CN110283223A - A kind of cation lipid molecule and its application in delivery of nucleic acids - Google Patents
A kind of cation lipid molecule and its application in delivery of nucleic acids Download PDFInfo
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Abstract
The invention discloses a kind of cation lipid molecule and its applications in delivery of nucleic acids.The structural formula of the cation lipid molecule is as shown in formula I, and the present invention also provides cationic-liposome, lipid complex, reagent, kit, preparation and pharmaceutical compositions on the basis of the cation lipid molecule.The synthesis technology of cation lipid molecule provided by the invention is simple, stability is good, and cationic-liposome has both efficient (can be presented as high transfection efficiency) and hypotoxicity；Stable uniform, easily prepared simultaneously；It can be used for various cell line transfections.To have outstanding transitivity, the high efficiency regulatory of active material can be realized by active material (such as exemplary siRNA) high-efficiency delivery to cell (such as exemplary lung carcinoma cell), tissue, organ.Solve the problems, such as that toxicity and the transmission efficiency of cationic-liposome existing in the prior art be not high.
Description
Technical field
The present invention relates to a kind of cation lipid molecule and its applications in delivery of nucleic acids, belong to delivery system for nucleic acids neck
Domain.
Background technique
Gene therapy is that external source normal gene is imported target cell, to correct or compensator gene missing or abnormal caused
Disease, to achieve the purpose that treatment.Gene silent technology is Typical Representative therein.In order to realize gene silencing, mediate
Molecule such as siRNA have to enter in target cell and play a role.However, naked siRNA is degradable, the half-life period in blood plasma
Less than one hour；And since size is very small, can be excreted in the circulating cycle by kidney rapidly；In addition siRNA cannot pass through carefully
After birth.Therefore, it in order to realize the gene silencing function of siRNA, needs to find effective delivery system protection siRNA, avoids dropping
Solution, while increasing the intake etc. of cell.
Cationic-liposome is the nucleic acid molecules delivery vector material of a kind of great potential, but the synthesis of conventional liposome is logical
Single optimization, and complex steps, synthesis cost relative difficulty and valuableness often are needed, frequently results in the price mistake for user
It is high.In addition the challenge of the problem of existing commercialization cationic-liposome still faces safety and high efficiency.Therefore, exploitation has
High efficiency, hypotoxicity prepare New Cationic Liposome simple and feasible, that property is stable and become urgent problem to be solved.
Summary of the invention
The object of the present invention is to provide a kind of novel cation lipid molecule and its in terms of application, institute
The cation lipid molecule of offer can be by active material (such as siRNA) high-efficiency delivery to cell (such as exemplary lung carcinoma cell), group
It knits, in organ, realizes the high efficiency regulatory of active material.
Cation lipid molecule provided by the present invention, structural formula is as shown in formula I:
In formula I, A indicates C14-28Acyl group or C14-28Alkoxy；
B is indicatedOr be not present, wherein j is whole between 1~5
Number, m are the integer between 1~4, and n is the integer between 2~4；
C is indicatedWherein k is the integer between 2~4；
D is indicatedWherein p is the integer between 1~4；
E is indicatedWherein R1And R2Independently indicate C1-4Alkyl or R1And R2It is condensed to form any substitution
C5-7Heterocycle, X are chlorine, bromine or iodine atom.
In formulaIndicate that the pyridine quaternary ammonium salt of different location substituent group, X are chlorine, bromine or iodine atom comprising
Specifically, compound shown in formula I can be following structures in table 1:
The structural formula of 1 Formulas I-VII of table
Wherein, A is the C for being optionally inserted into 0, one or more double bonds14-28Acyl group or C25-27Alkoxy, C14-28Acyl group
Be specifically including but not limited to: mace oil acyl group (C14:1, it is cis- 9), palmitoleoyl (C16:1, it is cis- 9), oleoyl (C18:
1, it is cis- 9), arachidonic acyl group (C20:4, cis- 5,8,11,14), mustard acyl group (C22:1, it is cis- 13), neural acyl group (C24:1,
It is cis- 15), myristoyl, palmityl, stearyl, 20 carbonic acyl radicals, 22 carbonic acyl radicals, tetracosa carbon acyl group, 20
Six carbonic acyl radicals or 28 carbonic acyl radicals.
Cation lipid molecule provided by the invention is specifically as shown in table 2:
The structural formula of each cation lipid molecule of table 2
Cation lipid molecule provided by the invention can be prepared by conventional method in the prior art.
Invention further provides a kind of cationic-liposomes, by the cation lipid molecule or by the lipid point
Sub and auxiliary rouge is made, and the auxiliary rouge is neutral lipid molecule and/or PEG lipid molecular；
Preferably, the neutral lipid molecule can be DOPE；The PEG lipid molecular can be DSPE-PEG.
The cation lipid molecule can be mixed with the neutral lipid molecule with equimolar ratio；
The molar ratio of the cation lipid molecule and the PEG lipid molecular can be 9:1~2.
The cationic-liposome is the nano particle that surface has net positive charge.
In the cationic-liposome, the molar content of the cation lipid molecule can be 30%~70%, preferably
45%~50%.
The cationic-liposome at least has one of (1) and (2) feature:
(1) partial size are as follows: 80~200nm, 89~130nm, 100~200nm, 80~120nm, 80~100nm or 80~
89nm；
(2) surface potential are as follows: 5~70mv, 30~70mv, 30~45mv, 5~45mv or 5~30mv.
Cationic-liposome of the present invention is uniform and stablizes, can be used as carrier system use, such as genophore, transfection examination
Agent.
The preparation method of subject cationic liposome includes but is not limited to ultrasonic method, reverse phase evaporation, injection method, cold
Freeze the methods of seasoning and high-pressure homogeneous, rotary evaporation, emulsion process.
Preferably, emulsion process also have passed through filtering after obtaining transparency emulsion.
The present invention still further provides a kind of lipid complex, by the cationic-liposome and electronegative active matter
Matter (wait load/deliver object) be made.
Preferably, the active material refers to the substance with biology or pharmacological activity, can be nucleic acid molecules or protein molecular；
When for the nucleic acid molecules, the N:P ratios of the cationic-liposome and electronegative nucleic acid molecules can for 20~
50:1.
The nucleic acid may include at least one nucleic acid analog by modification, or the nucleosides comprising nucleic acid derivative
Acid polymer.
The cationic-liposome and the active material pass through positive and negative charge electrostatic interaction formation lipid complex.
The lipid complex can form the nano particle that the partial size being dispersed in water phase is 80~600nm.The nano particle is uniform and steady
It is fixed.
The present invention also provides a kind of reagent, kit, preparation or pharmaceutical composition, being includes sun of the present invention
Cationic lipid molecule, cationic-liposome or lipid complex.
The present invention also provides above-mentioned cation lipid molecule, cationic-liposome, lipid complex, reagent, kit,
Preparation or pharmaceutical composition have following 1) -4 in preparation) in any function product in application:
1) encapsulating active substance；
2) by active delivery to cell, tissue or organ；
3) active material is made to play activity in cell, tissue or organ；
4) prevent, diagnose and/or treat disease.
The synthesis technology of cation lipid molecule provided by the invention is simple, stability is good, and cationic-liposome has both
Efficiently (it can be presented as high transfection efficiency) and hypotoxicity；Stable uniform, easily prepared simultaneously；It can be used for various cell line transfections.
It, can to cell, (such as exemplary lung cancer be thin by active material (such as exemplary siRNA) high-efficiency delivery to having outstanding transitivity
Born of the same parents), tissue, in organ, realize the high efficiency regulatory of active material.Solves the poison of cationic-liposome existing in the prior art
Property and the not high problem of transmission efficiency.
Detailed description of the invention
Fig. 1 is the cationic-liposome and corresponding liposome/siRNA compound partial size of rotary evaporation preparation
(Fig. 1 (A)) and surface potential (Fig. 1 (B)).
Fig. 2 is that the cationic-liposome of emulsion process preparation filters preceding and filtered partial size (Fig. 2 (A)) using 100nm film
With surface potential (Fig. 2 (B)).
Fig. 3 is long circulating cationic-liposome using before the filtering of 100nm film and filtered partial size (Fig. 3 (A)) and surface
Current potential (Fig. 3 (B)).
Fig. 4 is the liposome/siRNA compound partial size (Fig. 4 (A)) and surface potential (Fig. 4 of different nitrogen phosphorus (N/P) ratios
(B))。
Fig. 5 is that liposome/Cy3-siRNA compound transfects cell fluorescence microscope photograph.
Fig. 6 is the expression that liposome/siRNA compound transfects cell green fluorescent protein after cell.
Fig. 7 is unloaded liposome cytoactive detection.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Table 3 is present invention Chinese and English or the Chinese abridged referring to table, and table 4 show each cell line.
3 English of table or abbreviation Chinese are referring to table
4 cell line table of table
| A549 | Lu-csf-1 |
| MDA-MB-231 | Human breast cancer cell |
| MCF-7 | Human breast cancer cell line |
| H1299 | Non-small cell lung cancer cell |
The preparation of embodiment 1, compound I-1
(1) synthesis of compound 3
Oleic acid 1, HBTU (1.1eq), DIEA (1.2eq) are added in single port bottle and is dissolved in methylene chloride.2h is stirred at room temperature
Diethanol amine 2 (1.2eq) is added afterwards.It finishes, 18h is stirred at room temperature.Reaction solution uses 1N HCl, saturation Na respectively2CO3Solution, washing
After washing, add anhydrous sodium sulfate dry.Filtering, the filtrate was concentrated to dryness crude product.Crude product crosses silicagel column (petroleum ether: ethyl acetate V:V
=1:1), light yellow oil 3, yield 58.2% is made.1HNMR (400M, CDCl3) δ ppm:(5.305-5.388, m,
2H), (3.811-3.836, t, 2H), (3.761-3.787, t, 2H), (3.531-3.556, t, 2H), (3.482-3.508, t,
2H), (2.368-2.406, t, 2H), (2.005-2.019, m, 4H), (1.603-1.638, t, 2H), (1.272-1.308, m,
20H), (0.885-0.901, t, 3H).ESI-MS:m/z 392.93 [M+Na]+,761.74[2M+Na]+。
(2) synthesis of compound 5
N- hydroxyethyl morpholine 4 is added in single port bottle, carbon tetrabromide (1.1eq) is dissolved in methylene chloride.Then slowly add
Enter triphenylphosphine (1.1eq).It finishes, removes ice bath, 30min is stirred at room temperature.Reaction solution is concentrated to dryness.Petroleum ether is added
A large amount of solids are precipitated in 500mL.Filtering concentrates the filtrate to its dry crude product.Crude product cross silicagel column (petroleum ether: ethyl acetate V:
V=5:1), intermediate (2- (4- morpholine) bromic ether) is made, is directly used in next step.
Bottle with two necks separately is taken, compound 3 is added thereto, anhydrous tetrahydro furan under ice bath stirring, is slowly added to NaH
(4.0eq).It finishes, replaces nitrogen, be warming up to 60 DEG C of reaction 1h.After transparent oil obtained is dissolved with anhydrous tetrahydro furan
It is added into reaction solution.It finishes, continues 60 DEG C of reaction 16h.Add water quenching reaction, be extracted with ethyl acetate, anhydrous sodium sulfate is dry
It is dry.Filtering, the filtrate was concentrated to dryness crude product.Crude product crosses silicagel column, and light yellow oil 5, yield 37.3% is made.ESI-
MS:m/z 596.94 [M+H]+, 618.81 [M+Na]+, 1213.68 [2M+Na]+。
(3) synthesis of compound 6
4.0g compound 5 is added in single port bottle and iodomethane (10eq) is dissolved in acetonitrile.It is warming up to 70 DEG C of reaction 20h.It will
Reaction solution is concentrated to dryness, and obtains yellow crude.Take crude product C18Faint yellow solid 6, yield 40.6% is made in post separation.ESI-
313.08 [M-2I of MS:m/z-]/2,752.47 [M-I-]。1HNMR (400M, CDCl3) δ ppm:(5.321-5.332, d, 2H),
(4.066-4.136, m, 16H), (3.562-3.799, m, 22H), (2.309, s, 2H), (1.996-2.003, m, 4H),
(1.548, s, 2H), (1.232-1.298, m, 20H), (0.857-0.884, t, 3H).
(4) synthesis of compound I-1
Chlorine type exchanger resin is added in single port bottle, washs impurity with dehydrated alcohol, removes ethyl alcohol, it is spare.Separately take a list
Mouth bottle, is added compound 6 thereto, and dehydrated alcohol is stirred at room temperature.It is added after its dissolution into above-mentioned resin, finishes, in room
Temperature stirring 2h.Filtering, concentrates the filtrate to dry, obtains anhydrous transparent solid I-1, yield 96.0%.ESI-MS:m/z 313.12
[M-2Cl-]/2,660.57 [M-Cl-]。1HNMR (400M, CDCl3) δ ppm:(5.366, s, 2H), (4.079-4.134, m,
16H), (3.552-3.786, m, 22H), (2.322, s, 2H), (2.037, s, 4H), (1.579, s, 2H), (1.297-1.324,
M, 20H), (0.909, s, 3H).
The preparation of embodiment 2, compound I-2
(1) synthesis of compound 8
Erucic acid 7 is added in single port bottle, stirring and dissolving in methylene chloride,.HBTU (1.05eq) and DIEA (2eq) is added,
It finishes, 2h is stirred at room temperature.It is added diethanol amine (2eq).It finishes, 18h is stirred at room temperature.Reaction solution is washed with water and dilute hydrochloric acid respectively
Afterwards, dry with anhydrous sodium sulfate.Filtering, the filtrate was concentrated to dryness obtains crude product.Crude product crosses silicagel column, compound 8 is made, yield is about
83%.ESI-MS:m/z 873.22 [2M+Na]+。
(2) synthesis of compound 9
It is added N- hydroxyethyl morpholine 4 in single port bottle, carbon tetrabromide (1.1eq) and q. s. methylene chloride, after stirring and dissolving
It is slowly added to triphenylphosphine (1.1eq).It finishes, removes ice bath, 30min is stirred at room temperature.Reaction solution is concentrated to dryness.Petroleum is added
A large amount of solids are precipitated in ether.Filtering concentrates the filtrate to its dry crude product.Crude product crosses silicagel column, and intermediate (2- (4- is made
Quinoline) bromic ether), it is directly used in next step.
It is added compound 8 and anhydrous tetrahydro furan in bottle with two necks, at 0 DEG C, is slowly added to NaH (4.0eq).It finishes, sets
Nitrogen is changed, 60 DEG C of reaction 1h are warming up to.By intermediate obtained above, it is added after being dissolved with anhydrous tetrahydro furan to reaction solution
In.It finishes, continues 60 DEG C of reaction 18h.Add ammonium chloride quenching reaction, is extracted with ethyl acetate, saturated common salt water washing, anhydrous sulphur
Sour sodium is dry.Filtering, the filtrate was concentrated to dryness obtains crude product.Crude product crosses silicagel column, and brown oil 9, yield 47.0% is made.
ESI-MS:m/z 652.66 [M+H]+, 1225.50 [2M+Na]+。
(3) synthesis of compound 10
Compound 9 and anhydrous DMF are added in single port bottle, is added with stirring iodomethane.It is closed to be warming up to 100 DEG C of reactions
20h.Reaction solution is concentrated to dryness, its crude product is obtained.Crude product C18Product 10 can be made about in post separation, yield 48.8%.ESI-
341.05 [M-2I of MS:m/z-]/2,808.36 [M-I-]。1HNMR (400M, D2O) δ ppm:(5.27, s, 2H), (3.97-
4.02, m, 12H), (3.73-3.84, m, 6H), (3.54-3.61, m, 14H), (3.32, s, 3H), (3.30, s, 3H), (2.38,
S, 2H), (1.95-1.97, m, 4H), (1.50, s, 2H), (1.22-1.24, m, 28H), (0.81-0.84, t, 3H).
(4) synthesis of compound I-2
Anion exchange resin is added in single port bottle, washs impurity with dehydrated alcohol, removes ethyl alcohol, it is spare.Separately take one
Compound 10, dehydrated alcohol are added thereto, is stirred at room temperature for single port bottle.It is added after its dissolution into above-mentioned resin, finishes,
18h is stirred at room temperature.Filtering, and concentrate the filtrate to dry, obtain product I-2, yield 89.5%.341.05 [M- of ESI-MS:m/z
2Cl-]/2,716.49 [M-Cl-]。1HNMR (400M, D2O) δ ppm:(5.26-5.29, m, 2H), (3.90-4.70, m, 12H),
(3.65-3.73, m, 6H), (3.47-3.60, m, 14H), (3.23, s, 6H), (2.31-2.35, m, 2H), (1.93-1.96, m,
4H), (1.48, s, 2H), (1.22-1.25, m, 28H), (0.81-0.84, t, 3H).
The preparation of embodiment 3, compound I-3
(1) synthesis of compound 11
A single port bottle is taken, compound 5 and methanol are added thereto.Pd/C is added after its dissolution, in room after replacing hydrogen
Temperature stirring 18h.Diatomite filtering is spread, and concentrates the filtrate to dry, obtains compound 11, is directly used in next step.ESI-MS:
m/z 598.52[M+H]+, 620.50 [M+Na]+。1HNMR(CD3OD, ppm): δ (3.69-3.73, m, 8H), (3.61-3.66,
M, 12H), (2.53-2.65, m, 12H), (2.45, t, 2H), (1.61, t, 2H), (1.31-1.35, m, 28H), (0.92, t,
3H)。
(2) synthesis of compound 12
Compound 11 and DMF are added in single port bottle, iodomethane (10eq) is added after stirring and dissolving.It heats up under confined conditions
To 100 DEG C of reaction 20h.Reaction solution is concentrated to dryness, its crude product is obtained after being washed with ethyl acetate.Crude product C18Post separation obtains light
Yellow solid 12, yield 63.5%.ESI-MS:m/z 314.17 [M-2I-]/2,754.37 [M-I-].1HNMR (400M,
D2O) δ ppm:(3.98-4.05, m, 12H), (3.74-3.85, m, 6H), (3.55-3.68, m, 14H), (3.33, s, 3H),
(3.31, s, 3H), (2.39, s, 2H), (1.52, s, 2H), (1.24, s, 28H), (0.82-0.86, t, 3H).
(3) synthesis of compound I-3
Anion exchange resin is added in single port bottle, washs impurity with dehydrated alcohol, removes ethyl alcohol, it is spare.Separately take one
Single port bottle, is added compound 12, dehydrated alcohol thereto, and water is stirred at room temperature.It is added after its dissolution into above-mentioned resin, adds
Finish, 18h is stirred at room temperature.Filtering, washs filter cake with a small amount of dehydrated alcohol.It concentrates the filtrate to dry, obtains colorless and transparent solid (I-
3), yield 94.0%.ESI-MS:m/z 313.95 [M-2Cl-]/2,662.35 [M-Cl-].1HNMR (400M, D2O) δ ppm:
(3.92-4.04, m, 12H), (3.67-3.74, m, 6H), (3.47-3.62, m, 14H), (3.27, s, 3H), (3.24, s, 3H),
(2.33-2.36, t, 2H), (1.49-1.51, m, 2H), (1.24, s, 28H), (0.83-0.86, t, 3H).
The preparation of embodiment 4, compound ii -1
(1) synthesis of compound 14
Compound 13 and triethylamine (1.1eq) stirring and dissolving in THF are added in bottle with two necks, MsCl is added at 0 DEG C
(1.05eq).Keep 0 DEG C of reaction 1h.Add diatomite to filter, and wash filter cake with a small amount of THF, it is stand-by to obtain intermediate filtrate.
After separately a bottle with two necks, compound 3 being taken to be dissolved in THF, NaH (3eq) is added at 0 DEG C.It finishes, replaces nitrogen, heating
To 60 DEG C of reaction 1h.Mesosome filtrate (about 3eq) among the above is added with syringe.It finishes, continues 60 DEG C of reaction 2h.Water quenching is added to go out instead
It answers, is extracted with ethyl acetate, saturated common salt water washing, anhydrous sodium sulfate drying.Filtering, the filtrate was concentrated to dryness obtains crude product.Crude product
Through column chromatography for separation, compound 14 is made, is directly used in next step.ESI-MS:m/z 1125.20 [2M+Na]+。
(2) synthesis of compound 15
Compound 14 and anhydrous DMF are added in single port bottle, is added with stirring iodomethane.It is closed to be warming up to 100 DEG C of reactions
20h.Reaction solution is concentrated to dryness, its crude product is obtained.Crude product is through C18Product 15 is made in column chromatography for separation.ESI-MS:m/z 290.95
[M-2I-]/2,708.17 [M-I-]。1HNMR (400M, D2O) δ ppm:(8.76-8.78, d, 2H), (8.69-8.71, d, 2H),
(7.91-7.93, d, 4H), (5.22-5.24, m, 2H), (4.87, s, 2H), (4.80, s, 2H), (4.30, s, 3H), (4.28,
S, 3H), (3.63-3.81, m, 8H), (2.42-2.45, m, 2H), (1.89-1.91, m, 4H), (1.51, s, 2H), (1.16-
1.21, m, 20H), (0.76-0.79, m, 3H).
(3) synthesis of compound ii -1
Anion exchange resin is added in single port bottle, washs impurity with dehydrated alcohol, removes ethyl alcohol, it is spare.Separately take one
Single port bottle, is added compound 15 thereto, and dehydrated alcohol is stirred at room temperature.It is added after its dissolution into above-mentioned resin, finishes,
18h is stirred at room temperature.Filtering, and concentrate the filtrate to dry, obtain product (II -1), yield about 90.9%.ESI-MS:m/z
290.91[M-2Cl-]/2,615.99 [M-Cl-]。1HNMR (400M, D2O) δ ppm:(8.69-8.76, m, 4H), (7.89-
7.91, m, 4H), (5.23-5.24, m, 2H), (4.70-4.85, m, 2H), (4.32, s, 3H), (4.30, s, 3H), (3.64-
3.81, m, 8H), (2.42-2.46, m, 2H), (1.90-1.91, m, 4H), (1.51-1.54, m, 2H), (1.17-1.22, m,
20H), (0.77-0.80, t, 3H).
The preparation of embodiment 5, compound ii -2
(1) synthesis of compound 16
Compound 13 and triethylamine (1.1eq) stirring and dissolving in THF are added in bottle with two necks, MsCl is added at 0 DEG C
(1.05eq).Maintain 0 DEG C of reaction 1h.Add diatomite to filter, and wash filter cake with a small amount of THF, obtains intermediate filtrate and be directly used in
In next step.
Compound 8 is added in bottle with two necks, anhydrous tetrahydro furan under ice bath stirring, is slowly added to NaH (3.0eq).Add
Finish, replaces nitrogen, be warming up to 60 DEG C of reaction 1h.Mesosome filtrate among the above is added with syringe.It finishes, continues 60 DEG C of reaction 2h.
Add water quenching reaction, be extracted with ethyl acetate, anhydrous sodium sulfate is dry.Filtering, the filtrate was concentrated to dryness obtains crude product.Crude product is through column layer
Analysis, which separated column, can be made compound 16, yield 53.9%.ESI-MS:m/z 1237.33 [2M+Na]+。1HNMR(CDCl3,
Ppm): δ (8.35-8.60, m, 4H), (7.19-7.28, m, 4H), (5.34-5.36, m, 2H), (4.50-4.63, m, 4H),
(3.61-3.78, m, 8H), (2.38-2.42, m, 2H), (1.99-2.04, m, 4H), (1.62-1.65, m, 2H), (1.27-
1.35, m, 28H), (0.88, t, 3H).
(2) synthesis of compound 17
Compound 16 and anhydrous DMF are added in single port bottle, is added with stirring iodomethane.It is closed to be warming up to 100 DEG C of reactions
20h.Reaction solution is concentrated to dryness, obtains its crude product with petroleum ether.Crude product C18Faint yellow product is made in column chromatography for separation
17, yield 31.1%.318.96 [M-2I of ESI-MS:m/z-]/2,764.21 [M-I-]。1HNMR(D2O, ppm): δ (8.81-
8.83, d, 2H), (8.72-8.74, d, 2H), (7.94-7.97, dd, 4H), (5.26, s, 2H), (4.90, s, 2H), (4.83,
S, 2H), (4.34, s, 3H), (4.31, s, 3H), (3.66-3.85, m, 8H), (2.46, s, 2H), (1.92-1.96, m, 4H),
(1.54, s, 2H), (1.21-1.28, m, 28H), (0.82, t, 3H).
(3) synthesis of compound ii -2
Anion exchange resin is added in single port bottle, washs impurity with anhydrous methanol, removes methanol, it is spare.Separately take one
Compound 17 and anhydrous methanol are added thereto, is stirred at room temperature for single port bottle.It is added after its dissolution into above-mentioned resin, finishes,
18h is stirred at room temperature.Filtering, and concentrate the filtrate to dry, obtain light brown product II -2, yield 94.3%.ESI-MS:m/z
318.97[M-2Cl-]/2,672.08 [M-Cl-]。1HNMR(D2O, ppm): δ (8.69-8.78, m, 4H), (7.89-7.92, dd,
4H), (5.23-5.26, m, 2H), (4.85, s, 2H), (4.79, s, 2H), (4.33, s, 3H), (4.30, s, 3H), (3.63-
3.82, m, 8H), (2.44, t, 2H), (1.90-1.93, m, 4H), (1.53, s, 2H), (1.19-1.22, m, 28H), (0.80,
T, 3H).
The preparation of embodiment 6, compound III -1
(1) synthesis of compound 20
Compound 18, DMF and methylene chloride, stirring suspension are added in single port bottle.Oxalyl chloride (4.5eq) is added at 0 DEG C,
It finishes, is warming up to 50 DEG C of reaction 1h, dissolved clarification.It continues at 50 DEG C and reacts 3h.Reaction solution is concentrated to dryness to obtain yellow solid, is added
Methylene chloride obtains its solution of acid chloride, spare.
A single port bottle is separately taken, compound 19 (2.1eq), anhydrous pyridine and methylene chloride, stirring and dissolving is added.It is dripped at 0 DEG C
Enter above-mentioned solution of acid chloride.It finishes, reacts at room temperature 2h, there are a large amount of solids to be precipitated in whipping process.Mother liquor is poured out, residual crude product is obtained.
Blood red sticky oil object 20 is made through column chromatography for separation in crude product, is directly used in next step.464.48 [M+H of ESI-MS:m/z
]+。
(2) synthesis of compound 21
The stirring and dissolving in methyl alcohol of compound 20 is added in bottle with two necks.Pd/C is added, two bottlenecks put on hydrogen balloon respectively,
Replacing hydrogen.16h is reacted at room temperature, two hydrogen balloons are re-replaced.Continue room temperature reaction to fully reacting.Diatomite filtering is spread,
The filtrate was concentrated to dryness obtains crude product.Light brown sticky oil object 21, yield 95.2% is made through column chromatography for separation in crude product.ESI-MS:
m/z 434.44[M+H]+, 889.47 [2M+Na]+。1HNMR (400M, DMSO-d6) δ ppm:(8.447, s, 2H), (7.464, s,
1H), (7.110, s, 2H), (5.417, s, 2H), (3.256-3.271, m, 8H), (3.168, s, 4H), (2.419, s, 12H),
(1.682-1.715, m, 4H).
(3) synthesis of compound 22
Compound 1, DMF and methylene chloride are added in single port bottle.Oxalyl chloride is added at 0 DEG C after stirring and dissolving
(2.5eq).It finishes, is warming up to 50 DEG C of reflux 2h.Reaction solution is concentrated to dryness, methylene chloride is added and obtains its solution of acid chloride, it is spare.
A single port bottle is separately taken, compound 21 (0.8eq), anhydrous pyridine (3.0eq) and DMF is added.Yu Bing after stirring and dissolving
Bath is lower to instill above-mentioned solution of acid chloride.It finishes, in room temperature reaction 2h.Concentration removes solvent afforded crude material, and crude product is through column chromatography for separation, system
Obtain product 22, yield 65.6%.ESI-MS:m/z 698.78, [M+H]+, 1395.66, [2M+H]+。1HNMR(CDCl3, ppm):
δ (9.314, s, 1H), (8.357, s, 2H), (8.165, s, 2H), (8.018, s, 1H), (5.313-5.356, m, 2H),
(3.817, s, 8H), (3.482, s, 4H), (2.946, s, 12H), (2.433, s, 2H), (2.002-2.016, m, 8H),
(1.674, s, 2H), (1.270-1.304, m, 20H), (0.891, t, 3H).
(4) synthesis of compound 23
Compound 22 and anhydrous DMF are added in single port bottle, is added with stirring iodomethane.It is closed to be warming up to 100 DEG C of reactions
24h.Reaction solution is concentrated to dryness, washs to obtain its crude product with petroleum ether-ethyl acetate system.Crude product is made light through C18 post separation
Yellow solid 23, yield 21.3%.364.60 [M-2I of ESI-MS:m/z-]/2,854.59 [M-I-]。1HNMR(CDCl3,
Ppm): δ (8.090, s, 2H), (7.943, s, 1H), (5.335, s, 2H), (4.016, s, 8H), (3.513-3.644, m,
16H), (3.232, s, 6H), (2.454, s, 2H), (2.152, s, 4H), (2.007, s, 4H), (1.641, s, 2H), (1.273,
S, 20H), (0.883, s, 3H).
(5) synthesis of compound III -1
Anion exchange resin is added in single port bottle, washs impurity with dehydrated alcohol, removes ethyl alcohol, it is spare.Separately take one
Compound 23 is added in single port bottle thereto, and dehydrated alcohol and water are stirred at room temperature.It is added after its dissolution into above-mentioned resin, adds
Finish, 20h is stirred at room temperature.Filtering, and concentrate the filtrate to dry, obtain faint yellow solid III -1, yield about 88%.ESI-MS:m/
Z364.85, [M-2Cl-]/2,763.19, [M-Cl-]。1HNMR(CDCl3, ppm): δ (7.97, s, 2H), (7.85, s, 1H),
(5.23-5.24, d, 2H), (3.91, t, 8H), (3.39-3.51, m, 16H), (3.12, s, 6H), (2.33, s, 2H), (2.05,
S, 4H), (1.91, s, 4H), (1.54, s, 2H), (1.17-1.20, m, 20H), (0.79, t, 3H).
The preparation of embodiment 7, compound III -2
(1) synthesis of compound 24
A single port bottle is taken, compound 22 is added thereto, methanol stirring is added.Pd/C is added after its dissolution, replaces hydrogen
18h is stirred at room temperature after gas.Diatomite filtering is spread, and concentrates the filtrate to dry, obtains solid chemical compound 24, yield
90.6%.ESI-MS:m/z 700.55 [M+H]+。
(2) synthesis of compound 25
Compound 24 and anhydrous DMF are added in single port bottle, is added with stirring iodomethane.It is closed to be warming up to 100 DEG C of reactions
24h.Reaction solution is concentrated to dryness, washs to obtain its crude product with petroleum ether-ethyl acetate system.Half crude product is taken to chromatograph through C18 column
Product 25, yield 25.8% is made in separation.364.85 [M-2I of ESI-MS:m/z-]/2,856.48 [M-I-]。1HNMR
(D2O, ppm): δ (8.00, s, 2H), (7.86, s, 1H), (3.93, s, 8H), (3.43-3.57, m, 16H), (3.15, s, 6H),
(2.37, s, 2H), (2.07, s, 4H), (1.56, s, 2H), (1.21, s, 28H), (0.82, s, 3H).
(3) synthesis of compound III -2
Anion exchange resin is added in single port bottle, washs impurity with dehydrated alcohol, removes ethyl alcohol, it is spare.Separately take one
Compound 25 is added in single port bottle thereto, and dehydrated alcohol, water is stirred at room temperature.It is added after its dissolution into above-mentioned resin, adds
Finish, 18h is stirred at room temperature.Filtering, and concentrate the filtrate to dry, obtain product III -2, yield about 92%.ESI-MS:m/z
364.94 [M-2Cl-]/2,764.40 [M-Cl-]。1HNMR(D2O, ppm): δ (7.96, s, 2H), (7.85, s, 1H), (3.89-
3.92, t, 8H), (3.39-3.49, m, 16H), (3.12, s, 6H), (2.34, s, 2H), (2.04, s, 4H), (1.54, s, 2H),
(1.19, s, 28H), (0.80, t, 3H).
The preparation of embodiment 8, compounds Ⅳ -1
(1) synthesis of compound 27
Compound 1, DMF and methylene chloride are added in single port bottle.Thionyl chloride (2.0eq) is added under the conditions of 0 DEG C, adds
Finish, is warming up to room temperature reaction 2h.Reaction solution is concentrated to dryness, methylene chloride is added and obtains its solution of acid chloride, it is spare.
A single port bottle is separately taken, compound 26 (0.73eq), triethylamine and methylene chloride is added.It is instilled under ice bath stirring above-mentioned
Solution of acid chloride.It finishes, reacts at room temperature 1h.It is washed respectively with water, dilute sodium hydroxide and dilute hydrochloric acid.Anhydrous sodium sulfate is dry.Filtering,
The filtrate was concentrated to dryness obtains crude product.Product 27, yield 73.5% is made through column chromatography for separation in crude product.ESI-MS:m/z 969.15
[2M+Na]+。
(2) synthesis of compound 28
Compound 27, tetrahydrofuran and water are added in single port bottle.It is added with stirring anhydrous lithium hydroxide (4.0eq), is added
Finish, reacts at room temperature 18h.Concentration removes tetrahydrofuran, and residue adjusts pH to 2 with 4N HCl, a large amount of solids are precipitated.Filtering, filter
Cake is concentrated to dryness, and off-white powder product 28 is made.ESI-MS:m/z 889.74 [2M-H]-。
(3) synthesis of compound 30
Compound 28, HBTU (2.2eq), DIEA (3.0eq) and DMF are added in single port bottle.1h is stirred at room temperature.Additionization
It closes object 29 (2.2eq).It finishes, 2h is stirred at room temperature.Add water quenching reaction, and is extracted with dichloromethane.Organic phase is washed with water 3
It is secondary, add anhydrous sodium sulfate dry.Filtering, the filtrate was concentrated to dryness crude product.Crude product crosses column with methylene chloride-methanol system, is made
Pale orange solid 30, yield about 92%.ESI-MS:m/z 1173.33 [2M+Na]+。
(4) synthesis of compound 31
Compound 30 and anhydrous methanol are added in single port bottle, is added with stirring DMF and iodomethane.It is closed to be warming up to 60 DEG C
Reaction is for 24 hours.Reaction solution is concentrated to dryness, washs to obtain its crude product with petroleum ether-ethyl acetate system.Crude product is through C18 column chromatography point
From, obtained faint yellow solid product 31, yield 24.8%.327.95 [M-2I of ESI-MS:m/z-]/2,782.17 [M-I-]。1HNMR(D2O, ppm): δ (8.56-8.58, t, 4H), (8.03, s, 2H), (7.95, s, 1H), (7.84-7.85, d, 4H),
(5.24, s, 2H), (4.64, s, 4H), (4.17, s, 6H), (2.38, s, 2H), (1.92, s, 4H), (1.56, s, 2H),
(1.18-1.21, m, 20H), (0.78-0.79, t, 3H).
(5) synthesis of compounds Ⅳ -1
Anion exchange resin is added in single port bottle, washs impurity with dehydrated alcohol, removes ethyl alcohol, it is spare.Separately take one
Compound 31 is added in single port bottle thereto, and dehydrated alcohol, water is stirred at room temperature.It is added after its dissolution into above-mentioned resin, adds
Finish, 18h is stirred at room temperature.Filtering, and concentrate the filtrate to dry, obtain product IV -1, yield about 93%.ESI-MS:m/z
327.94[M-2Cl-]/2,690.23 [M-Cl-]。1HNMR(D2O, ppm): δ (8.56-8.58, m, 4H), (7.81-8.02, m,
7H), (5.22, s, 2H), (4.66-4.71, m, 4H), (4.18-4.20, m, 6H), (2.34, s, 2H), (1.90, s, 4H),
(1.55, s, 2H), (1.16-1.23, m, 20H), (0.76-0.79, t, 3H).
The preparation of embodiment 9, compound V -1
(1) synthesis of compound 33
Compound 32, DMF and methylene chloride, stirring and dissolving are added in single port bottle.Oxalyl chloride (2.0eq) is added at 0 DEG C,
It finishes, is warming up to 50 DEG C of reflux 2h.Reaction solution is concentrated to dryness, methylene chloride is added and obtains its solution of acid chloride, it is spare.
A single port bottle is separately taken, compound 21 (0.95eq), anhydrous pyridine (3.0eq), DMF, stirring and dissolving is added.Zero degree
It is lower to instill above-mentioned solution of acid chloride.It finishes, reacts at room temperature 2h.Concentration removes solvent, is separated with silica gel column chromatography, obtains faint yellow oily
Object 33, yield 71.7%.ESI-MS:m/z 576.64 [M+H]+, 1151.60 [2M+H]+, 1173.55 [2M+Na]+。
(2) synthesis of compound 34
Compound 33, tetrahydrofuran and water, stirring and dissolving are added in single port bottle.It is added anhydrous lithium hydroxide (4.0eq),
It finishes, reacts at room temperature 20h.Concentration removes tetrahydrofuran, and water supplement washed with methylene chloride, water phase 6N HCl adjust pH to
6.Water phase is concentrated to dryness, the crude oil 34 containing inorganic salts is made, is directly used in next step.562.51 [M+ of ESI-MS:m/z
H]+, 568.59 [M+Li]+。
(3) synthesis of compound 36
In a single port bottle methylene chloride dissolution is added, for use in Weigh Compound 35.Separately take a single port bottle, thereto plus
Enter compound 34 and anhydrous DMF, is added with stirring the dichloromethane solution of HOBT (1.5eq), DIEA (3eq) and compound 35.
It finishes, half an hour is stirred at room temperature.HBTU (1.5eq) is added, is finished, 16h is reacted at room temperature.Reaction solution is concentrated to dryness, is added
It is dry that anhydrous sodium sulfate is added in methylene chloride dissolution residual substance after being washed with water.Filtering, and concentrate the filtrate to dry.Cross silica gel
Column obtains light yellow oil 36, yield 47.7%.ESI-MS:m/z 1106.14 [M+H]+, 1128.07 [M+Na]+。1HNMR
(400M, CDCl3) δ ppm:(9.581, s, 1H), (8.294-8.297, d, 2H), (8.033-8.058, t, 2H), (7.969, s,
1H), (6.948, s, 1H), (5.305-5.326, m, 1H), (3.681-3.704, t, 8H), (3.616-3.645, m, 12H),
(3.512-3.562, m, 6H), (3.442-3.469, t, 2H), (3.106-3.211, m, 1H), (2.483-2.541, m, 14H),
(2.248-2.385, m, 4H), (1.725-2.015, m, 12H), (0.995-1.582, m, 22H), (0.970, s, 3H),
(0.898-0.915, d, 3H), (0.850-0.871, dd, 6H), (0.664, s, 3H).
(4) synthesis of compound 37
Compound 36 and anhydrous DMF are added in single port bottle, is added with stirring iodomethane.It is closed to be warming up to 100 DEG C of reactions
24h.Iodomethane is added, continues 100 DEG C of confined reactions for 24 hours.Reaction solution is concentrated to dryness, is washed with petrol ether/ethyl acetate system
Wash to obtain its crude product.Crude product crosses C18Column obtains faint yellow solid 23, yield 25.5%.568.36 [M-2I of ESI-MS:m/z-]/2,
1261.99[M-I-]。
(5) synthesis of compound V -1
Anion exchange resin is added in single port bottle, washs impurity with dehydrated alcohol, removes ethyl alcohol, it is spare.Separately take one
Single port bottle, is added compound 37 thereto, and dehydrated alcohol is stirred at room temperature.It is added after its dissolution into above-mentioned resin, finishes,
18h is stirred at room temperature.Filtering, washs filter cake with a small amount of dehydrated alcohol.It concentrates the filtrate to dry, obtains colorless and transparent solid 18, receive
Rate 98.0%.568.17 [M-2Cl of ESI-MS:m/z-]/2,1169.87 [M-Cl-]。1HNMR (400M, D2O) δ ppm:(7.93,
S, 2H), (7.85, s, 1H), (5.47, s, 1H), (4.01, s, 8H), (3.37-3.69, m, 34H), (3.21, s, 6H),
(0.73-2.36, m, 54H).
The preparation of embodiment 10, compound VI -1
(1) synthesis of compound 40
Benzyl alcohol 38 and triethylamine (1.1eq) stirring and dissolving are added in single port bottle in methylene chloride.Weigh chloro-carbonic acid benzene
Ester 39 (1.0eq), is slowly dropped at 0 DEG C.It finishes, 18h is stirred at room temperature.Reaction solution is washed with water and dilute hydrochloric acid respectively, is used in combination
NaHCO3Adjust pH to 8.Add the dry organic phase of anhydrous sodium sulfate.Filtering, concentrates the filtrate to dry, obtains its crude product.Crude product crosses silica gel
Transparent oil 40 is made in column, and yield is about 82%.1HNMR (400M, CDCl3) δ ppm:(7.38-7.49, m, 7H), (7.20-
7.32, m, 3H), (5.30, s, 2H).
(2) synthesis of compound 42
Compound 40 and methylene chloride, stirring and dissolving are added in single port bottle.Weigh Compound 41 (1.0eq) instills extremely
It in reaction solution, finishes, in room temperature reaction 40h.Reaction solution is washed with water and dilute sodium hydroxide respectively, and organic phase is concentrated into closely
It is dry, add hydrochloric acid at salt and with ethyl alcohol recrystallization, the hydrochloride of obtained white solid 42.It is free with sodium carbonate, it is solid that off-white color is made
Body 42, yield 53.0%.ESI-MS:m/z 372.09 [M+H]+。1HNMR (400M, CD3OD) δ ppm:(7.34-7.37, m,
10H), (5.12, s, 4H), (3.45-3.48, m, 4H), (3.18-3.21, m, 4H).
(3) synthesis of compound 43
Addition compound 32 (1.0eq), HBTU (1.1eq), DIEA (2.0eq) are stirred in methylene chloride in single port bottle
Dissolution.Reaction 1h is stirred at room temperature.It is added compound 42 (0.95eq), finishes and reaction 20h is stirred at room temperature.Reaction solution use respectively water,
Dilute hydrochloric acid and sodium bicarbonate washing, it is dry to be added anhydrous sodium sulfate.Filtering, the filtrate was concentrated to dryness, obtains crude product.Crude product crosses silica gel
Column obtains grease 43, yield 91.6%.1HNMR(CDCl3, ppm): δ (7.34-7.36, m, 10H), (5.08-5.12, d,
4H), (3.66, s, 3H), (3.29-3.48, m, 8H), (2.29-2.37, m, 4H), (1.60-1.65, m, 4H).
(4) synthesis of compound 44
The stirring and dissolving in tetrahydrofuran of compound 43 is added in single port bottle.Weigh anhydrous lithium hydroxide (4.0eq) in
It is added after being dissolved in water into above-mentioned reaction solution, finishes, react at room temperature 6h.Concentration removes tetrahydrofuran, with petroleum ether-acetic acid second
Ester system washes twice, and water phase adjusts pH to 2 with 4N HCl.It is extracted with dichloromethane, anhydrous sodium sulfate is dry.Filtering, filtrate
It is concentrated to dryness to obtain compound 8, yield about 82.3%.ESI-MS:m/z 498.09 [M-H]-。
(5) synthesis of compound 45
Compound 44 (1.0eq) is added in single port bottle, anhydrous DMF, methylene chloride, stirring and dissolving.It is slowly added to chlorination
Sulfoxide (2.0eq), finishes, and is warming up to 40 DEG C of reflux 2h.Reaction solution is concentrated to dryness, methylene chloride is added and obtains its solution of acid chloride,
It is spare.
A single port bottle is separately taken, addition compound 26 (0.9eq) and triethylamine (2.0eq) stir molten in methylene chloride
Solution.Above-mentioned solution of acid chloride is instilled at 0 DEG C.It finishes, reacts at room temperature 2h.Reaction solution uses 1M sodium hydroxide, 1M hydrochloric acid and saturation respectively
Sodium bicarbonate washing, anhydrous sodium sulfate are dry.Filtering, the filtrate was concentrated to dryness, obtains crude product.Crude product crosses silicagel column, and it is solid to obtain off-white color
Body 45, yield 80.6%.ESI-MS:m/z 713.32 [M+Na]+, 1402.80 [2M+Na]+。1HNMR (400M, CDCl3)δ
Ppm:(8.48-8.49, d, 2H), (8.31-8.32, t, 1H), (7.27-7.33, m, 10H), (5.04-5.08, d, 4H),
(3.93, s, 6H), (3.42-3.47, m, 4H), (3.27-3.33, m, 4H), (2.37-2.42, m, 4H), (1.64-1.70, m,
4H)。
(6) synthesis of compound 46
Compound 45, methanol and ethyl acetate, stirring and dissolving are added in bottle with two necks.After Pd/C is added, two bottlenecks are equal
Put on hydrogen balloon, replacing hydrogen.16h is reacted at room temperature, two hydrogen balloons are re-replaced.Continue to react at room temperature 4h, spreads diatomite mistake
Filter, the filtrate was concentrated to dryness, obtains transparent oil 46, yield 70.3%.ESI-MS:m/z 421.38 [M-H]-。
(7) synthesis of compound 47
Compound 1, DMF, methylene chloride, stirring and dissolving are added in single port bottle.Thionyl chloride is added under ice bath
(2.0eq), finishes, and is warming up to 40 DEG C of reaction 2h.Reaction solution is concentrated to dryness, methylene chloride is added and obtains its solution of acid chloride, it is standby
With.
A single port bottle is separately taken, compound 46 (0.45eq) is added, triethylamine (2.0eq) is dissolved in methylene chloride.Ice bath stirs
Mix the lower above-mentioned solution of acid chloride of instillation.It finishes, reacts at room temperature 1h.
After reaction solution is washed with water, dilute sodium hydroxide, dilute hydrochloric acid and saturated sodium chloride solution respectively plus anhydrous sodium sulfate is dry
It is dry.Filtering, filtrate are concentrated to dryness.Column chromatography for separation is carried out after methylene chloride dissolution, product about 17.4g is made.ESI-MS:m/z
973.77[M+Na]+,1HNMR(CD3OD, ppm): δ (8.51-8.52, d, 2H), (8.34, t, 1H), (5.29-5.37, m, 4H),
(3.95, s, 6H), (3.48-3.51, m, 4H), (3.33-3.39, m, 4H), (2.45-2.50, m, 4H), (2.14-2.22, m,
4H), (1.99-2.05, m, 8H), (1.71-1.78, m, 4H), (1.55-1.61, m, 4H), (1.27-1.33, m, 40H),
(0.89-0.92, m, 6H).
(8) synthesis of compound 48
Compound 47 is added in single port bottle and is dissolved in tetrahydrofuran.Anhydrous lithium hydroxide (8.0eq) is weighed, is dissolved with water
After be added into above-mentioned reaction solution, finish, react at room temperature 18h.Concentration removes tetrahydrofuran, and water phase adjusts pH to 2 with 4N HCl.
It is extracted with dichloromethane, anhydrous sodium sulfate is dry.Filtering, filtrate are concentrated to dryness to obtain product 48, yield about 95.0%.ESI-MS:
m/z 922.03[M-H]-。
(9) synthesis of compound 49
Compound 48 is added in single port bottle, HBTU (2.4eq), DIEA (3.0eq) are dissolved in methylene chloride, are stirred at room temperature
1h.It is added compound 16 (2.4eq).It finishes, 3h is stirred at room temperature.After reaction solution is washed with water, 1N sodium hydroxide respectively, add anhydrous
Sodium sulphate is dry.Filtering, the filtrate was concentrated to dryness obtains crude product.Crude product is chromatographed with methylene chloride-methanol system column, is made faint yellow
Grease 49, yield 46.6%.ESI-MS:m/z 1175.92 [M+H]+。1HNMR(CDCl3, ppm): δ (10.59, d, 1H),
(8.70, s, 2H), (7.94-8.07, m, 4H), (7.53, s, 1H), (5.31-5.38, m, 4H), (3.70-3.72, m, 8H),
(3.49-3.63, m, 12H), (2.41-2.56, m, 15H), (2.22-2.26, m, 2H), (1.95-2.10, m, 11H), (1.48-
1.84, m, 12H), (1.08-1.36, m, 40H), (0.87-0.91, m, 6H).
(10) synthesis of compound 50
Compound 49 and anhydrous DMF are added in single port bottle, is added with stirring iodomethane.It is closed to be warming up to 100 DEG C of reactions
18h.Reaction solution is concentrated to dryness, obtains its crude product with petroleum ether.Crude product C18Column chromatography for separation is made faint yellow solid and produces
Object 50, yield 50.2%.602.67 [M-2I of ESI-MS:m/z-]/2,1331.73 [M-I-]。1HNMR(D2O, ppm): δ
(8.29-8.30, d, 2H), (8.21-8.22, m, 1H), (5.34-5.37, m, 4H), (4.02-4.05, m, 8H), (3.68-
3.71, m, 4H), (3.48-3.66, m, 16H), (3.34-3.40, m, 4H), (3.28, s, 6H), (2.47-2.52, m, 4H),
(2.15-2.26, m, 8H), (2.01-2.08, m, 8H), (1.70-1.81, m, 4H), (1.59-1.63, m, 4H), (1.30-
1.34, m, 40H), (0.90-0.94, m, 6H).
(11) synthesis of compound VI -1
Anion exchange resin is added in single port bottle, washs impurity with acetonitrile, removes acetonitrile, it is spare.Separately take a single port
Compound 50 is added in bottle thereto, and acetonitrile, water is stirred at room temperature.It is added after its dissolution into above-mentioned resin, finishes, in room temperature
Stir 18h.Filtering, washs filter cake with a small amount of acetonitrile.Concentrate the filtrate to it is dry, and add a small amount of toluene be spin-dried for obtaining product VI -1, receive
Rate about 73%.602.66 [M-2Cl of ESI-MS:m/z-]/2,1239.86 [M-Cl-]。1HNMR(D2O, ppm): δ (8.29-
8.29, d, 2H), (8.23-8.23, t, 1H), (5.34-5.37, m, 4H), (4.02-4.04, m, 8H), (3.62-3.67, m,
4H), (3.47-3.60, m, 16H), (3.34-3.39, m, 4H), (3.26, s, 6H), (2.47-2.51, m, 4H), (2.15-
2.24, m, 8H), (2.01-2.10, m, 8H), (1.70-1.81, m, 4H), (1.57-1.63, m, 4H), (1.30-1.41, m,
40H), (0.90-0.94, m, 6H).
Embodiment 11,
The liposomal cationic compound of I-3, III -1, III -2 and DOPE (molar ratio: 1:1) that the present invention synthesizes are dissolved in
In chloroform/methanol mixed system (10mL, 1:1, v/v), pour into the round-bottomed flask of Rotary Evaporators, in 50 DEG C of constant temperature water baths,
2h is evaporated under vacuum conditions.Then in 40 DEG C of vacuum drying 4h of vacuum oven, organic solvent is completely removed.Physiology salt is added
Water (6mL), normal pressure, 50 DEG C of constant temperature water bath concussion 1h obtain white emulsion, then white emulsion are transferred in vial, is surpassed
Sound 30min is to lotion substantially transparent.Final emulsion carries out 220 μm of filter (Millipore Corp.) filtering head degermings, is transferred to
New sample bottle is saved in 4 DEG C.The survey of partial size, polydispersity and surface charge is carried out using Zetasizer Nano particle instrument
Fixed (Fig. 1).
Embodiment 12,
With the dehydrated alcohol cationic compound I -1 that compound concentration is 0.01mol/L respectively, I -2, I -3, II -1, II -2,
III -1, III -2, VI -1, VI -2, V -1 and DOPE solution.By cationic compound and DOPE equimolar ratio (50%:50%,
Mol/mol) the 0.2M sodium acetate buffer aqueous solution of pH=5 is added in alcohol mixeding liquid, is vortexed uniformly, and ventilation magnetic agitation extremely mixes
Ethyl alcohol completes volatilization in system, adds equal amount of distilled water, obtains transparency emulsion.Take partial emulsion liposome mini-extruder extrusion instrument (Mini-
Extruder, Avanti Lipids Polar, Inc, USA) it is filtered using 100nm filter membrane, use Zetasizer Nano
Diameter instrument carries out partial size and the surface potential variation (Fig. 2) of partial size comparison filtering front and back.
Embodiment 13,
Cationic compound I -1, DOPE (dioleoylphosphatidylethanolamine) and DSPE-PEG (distearyl acyl group phosphatidyl
Ethanol amine-polyethylene glycol) respectively according to molar ratio 45%:45%；5% and 40%:40%:10% configures ethanol solution, then
The 0.2M sodium acetate buffer aqueous solution of pH=5 is added, is vortexed uniformly, magnetic agitation of divulging information ethyl alcohol completion into mixed system is waved
Hair, adds equal amount of distilled water, obtains transparency emulsion.It takes partial emulsion liposome mini-extruder extrusion instrument 100nm filter membrane to filter, uses
Zetasizer Nano particle instrument carries out partial size and the surface potential variation (Fig. 3) of partial size comparison filtering front and back.
As embodiment 11,12 and 13 it is found that cationic compound synthesized by the present invention can by rotary evaporation and
Sterile cationic-liposome is prepared in emulsion process.In fact, the method that various routines prepare liposome, such as ultrasonic method, instead
Phase evaporation, injection method, freeze-drying and high-pressure homogeneous hair etc. are suitable for cation lipid molecule preparation of the present invention and receive
Mizhi plastid.
It is the associated cation liposome prepared using Zetasizer Nano particle instrument to rotary evaporation shown in Fig. 1
Partial size and surface potential detection result.For resulting liposomal particle size range in 89~130nm, surface potential range is 30-45mV
Left and right.The cationic-liposome that Fig. 2 show emulsion process preparation filters front and back partial size and table using liposome mini-extruder extrusion instrument 100nm film
Face potential measurement result.The liposome directly prepared with emulsion process, before not filtered using liposome mini-extruder extrusion instrument 100nm film,
Partial size is in 100-200nm etc., and surface charge is in 30~70mV range.After being filtered by liposome mini-extruder extrusion instrument 100nm film, this hair
Bright all liposomal particle sizes become more uniform, and in 80~120nm range, and its surface potential also accordingly reduces, 5~
45mV range, individual samples, which exist, sharply reduces phenomenon.Fig. 3 is shown to be added in I -1 and DOPE liposome as main component
Add helper lipid molecule DSPE-PEG mini-extruder extrusion instrument (100nm film) filtering front and back partial size and surface potential detection result.DSPE-PEG
Addition, can increase surface of liposome hydrophily, increase circulation time in vivo.The increase of DSPE-PEG, liposomal particle size is slightly
Micro- increase (Fig. 3 (A)).But its surface potential is reduced with the increase of DSPE-PEG content, is down to from 53.6 ± 1.6mV
20.0 ± 0.6mV (Fig. 3 (B)) of 10%DSPE-PEG.
From the above it is found that the present invention uses the preparation method of different liposome, different helper lipids point are added
Son, the stable nano-lipid liquid solution of available uniform particle sizes, and also surface potential is positive, it was demonstrated that surface of liposome band
There is net charge.
Embodiment 14,
The preparation and characterization of I -1/DOPE liposome and siRNA compound, specific method is: 10ul sample being taken to be diluted in
After the ultrapure water of 1ml, the measurement of partial size and surface charge is carried out using Zetasizer Nano.
I -1/DOPE, the I -3/DOPE, III -1/DOPE, III -2/DOPE liposome solutions for taking implementation example 12 to prepare respectively
20 μ L Opti-MEM culture solutions are added in 5 μ L, mix, and are solution 1.24 μ L Opti- are added in 1 μ L siRNA (20 μM of concentration)
MEM culture solution is solution 2.Solution 2 is added drop-wise to solution 1, is mixed, the static 30min of room temperature obtains I -1/DOPE, I -3/
DOPE, III -1/DOPE, III -2/DOPE liposome and siRNA nano-complex.Then Zetasizer Nano partial size is used
Instrument carries out measurement (I -3/DOPE, III -1/DOPE, the III -2/DOPE rouge of nano-complex partial size, polydispersity and surface potential
The nano-complex measurement result of plastid and siRNA are shown in Fig. 1, the nano-complex measurement knot of I -1/DOPE liposome and siRNA
Fruit sees Fig. 4.)
Fig. 4 show I -1 liposome of different nitrogen phosphorus (N/P) ratios and the partial size and Surface potential measurement of siRNA compound
As a result.Cationic-liposome and siRNA nucleic acid form composite nanoparticle by electrostatic interaction.From Fig. 4 it is observed that with
The increase of N/P ratio, composite surface charge increase.When the N/P ratio of cationic-liposome I -1 and siRNA are 10:1,
Its partial size is 581 ± 44nm, and it is negative value that surface potential, which is -18.7 ± 5.6mV,.Illustrate that liposome cannot be fine in this N/P ratio
SiRNA is compressed, form good compound.When N/P ratio is 20:1, partial size is 218 ± 61nm, surface potential
For 13.3 ± 2.4mV.The quantity of electric charge entrained by liposome and siRNA composite surface be influence one of transfection efficiency it is important because
Element.Surface potential is for measuring the physical quantity of gravitation or repulsion intensity between material, and surface potential is too low, and nanoparticle is easier
In cohesion, the stability of system not only will affect, and its ability for wrapping up nucleic acid molecules is also weaker, and then leads to transfection efficiency
Decline.And since cell membrane is negatively charged, carrier material and nucleic acid are formed by compound and electropositive are presented, and can help to receive
Rice compound more easily enters cell interior.Therefore, for liposome siRNA compound, suitable surface potential will
It helps to ensure that compound has higher transfection efficiency, and is stabilized in body fluid transport.
Cationic-liposome of the invention can be interacted by positive and negative charge it can be seen from the present embodiment 14, with
Nucleic acid spontaneously forms liposome/nucleic acid complexes.In fact, the method that various routines prepare liposome-nucleic acid compound, such as rouge
The methods of plastid film and nucleic acid solution hydration hair, freeze-drying-rehydrated method, are adapted to liposome-nucleic acid of the present invention
The preparation of compound.Equally, also properly the various nucleic acid in addition to siRNA wrap up compound reality to cationic-liposome of the invention
It tests, these nucleic acid include but be not limited to that DNA, messenger mrna, microRNA etc., and it is small to effectively form particle,
One stabilized liposome/nucleic acid complexes.
Embodiment 15,
I -1, I -3, III -1, III -2, V -1, VI -1 and VI -2 liposome and Cy3 fluorescent marker siRNA (Cy3-siRNA)
Compound transfects A549 cell.By A549 cell with 2 × 105The density of a cells/well is taped against on 24 orifice plates, and addition contains 10%
The DMEM culture solution culture 24 hours of calf serum to cell density reaches 70% or so.Culture solution is removed, is washed twice with PBS.
Liposome/siRNA compound prepared by embodiment 14 is diluted to 500 μ L Opti-MEM culture solutions, is added in 24 orifice plates.37
DEG C, 5%CO2Under the conditions of incubator be incubated for 4h.It removes and contains liposome/siRNA compound culture solution, be added small containing 10%
The DMEM culture solution of cow's serum is incubated for 24 hours.Then the transfected condition of fluorescence microscope Cy3-siRNA, exciting light are used
550nm emits light 570nm.Fig. 5 show liposome/Cy3-siRNA compound transfection results fluorescence microscope picture, red
Color fluorescence is the Cy3-siRNA for being entered cell by liposome transfection.The lipofatamine2000 sold on the market
(lipo2000) transfection reagent can be transmitted to Cy3-siRNA inside cell, and liposome of the invention equally can be Cy3-
SiRNA is transfected into cytoplasm.And compare, its red fluorescence intensity of the cell of the certain liposome transfections of the present invention is much stronger than
Lipo2000 illustrates that certain liposomes of the invention can more efficiently transfect Cy3-siRNA, such as III -1, V -1 and VI -
1 liposome.
Embodiment 16,
By the A549 cell of expressing green fluorescent protein (GFP) with 2 × 105The density of a cells/well is taped against on 24 orifice plates,
The DMEM culture solution culture containing 10% calf serum is added and reaches 70% or so to cell density for 24 hours.Culture solution is removed, is used
PBS is washed twice.Liposome/siRNA compound prepared by the method for implementation example 14 is diluted to 500 μ L Opti-MEM culture
Liquid is added in 24 orifice plates.37 DEG C, 5%CO2Under the conditions of incubator be incubated for 4h.It removes and contains the training of liposome/siRNA compound
Nutrient solution is added the DMEM culture solution containing 10% calf serum and is incubated for 48h.Culture solution is removed, is washed twice with PBS, it will with pancreatin
Cell dissociation gets off, and 4 DEG C, 1000rpm centrifugation 5min obtains cell precipitation, washs cell precipitation with PBS, 4 DEG C, 1000rpm from
Heart 5min abandons supernatant.It washes twice repeatedly, cell precipitation is resuspended in plasma-free DMEM medium, uses fluidic cell
Instrument (BD, FACScalibur, BD Biosciences), detection every 1 × 104The relative intensity of fluorescence of cell.
It can be come out, be operated by the cell transfecting similar with embodiment 16 of embodiment 15, institute of the present invention by embodiment 16
The New Cationic Liposome stated/siRNA compound may be implemented to pass into siRNA including but not limited to
The various types of cells such as A549, MDA-MB-231, MCF-7 cell.In fact, for certain specific cells, sun of the present invention
Specific liposome is had in cationic liposomal combinatorial libraries can efficiently transmit siRNA into these cells.Fig. 6 show act
The expression of cell green fluorescent protein after the liposome of example/siRNA compound transfection cell.Liposome of the present invention
The liposome being prepared/siRNA complexes upon cell is transfected, and the expression that siRNA can be corresponded to gene is effectively dropped
It is low.With the N/P ratio of adjustment liposome and siRNA, liposome of the invention/downward of the siRNA compound to target gene
It is equivalent to transfection reagent mark post reagent Lipo2000 even better than on the market.
Embodiment 17,
By A549 cell inoculation in 96 orifice plates (about 1.5 × 104A cells/well), every hole is added 100 μ L and newly matches cell suspension
(culture medium containing 10%FBS), at 37 DEG C, 5%CO2Environment in be incubated for.After 24 hours, culture medium is discarded, and cleaned with PBS
Twice, the liposome particles of the 100 μ L embodiment of the present invention 12 preparation are added in every hole, and 3 parallel groups are arranged in each sample.Cell exists
After being incubated for 24 hours again at 37 DEG C, 20 μ L CCK-8 reagents are added, 2h is cultivated in incubator, read by microplate reader
Absorbance at 450nm wavelength carries out the detection of cell toxicity.Not stimulate cell to deposit in the absorbance of 450nm wavelength as cell
Motility rate 100% indicates the cell survival rate under various cationic-liposome stimulations.
It is operated by carrying out with the detection of the similar cytotoxicity of embodiment 17 it can be seen from embodiment 17, it can be real
Unloaded cationic-liposome of the present invention is applied to including but not limited to A549, MDA-MB-231, MCF-7 and H1299 cell
Toxicity etc. various cell lines is detected and is evaluated.What Fig. 7 was indicated is the various cationic-liposomes pair of zero load of the invention
The methanol of A549 cytotoxicity.Under same dosage, the zero load cation enumerated has very small toxicity to cell line, has
Liposome even can to cell grow there are facilitations.The Lipo2000 transfection reagent of comparison in the market, sun of the invention
Cationic liposomal shows more good biocompatibility.
Although the present invention describes preferred embodiment as above, the content that however, it is not to limit the invention is any ripe
Researcher in this field is known, is not departing from main spirits and context of the invention, various changes and retouching can be done,
Therefore protection scope of the present invention should be subject to the actual requirement range applied for a patent.
Claims (10)
1. compound shown in formula I,
In formula I, A indicates C14-28Acyl group or C14-28Alkoxy；
B is indicatedOr be not present, wherein j is the integer between 1~5, m
For the integer between 1~4, n is the integer between 2~4；
C is indicatedWherein k is the integer between 2~4；
D is indicatedWherein p is the integer between 1~4；
E is indicatedWherein R1And R2Independently indicate C1-4Alkyl or R1And R2It is condensed formed it is any substituted
C5-7Heterocycle, X are chlorine, bromine or iodine atom.
2. compound according to claim 1, it is characterised in that: in formula I, the A indicates to include 0~3 double bond
C14-28Acyl group or C25-27Alkoxy.
3. compound according to claim 2, it is characterised in that: the C14-28Acyl group is mace oil acyl group, palm oil
Acyl group, oleoyl, arachidonic acyl group, mustard acyl group, neural acyl group, myristoyl, palmityl, stearyl, 20 carbon
Acyl group, 22 carbonic acyl radicals, tetracosa carbon acyl group, 26 carbonic acyl radicals or 28 carbonic acyl radicals.
4. compound according to any one of claim 1-3, it is characterised in that: the structural formula of the compound such as formula I
Or shown in formula II:
In formula I and formula II, the definition of A and X are the same as formula I；
The value of k and p is the same as formula I.
5. compound according to any one of claim 1-3, it is characterised in that: the structural formula of the compound such as formula III
Or shown in formula IV:
In formula III and formula IV, the definition of A and X are the same as formula I；
The value of p is the same as formula I.
6. compound according to any one of claim 1-3, it is characterised in that: the structural formula of the compound such as formula
V, shown in formula VI or formula VII:
In formula V, the value of j and p are the same as formula I；
In formula VI and formula VII, the definition of A and X are the same as formula I；
M, the value of n and p is the same as formula I.
7. a kind of cationic-liposome, for it is following 1) or 2):
1) compound described in any one of claim 1-5 is made；
2) compound described in any one of claim 1-5 and auxiliary rouge are made；
The auxiliary rouge is neutral lipid molecule and/or PEG lipid molecular.
8. a kind of lipid complex, the cationic-liposome described in claim 7 and negatively charged active material are made；
The negatively charged active material is nucleic acid molecules or protein molecular.
9. compound described in any one of a kind of reagent, kit, preparation or pharmaceutical composition, including claim 1-6, right
It is required that lipid complex described in 7 cationic-liposomes or claim 8.
10. compound described in any one of claim 1-6, described in cationic-liposome, claim 8 described in claim 7
Reagent, kit described in lipid complex or claim 9, preparation or pharmaceutical composition have following 1) -4 in preparation) in appoint
A kind of application in the product of function:
1) encapsulating active substance；
2) by active delivery to cell, tissue or organ；
3) active material is made to play activity in cell, tissue or organ；
4) prevent, diagnose and/or treat disease.
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| CN114904003B (en) * | 2021-02-09 | 2023-09-29 | 广州立得生物医药科技有限公司 | Use of ionizable cationic lipid analog materials as nucleic acid drug delivery vehicles or transfection reagents |
| CN115872893A (en) * | 2021-09-28 | 2023-03-31 | 合肥阿法纳生物科技有限公司 | Cationic lipid compound and preparation method and application thereof |
| CN115872893B (en) * | 2021-09-28 | 2024-03-19 | 合肥阿法纳生物科技有限公司 | Cationic lipid compound, and preparation method and application thereof |
| CN114163345A (en) * | 2021-12-15 | 2022-03-11 | 武汉滨会生物科技股份有限公司 | Ionizable lipid compound and nucleic acid in-vitro cell transfection reagent |
| WO2023109243A1 (en) * | 2021-12-15 | 2023-06-22 | 武汉滨会生物科技股份有限公司 | Ionizable lipid compound and nucleic acid in vitro cell transfection reagent |
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