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CN114763324A - Lipid compound, liposome and pharmaceutical composition - Google Patents

Lipid compound, liposome and pharmaceutical composition Download PDF

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Publication number
CN114763324A
CN114763324A CN202110057708.0A CN202110057708A CN114763324A CN 114763324 A CN114763324 A CN 114763324A CN 202110057708 A CN202110057708 A CN 202110057708A CN 114763324 A CN114763324 A CN 114763324A
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integer
reaction
liposome
lipid compound
synthesis
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Inventor
刘兆贵
张海霖
宋玉宝
姜亦宝
孙婷婷
王正勇
陈璞
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Naptide Qingdao Biomedical Co ltd
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Naptide Qingdao Biomedical Co ltd
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Abstract

The invention relates to a lipid compound, and particularly provides a lipid compound with a structure shown in a formula (I), wherein the formula (I) is shown as follows:

Description

Lipid compound, liposome and pharmaceutical composition
Technical Field
The invention relates to a lipid compound, in particular to a lipid compound, a liposome and a pharmaceutical composition.
Background
siRNA technology is one of the most widely used biological technologies, and is widely used in many fields such as functional genomics, genetics, gene therapy, viral disease therapy, etc. due to its high specificity and high efficiency expression control of specific gene mRNA. However, both in vivo and in vitro, siRNA is easily degraded by ubiquitous rnases and siRNA cannot induce RNAi effects through cell membranes alone, and therefore siRNA technology is not conducive to highly efficient, low-toxicity siRNA carriers. Commonly used siRNA transfection carriers include viral vectors, liposomes, high polymers, inorganic nanoparticles, polypeptide biological vectors, and the like.
The prior siRNA transfection carrier has the defects of low entrapment capacity, poor penetrability, high toxicity, difficult quality control and the like, and further improvement and improvement of the related technology are needed.
Disclosure of Invention
On the basis of the common general knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily without departing from the concept and the protection scope of the invention.
In order to solve the above-mentioned technical problems, a first aspect of the present invention provides a lipid compound having a structure represented by formula (i), wherein formula (i) is as follows:
Figure BDA0002901221770000011
wherein A is N or CH; x, Y, Z are each independently selected from chemical groups attached to A with a carbon atom or carbon chain.
As a preferred embodiment, the lipid compound is such that when A is N, X is
Figure BDA0002901221770000012
And n is1Is an integer of 1 to 4; y is selected from
Figure BDA0002901221770000013
Figure BDA0002901221770000014
And n is2Is an integer of 2 to 8, m1Is an integer of 4 to 12, n3Is an integer of 2 to 8, m2Is an integer of 4 to 12, p1Is an integer of 1 to 5, n4Is an integer of 2 to 6, m3Is an integer of 4 to 10, n5Is an integer of 2 to 6, m4Is an integer of 1 to 5, p2Is an integer of 3 to 8, n6Is an integer of 4 to 8, m5Is an integer of 7 to 9, R1Is an oxygen atom or a sulfur atom;
z is selected from
Figure BDA0002901221770000021
And n is7Is an integer of 4 to 8, m6、m7Are all integers of 6 to 10, n8M is an integer of 5 to 78、m9Is an integer of 6 to 10.
As a preferred technical scheme, the lipid compound, when A is CH, X is selected from
Figure BDA0002901221770000022
And n is9Is an integer of 1 to 4, R2Is methyl or hydrogen, n10Is an integer of 1 to 4;
y is selected from
Figure BDA0002901221770000023
And n is11Is an integer of 2 to 8, m10Is an integer of 4 to 12, n12Is an integer of 2 to 8, m11Is an integer of 4 to 12;
z is selected from
Figure BDA0002901221770000024
And n is13Is an integer of 4 to 8, m12、m13Is an integer of 6 to 10, n14Is an integer of 5 to 7, m14、m15Is an integer of 6 to 10.
In a preferred embodiment, the lipid compound has a structure represented by formula (i) selected from:
numbering formula (I) structure
LP-C2：
Figure BDA0002901221770000025
LP-C3：
Figure BDA0002901221770000031
LP-C4：
Figure BDA0002901221770000032
LP-C5：
Figure BDA0002901221770000033
LP-C6：
Figure BDA0002901221770000034
LP-C7：
Figure BDA0002901221770000035
LP-C8：
Figure BDA0002901221770000036
LP-C9：
Figure BDA0002901221770000037
LP-C10：
Figure BDA0002901221770000041
LP-C11：
Figure BDA0002901221770000042
LP-C12：
Figure BDA0002901221770000043
LP-C13：
Figure BDA0002901221770000044
LP-C14：
Figure BDA0002901221770000045
LP-C15：
Figure BDA0002901221770000046
LP-C16：
Figure BDA0002901221770000047
LP-C17：
Figure BDA0002901221770000051
LP-C18：
Figure BDA0002901221770000052
LP-C19：
Figure BDA0002901221770000053
LP-C20：
Figure BDA0002901221770000054
LP-C21：
Figure BDA0002901221770000055
LP-C22：
Figure BDA0002901221770000056
LP-C23：
Figure BDA0002901221770000057
LP-C24：
Figure BDA0002901221770000061
LP-C25：
Figure BDA0002901221770000062
LP-C26：
Figure BDA0002901221770000063
LP-C27：
Figure BDA0002901221770000064
LP-C28：
Figure BDA0002901221770000065
As a preferred embodiment, wherein the lipid compound is in the form of a chemically acceptable salt.
As a preferred embodiment, wherein the lipid compound is in the form of a cationic lipid.
In a second aspect of the invention there is provided a liposome comprising said lipid compound.
As a preferred embodiment, the liposome further comprises other non-cationic forms of lipid compounds.
As a preferred embodiment, the other non-cationic lipid compounds are selected from phospholipids and cholesterol derivatives.
In a preferred embodiment, the phospholipid is selected from dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine and dioleoylphosphatidylethanolamine.
In a preferred embodiment, the cholesterol derivative is cholesterol.
As a preferred embodiment, the liposome further comprises a lipid conjugate.
As a preferred embodiment, the lipid conjugate is selected from the group consisting of PEG-diacylglycerol conjugate, PEG-dialkoxypropyl conjugate.
As a preferable technical scheme, the median diameter of the liposome is 30-150 nm; preferably, the median diameter of the liposome is 50-100 nm; more preferably, the liposomes have a median diameter of from 60 to 90 nm.
In a third aspect of the invention, there is provided a pharmaceutical composition comprising said lipid compound and a therapeutic agent.
As a preferred technical scheme, the molar ratio of the lipid compound to the therapeutic agent is 20: 1-200: 1; preferably, the molar ratio of the lipid compound to the therapeutic agent is 50:1 to 150: 1; more preferably, the lipid compound and therapeutic agent are present in a molar ratio of 100: 1.
In a fourth aspect of the invention, a pharmaceutical composition is provided, which consists of the liposome-encapsulated therapeutic agent.
In a preferred embodiment, the therapeutic agents are selected from the group consisting of siRNA, antisense oligonucleotide, microrna, mRNA, and DNA.
As a preferred technical scheme, the pharmaceutical compositions are respectively and independently used for treating diseases caused by gene abnormality.
Compared with the prior art, the invention has the following remarkable advantages and effects:
the invention provides a lipid compound and a liposome for transfecting cells, application thereof in carrying nucleic acid and other therapeutic agents and preparing medicaments, and a medicinal composition. In addition, the lipid compound has low toxicity, small damage to cells and good application prospect.
Detailed Description
The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. The reagents and starting materials used in the present invention are commercially available. The technical solutions of the present invention are described in detail below with reference to examples, but the present invention is not limited to the scope of the examples.
The meanings of the various abbreviations used in the following examples are explained as follows:
DCM: methylene dichloride
DCE (DCE): 1, 2-dichloroethane
MeOH: methanol
DMF: n, N-dimethylformamide
EtOAc: acetic acid ethyl ester
THF: tetrahydrofuran (THF)
EDCI: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride
DCC: dicyclohexylcarbodiimide
PyBOP: 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphates
DMAP: 4-dimethylaminopyridine
DIEA: n, N-diisopropylethylamine
TFA: trifluoroacetic acid
PPh3: triphenylphosphine
CBr4: carbon tetrabromide
NaH: sodium hydride
NaOH: sodium hydroxide
LiOH · H2O: lithium hydroxide monohydrate
PDC: pyridine dichromate
TEA: triethylamine
Pd/C: palladium on carbon
BnBr: benzyl bromide
Boc2O: di-tert-butyl dicarbonate
NaBH4: sodium borohydride
TLC: thin layer chromatography
DOPE: 1, 2-dioleoyl-SN-glycerol-3-phosphorylethanolamine
EXAMPLE 1 Synthesis of Compound LP-C1
(1) Synthesis of LP-Z1-01
Figure BDA0002901221770000081
The starting material 8-bromooctanoic acid (8.66g, 39.0mmol) was dissolved in DCM (100mL), and 9-heptadecanol (10.00g, 39.0mmol), EDCI (11.20g, 58.4mmol), and DMAP (1.00g, 8.2mmol) were added to the reaction flask in this order with stirring and reacted for 12h at ambient temperature with stirring. TLC detection shows that the raw material disappears and a new spot is generated. The reaction solution was washed 2 times with dichloromethane and water, the organic phase was retained, the organic phase was washed once with saturated brine, the liquid was separated, the organic phase was retained, the organic phase was dried over anhydrous sodium sulfate, filtered, the organic phase was retained, the organic phase was concentrated by distillation under reduced pressure, and the residue was purified by column chromatography to give intermediate LP-Z1-01(26.24g, pale yellow oil, yield 82%).
(2) Synthesis of LP-Z1
Figure BDA0002901221770000091
Intermediate LP-Z1-01(26.00g, 56.3mmol) was dissolved in absolute ethanol (20mL), 2-hydroxyethylamine (103mL, 1.77mol) was added, and the reaction was heated to 65 ℃ and stirred for 18 h. New spots were formed by TLC detection. The reaction was cooled to room temperature, diluted with ethyl acetate, the organic phase was washed with water 2 times, saturated brine 1 time, the organic phase was dried over anhydrous sodium sulfate, concentrated by distillation under reduced pressure, and the residue was purified by column chromatography to give intermediate LP-Z1(13.0g, pale yellow oil, yield 52%). MS (ESI) M/z [ M + H]+822.97。
(3) Synthesis of LP-C1
Figure BDA0002901221770000092
Intermediate LP-Z1-01(26.00g, 56.3mmol) was dissolved in absolute ethanol (20mL), 2-hydroxyethylamine (103mL, 1.77mol) was added and the reaction was stirred at 65 ℃ for 18 h. The product was formed by TLC. After completion of the reaction, the reaction was cooled to room temperature, diluted with ethyl acetate, and the organic phase was washed with water 2 times, saturated brine 1 time, and the organic phase was dried over anhydrous sodium sulfate, concentrated by distillation under reduced pressure, and the residue was purified by column chromatography to give intermediate LP-C1(140mg, pale yellow oil, yield 0.3%). MS (ESI) M/z [ M + H]+442.43。
1HNMR(500MHz,CDCl3)δ4.87(m,2H),3.72(br,2H),2.78-2.67(br,6H),2.28(m,4H),1.65-1.26(m,76H),0.88(m,12H)。
EXAMPLE 2 Synthesis of Compound LP-C2
Synthesis of LP-C2
Figure BDA0002901221770000093
Intermediate LP-Z1(250mg, 0.56mmol) was dissolved in absolute ethanol (3mL), and the starting material octyl 3-bromoalkanoate (174mg, 0.62mmol) and DIEA (120mg, 0.62mmol) were added sequentially and heated to 65 deg.C for 16 h. TLC detection raw material remained, and new spots were generated. Cooled to room temperature, concentrated under reduced pressure to remove the solvent, and the residue was purified by column chromatography to give the product LP-C2(70mg, colorless oil, yield 20%). MS (ESI) M/z [ M + H]+626.71。
1HNMR(500MHz,CDCl3)δ4.86(m,1H),4.07(t,2H),3.56(br2H),2.82-2.47(br,8H),2.28(t,2H),1.32-1.26(m,50H),0.88(m,9H)。
EXAMPLE 3 Synthesis of Compound LP-C3
Synthesis of LP-C3
Figure BDA0002901221770000101
Intermediate LP-Z1(500mg, 1.13mmol) was dissolved in anhydrous ethyl acetateTo an alcohol (5mL), the starting 3-bromopropane undecyl ester (382mg, 1.24mmol) and DIEA (161mg, 1.24mmol) were added in this order, and the mixture was heated to 65 ℃ to react for 16 hours. A small amount of starting material remained upon TLC detection, and a new spot was formed. Cooled to room temperature, concentrated under reduced pressure to remove the solvent, and the residue was purified by column chromatography to give the product LP-C3(410mg, colorless oil, yield 54%). MS (ESI) M/z [ M + H]+668.64。
1HNMR(500MHz,CDCl3)δ4.87(m,1H),4.08(t,2H),3.58(br2H),2.84-2.47(br,8H),2.28(t,2H),1.62-1.29(m,56H),0.88(m,9H)。
EXAMPLE 4 Synthesis of Compound LP-C4
(1) Synthesis of LP-C4-01
Figure BDA0002901221770000102
The raw material LP-Z1(500mg, 1.1mmol, 1.0eq) was dissolved in absolute ethanol (30mL), 2-bromoethanol (560mg, 4.4mmol, 4.0eq) and DIEA (560mg, 4.4mmol, 4.0eq) were added, and the temperature was raised to 65 ℃ under nitrogen protection for 16h reaction. TLC detection found a small amount of starting material remaining and a new spot was produced. The mixture was concentrated under reduced pressure to remove the solvent, the remaining part was stirred with silica gel and purified by column chromatography (DCM/MeOH 20:1, 5% ammonia), and after concentration, the product LP-C4-01(230mg, pale yellow oil, yield 42%) was obtained as a product, MS (ESI): M/z [ M + H ]: M/z]+486.34。
(2) Synthesis of LP-C4
Figure BDA0002901221770000103
Raw material LP-C4-01(100mg, 0.2mmol, 1.0eq) was dissolved in DCM (20mL), n-decanoic acid (35mg, 0.2mmol, 1.0eq) was added, EDCI (40mg, 0.22mmol, 1.1eq), DMAP (5mg, 0.04mmol, 0.2eq) and DIEA (80mg, 0.6mmol, 3.0eq) were added, and the mixture was reacted at room temperature for 16h under nitrogen protection. TLC detection raw material disappeared, and new spot was generated. The reaction was diluted with DCM (30mL), the organic phase washed with water (2 × 20mL), saturated brine (20mL), dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to give the productLP-C4(41mg, yellow oil, yield 32%), MS (ESI) M/z [ M + H]+640.51。
1HNMR(500MHz,CDCl3)δ4.86(q,J＝6.5Hz,1H),4.15(s,2H),3.55(s,2H),2.83–2.46(m,6H),2.33-2.26(m,4H),1.65-1.59(m,4H),1.55–1.41(m,6H),1.36–1.18(m,38H),0.88(t,J＝6.9Hz,9H)。
EXAMPLE 5 Synthesis of Compound LP-C5
(1) Synthesis of LP-C5-01
Figure BDA0002901221770000111
Starting material 6- (4-hydroxybutoxy) hexanoic acid (0.25g, 0.9mmol) was dissolved in DCM (5mL) and carbon tetrabromide (0.34g, 1.0mmol) and triphenylphosphine (0.36g, 1.4mmol) were added sequentially under ice bath. After the addition, the temperature is raised to room temperature for reaction for 2 hours. TLC detection shows that the raw material disappears and a new spot is generated. The reaction solution was concentrated, and the residue was purified by column chromatography to give the product LP-C5-01(170mg, colorless oil, yield 71%).
(2) Synthesis of LP-C5-02
Figure BDA0002901221770000112
The starting material LP-C5-01(170mg, 0.6mmol) was dissolved in DCM (10mL), and n-octanol (91mg, 0.07mmol), TEA (83mg, 0.8mmol)) and PyBOP (364mg, 0.7mmol) were added sequentially, followed by reaction at room temperature for 2 h. TLC monitored the end of the reaction. The reaction mixture was concentrated under reduced pressure, and the residue was purified by column chromatography to give intermediate LP-C5-02(70mg, colorless oil, yield 31%).
(3) Synthesis of LP-C5
Figure BDA0002901221770000113
The starting material LP-Z1(90mg, 0.20mmol) was dissolved in absolute ethanol (5mL), and intermediate LP-C5-02(70mg, 0.18mmol) and DIEA (40mg, 0.21mmol) were added sequentially, followed by warming to 65 deg.C and reactionAnd the time is 16 hours. The TLC detection shows that the raw material is remained and new spots are generated. Cooled to room temperature, concentrated under reduced pressure to remove the solvent, and the residue was purified by column chromatography to give the product LP-C5(37mg, colorless oil, yield 27%). MS (ESI) M/z [ M + H]+740.92。
1HNMR(500MHz,CDCl3)δ4.86(m,1H),4.06(dd,2H),3.90(br,2H),3.42(m,4H),3.01(br,6H),2.30(m,4H),1.6-1.26(m,60H),0.89(m,9H)。
EXAMPLE 6 Synthesis of Compound LP-C6
(1) Synthesis of LP-C6-01
Figure BDA0002901221770000121
The starting material, 1, 4-butanediol (5.0g, 55.6mmol, 1.0eq) was dissolved in anhydrous DMF (50mL) and NaH (40% inoil) (2.2g, 55.6mmol, 1.0eq) was added slowly in portions to the system at 0 deg.C and maintained at 0 deg.C for 30 min. Then BnBr (9.5g, 55.6mmol, 1.0eq) was slowly added dropwise to the system under nitrogen. After the addition, the reaction is carried out for 16h at room temperature. TLC detection has a small amount of starting material remaining and new spots formed. The reaction was quenched with water, the aqueous phase extracted with EtOAc (200mL), the organic phase washed with water (3 × 100mL), saturated brine (100mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (PE/EtOAc ═ 3:1) to give product LP-C6-01(7.4g, colorless oil, 74% yield). MS (ESI) M/z [ M + H]+181.21。
(2) Synthesis of LP-C6-02
Figure BDA0002901221770000122
Starting material LP-C6-01(5.0g, 27.8mmol, 1.0eq) was dissolved in DCM (80mL) and CBr was added4(13.8g, 41.7mmol, 1.5eq) and then PPh was added slowly at 0 deg.C3(12.3g, 47.26mmol, 1.7 eq). After the addition, the reaction is carried out for 1h at room temperature. TLC detection shows that the raw material disappears and a new spot is formed. The reaction was poured into MTBE and the solid was washed out, filtered to remove the solid, the filtrate was concentrated and purified by column chromatography (PE/EtOAc 20:1),the product LP-C6-02(6.1g, pale yellow oil, 91% yield) was obtained.
(3) Synthesis of LP-C6-03
Figure BDA0002901221770000123
NaH (1.2g, 31mmol, 1.5eq) was slowly added to a solution of 1, 4-butanediol (3.7g, 41.32mmol, 2.0eq) in DMF (70mL) at 0 ℃ and the reaction was maintained at 0 ℃ for 30 min. Then, under nitrogen protection, a solution of LP-C6-02(5.0g, 20.66mmol, 1.0eq) in DMF (10mL) was slowly added dropwise to the system. After the addition, the reaction is carried out for 16h at room temperature. TLC detection starting material disappeared and a new spot was generated. The reaction was quenched with water, the aqueous phase was extracted with ethyl acetate (200mL), the organic phase was washed with water (3X100mL), saturated brine (100mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (PE/EtOAc ═ 3:2) to give product LP-C6-03(3.4g, light yellow oil, yield 65%), MS (ESI): M/z [ M + H ]: M/z]+253.20。
(4) Synthesis of LP-C6-04
Figure BDA0002901221770000131
The starting material LP-C6-03(3.4g, 13.5mmol, 1.0eq) was dissolved in DMF (60mL), PDC (8.1g, 21.6mmol, 1.6eq) was added, and the reaction was carried out at room temperature for 24 h. A small amount of raw material remains in TLC detection, and a new spot is generated. The reaction was quenched with water and the reaction was extracted with EtOAc (3x150mL), the organic phases combined, dried over anhydrous sodium sulfate, concentrated by filtration and purified by column chromatography (PE/EtOAc ═ 1:1) to give the product LP-C6-04(900mg, light yellow oil, 25% yield). MS (ESI) M/z [ M-H]-265.26。
(5) Synthesis of LP-C6-05
Figure BDA0002901221770000132
Stock LP-C6-04(500mg, 1.88mmol, 1.0eq) and stock 1-nonanol (540mg, 3.76mmol, 2.0eq) were dissolved in DCM (20mL)EDCI (400mg, 2.07mmol, 1.1eq), DMAP (46mg, 0.38mmol, 0.2eq) and DIEA (730mg, 5.64mmol, 3.0eq) were added successively and reacted at room temperature under nitrogen for 16 h. TLC detection raw material disappeared, and new spot was generated. Water and DCM were added to the reaction solution for liquid separation, the organic phase was washed with water, Brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (PE/EtOAc 15:1) to give product LP-C6-05(430mg, colorless oil, yield 58%), MS (ESI) M/z [ M + Na ])]+415.24。
(6) Synthesis of LP-C6-06
Figure BDA0002901221770000133
The starting material LP-C6-05(300mg, 0.77mmol, 1.0eq) was dissolved in ethyl acetate (30mL), palladium on carbon (60mg, 20% wt) was added, the mixture was replaced with hydrogen three times, and the reaction was carried out at room temperature for 2h under the protection of a hydrogen balloon. TLC detection raw material disappeared, and new spot was generated. The insoluble catalyst was removed by filtration through a filter. Concentrating the filtrate to obtain LP-C6-06(210mg, light gray oil, yield 91%), MS (ESI) M/z [ M + Na ]]+325.14。
(7) Synthesis of LP-C6-07
Figure BDA0002901221770000141
Starting material LP-C6-06(210mg, 0.7mmol, 1.0eq) was dissolved in DCM (20mL) and CBr was added4(350mg, 1.05mmol, 1.5eq) and then PPh was added to the system in portions3(310mg, 1.19mmol, 1.7eq), after addition the reaction was carried out at room temperature for 1 h. TLC detection raw material disappeared, and new spot was generated. The reaction was stirred directly on silica gel and purified by column chromatography (PE/EtOAc ═ 20:1) to give LP-C6-07(245mg, colorless oil, 96% yield), MS (ESI) M/z [ M + Na ], (ESI)]+387.03。
(8) Synthesis of LP-C6
Figure BDA0002901221770000142
Raw material LP-C6-07(190mg, 0.52mmol) and LP-Z1(230mg, 0.52mmol) as starting material were dissolved in ethanol (20mL), DIEA (200mg, 1.56mmol) was added and the reaction was warmed to 65 ℃ for 16 h. TLC detection had starting material remaining, but major new spots were generated. In MS (ESI), m/z shows that there are products and raw materials. The solvent was removed by concentration under reduced pressure, the remaining portion was diluted with DCM, the organic phase was washed twice with water, with saturated brine, dried over anhydrous sodium sulfate, concentrated by filtration and purified by column chromatography to give the product LP-C6(65mg, yellow oil, yield 16%). MS (ESI) M/z [ M + H]+726.66。
1HNMR(400MHz,CDCl3)δ4.85(d,J＝8.5Hz,1H),4.04(t,J＝7.5Hz,2H),3.65(br,1H),3.44–3.37(m,4H),2.68(br,4H),2.37(t,J＝9.5Hz,2H),2.26(t,J＝9.5Hz,2H),1.88(d,J＝7.5Hz,2H),1.67–1.44(m,14H),1.30-1.24(m,46H),0.89-0.84(s,9H)。
EXAMPLE 7 Synthesis of Compound LP-C7
(1) Synthesis of LP-C7-01
Figure BDA0002901221770000143
Starting material 6-bromohexanoic acid (2.0g, 13.2mmol) was dissolved in DMF (60mL) at 0 deg.C and NaH (1.1g, 27.7mmol) was added for reaction at room temperature for 30 min. Then, 2-benzyloxyethanol (2.6g, 13.2mmol) was added to the system, and the mixture was reacted at room temperature overnight under nitrogen atmosphere. The reaction was carried out for 3h at an elevated temperature of 50 ℃. New spots were generated by TLC detection. The reaction solution was diluted with EtOAc, quenched with water, separated, the aqueous phase was adjusted to pH 2-3 with dilute hydrochloric acid, the aqueous phase was extracted three times with DCM, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to give LP-C7-01(840mg, yellow oil, 24% yield).
(2) Synthesis of LP-C7-02
Figure BDA0002901221770000151
Starting material LP-C7-01(840mg, 3.16mmol) and starting material 1-nonanol (500mg, 3.48mmol) were dissolved in DCM (30mL), EDCI (668mg, 3.48mmol), DIEA (1.2g, 9.48mmol) and DMAP (77mg, 0.63mmol) were added sequentially, and reacted at room temperature under nitrogen for 16 h. New spots were formed by TLC detection, and the reaction solution was directly added to silica gel and stirred, followed by purification by column chromatography to give LP-C7-02(780mg, pale yellow oil, yield 63%).
(3) Synthesis of LP-C7-03
Figure BDA0002901221770000152
The starting material LP-C7-02(370mg, 0.94mmol) was dissolved in ethyl acetate (20mL), palladium on carbon (74mg, 20% wt) was added, hydrogen was substituted three times, and the reaction was carried out at room temperature for 3h under the protection of a hydrogen balloon. TLC detection raw material disappeared, and new spot was generated. The palladium-carbon and insoluble substances are removed by filtration through a filter membrane, and the filtrate is concentrated to obtain a product LP-C7-03(285mg, colorless oil, quantitative) which is directly used for the next reaction.
(4) Synthesis of LP-C7-04
Figure BDA0002901221770000153
Starting materials LP-C7-03(285mg, 0.94mmol) and CBr4(1.09g, 3.29mmol) was dissolved in DCM and PPh was added to the system in portions at 0 deg.C3(616mg, 2.35mmol) and then reacted at room temperature for 1 h. TLC detection raw material disappeared, and new spot was generated. Silica gel was directly added to the reaction mixture, and the mixture was purified by column chromatography to give LP-C7-04(310mg, colorless oil, yield 91%).
(5) Synthesis of LP-C7
Figure BDA0002901221770000154
The starting material LP-C7-04(150mg, 0.41mmol) and the starting material LP-Z1(182mg, 0.45mmol) were dissolved in absolute ethanol (10mL), DIEA (58mg, 0.45mmol) was added and the temperature was raised to 65 ℃ for reaction for 16 h. TLC detection leaves a small amount of starting material, mainly new spots. The reaction solution was directly added to silica gel and stirred, purified by column chromatography to give LP-C7(70mg, yellow oil, yield 2)4％)。MS(ESI):m/z[M+H]+726.78。
1HNMR(500MHz,CDCl3)δ4.85(q,J＝6.5Hz,1H),4.45(br,1H),4.05(t,J＝6.5Hz,2H),4.03-3.95(m,4H),3.48(t,J＝6.5Hz,2H),3.37-3.17(m,6H),2.29(dt,J＝11.5,7.5Hz,4H),1.93-1.85(m,2H),1.72–1.45(m,14H),1.43–1.18(m,40H),0.90-0.87(m,9H)。
EXAMPLE 8 Synthesis of Compound LP-C8
(1) Synthesis of LP-C8-01
Figure BDA0002901221770000161
1, 4-butanediol (3.14g, 34.9mmol) as a raw material is dissolved in DMF (50mL), NaH (0.7g, 17.5mmol) is added under the condition of ice-water bath, after stirring and reacting for 0.5h ((2-bromoethoxy) methyl) benzene (2.5g, 11.6mmol) is added into a reaction bottle, and the temperature is raised to room temperature for reacting for 16 h. TLC monitored the reaction completion, water was added to quench the reaction, and the reaction solution was extracted with 100mL of ethyl acetate, the organic phase was washed 3 times with saturated brine, the organic phase was retained, dried over anhydrous sodium sulfate, concentrated by vacuum distillation, and the residue was purified by column chromatography to give product LP-C8-01(1.9g, colorless oil, yield 73%).
(2) Synthesis of LP-C8-02
Figure BDA0002901221770000162
The starting material LP-C8-01(0.4g, 1.8mmol) was dissolved in DMF (15mL), PDC (1.0g, 2.6mmol) was added with stirring, and the reaction was carried out at room temperature for 16 h. TLC monitored the reaction completion, water was added to quench the reaction, the reaction was extracted with ethyl acetate, the combined organic phases were washed with saturated brine 1 time, dried over anhydrous sodium sulfate, concentrated by distillation under reduced pressure, and the residue was purified by column chromatography to give product LP-C8-02(360mg, colorless oil, yield 84%).
(3) Synthesis of LP-C8-03
Figure BDA0002901221770000163
Starting material LP-C8-02(360mg, 1.5mmol) was dissolved in DCM (10mL) and n-nonanol (280mg, 1.9mmol), TEA (197mg, 2.0mmol) and PyBOP (858mg, 1.6mmol) were added sequentially. Then the reaction was carried out at room temperature for 2 hours. TLC monitored the completion of the reaction. The reaction solution was concentrated, and the residue was purified by column chromatography to give product LP-C8-03(80mg, colorless oil, yield 15%).
(4) Synthesis of LP-C8-04
Figure BDA0002901221770000171
The starting material LP-C8-03(76mg, 0.21mmol) was dissolved in ethyl acetate (10mL), Pd/C (40mg, 20% wt) was added, hydrogen replaced three times, and the reaction was carried out at room temperature for 2h under the protection of a hydrogen balloon. The reaction was complete by TLC. Palladium on carbon was removed by filtration through Celite, and the filtrate was concentrated to give LP-C8-04(24mg, pale gray oil, yield 42%).
(5) Synthesis of LP-C8-05
Figure BDA0002901221770000172
Starting material LP-C8-04(24mg, 0.09mmol) was dissolved in DCM (10mL) and carbon tetrabromide (44mg, 0.13mmol) and triphenylphosphine (34mg, 0.13mmol) were added sequentially. After the addition, the reaction was carried out at room temperature for 2 hours. The reaction was completed by TLC detection. Silica gel was directly added to the reaction mixture to mix with a sample, and the mixture was purified by column chromatography to give LP-C8-05(29mg, colorless oil, yield 99%).
(6) Synthesis of LP-C8
Figure BDA0002901221770000173
The starting materials LP-C8-05(29mg, 0.09mmol) and LP-Z1(49mg, 0.11mmol) were dissolved in absolute ethanol (5mL) and DIEA (19mg, 0.10mmol) was added. Heated to 65 ℃ and reacted for 16 h. The reaction was completed by TLC detection. Is cooled toThe solvent was removed by concentration under reduced pressure at room temperature, and the remaining portion was purified by column chromatography to give the product LP-C8(27mg, pale yellow oil, yield 42%). MS (ESI) M/z [ M + H]+698.61。
1HNMR(500MHz,CDCl3)δ4.84(m,1H),4.04(dd,2H),3.51(br,2H),3.45(t,2H),2.60(br,4H),2.37(t,4H),2.25(t,2H),1.88(m,2H),1.65-1.26(m,54H),0.86(m,9H)。
EXAMPLE 9 Synthesis of Compound LP-C9
(1) Synthesis of LP-C9
Figure BDA0002901221770000181
The starting materials LP-C9-01(200mg, 0.55mmol) and LP-Z1(266mg, 0.60mmol) were dissolved in anhydrous ethanol (5mL) and DIEA (116mg, 0.60mmol) was added. Heated to 65 ℃ and reacted for 16 h. The reaction was complete by TLC. Cooled to room temperature, concentrated under reduced pressure to remove the solvent, and the remaining portion was purified by column chromatography to give product LP-C9(47mg, pale yellow oil, yield 12%). MS (ESI) M/z [ M + H]+726.66。
1HNMR(500MHz,CDCl3)δ4.87(m,1H),4.05(dd,2H),3.65(br,2H),3.41(m,4H),2.63(br,6H),2.29(m,4H),1.66-1.29(m,58H),0.89(m,9H)。
EXAMPLE 10 Synthesis of Compound LP-C10
(1) Synthesis of LP-C10
Figure BDA0002901221770000182
The starting materials LP-C10-01(154mg, 0.50mmol) and LP-Z1(200mg, 0.45mmol) were dissolved in anhydrous ethanol (5mL) and DIEA (96mg, 0.50mmol) was added. Heated to 65 ℃ and reacted for 16 h. The reaction was completed by TLC detection. Cooled to room temperature, concentrated under reduced pressure to remove the solvent, and the remaining portion was purified by column chromatography to give product LP-C10(76mg, pale yellow oil, yield 24%). MS (ESI) M/z [ M + H]+698.83。
1HNMR(500MHz,CDCl3)δ4.86(m,1H),4.09(t,2H),3.70(br,2H),3.41(m,4H),2.70(br,6H),2.30(m,4H),1.63-1.26(m,54H),0.89(m,9H)。
EXAMPLE 11 Synthesis of Compound LP-C11
(1) Synthesis of LP-C11
Figure BDA0002901221770000183
The starting materials LP-C11-01(450mg, 1.34mmol) and LP-Z1(531mg, 1.20mmol) were dissolved in absolute ethanol (5mL) and DIEA (282mg, 1.47mmol) was added. Heated to 65 ℃ and reacted for 16 h. The reaction was completed by TLC detection. Cooled to room temperature, concentrated under reduced pressure to remove the solvent, and the remaining portion was purified by column chromatography to give product LP-C11(340mg, pale yellow oil, yield 40%). MS (ESI) M/z [ M + H]+698.66。
1HNMR(500MHz,CDCl3)δ4.87(m,1H),4.10(t,2H),3.55(br,2H),3.41(m,4H),2.70(br,6H),2.30(m,4H),1.63-1.26(m,54H),0.89(m,9H)。
EXAMPLE 12 Synthesis of Compound LP-C12
(1) Synthesis of LP-C12
Figure BDA0002901221770000191
The starting materials LP-C12-01(363mg, 0.99mmol) and LP-Z1(400mg, 0.90mmol)) were dissolved in absolute ethanol (5mL) and DIEA (191mg, 1.00mmol) was added. Heated to 65 ℃ and reacted for 16 h. The reaction was completed by TLC detection. Cooled to room temperature, concentrated under reduced pressure to remove the solvent, and the remaining portion was purified by column chromatography to give product LP-C12(253mg, pale yellow oil, yield 38%). MS (ESI) M/z [ M + H]+726.77。
1HNMR(500MHz,CDCl3)δ4.86(m,1H),4.06(t,2H),3.97(br,2H),3.40(m,4H),3.07(br,6H),2.30(m,4H),1.63-1.26(m,58H),0.89(m,9H)。
EXAMPLE 13 Synthesis of Compound LP-C13
(1) Synthesis of LP-C13
Figure BDA0002901221770000192
The starting materials LP-C13-01(273mg, 0.75mmol) and LP-Z1(300mg, 0.68mmol) were dissolved in absolute ethanol (5mL) and DIEA (143mg, 0.74mmol) was added. Heated to 65 ℃ and reacted for 16 h. The reaction was completed by TLC detection. Cooled to room temperature, concentrated under reduced pressure to remove the solvent, and the remaining portion was purified by column chromatography to give product LP-C13(200mg, pale yellow oil, yield 40%). MS (ESI) M/z [ M + H]+726.77。
1HNMR(500MHz,CDCl3)δ4.86(m,1H),4.22(m,2H),3.95(br,2H),3.62(m,4H),3.46(t,2H),3.04(br,6H),2.37(t,2H),2.28(t,2H),1.63-1.26(m,56H),0.88(m,9H)。
EXAMPLE 14 Synthesis of Compound LP-C14
(1) Synthesis of LP-C14
Figure BDA0002901221770000201
The starting materials LP-C14-01(167mg, 0.50mmol) and LP-Z1(200mg, 0.45mmol) were dissolved in anhydrous ethanol (5mL) and DIEA (96mg, 0.50mmol) was added. Heated to 65 ℃ and reacted for 16 h. The reaction was completed by TLC detection. Cooled to room temperature, concentrated under reduced pressure to remove the solvent, and the remaining portion was purified by column chromatography to give the product LP-C14(100mg, pale yellow oil, yield 30%). MS (ESI) M/z [ M + H]+726.77。
1HNMR(500MHz,CDCl3)δ4.86(m,1H),4.22(m,2H),3.91(br,2H),3.62(m,4H),3.46(t,2H),2.97(br,6H),2.34(t,2H),2.28(t,2H),1.70-1.26(m,54H),0.88(m,9H)。
EXAMPLE 15 Synthesis of Compound LP-C15
(1) Synthesis of LP-C15
Figure BDA0002901221770000202
The raw material LP-C15-01(660mg, 1.81mmol) and LP-Z1(721mg, 1.63mmol) were dissolved in absolute ethanol (5mL) and DIEA (381mg, 1.98mmol) was added. Heated to 65 ℃ and reacted for 16 h. The reaction was completed by TLC detection. Cooled to room temperature, concentrated under reduced pressure to remove the solvent, and the remaining portion was purified by column chromatography to give the product LP-C15(390mg, pale yellow oil, yield 33%). MS (ESI) M/z [ M + H]+726.75。
1HNMR(500MHz,CDCl3)δ4.86(m,1H),4.09(t,2H),3.94(br,2H),3.41(m,4H),3.03(br,6H),2.30(dt,4H),1.72-1.26(m,54H),0.88(m,9H)。
EXAMPLE 16 Synthesis of Compound LP-C16
(1) Synthesis of LP-C16-01
Figure BDA0002901221770000203
Starting material 1, 7-heptanediol (3.8g, 29.1mmol) was dissolved in DMF (70mL) at 0 deg.C, and NaH (580mg, 14.6mmol) was added for reaction for 30 min. Then, 1-bromononane (2.0g, 9.7mmol) was added, and the reaction was carried out at room temperature for 16 hours. New spots were generated by TLC detection. The reaction was diluted with EtOAc, quenched with water, washed three times with water, once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to give the product LP-C6-01(1.5g, pale yellow oil, yield 60%).
(2) Synthesis of LP-C16-02
Figure BDA0002901221770000211
Starting materials LP-C6-01(600mg, 2.3mmol) and CBr4(2.7g, 8.1mmol) was dissolved in DCM (20mL) and PPh was added slowly in portions3(1.5g, 5.75mmol), and after the addition was complete, the reaction was carried out at room temperature for 1 hour. TLC detection raw material disappeared, and new spot was generated. The reaction mixture was directly added to silica gel and stirred, and purified by column chromatography to obtain LP-C6-02(680mg, colorless oil, yield 91%).
(3) Synthesis of LP-C16
Figure BDA0002901221770000212
The starting material LP-C6-02(200mg, 0.625mmol) and the starting material LP-Z1(304mg, 0.69mmol) were dissolved in ethanol (20mL), DIEA (89mg, 0.69mmol) was added, and the temperature was raised to 65 ℃ for reaction for 16 h. TLC detection has a small amount of starting material remaining and new spots are generated. The reaction mixture was directly added to silica gel and stirred, followed by column chromatography purification to give LP-C6(40mg, yellow oil, yield 10%). MS (ESI) M/z [ M + H]+682.68。
1HNMR(500MHz,CDCl3)δ4.87(t,J＝6.5Hz,1H),3.71(s,2H),3.39(t,J＝6.5Hz,4H),2.71(s,6H),2.28(t,J＝7.5Hz,2H),1.71–1.46(m,14H),1.39–1.19(m,44H),0.88(t,J＝7.0Hz,9H)。
EXAMPLE 17 Synthesis of Compound LP-C17
(1) Synthesis of LP-C17
Figure BDA0002901221770000213
The starting materials LP-C17-01(218mg, 0.75mmol) and LP-Z1(300mg, 0.68mmol) were dissolved in absolute ethanol (5mL) and DIEA (143mg, 0.74mmol) was added. Heated to 65 ℃ and reacted for 16 h. The reaction was completed by TLC detection. Cooled to room temperature, concentrated under reduced pressure to remove the solvent, and the remaining portion was purified by column chromatography to give product LP-C17(101mg, pale yellow oil, yield 23%). MS (ESI) M/z [ M + H]+655.15。
1HNMR(500MHz,CDCl3)δ4.86(m,1H),3.88(br,2H),3.40(dt.,4H),2.92(br,6H),2.28(t,2H),1.60-1.20(m,58H),0.88(m,9H)。
EXAMPLE 18 Synthesis of Compound LP-C18
(1) Synthesis of LP-C18
Figure BDA0002901221770000221
The starting materials LP-C18-01(28mg, 0.08mmol) and LP-C19-03(30mg, 0.07mmol) were dissolved in absolute ethanol (5mL) and DIEA (R) (0.3 mmol) was added143mg, 0.74 mmol). Heated to 65 ℃ and reacted for 16 h. The reaction was complete by TLC. Cooled to room temperature, concentrated under reduced pressure to remove the solvent, and the remaining portion was purified by column chromatography to give product LP-C18(17mg, pale yellow oil, yield 34%). MS (ESI) M/z [ M + H]+682.75。
1HNMR(500MHz,CDCl3)δ4.85(m,1H),4.04(t,2H),3.45(br,5H),2.60(br,4H),2.37(t,2H),2.26(t,2H),1.88(m,2H),1.70-1.26(m,60H),0.88(m,9H)。
EXAMPLE 19 Synthesis of Compound LP-C19
(1) Synthesis of LP-C19-01
Figure BDA0002901221770000222
The starting material, 1, 7-heptanediol (3.74g, 28.3mmol) was dissolved in DMF (50mL), NaH (0.95g, 23.8mmol) was added under ice-water bath, and the reaction was stirred for 1 h. 9-Bromopentadecane (890mg, 2.8mmol) was then added, and the reaction was allowed to warm to room temperature for 16 h. TLC monitored the completion of the reaction. The reaction was quenched with water, the aqueous phase was extracted with ethyl acetate, the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to give the product LP-C19-01(251mg, colorless oil, yield 25%).
(2) Synthesis of LP-C19-02
Figure BDA0002901221770000223
The starting material LP-C19-01(250mg, 0.7mmol) was dissolved in DCM (5mL), carbon tetrabromide (0.34g, 1.0mmol) and triphenylphosphine (0.27g, 1.0mmol) were added sequentially with stirring, and the reaction was stirred at RT for 1 h. The reaction was completed by TLC detection. The reaction solution was concentrated, and the residue was purified by column chromatography to give product LP-C19-02(31mg, colorless oil, yield 11%).
(3) Synthesis of LP-C19-03
Figure BDA0002901221770000231
The starting material LP-C19-02(31mg, 0.07mmol) was dissolved in absolute ethanol (5mL), 2-aminoethanol (436mg, 7.2mmol) was added, and the reaction was stirred for 16h while heating to 62 ℃. TLC monitored the end of the reaction. The reaction was cooled to room temperature, diluted with ethyl acetate and washed 3 times with water, separated, the organic phase retained, dried over anhydrous sodium sulfate and concentrated to give the product LP-C19-03(31mg, pale yellow oil, crude) which was used directly in the next reaction.
(4) Synthesis of LP-C19
Figure BDA0002901221770000232
The starting material LP-C19-03(31mg, 0.08mmol) was dissolved in absolute ethanol (5mL), the starting material LP-C19-01(36mg, 0.11mmol) and DIEA (17mg, 0.09mmol) were added, and the temperature was raised to 65 ℃ for reaction for 16 h. The reaction was completed by TLC detection. After cooling to room temperature, silica gel was directly added to the reaction solution to stir it, followed by purification by column chromatography to give product LP-C19(20mg, brown yellow oil, yield 19%). MS (ESI) M/z [ M + H]+654.58。
1HNMR(500MHz,CDCl3)δ3.79(s,2H),3.39(t,2H),3.18(p,1H),2.84(d,6H),1.70-1.26(m,60H),0.88(m,9H)。
EXAMPLE 20 Synthesis of Compound LP-C20
(1) Synthesis of LP-C20-01
Figure BDA0002901221770000233
The starting material, 1, 7-heptanediol (2.0g, 15.15mmol) was dissolved in DMF (20mL), and NaH (606mg, 15.15mmol) was added to react at room temperature for 20 min. Then, under the protection of nitrogen, BnBr (2.59g, 15.15mmol) was slowly added to the system. After the addition was completed, the reaction was maintained at room temperature for 16 hours. TLC detection raw material disappeared, and new spot was generated. Diluting the reaction solution with EtOAc, adding water for quenching, washing the organic phase with water for three times, washing with saturated salt water for one time, drying with anhydrous sodium sulfate, filtering, concentrating, and purifying by column chromatography to obtain the final productLP-C20-01(1.2g, colorless oil, yield 36%). MS (ESI) M/z [ M + H]+222.82。
(2) Synthesis of LP-C20-02
Figure BDA0002901221770000234
Starting materials LP-C20-01(1.2g, 5.4mmol) and CBr4(2.7g, 8.1mmol) was dissolved in DCM (20mL) and PPh was added slowly in portions3(2.4g, 9.18mmol), and after the addition was complete, the reaction was carried out for 1h at room temperature. TLC detection the disappearance of the starting material and new spots were generated. The reaction solution was directly added to silica gel and stirred, followed by purification by column chromatography to give LP-C20-02(1.6g, colorless oil, yield 99%). MS (ESI) M/z [ M + K]+322.94。
(3) Synthesis of LP-C20-03
Figure BDA0002901221770000241
Starting material 1-nonanethiol (450mg, 2.8mmol) was dissolved in DMF (10mL) and NaH (115mg, 2.8mmol) was added for reaction at room temperature for 30 min. LP-C20-02(800mg, 2.8mmol) was then added and the reaction was allowed to warm to 70 ℃ under nitrogen for 16 h. TLC showed the starting material was substantially lost and new spots were generated. The reaction was diluted with EtOAc, quenched with water, washed three times with water, once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to give product LP-C20-03(750mg, colorless oil, 73% yield). MS (ESI) M/z [ M + H]+365.40。
(4) Synthesis of LP-C20-04
Figure BDA0002901221770000242
Raw material LP-C20-03(750mg, 2.06mmol) was dissolved in methanol (20mL), palladium on carbon (750mg, 100% wt) was added, hydrogen was substituted three times, and the mixture was heated to 50 ℃ under the protection of a hydrogen balloon and reacted for 16 h. TLC detection has residual material and mainly produces new spots. Filtering with diatomaceous earth to remove palladium carbon, concentrating the filtrate, and passing through column layerThe product was isolated and purified LP-C20-04(170mg, white solid, yield 30%). MS (ESI) M/z [ M + H]+275.36。
(5) Synthesis of LP-C20-05
Figure BDA0002901221770000243
Starting materials LP-C20-04(170mg, 0.62mmol) and CBr4(309mg, 0.93mmol) was dissolved in DCM (10mL) and PPh was added slowly in portions3(276mg, 1.05mmol), and after the addition was complete, the reaction was carried out at room temperature for 1 hour. TLC detection raw material disappeared, and new spot was generated. Silica gel was directly added to the reaction mixture to mix the mixture, and the mixture was purified by column chromatography to obtain LP-C20-05(190mg, colorless oil, yield 91%).
(6) Synthesis of LP-C20
Figure BDA0002901221770000251
The starting material LP-Z1(249mg, 0.565mmol) and the starting material LP-C20-05(190mg, 0.565mmol) were dissolved in absolute ethanol (10mL), DIEA (109mg, 0.85mmol) was added, and the reaction was allowed to proceed overnight at 70 ℃. A part of the raw material remained in TLC detection, and a new spot was generated. Directly adding silica gel into the reaction solution for sample stirring. Purification by column chromatography gave the product LP-C20(79mg, yellow oil, 20% yield). MS (ESI) M/z [ M + H]+698.60。
1HNMR(500MHz,CDCl3)δ4.86(t,J＝6.5Hz,1H),3.74(s,2H),2.84(s,2H),2.72(s,4H)2.50(td,J＝7.5,2.0Hz,4H),2.28(t,J＝7.5Hz,2H),1.74–1.46(m,14H),1.43–1.18(m,44H),0.92–0.84(m,9H)。
EXAMPLE 21 Synthesis of Compound LP-C21
(1) Synthesis of LP-C21-01
Figure BDA0002901221770000252
The starting 4- (methylamino) butanoic acid hydrochloride (2.0g, 13mmol) was dissolved in THF (30mL) and addedNaOH (1.56g, 39mmol) in water (10mL) and Boc2O (5.7g, 26mmol) and then heated to 50 ℃ under nitrogen for 16 h. After cooling to room temperature, THF was removed by concentration under reduced pressure, the reaction solution was diluted with ethyl acetate, the pH was adjusted to 3-4 with dilute hydrochloric acid, the organic phase was separated, washed once with water, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to give LP-C21-01(1.8g, colorless oil, yield 64%).
(2) Synthesis of LP-C21-02
Figure BDA0002901221770000253
The starting materials LP-C27-06(260mg, 0.38mmol) and LP-C21-01(249mg, 1.14mmol) were dissolved in DCE (20mL), DCC (313mg, 1.52mmol), TEA (192mg, 1.9mmol) and DMAP (12mg, 0.1mmol) were added, and the temperature was raised to 50 ℃ for reaction for 16 h. TLC detection had residual starting material, new spots were generated, and no progress was made with additional and extended time. The reaction mixture was directly sampled and purified by column chromatography to give LP-C21-02(135mg, colorless oil, yield 40%). MS (ESI) M/z [ M-Boc]+780.69 and MS (ESI) M/z [ M + NH4 +]+897.88 (ammoniation)
(3) Synthesis of LP-C21-03
Figure BDA0002901221770000261
Starting material LP-C21-02(120mg, 0.14mmol) was dissolved in DCM (10mL), TFA (2mL) was added and reacted at room temperature for 2 h. The reaction was completed by TLC detection. Concentrating under reduced pressure to remove solvent and TFA, and pumping to dryness to obtain product LP-C21-03(180mg, TFAsalt, quantitative), MS (ESI): M/z [ M + H ]]+780.63. Directly used for the next reaction.
(4) Synthesis of LP-C21-04
Figure BDA0002901221770000262
Raw material LP-C21-03(180mg, creudeTFAsalt) was dissolved in DCM (20mL), and TEA (282mg, 2.8mmol) and 1, 3-di-Boc-2- (trifluoromethylsulfonyl) guanidine (328mg, 0.84mmol) were added and reacted at room temperature for 48 h. In MS (ESI), m/z detection shows that a small amount of raw materials are remained, and mainly products are generated. The reaction solution is directly mixed with a sample and purified by column chromatography to obtain the product LP-C21-04(120mg, crude product, yellow oily, yield 84%), MS (ESI) M/z [ M + H ]]+1022.89. The crude product was used directly in the next reaction.
(5) Synthesis of LP-C21
Figure BDA0002901221770000263
Starting material LP-C21-04(120mg, crude, 0.12mmol) was dissolved in DCM (10mL), TFA (5mL) was added, and reacted at room temperature for 16 h. New spots were generated by TLC detection. The solvent and excess TFA were removed by concentration under reduced pressure, followed by purification by column chromatography to give the product LP-C21(13mg, yellow oil, 14% yield). MS (ESI) M/z [ M + H]+822.75。
EXAMPLE 22 Synthesis of Compound LP-C22
(1) Synthesis of LP-C22-01
Figure BDA0002901221770000264
The starting material LP-C27-06(300mg, 0.44mmol) and the starting material Boc-beta-alanine (166mg, 0.88mmol) were dissolved in DCE (20mL), DCC (227mg, 1.1mmol), TEA (222mg, 2.2mmol) and DMAP (11mg, 0.09mmol) were added, and the temperature was raised to 50 ℃ under nitrogen for 16 h. The TLC detection has residual raw material and new spots are generated. Cooling to room temperature, adding silica gel into the direct reaction solution, stirring, and purifying by column chromatography to obtain LP-C22-01(185mg, crude product, colorless oil, yield 49%), MS (ESI) M/z [ M-Boc%]+752.66. Directly used for the next reaction.
(2) Synthesis of LP-C22-02
Figure BDA0002901221770000271
Starting material LP-C22-01(185mg, 0.22mmol) was dissolved in DCM (10mL), TFA (3mL) was added and reacted at room temperature for 1 h. The reaction was completed by TLC detection. The solvent and TFA were removed by concentration under reduced pressure, the remainder was taken up twice with DCM to further remove TFA, and the product LP-C22-02(260mg, credTFAsalt) was obtained by oil pump drying and used directly in the next reaction.
(3) Synthesis of LP-C22-03
Figure BDA0002901221770000272
Starting material LP-C22-02(260mg) was dissolved in DCM (10mL), and TEA (1mL) in excess and starting material 1, 3-bis-BOC-2- (trifluoromethylsulfonyl) guanidine (95mg, 0.242mmol) were added and reacted at room temperature under nitrogen for 16 h. MS (ESI) in which m/z shows that the product was formed. The reaction mixture was directly stirred and purified by column chromatography to give LP-C22-03(156mg, crude, colorless oil, yield 72%). MS (ESI) M/z [ M + H]+752.66 (raw material) and MS (ESI) M/z [ M + H]+995.14 (product), TLC showed one spot. The crude product was used directly in the next reaction.
(4) Synthesis of LP-C22
Figure BDA0002901221770000273
Starting material LP-C22-03(156mg, crede, 0.16mmol) was dissolved in DCM (10mL), TFA (4mL) was added and reacted at room temperature for 16 h. MS (ESI) m/z detection reaction is complete. The solvent and TFA were removed by concentration under reduced pressure, the remaining portion was taken up twice with acetonitrile, TFA was further removed, and then purification by column chromatography gave the product LP-C22(40mg, colorless oil, yield 23%), MS (ESI): M/z [ M + H ]]+794.71。
EXAMPLE 23 Synthesis of Compound LP-C27
(1) Synthesis of LP-C27-01
Figure BDA0002901221770000281
To a solution of sodium ethoxide (35.7g, 0.21mol) in anhydrous ethanol (60mL) was added diethyl 1, 3-acetonedicarboxylate (21.3g, 0.105mol) as a starting material, the mixture was heated to reflux, ethyl 8-bromooctanoate (25g, 0.105mol) was slowly added, and the reaction was refluxed for 2 hours. Sodium ethoxide (35.7g, 0.105mol) was added again to the reaction and ethyl 8-bromooctanoate (25g, 0.105mol) was added again slowly, after which the reaction was refluxed for 16 h. The solvent was removed by concentration under reduced pressure, the remaining portion was diluted with EtOAc, the organic phase was washed twice with saturated ammonium chloride solution, once with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give product LP-C27-01(58g, crude, yellow oil, quantitative). The crude product was used directly in the next reaction.
(2) Synthesis of LP-C27-02
Figure BDA0002901221770000282
Starting material LP-C27-01(58g, crude, 0.11mol) was dissolved in concentrated HCl/glacial acetic acid (110mL/60mL) and heated to reflux for 24 h. MS (ESI) in which m/z shows that the product was formed. Cooling to room temperature to precipitate solid, concentrating under reduced pressure to remove hydrochloric acid and acetic acid, washing the obtained solid with water twice, concentrating under reduced pressure to remove excessive water, and recrystallizing the rest with acetone to obtain product LP-C27-02(8.3g, white solid, yield 24%), MS (ESI): M/z [ M-H ]]-313.18。
(3) Synthesis of LP-C27-03
Figure BDA0002901221770000283
Starting material LP-C27-02(10.2g, 32.48mmol) and starting material 1-nonanol (10.3g, 71.46mmol) were dissolved in DCM (260mL), DCC (26.7g, 129.92mmol) and DMAP (11.9g, 97.44mmol) were added sequentially, and then reacted at room temperature for 16 h. TLC detection raw material disappeared, and new spot was generated. Filtering the reaction solution to remove insoluble solid, concentrating the filtrate, purifying by column chromatography, pulping the crude product with PE/EtOAc to obtain LP-C27-03(9.4g, white solid, yield 51%), MS (ESI) M/z [ M + Na ]]+589.36。
(4) Synthesis of LP-C27-04
Figure BDA0002901221770000291
The starting material LP-C27-03(15.5g, 27.39mmol) was dissolved in THF (150mL), and a solution of LiOH. H2O (1.38g, 32.87mmol) in water (30mL) was added, followed by reaction at room temperature overnight. TLC detection has residual raw material, product generation and double hydrolysis product. The solvent was removed by concentration under reduced pressure, the remaining part was diluted with water, and the pH was adjusted to 2-3 with dilute hydrochloric acid, and the mixture was extracted four times with DCM, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, purified by column chromatography, and the recovered starting material was hydrolyzed again and purified to finally obtain LP-C27-04(2.6g, white solid, yield 22%). MS (ESI) M/z [ M + Na%]+463.22。
(5) Synthesis of LP-C27-05
Figure BDA0002901221770000292
Starting material LP-C27-04(2.6g, 5.9mmol) and starting material 9-heptadecanol (1.8g, 7.08mmol) were dissolved in DCM (100mL) and DCC (2.43g, 11.8mmol) and DMAP (1.1g, 8.85mmol) were added. Then reacted at room temperature for 16 h. TLC detection raw material disappears, and a new spot is generated. Filtering to remove insoluble solid, concentrating the filtrate, purifying by column chromatography to obtain product LP-C27-05(4.47g, crude product, light yellow solid, main impurity is 9-heptadecanol), and directly using the crude product in the next step, MS (ESI) M/z [ M + Na ]]+701.64。
(6) Synthesis of LP-C27-06
Figure BDA0002901221770000293
Starting material LP-C27-05(4.47g, crude, 6.59mmol) was dissolved in MeOH (40mL) and NaBH was added slowly in portions4(376mg, 9.9mmol), and after the addition was completed, the reaction was carried out at room temperature for 30 min. TLC detection raw material disappeared, and new spot was generated. Adding a small amount of water to quench the reaction, and directly using anhydrous sulfur as reaction liquidSodium sulfate drying, filtering, concentrating the filtrate, and purifying by column chromatography to obtain LP-C27-06(2.5g, pale yellow oily substance, yield 62%), MS (ESI) M/z [ M + Na ]]+703.59。
(7) Synthesis of LP-C27
Figure BDA0002901221770000301
The starting materials LP-C27-06(100mg, 0.15mmol) and 4-dimethylaminobutyric acid hydrochloride (50mg, 0.3mmol) were dissolved in DCM (15mL), DCC (68mg, 0.33mmol), TEA (76mg, 0.75mmol) and DMAP (4mg, 0.03mmol) were added sequentially, reacted at room temperature for 16h under nitrogen protection, and reacted at elevated temperature to 40 ℃ for 3 h. TLC detection raw material basically disappears, and new spots are generated. Silica gel is directly added into the reaction solution for sample stirring and purification by column chromatography is carried out to obtain the product LP-C27(40mg, yellow oily substance, yield 34%), MS (ESI) M/z [ M + H ]]+794.60。
1HNMR(400MHz,CDCl3)δ4.84(t,J＝6.4Hz,2H),4.08–4.00(m,2H),2.45–2.16(m,15H),1.90–1.73(m,4H),1.58(d,J＝8.4Hz,7H),1.48(s,9H),1.25(d,J＝9.1Hz,62H),0.87(d,J＝6.0Hz,9H).
Example 23 preparation of a pharmaceutical composition: the liposome is coated with siRNA therapeutic agent.
(1) Solution preparation
Preparing an acetate buffer solution with the concentration of 20mM and the pH value of 4.0 by adopting a standard method by taking 15% ethanol as a medium; a 10mM cholesterol ethanol solution (A); DOPE ethanol solution (B) with a concentration of 10 mM; a 10mM ethanol solution of DMG-PEG2000 (C); mix solution with ratio of solution (a)/solution (B)/solution (C) 38.5/10/1.5(v: v: v); a cationic lipid compound solution at a concentration of 10 mM; siRNA solution with concentration of 1 OD/100. mu.L.
Wherein 1 OD/100. mu.L means that the solution contains siRNA of 1OD in 100. mu.L, and siRNA of 1OD is about 33. mu.g.
(2) Liposomes encapsulating siRNA therapeutics are formed
The encapsulation of siRNA with cationic liposomes follows the following steps,
step S1: mixing 15 μ L of cationic lipid compound solution and 15 μ Lmix solution uniformly;
step S2: diluting the mixture obtained in the step S1 to 1.12mg/mL by using ethanol;
step S3: adding 150 μ L of the S2 solution into 450 μ L of acetate buffer solution with pH4.0, and mixing well;
step S4: adding 0.2ODsiRNA to 300 μ L step S3 mixture;
step S5: incubating at 35 ℃ for 30 min;
step S6: add 180. mu.L 1 XPBS to dilute to 0.5 mL.
Wherein the preparation of the pharmaceutical composition: the lipid compound is selected from LP-C2-LP-C20 with the structure of formula (I), and is prepared from example 1 to example 22 respectively.
Example 24 evaluation of Gene silencing efficacy of Liposome formulations in vitro
This example compares the effectiveness of the cationic liposomal siRNA formulations of the invention in an in vitro NSFB cell (human skin fibroblast) model.
The NSFB cell model was plated 24h before transfection with cells with confluency of 80% -90%, and the number of plated cells was 100000 cells/well. Experiment group at transfection, cationic liposome siRNA formulation was diluted with 1 XPBS to final concentrations of 10nM and 15nM, while siRNA was set to scramblesiRNA (sense strand: 5'-UUCUCCGAACGUGUCACGUTT-3', antisense strand: 5'-ACGUGACACGUUCGGAGAATT-3') negative control group and blank control group without liposome siRNA formulation, and siRNA (sense strand: 5'-GACAUCAAGAAGGUGGUGATT-3', antisense strand: 5'-UCACCACCUUCUUGAUGUCTT-3') target gene of experiment group was GAPDH (glyceraldehyde-3-phosphate dehydrogenase). And (3) collecting cells after 24h, extracting siRNA by a Trizol method, detecting the mRNA expression level of a target gene by reverse transcription and PCR, and taking human PPIBsiRNA as an internal reference.
Figure BDA0002901221770000311
To compare to the positive control, the cationic lipid of the invention was compared to the potent cationic lipid MC3 (known to be effective for in vivo delivery of nucleic acids, angelw. chem. int. ed.2012,51, 8529-. MC3 has the following structure:
Figure BDA0002901221770000312
as shown in Table 1, most of the partially cationic lipids of the present invention exhibited greater potency than MC3 at doses of 10nM and 50nM, respectively.
Table 1 in vitro silencing efficiency of liposomal siRNA-loaded therapeutics against GAPDH
Figure BDA0002901221770000313
Figure BDA0002901221770000321
Figure BDA0002901221770000331
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such modifications are intended to be included in the scope of the present invention.

Claims (19)

1. A lipid compound having a structure represented by formula (i), wherein formula (i) is as follows:
Figure FDA0002901221760000011
wherein A is N or CH; x, Y, Z are each independently selected from chemical groups attached to A with a carbon atom or carbon chain.
2. A lipid compound according to claim 1, wherein when A is N, X is
Figure FDA0002901221760000012
And n is1Is an integer of 1 to 4; y is selected from
Figure FDA0002901221760000013
Figure FDA0002901221760000014
And n is2Is an integer of 2 to 8, m1Is an integer of 4 to 12, n3Is an integer of 2 to 8, m2Is an integer of 4 to 12, p1Is an integer of 1 to 5, n4Is an integer of 2 to 6, m3Is an integer of 4 to 10, n5Is an integer of 2 to 6, m4Is an integer of 1 to 5, p2Is an integer of 3 to 8, n6Is an integer of 4 to 8, m5Is an integer of 7 to 9, R1Is an oxygen atom or a sulfur atom;
z is selected from
Figure FDA0002901221760000015
And n is7Is an integer of 4 to 8, m6、m7Are all integers of 6 to 10, n8Is an integer of 5 to 7, m8、m9Is an integer of 6 to 10.
3. A lipid compound according to claim 1, wherein when A is CH, X is selected from
Figure FDA0002901221760000016
And n is9Is an integer of 1 to 4, R2Is methyl or hydrogen, n10Is an integer of 1 to 4;
y is selected from
Figure FDA0002901221760000017
And n is11Is an integer of 2 to 8, m10Is an integer of 4 to 12，n12Is an integer of 2 to 8, m11Is an integer of 4 to 12;
z is selected from
Figure FDA0002901221760000021
And n is13Is an integer of 4 to 8, m12、m13Is an integer of 6 to 10, n14Is an integer of 5 to 7, m14、m15Is an integer of 6 to 10.
4. A lipid compound according to claim 1, wherein the structure of formula (i) is selected from:
Figure FDA0002901221760000022
Figure FDA0002901221760000031
Figure FDA0002901221760000041
Figure FDA0002901221760000051
Figure FDA0002901221760000061
5. a lipid compound according to any one of claims 1 to 4 wherein the lipid compound is in the form of a chemically acceptable salt.
6. A lipid compound according to any one of claims 1 to 4 wherein the lipid compound is in the form of a cationic lipid.
7. A liposome comprising the lipid compound according to any one of claims 1 to 6.
8. The liposome of claim 7, further comprising an additional lipid compound in a non-cationic form.
9. The liposome of claim 8, wherein the other non-cationic form of the lipid compound is selected from the group consisting of phospholipids, cholesterol derivatives.
10. The liposome of claim 9, wherein the phospholipid is selected from dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylethanolamine.
11. The liposome of claim 9, wherein the cholesterol derivative is cholesterol.
12. The liposome of any one of claims 7-11, wherein the liposome further comprises a lipid conjugate.
13. The liposome of any one of claim 12, wherein the lipid conjugate is selected from the group consisting of a PEG-diacylglycerol conjugate, a PEG-dialkoxypropyl conjugate.
14. The liposome of claim 12, wherein the liposome has a median diameter of 30 to 150 nm.
15. A pharmaceutical composition comprising a lipid compound according to any one of claims 1 to 6 and a therapeutic agent.
16. A pharmaceutical composition consisting of the liposome-encapsulated therapeutic agent of any one of claims 7-14.
17. The pharmaceutical composition of claim 15 or 16, wherein the therapeutic agents are each independently selected from the group consisting of siRNA, antisense oligonucleotide, microrna, mRNA, DNA.
18. The use of a pharmaceutical composition according to claim 15 or 16, wherein the pharmaceutical compositions are each independently for the treatment of a disease caused by a genetic abnormality.
19. The pharmaceutical composition of claim 15, wherein the lipid compound and the therapeutic agent are present in a molar ratio of 20:1 to 200: 1.
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