35(m,2H),4.21(m,2H),3.51(m,4H),2.67-2.54(m,6H),2.19(m,4H),1.68(m,4H),1.55-1.23(m,38H),0.88-0.84(m,6H).ESI-MS calculated for C 36H 67N 4OS 2 +[M+H] +635.5,found 635.7.
实施例2：胺基脂质化合物制备的脂质纳米颗粒的荧光素酶mRNA体内递送性能评价
1.脂质纳米颗粒的制备
制剂方法1：
将本发明的胺基脂质化合物与DOPE、胆固醇、PEG2000-DMG按45:10:42.5:2.5的摩尔比混合并溶解在无水乙醇中，使胺基脂质化合物的摩尔浓度为0.001-0.01mmol/L。使用微量注射泵，使所得的乙醇溶液和溶解有荧光素酶mRNA(Fluc mRNA,TriLink)的醋酸钠溶液(50mM,pH＝4.0)以1:3的体积比在微流道芯片中混合以制得脂质纳米颗粒的粗溶液，然后将其用透析盒(Fisher，MWCO 20,000)在1X PBS、控温4℃下透析6h，在使用前通过0.22μm的微孔滤膜过滤。胺基脂质化合物与Fluc mRNA的质量比约为10:1。所得的脂质纳米颗粒(LNP)溶液通过皮下给药方式施用于受试动物。
脂质纳米颗粒的表征：
粒径的表征：所制备的脂质纳米颗粒的粒径和PDI通过Nano-ZSZEN3600(Malvern)测定。取LNP溶液40uL进行粒径测量，循环三次，每次循环30s。
包封率检测：使用
Figure PCTCN2021077852-appb-000070
RNA HS Assay试剂盒检测LNP RNA浓度。理论RNA浓度为投入的总RNA量除以最终溶液的总体积。
Figure PCTCN2021077852-appb-000071
表4：使用制剂方法1用代表性胺基脂质化合物制备的LNP的表征数据。
Figure PCTCN2021077852-appb-000072
Figure PCTCN2021077852-appb-000073
Figure PCTCN2021077852-appb-000074
制剂方法2：
制备方法同制剂方法1，除了使用摩尔比为50:10:38.5:1.5的胺基脂质化合物、DSPC、胆固醇和PEG2000-DMG。所得的脂质纳米颗粒(LNP)溶液通过尾静脉和肌肉注射的给药方式施用于受试动物。
表5：使用制剂方法2用代表性胺基脂质化合物制备的LNP的表征数据。
Figure PCTCN2021077852-appb-000075
2.动物实验
动物准备：选取体重约20g的6周龄的雌性BALB/c小鼠，于SPF级的饲养室中饲养。动物试验严格按照国家健康机构的指南以及动物伦理要求进行。
体内递送：每组随机选取9只小鼠，按0.5mg/kg Fluc mRNA的用量，分别使用皮下、肌肉和尾静脉注射三种给药方式注射脂质纳米颗粒溶液(每种给药方式3只小鼠)。12小时后，往每只小鼠体内通过尾静脉注射200μL 10mg/mL的D-荧光素钾盐(PerkinElmer)，10分钟后，将小鼠放置于活体成像系统(IVIS-200,Xenogen)下，观察每只小鼠总的萤光强度，并拍照记录下来。代表性胺基脂质化合物通过3种给药方式递送的Fluc mRNA的表达强度见表5-7。DLin-MC3作为对照。
表5：代表性胺基脂质化合物皮下给药递送的Fluc mRNA的表达强度。
胺基脂质编号 萤光强度
S16N9D3 2.7E+07
S15N11D5 1.9E+08
S12N13D6 8.6E+06
S16N7D9 1.4E+07
CD10 3.7E+08
S7S18D3 2.9E+08
S9S16D5 4.2E+07
S11S12D8 1.1E+07
S8S16D9 4.2E+06
S10S16D10 4.7E+07
S19S9O8 1.1E+07
S19S9O9 2.8E+07
DLin-MC3(对照) 3.1E+06
表6：代表性胺基脂质化合物肌注给药递送的Fluc mRNA的表达强度。
编号 萤光强度
S16N9D3 4.3E+06
S15N11D5 3.7E+06
S12N13D6 1.2E+06
S16N7D9 4.3E+07
S16N8D10 3.9E+06
S7S18D3 8.7E+07
S9S16D5 5.2E+06
S11S12D8 4.7E+06
S8S16D9 8.2E+06
S10S16D10 3.2E+06
S19S9O8 9.1E+07
S19S9O9 1.8E+07
DLin-MC3(对照) 2.7E+07
表7：代表性胺基脂质化合物尾静脉给药递送的Fluc mRNA的表达强度。
编号 萤光强度
S16N9D3 5.3E+06
S15N11D5 3.7E+06
S12N13D6 3.9E+06
S16N7D9 5.1E+07
S16N8D10 3.2E+06
S7S18D3 8.1E+07
S9S16D5 7.5E+06
S11S12D8 3.8E+06
S8S16D9 6.5E+06
S10S16D10 2.9E+06
S19S9O8 1.1E+08
S19S9O9 3.1E+07
DLin-MC3(对照) 2.7E+07
实施例3：胺基脂质化合物制备的脂质纳米颗粒的卵清蛋白mRNA体内递送及免疫性能评价
制剂方法：
将本发明的胺基脂质化合物与DOPE、胆固醇、PEG2000-DMG按50:10:38.5:1.5的摩尔比混合并溶解在无水乙醇中，使胺基脂质化合物的摩尔浓度为0.001-0.01mmol/L。卵清蛋白mRNA(OVA mRNA,TriLink)溶解在醋酸钠溶液(50mM,pH＝4.0)中。使用微量注射泵，使所得的乙醇溶液和醋酸钠溶液(50mM,pH＝4.0)以1:3的体积比在微流道芯片中混合以制得脂质纳米颗粒的粗溶液，然后使用透析盒(Fisher，MWCO 20,000)在1X PBS、控温4℃下透析6h，在使用前通过0.22μm的微孔滤膜过滤。胺基脂质化合物与卵清蛋白mRNA(OVA mRNA)的质量比约为10:1。
动物准备：选取体重约20g的6周龄的雌性BALB/c小鼠，于SPF级的饲养室中饲养。动物试验严格按照国家健康机构的指南以及动物伦理要求进行。
体内递送：每组随机选取3只小鼠，按0.5mg/kg mRNA的用量，在腿部肌肉注射脂质纳米颗粒溶液(Day 0)。7天后，使用相同的量再加强一次(Day 7)。在第21天尾静脉取血，用于血清学分析。DLin-MC3作为对照(图1)
酶联免疫吸附测定(ELISA)：对平底96孔板(Nunc)进行预涂在50mM碳酸盐缓冲液中，OVA蛋白的浓度为每孔0.5μg蛋白(pH 9.6)在4℃过夜，然后用5％甘氨酸封闭。将获得自接受了含有mRNA的脂质纳米颗粒溶液的动物的血清用pH 7.4的PBS-0.05％Tween(PBS-T)从10 2稀释至10 6 倍，并添加到孔中并在室温下孵育在37℃下放置1小时。辣根过氧化物酶(HRP,Invitrogen)偶联的山羊抗小鼠IgG在PBS-T-1％BSA中以1：10,000的稀释度进行标记。加入HRP底物后，在ELISA酶标仪(Bio-Rad)中测定450nm下的吸光度。如图1所示，S7S18D3、S16N7D9、S19S9O8和S19S9O9的IgG抗体滴定显著优于对照组。
实施例4：胺基脂质化合物作为DNA载体的初步筛选
细胞系：HEK293细胞(ATCC CRL-1573 TM)
培养基：补充了10％胎牛血清的DMEM(Invitrogen)
筛选形式：96孔板细胞转染
检测(读出)：相对于总细胞数(使用核染料Hoechst测定总细胞数－参见图2)的GFP荧光细胞数百分比例。根据制造商的说明，Lipofectamine2000(Invitrogen)用作阳性对照组。
方法：使用8通道移液管加样。所示的含量为96孔平板的单孔。
1.将实施例1中制备得到的化合物(0.01mmol)溶解在1mL的无水乙醇中，超声溶解后，从中取出10μL所得的胺基脂质的乙醇溶液和5μL的DOPE乙醇溶液(0.01M)混合，然后向所得的混合物中加入35μL的0.2M醋酸钠缓冲液(pH 5.6)，使用恒定的转速涡旋30s，制得50μL脂质体溶液。再从50μL脂质体溶液中取出4μL添加至46μL的0.02M乙酸钠缓冲液(pH 5.6)中，使用恒定的转速涡旋30s以形成最终的脂质体溶液。从上述最终的脂质体溶液中取出5μL与溶于5μL的0.02M醋酸钠缓冲液的75ng质粒DNA(pSin-EGFP-IRES-Puro,Clontech)混合，室温下放置30min，以形成脂质/DNA转染复合物。
2. 10μL的脂质/DNA转染复合物在室温下孵育30min后，加入90μL新鲜重悬浮的细胞(3-5×10 4细胞)，并用移液管混合。将100μL的细胞+脂质/DNA复合物立即转移至96-孔培养平板的分开孔中，并且置于37℃的含有5％CO 2的培养箱中。
3.细胞初始转染后20至24小时，将Hoechst33258(Invitrogen)以0.2μg/ml的终浓度加入细胞中，在37℃黑暗下培养15min。然后用PBS溶液清洗细胞1次，再加入培养基培养20至24小时。
4.将细胞置于高通量共聚焦显微镜(Molecular Devices ImageXpress)中，从每个孔捕获细胞的4个图像视野，对于每个样品，捕获3种激光波长图像：细胞的亮视野图像(图2(a)和图2(b))，显示总细胞核的Hoechst染色图像(图2(c)和图2(d))和显示用质粒DNA成功转染并表达GFP的GFP图像(图2(e)和图2(f))。用MetaXpress软件对获得的Hoechst染色图像和GFP图像分别进行细胞计数，再将表达GFP的细胞数除以总细胞核数，即为细胞绝对转染效率。绝对转染效率如下计算：
Figure PCTCN2021077852-appb-000076
结果：2530种化合物对HEK293细胞的DNA的转染效率示于表8中。
表8：2530种化合物对HEK293细胞的DNA的绝对转染效率。
Figure PCTCN2021077852-appb-000077
Figure PCTCN2021077852-appb-000078
Figure PCTCN2021077852-appb-000079
Figure PCTCN2021077852-appb-000080
Figure PCTCN2021077852-appb-000081
Figure PCTCN2021077852-appb-000082
Figure PCTCN2021077852-appb-000083
实施例5：胺基脂质化合物作为mRNA载体的初步筛选
细胞系：HEK293细胞(ATCC CRL-1573 TM)
培养基：补充了10％胎牛血清的DMEM(Invitrogen)
筛选形式：96孔板细胞转染
检测(读出)：相对于总细胞数(使用核染料Hoechst测定总细胞数)的GFP荧光细胞数百分比例。Lipofectamine2000(Invitrogen)用作阳性对照组。
方法：实验方法与实施例4基本相同，转染的EGFP mRNA(TriLink)质量为50ng每孔。
结果：2530种化合物对HEK293细胞的mRNA绝对转染效率示于表9中。
表9：2530种化合物对HEK293细胞的mRNA绝对转染效率。
Figure PCTCN2021077852-appb-000084
Figure PCTCN2021077852-appb-000085
Figure PCTCN2021077852-appb-000086
Figure PCTCN2021077852-appb-000087
Figure PCTCN2021077852-appb-000088
Figure PCTCN2021077852-appb-000089
Figure PCTCN2021077852-appb-000090
实施例6：胺基脂质化合物作为DNA载体的对Hela细胞株的转染
细胞系：Hela细胞(ATCC 35241)
培养基：补充了10％胎牛血清的DMEM(Invitrogen)
筛选形式：96孔板细胞转染
检测(读出)：相对于总细胞数(使用核染料Hoechst测定总细胞数)的GFP荧光细胞数百分比例。根据制造商的说明，Lipofectamine2000(Invitrogen)用作阳性对照组。
方法：实验方法与实施例4基本相同。
结果：170种化合物对Hela细胞的DNA绝对转染效率示于表10中。
表10：170种化合物对Hela细胞的DNA绝对转染效率。
Figure PCTCN2021077852-appb-000091
实施例7：胺基脂质化合物作为mRNA载体的对Hela细胞株的转染
细胞系：Hela细胞(ATCC 35241)
培养基：补充了10％胎牛血清的DMEM(Invitrogen)
筛选形式：96孔板细胞转染
检测(读出)：相对于总细胞数(使用核染料Hoechst测定总细胞数)的GFP荧光细胞数百分比例。根据制造商的说明，将可购得的脂质体转染试剂Lipofectamine2000(Invitrogen)用作阳性对照组。
方法：实验方法与实施例4基本相同，转染的EGFP mRNA质量为50ng每孔。
结果：170种化合物对Hela细胞的mRNA绝对转染效率示于表11中。
表11：170种化合物对Hela细胞的mRNA绝对转染效率。
Figure PCTCN2021077852-appb-000092
实施例8：胺基脂质化合物作为DNA载体的对MCF7细胞株的转染
细胞系：MCF7细胞(ATCC HTB-22)
培养基：补充了10％胎牛血清的DMEM
筛选形式：96孔板细胞转染
检测(读出)：相对于总细胞数(使用核染料Hoechst测定总细胞数)的GFP荧光细胞数百分比例。根据制造商的说明，Lipofectamine2000用作阳性对照组。
方法：实验方法与实施例4基本相同。
结果：102种化合物对MCF7细胞的DNA绝对转染效率示于表12中。
表12：102种化合物对MCF7细胞的DNA绝对转染效率
Figure PCTCN2021077852-appb-000093
Figure PCTCN2021077852-appb-000094
实施例9：胺基脂质化合物作为mRNA载体的对MCF7细胞株的转染
细胞系：MCF7细胞(ATCC HTB-22)
培养基：补充了10％胎牛血清的DMEM(Invitrogen)
筛选形式：96孔板细胞转染
检测(读出)：相对于总细胞数(使用核染料Hoechst测定总细胞数)的GFP荧光细胞数百分比例。Lipofectamine2000(Invitrogen)用作阳性对照组。
方法：实验方法与实施例4基本相同，转染的EGFP mRNA(TriLink)质量为50ng每孔。
结果：102种化合物对MCF7细胞的mRNA绝对转染效率示于表13中。
表13：102种化合物对MCF7细胞的mRNA绝对转染效率。
Figure PCTCN2021077852-appb-000095
实施例10：胺基脂质化合物作为DNA载体在难转染细胞系(胚胎干细胞)的筛选
细胞系：胚胎干细胞(hESC2，按已报道的方法培养Stem Cells,2005,23,544-549)
培养基：mTeSR1(STEMCELL)
筛选形式：96孔板细胞转染
检测(读出)：相对于总细胞数的GFP荧光细胞数百分比例。Lipofectamine Stem用作阳性对照组。
方法：使用8通道移液管加样。所示的含量为96孔平板的单孔。
1.在基质胶处理过的96孔板中培养干细胞至每孔3-5×10 4细胞。
2.将实施例1中制备得到的化合物(0.01mmol)溶解在1mL的无水乙醇中，超声溶解后，取10μL所得的胺基脂质的乙醇溶液和5μL的DOPE乙醇溶液(0.01M)混合，然后向混合物中加入35μL的0.2M醋酸钠缓冲液(pH 5.6)，使用恒定的转速涡旋30s。再从所得的产物中取出4μL添加 至46μL的0.02M醋酸钠缓冲液(pH 5.6)中，使用恒定的转速涡旋30s以形成脂质体溶液。将5μL该脂质体溶液与溶于5μL的0.02M醋酸钠缓冲液的75ng质粒DNA(pSin-EGFP-IRES-Puro)混合，室温下放置30min，以形成脂质/DNA转染复合物。
3. 10μL的脂质/DNA转染复合物在室温下孵育30min后，加入90μL mTeSR1培养基，并用移液管混合。将100μL的混合物加至96-孔培养平板的分开孔中，并且置于37℃的含有5％CO 2的培养箱中。
4.细胞转染后48小时，用0.05％的Trypsin-EDTA消化细胞，并且将细胞在PBS缓冲液中重悬，过滤。将细胞悬液放入流式细胞仪中，测量不少于2000个细胞中表达GFP的细胞数量，从而得到细胞转染的绝对效率。将细胞转染效率与阳性对照组Lipofectamine Stem作对比，得到细胞转染的相对效率。
结果：60种化合物对胚胎干细胞的DNA绝对转染效率示于表14中。
表14：60种化合物对胚胎干细胞的DNA绝对转染效率。
Figure PCTCN2021077852-appb-000096
实施例11：胺基脂质化合物作为mRNA载体在难转染细胞系(胚胎干细胞)的筛选
细胞系：胚胎干细胞(hESC2，按已报道方法培养Stem Cells,2005,23,544-549)
培养基：mTeSR1(STEMCELL)
筛选形式：96孔板细胞转染
检测(读出)：相对于总细胞数的GFP荧光细胞数百分比例。Lipofectamine Stem用作阳性对照组。
方法：实验方法与实施例10基本相同，转染的EGFP mRNA质量为50ng每孔。
结果：60种化合物对胚胎干细胞的mRNA绝对转染效率示于表15中。
表15：60种化合物对胚胎干细胞的mRNA绝对转染效率。
Figure PCTCN2021077852-appb-000097
实施例12：胺基脂质化合物作为DNA载体在难转染细胞系(心肌细胞)的筛选
细胞系：心肌细胞(按已报道的方法由hESC诱导分化而来,J.Mol.Cell.Cardiol.2011,51,288-298)
培养基：补充了10％胎牛血清的DMEM(Invitrogen)
筛选形式：96孔板细胞转染
检测(读出)：相对于总细胞数(使用核染料Hoechst测定总细胞数)的GFP荧光细胞数百分比例。根据制造商的说明，Lipofectamine Stem(Invitrogen)用作阳性对照组。
方法：实验方法与实施例4基本相同。
结果：60种化合物对心肌细胞的DNA绝对转染效率示于表16中。
表16：60种化合物对心肌细胞的DNA绝对转染效率。
Figure PCTCN2021077852-appb-000098
实施例13：胺基脂质化合物作为mRNA载体在难转染细胞系(心肌细胞)的筛选
细胞系：心肌细胞(按已报道的方法由hESC诱导分化而来,J.Mol.Cell.Cardiol.2011,51,288-298)
培养基：补充了10％胎牛血清的DMEM(Invitrogen)
筛选形式：96孔板细胞转染
检测(读出)：相对于总细胞数(使用核染料Hoechst测定总细胞数)的GFP荧光细胞数百分比例。根据制造商的说明，Lipofectamine Stem(Invitrogen)用作阳性对照组。
方法：实验方法与实施例4基本相同，转染的EGFP mRNA质量为50ng每孔。
结果：60种化合物对心肌细胞的mRNA绝对转染效率示于表17中。
表17：60种化合物对心肌细胞的mRNA绝对转染效率。
Figure PCTCN2021077852-appb-000099
缩写列表：
DMEM         基础高糖培养基
DNA          脱氧核糖核酸
DOPE         二油酰基磷脂酰乙醇胺
DSPC         二硬脂酰磷脂酰胆碱
PEG2000-DMG  (1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇2000(1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000))；
GFP          绿色荧光蛋白
EGFP         增强的GFP
kD           千道尔顿
RNA          核糖核酸
THF          四氢呋喃
DIPEA        N,N-二异丙基乙胺
PBS          磷酸盐缓冲溶液。
除本文中描述的那些外，根据前述描述，本发明的各种修改对本领域技术人员而言会是显而易见的。这样的修改也意图落入所附权利要求书的范围内。本申请中所引用的各参考文献(包括所有专利、专利申请、期刊文章、书籍及任何其它公开)均以其整体援引加入本文。

Claims (16)

  1. 胺基脂质化合物，其中所述胺基脂质化合物是由以下式I表示的化合物：
    Figure PCTCN2021077852-appb-100001
    其中：
    R 1和R 2彼此相同或不同，并且各自独立地选自C 6-C 24烷基、C 6-C 24烯基、C 6-C 24炔基和C 4-C 24酰基，其中所述C 6-C 24烷基、所述C 6-C 24烯基、所述C 6-C 24炔基和所述C 4-C 24酰基任选地被C 1-C 6烃基取代；
    X 1、X 2和X 3彼此相同或不同，并且各自独立地选自C、N、O、S、S＝O、S(＝O) 2和S-S；
    当X 1是C时，m＝2，并且两个R 6彼此相同或不同；当X 1是N时，m＝1；当X 1是O、S、S＝O、S(＝O) 2或S-S时，m＝0；
    当X 2是C时，n＝2，并且两个R 7彼此相同或不同；当X 2是N时，n＝1；当X 2为O、S、S＝O、S(＝O) 2或S-S时，n＝0；
    当X 3是C时，p＝2，并且两个R 8彼此相同或不同；当X 3是N时，p＝1；当X 3为O、S、S＝O、S(＝O) 2或S-S时，p＝0；
    R 3和R 4彼此相同或不同，并且各自独立地选自C 1-C 12烷基、C 2-C 12烯基和C 2-C 12炔基，其中所述C 1-C 12烷基、所述C 2-C 12烯基和所述C 2-C 12炔基任选地被C 1-C 6烃基取代，或者R 3和R 4彼此结合以形成包含选自氮、硫和氧中的1至6个杂原子的任选地取代的4至10元杂环；
    R 5不存在，或者R 5是氢或C 1-C 12烷基以提供季胺；
    R 6、R 7、R 8是氢或C 1-C 12烷基；
    L是C 1-C 12亚烷基、C 2-C 12亚烯基或C 2-C 12亚炔基，其中所述C 1-C 12亚烷基、所述C 2-C 12亚烯基和所述C 2-C 12亚炔基任选地被选自烃基、羧基、酰基和烷氧基中的一个或多个取代基取代，或者L是包含选自氮、硫和氧中的杂原子的任选地取代的4至10元杂环。
  2. 根据权利要求1所述的胺基脂质化合物，其中：
    X 1为C，R 6为H，m＝2；或者X 1为N，R 6为H，m＝1；或者X 1为O、S、S＝O、S(＝O) 2或S-S，m＝0；和/或
    X 2为C，R 7为H，n＝2；或者X 2为N，R 7为H，n＝1；或者X 2为O、S、S＝O、S(＝O) 2或S-S，n＝0；和/或
    X 3为C，R 8为H，p＝2；或者X 3为N，R 8为H，p＝1；或者X 3为O、S、S＝O、S(＝O) 2或S-S，p＝0。
  3. 根据权利要求1所述的胺基脂质化合物，其中：
    X 1和X 2彼此相同或不同，并且各自独立地为N或S；
    当X 1是N时，m＝1；当X 1为S时，m＝0；
    当X 2是N时，n＝1；当X 2为S时，n＝0；和/或
    X 3为N且p＝1，或者X 3为O且p＝0；和/或
    R 5不存在；和/或
    L是C 1-C 12亚烷基，所述C 1-C 12亚烷基任选地被C 1-C 6烃基取代，例如L是(CH 2) q，其中q是1至12的整数，例如1至8或1至6的整数。
  4. 根据权利要求3所述的胺基脂质化合物，其中：
    当m＝1时，R 6是氢；和/或
    当n＝1时，R 7是氢；和/或
    R 8是氢。
  5. 根据权利要求1、3或4所述的胺基脂质化合物，其中：
    R 1和R 2彼此相同或不同，并且各自独立地是C 6-C 24烷基或C 6-C 24烯基；
    X 1为N，R 6为H，m＝1；或者X 1为S，m＝0；
    X 2为N，R 7为H，n＝1；或者X 2为S，n＝0；
    X 3为N，R 8为H，p＝1；
    R 3和R 4彼此相同或不同，并且各自独立地是C 1-C 12烷基，其中，所述C 1-C 12烷基任选地被C 1-C 6烃基取代，或者R 3和R 4彼此结合以形成包含选自氮、硫和氧中的1至6个杂原子的任选地取代的4至10元杂环；并且
    R 5不存在。
  6. 根据权利要求1至5中任意一项所述的胺基脂质化合物，其中：
    L是C 1-C 4亚烷基，其中所述C 1-C 4亚烷基任选地被C 1-C 6烃基取代，例如L是C 2-C 4亚烷基，如(CH 2) q，其中q是1、2、3或4。
  7. 根据权利要求1至6中任意一项所述的胺基脂质化合物，其中：
    R 1和R 2彼此相同或不同，并且各自独立地是C 6-C 18烷基或C 6-C 18烯基。
  8. 根据权利要求1和3至7中任一项所述的胺基脂质化合物，其中：
    m＝0或1，n＝0或1，并且
    Figure PCTCN2021077852-appb-100002
    彼此相同或不同，并且各自独立地是选自以下的S6、S7、S8、S9、S10、S11、S12、S14、S15、S16、S18、S19、S20、N6、N7、N8、N9、N11、N12、N13、N15、N16、N18和 2N 12中的一种：
    S6:CH 3(CH 2) 5S-；S7:CH 3(CH 2) 6S-；S8:CH 3(CH 2) 7S-；
    S9:CH 3(CH 2) 8S-；S10:CH 3(CH 2) 9S-；S11:CH 3(CH 2) 10S-；
    S12:CH 3(CH 2) 11S-；S14:CH 3(CH 2) 13S-；S15:CH 3(CH 2) 14S-；
    S16:CH 3(CH 2) 15S-；S18:CH 3(CH 2) 17S-；
    S19：
    Figure PCTCN2021077852-appb-100003
    S20：
    Figure PCTCN2021077852-appb-100004
    N6:CH 3(CH 2) 5NH-；N7:CH 3(CH 2) 6NH-；N8:CH 3(CH 2) 7NH-；
    N9:CH 3(CH 2) 8NH-；N11:CH 3(CH 2) 10NH-；N12:CH 3(CH 2) 11NH-；
    N13:CH 3(CH 2) 12NH-；N15:CH 3(CH 2) 14NH-；N16:CH 3(CH 2) 15NH-；
    N18:CH 3(CH 2) 17NH-； 2N 12:(CH 3(CH 2) 11) 2N-。
  9. 根据权利要求1和3至8中任一项所述的胺基脂质化合物，其中：
    p＝1，并且
    Figure PCTCN2021077852-appb-100005
    是选自以下的D1、D2、D3、D4、D5、D6、D7、D8、D9和D10中的一种：
    D1：
    Figure PCTCN2021077852-appb-100006
    D2：
    Figure PCTCN2021077852-appb-100007
    D3：
    Figure PCTCN2021077852-appb-100008
    D4：
    Figure PCTCN2021077852-appb-100009
    D5：
    Figure PCTCN2021077852-appb-100010
    D6：
    Figure PCTCN2021077852-appb-100011
    D7：
    Figure PCTCN2021077852-appb-100012
    D8：
    Figure PCTCN2021077852-appb-100013
    D9：
    Figure PCTCN2021077852-appb-100014
    D10：
    Figure PCTCN2021077852-appb-100015
  10. 根据权利要求1和3至9中任一项所述的胺基脂质化合物，其中：
    X 3是O；
    p是0；
    R 5不存在；并且
    Figure PCTCN2021077852-appb-100016
    是选自以下的O1、O2、O3、O4、O5、O6、O7、O8、O9和O10中的一种：
    O1：
    Figure PCTCN2021077852-appb-100017
    O2：
    Figure PCTCN2021077852-appb-100018
    O3：
    Figure PCTCN2021077852-appb-100019
    O4：
    Figure PCTCN2021077852-appb-100020
    O5：
    Figure PCTCN2021077852-appb-100021
    O6：
    Figure PCTCN2021077852-appb-100022
    O7：
    Figure PCTCN2021077852-appb-100023
    O8：
    Figure PCTCN2021077852-appb-100024
    O9：
    Figure PCTCN2021077852-appb-100025
    O10：
    Figure PCTCN2021077852-appb-100026
  11. 制备根据权利要求1至10中任意一项所述的胺基脂质化合物的制备方法，所述方法包括以下步骤：
    (1)在-40℃至30℃(优选-30℃至30℃)的温度下，在作为缚酸剂的碱的存在下，使三聚氯氰与由R 1(R 6) m-X 1H表示的化合物进行第一反应，得到式I-1的第一中间体；
    Figure PCTCN2021077852-appb-100027
    (2)在分离或(优选)不分离所述第一中间体的情况下，在室温或加热条件下，使所述第一中间体与由R 2(R 7) n-X 2H表示的化合物在作为缚酸剂的碱的存在下进行第二反应，得到式I-2的第二中间体；
    Figure PCTCN2021077852-appb-100028
    (3)在分离或(优选)不分离所述第二中间体的情况下，在加热条件下，优选还在作为缚酸剂的碱的存在下，使所述第二中间体与由HX 3(R 8) p-L-NR 3R 4R 5表示的二胺进行第三反应，得到所述式I的胺基脂质化合物；
    其中，X 1、X 2、X 3、R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、L、m、n和p的定义与权利要求1至10中任意一项中它们的定义相同。
  12. 根据权利要求1至10中任意一项所述的胺基脂质化合物用于制备脂质颗粒的用途，其中所述脂质颗粒为脂质纳米粒、脂质体、多层囊泡或胶束；更优选地，所述脂质颗粒为脂质纳米粒。
  13. 脂质颗粒，其包含权利要求1至10中任意一项所述的胺基脂质化合物；优选地，所述脂质颗粒为脂质纳米粒、脂质体、多层囊泡或胶束；更优选地，所述脂质颗粒为脂质纳米粒。
  14. 根据权利要求13所述的脂质颗粒，其中所述脂质颗粒进一步含有辅助脂质、固醇和生物活性剂中的一种或多种；
    优选地，所述脂质颗粒进一步含有聚乙二醇脂质(PEG)-脂质；和/或
    优选地，在所述脂质颗粒中，所述胺基脂质化合物与所述辅助脂质的摩尔比为约(2至10):1，优选约(3至8):1，更优选约(4至6):1，例如约4:1、约4.5:1或约5:1；和/或
    优选地，在所述脂质颗粒中，所述胺基脂质化合物与所述固醇的摩尔比为约(0.5至1.5):1，优选约(1至1.4):1，例如约(1至1.3):1；和/或
    优选地，在所述脂质颗粒中，所述胺基脂质化合物与所述PEG-脂质的摩尔比为约(9至42):1，优选约(12至38):1，更优选约(16至36):1，例如约(18至34):1；和/或
    优选地，所述辅助脂质为非阳离子脂质，更优选地，所述辅助脂质是非阳离子磷脂，进一步优选地，所述非阳离子脂质是DOPE、DSPC或其组合；和/或
    优选地，所述固醇为选自胆固醇、谷甾醇、豆甾醇和麦角固醇的一种或多种，优选为胆固醇；和/或
    优选地，所述PEG-脂质为选自PEG1000-DMG、PEG5000-DMG、PEG2000-DMG和PEG2000-DSPE中的一种或多种，优选为PEG2000-DMG；和/或
    优选地，所述生物活性剂为核酸、抗肿瘤剂、抗生素、免疫调节剂、抗炎剂、作用于中枢神经系统的药剂、抗原或其片段、肽、蛋白、抗体、疫苗和小分子中的一种或多种；更优选地，所述核酸为RNA、信使RNA(mRNA)、反义寡核苷酸、DNA、质粒、核糖体RNA(rRNA)、微RNA(miRNA)、转移RNA(tRNA)、小的抑制RNA(siRNA)和小的核RNA(snRNA)。
  15. 根据权利要求12或13所述的脂质颗粒在药物的制备中的用途，所述药物是用于基因治疗、基因疫苗接种、反义治疗或通过干扰RNA的治疗的药物；
    优选地，所述脂质颗粒用作包封生物活性剂的载体；
    优选地，所述基因治疗可用于癌症和遗传疾病的治疗；更优选地，所述癌症为肺癌、胃癌、肝癌、食管癌、结肠癌、胰腺癌、脑癌、淋巴癌、血癌或前列腺癌中的一种或多种，所述遗传疾病为血友病，地中海贫血、高雪氏病中的一种或多种；
    优选地，所述基因疫苗接种用于治疗癌症、过敏、毒性和病原体感染；更优选地，所述病原体为病毒、细菌或真菌中的一种或多种。
  16. 根据权利要求12或13所述的脂质颗粒在制备用于核酸转移的药物中的用途，优选地，所述核酸为RNA、信使RNA(mRNA)、反义寡核苷酸、DNA、质粒、核糖体RNA(rRNA)、微RNA(miRNA)、转移RNA(tRNA)、小的抑制RNA(siRNA)和小的核RNA(snRNA)。
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