CN108164419B - Preparation and application of monodisperse polyethylene glycol monomethyl ether modified propofol prodrug - Google Patents
Preparation and application of monodisperse polyethylene glycol monomethyl ether modified propofol prodrug Download PDFInfo
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- CN108164419B CN108164419B CN201711422744.2A CN201711422744A CN108164419B CN 108164419 B CN108164419 B CN 108164419B CN 201711422744 A CN201711422744 A CN 201711422744A CN 108164419 B CN108164419 B CN 108164419B
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- C07C69/96—Esters of carbonic or haloformic acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
- C07C69/66—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
- C07C69/67—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of saturated acids
- C07C69/708—Ethers
Abstract
The invention discloses preparation and application of a monodisperse polyethylene glycol monomethyl ether modified propofol prodrug with a novel structure, and belongs to the field of organic chemistry and pharmaceutical chemical industry. The monodisperse polyethylene glycol monomethyl ether is connected with propofol through chemical bonds which can be degraded in vivo to respectively prepare a series of carbonate-type and acetate-type propofol prodrugs. The method can synthesize the corresponding water-soluble propofol prodrug by adjusting the length of the monodisperse polyethylene glycol monomethyl ether chain, the longer the monodisperse polyethylene glycol monomethyl ether chain is, the better the water solubility is, the water solubility can be improved, the preparation can be prepared into an aqueous solution preparation, and the defect of fat emulsion in the propofol fat emulsion is avoided. The method has the advantages of simple reaction, mild condition, low cost and convenient industrial production.
Description
Technical Field
The invention belongs to the field of organic chemical synthesis and pharmaceutical chemistry, and particularly relates to preparation and application of a monodisperse polyethylene glycol monomethyl ether modified propofol prodrug with a novel structure.
Background
Propofol (2, 6-diisopropyl phenol) is an alkylphenol intravenous injection whole anesthetic, has been widely accepted as a short-acting intravenous anesthetic, propofol intravenous anesthesia has the advantages of quick response, short action time and NO side effects such as cough, hiccup and the like of patients, propofol has a series of other valuable applications besides various advantages as an anesthetic, and propofol is reported to have the effects of antiemetic, antianxiety, neuroprotection, itching relieving, pain relieving, platelet aggregation inhibition, induced NO generation inhibition and the like, but propofol is highly fat-soluble and hardly soluble in water and is clinically prepared into an oil-in-water fatty oil emulsion, but the preparation has the defects of short shelf life, sensitivity to bacterial and fungal pollution, injection pain induction and low blood pressure and instantaneous apnea caused by propofol due to lipid components, long-term use may lead to hyperlipidemia and the like, and these characteristics greatly limit its clinical application.
Disclosure of Invention
In view of the above, it is an object of the present invention to provide a novel water-soluble propofol prodrug, which reduces its side effects in clinical applications.
The technical scheme of the invention can be realized by the following technical measures:
a monodisperse polyethylene glycol monomethyl ether modified propofol prodrug has a structure shown in a general structural formula I:
wherein Y is-OC-or-OCCH2—。
The synthesis method of the propofol prodrug when Y is-OC-in the general formula I comprises the following steps:
(1) reacting monodisperse polyethylene glycol monomethyl ether with N, N' -carbonyldiimidazole to obtain a monodisperse polyethylene glycol monomethyl ether carbonyl imidazole derivative;
(2) adding propofol, alkali and solvent into the obtained monodisperse polyethylene glycol monomethyl ether carbonyl imidazole derivative, and reacting at 60 ℃ to obtain a carbonate type propofol prodrug shown in a formula II:
preferably, the base comprises 4-dimethylaminopyridine, pyridine, triethylamine, N-diisopropylethylamine, sodium carbonate and potassium carbonate.
Preferably, the solvent comprises N, N-dimethylformamide, dimethyl sulfoxide, N-dimethylaniline, dichloromethane and tetrahydrofuran.
In the general formula I, Y is-OCCH2The synthesis method of the propofol prodrug comprises the following steps:
(1) reacting monodisperse polyethylene glycol monomethyl ether with tert-butyl bromoacetate to obtain a monodisperse polyethylene glycol monomethyl ether tert-butyl acetate derivative;
(2) reacting the obtained monodisperse polyethylene glycol monomethyl ether acetic acid tert-butyl ester derivative with trifluoroacetic acid, and removing tert-butyl to obtain corresponding acid;
(3) the obtained acid reacts with propofol under the action of alkali and a condensing agent to obtain an acetate type propofol prodrug shown in a formula III:
preferably, the base is 4-dimethylaminopyridine, pyridine, triethylamine, N-diisopropylethylamine, sodium carbonate and potassium carbonate.
Preferably, the condensing agent comprises dicyclohexylcarbodiimide, diisopropylcarbodiimide, (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, N, N, N ', N' -tetramethylchloroformamidine hexafluorophosphate.
Preferably, the monodisperse polyethylene glycol monomethyl ether is carried out according to the technical scheme disclosed in CN 201310710498.6.
The use of the propofol prodrug as described above for the preparation of centrally acting depressant drugs for producing sedative-hypnotic and/or anaesthetic effects in animals or humans by intravenous or parenteral routes.
Drawings
The invention is further illustrated by means of the attached drawings, the examples of which are not to be construed as limiting the invention in any way.
FIG. 1 is a graph of the results of an assay of the effect of propofol and the compounds 1a-1f and 2a-2f obtained in the examples on the cell viability of A549 cells;
FIG. 2 is a graph showing the results of a determination of the effect of propofol and the compounds 1a-1f and 2a-2f obtained in the examples on the cell viability of L929 cells.
Detailed Description
In order that the invention may be more readily understood, specific embodiments thereof will be described further below.
EXAMPLE 1 Synthesis of carbonate-based Propofol prodrug of formula II
(1) Synthesis of Compound 1a (n ═ 2)
Dimer ethylene glycol monomethyl ether carbonyl imidazole derivative (1.17g,5.46mmol) was dissolved in N, N-dimethylformamide (50mL), propofol (1.95g,10.92mmol) and 4-dimethylaminopyridine (0.14g,1.10mmol) were added and reacted at 60 ℃ for 48 hours, after the reaction was completed, the N, N-dimethylformamide solution was evaporated off and purified by column chromatography to give compound 1a (1.42g, 80%).1H NMR(400MHz,CDCl3)δ7.25-7.12(m,3H),4.51-4.35(m,2H),3.85-3.75(m,2H),3.70-3.68(m,2H),3.59-3.57(m,2H),3.41(s,3H),3.09-2.98(m,2H),1.21(d,J＝6.9Hz,12H).13C NMR(100MHz,CDCl3)δ153.9,145.7,140.5,126.8,124.1,71.9,70.6,69.0,67.7,59.1,27.3,23.3.HRMS(ESI)calcd for C18H28NaO5 +([M+Na]+),347.1829；found,347.1833。
(2) Synthesis of Compound 1b (n ═ 3)
Trimeric ethylene glycol monomethyl ether carbonyl imidazole derivative (1.15g,5.45mmol) is dissolved in N, N-dimethylformamide (50mL), propofol (3.17g,17.81mmol) and 4-dimethylaminopyridine (0.27g,2.23mmol) are added and reacted at 60 ℃ for 48h, after the reaction is finished, the N, N-dimethylformamide solution is evaporated and purified by column chromatography to obtain compound 1b (1.36g, 83%).1H NMR(400MHz,CDCl3)δ7.25-7.13(m,3H),4.48-4.32(m,2H),3.83-3.78(m,2H),3.73-3.64(m,6H),3.59-3.54(m,2H),3.39(s,3H),3.09-2.98(m,2H),1.21(d,J＝6.9Hz,12H).13C NMR(100MHz,CDCl3)δ153.9,145.7,140.5,126.8,124.1,71.9,70.8-70.5(m),68.9,67.7,59.0,27.2,23.3.HRMS(ESI)calcd for C20H32NaO6+([M+Na]+),391.2091；found,391.2084.HRMS(ESI)calcd for C20H32NaO6+([M+Na]+),391.2091；found,391.2084。
(3) Synthesis of Compound 1c (n ═ 4)
Tetramethoxypolyethylene glycol carbonylimidazole derivative (0.42g,1.38mmol) was dissolved in N, N-dimethylformamide (50mL), and propofol (0.99g,5.54mmol) and 4-Dimethylaminopyridine (0.08g,0.69mmol) was reacted at 60 ℃ for 48 hours, after completion of the reaction, the N, N-dimethylformamide solution was distilled off, and the resulting product was purified by column chromatography to give compound 1c (0.40g, 70%).1H NMR(400MHz,CDCl3)δ7.25-7.12(m,3H),4.44-4.39(m,2H),3.83-3.75(m,2H),3.72-3.64(m,10H),3.59-3.52(m,2H),3.38(s,3H),3.09-2.98(m,2H),1.21(d,J＝6.9Hz,12H).13C NMR(100MHz,CDCl3)δ153.9,145.7,140.5,126.8,124.1,71.9,70.8-70.3(m),68.9,67.7,59.0,27.3,23.3.HRMS(ESI)calcd for C22H36NaO7 +([M+Na]+),435.2353；found,435.2347。
(4) Synthesis of Compound 1d (n ═ 6)
Hexa-polyethylene glycol monomethyl ether carbonyl imidazole derivative (1.70g,4.35mmol) is dissolved in N, N-dimethylformamide (50mL), propofol (1.55g,8.70mmol) and 4-dimethylaminopyridine (0.11g,0.87mmol) are added to react at 60 ℃ for 48h, after the reaction is finished, the N, N-dimethylformamide solution is evaporated, and the compound 1d (1.47g, 68%) is obtained by column chromatography purification.1H NMR(400MHz,CDCl3)δ7.25-7.10(m,3H),4.47-4.38(m,2H),3.82-3.78(m,2H),3.72-3.62(m,18H),3.57-3.53(m,2H),3.38(s,3H),3.09-2.98(m,2H),1.21(d,J＝6.9Hz,12H).13C NMR(100MHz,CDCl3)δ153.9,145.7,140.4,126.8,124.1,71.9,70.8-70.4(m),68.9,67.7,59.0,27.2,23.3.HRMS(ESI)calcd for C26H44NaO9 +([M+Na]+),523.2878；found,523.2885。
(5) Synthesis of Compound 1e (n ═ 7)
Hepta-polyethylene glycol monomethyl ether carbonyl imidazole derivative (2.05g,4.72mmol) is dissolved in N, N-dimethylformamide (50mL), propofol (3.36g,18.88mmol) and 4-dimethylaminopyridine (0.28g,2.36mmol) are added and reacted at 60 ℃ for 48h, after the reaction is finished, the N, N-dimethylformamide solution is evaporated and purified by column chromatography to obtain compound 1e (1.89g, 74%).1H NMR(400MHz,CDCl3)δ7.26-7.12(m,3H),4.47-4.35(m,2H),3.83-3.78(m,2H),3.73-3.63(m,22H),3.58-3.51(m,2H),3.38(s,3H),3.09-2.98(m,2H),1.22(d,J＝6.9Hz 12H).13C NMR(100MHz,CDCl3)δ153.9,145.7,140.4,126.8,124.1,71.9,70.8-70.3(m),68.9,67.7,59.0,27.2,23.3.HRMS(ESI)calcd for C28H52NO10 +([M+NH4]+),562.3586；found,562.3582。
(6) Synthesis of Compound 1f (n ═ 10)
Decaethylene glycol monomethyl ether carbonylimidazole derivative (2.22g,3.91mmol) was dissolved in N, N-dimethylformamide (50mL), propofol (1.39g,7.82mmol) and 4-dimethylaminopyridine (0.14g,1.10mmol) were added and reacted at 60 ℃ for 48 hours, after the reaction was completed, the N, N-dimethylformamide solution was evaporated off and purified by column chromatography to give compound 1f (2.01g, 74%).1H NMR(400MHz,CDCl3)δ7.26-7.12(m,3H),4.49-4.35(m,2H),3.83-3.77(m,2H),3.72-3.63(m,34H),3.58-3.52(m,2H),3.38(s,3H),3.09-2.98(m,2H),1.21(d,J＝6.9Hz,12H).13C NMR(100MHz,CDCl3)δ153.8,145.7,140.4,126.8,124.0,71.9,70.8-70.2(m),68.9,67.7,59.0,27.2,23.3.HRMS(ESI)calcd for C34H64NO13 +([M+NH4]+),694.4372；found,694.4368。
EXAMPLE 2 Synthesis of Acetoacetate propofol prodrug of formula III
(1) Synthesis of Compound 2a (n ═ 2)
Dimer ethylene glycol monomethyl ether tert-butyl acetate derivative (1.80g,7.68mmol) was dissolved in dichloromethane (30mL), trifluoroacetic acid (14.2mL,192.00mmol) and anisole (0.96mL,8.83mmol) were added, and the mixture was stirred at room temperature for 5h and then drained to give the corresponding acid. Dicyclohexylcarbodiimide (1.90g,9.22mmol) was added to a solution of the acid obtained in the previous step and 4-dimethylaminopyridine (0.47g,3.84mmol) in dichloromethane (50mL) under nitrogen, and after stirring for 10 minutes, propofol (2.05g,11.52mmol) was dissolved in dichloromethane (10mL) and slowly added dropwise to the reaction mixture, and after the dropwise addition was completed, the reaction mixture was allowed to return to room temperature and stirred for 16 hours. After the reaction was completed, suction filtration was performed, the filtrate was washed twice with dilute hydrochloric acid, the organic phase was spin-dried, and purification by column chromatography gave compound 2a (1.47g, 57%).1H NMR(400MHz,CDCl3)δ7.25-7.15(m,3H),4.50(s,2H),3.88-3.83(m,2H),3.78-3.73(m,2H),3.71-3.66(m,2H),3.60-3.55(m,2H),3.39(s,3H),2.94-2.82(m,2H),1.19(d,J＝6.9Hz,12H).13C NMR(100MHz,CDCl3)δ169.2,145.0,140.3,126.7,124.0,71.9,71.1,70.7,70.6 68.4,59.1,27.6,23.3.HRMS(ESI)calcd for C19H30NaO5 +([M+Na]+),361.1985；found,361.1981。
(2) Synthesis of Compound 2b (n ═ 3)
Tert-butyl trimeric ethylene glycol monomethyl ether acetate derivative (1.30g,4.67mmol) is dissolved in dichloromethane (30mL), trifluoroacetic acid (7.0mL,93.40mmol) and anisole (0.6mL,5.60mmol) are added, and after stirring for 5h at room temperature, the mixture is drained to obtain the corresponding acid. Dicyclohexylcarbodiimide (1.90g,9.22mmol) was added to a solution of the acid obtained in the previous step and 4-dimethylaminopyridine (0.29g,2.34mmol) in dichloromethane (50mL) under nitrogen, and after stirring for 10 minutes, propofol (1.15g,5.60mmol) was dissolved in dichloromethane (10mL) and slowly added dropwise to the reaction mixture, after dropwise addition, the reaction mixture was allowed to return to room temperature and stirred for 16 hours. After the reaction, suction filtration was performed, the filtrate was washed twice with dilute hydrochloric acid, the organic phase was spin-dried, and column chromatography purification was performed to obtain compound 2b (1.10g, 62%).1H NMR(400MHz,CDCl3)δ7.25-7.15(m,3H),4.49(s,2H),3.87-3.82(m,2H),3.78-3.73(m,2H),3.73-3.64(m,6H),3.58-3.54(m,2H),3.38(s,3H),2.94-2.83(m,2H),1.19(d,J＝6.9Hz,12H).13C NMR(100MHz,CDCl3)δ169.2,145.0,140.3,126.8,124.1,71.9,71.1,70.7-70.4(m),68.4,59.1,27.6,23.3.HRMS(ESI)calcd for C21H34NaO6 +([M+Na]+),405.2248；found,405.2243。
(3) Synthesis of Compound 2c (n ═ 4)
Tetramethoxypolyethylene glycol tert-butyl acetate derivative (1.50g,4.65mmol) was dissolved in dichloromethane (30mL), trifluoroacetic acid (8.6mL,116.32mmol) and anisole (0.6mL,5.58mmol) were added, and the mixture was stirred at room temperature for 5h and then drained to give the corresponding acid. Dicyclohexylcarbodiimide (1.15g,5.58mmol) was added to a solution of the acid obtained in the previous step and 4-dimethylaminopyridine (0.28g,2.33mmol) in dichloromethane (50mL) under nitrogen, stirred for 10 min and then propylparaben was addedPhenol (1.24g,6.98mmol) was dissolved in dichloromethane (10mL) and added slowly dropwise to the reaction mixture, after which time stirring was allowed to resume at room temperature for 16 h. After the reaction, suction filtration was performed, the filtrate was washed twice with dilute hydrochloric acid, the organic phase was spin-dried, and purification by column chromatography gave compound 2c (0.87g, 44%).1H NMR(400MHz,CDCl3)δ7.26-7.13(m,3H),4.49(s,2H),3.86-3.81(m,2H),3.78-3.73(m,2H),3.70-3.63(m,10H),3.57-3.52(m,2H),3.38(s,3H),2.98-2.74(m,2H),1.19(d,J＝6.9Hz,12H).13C NMR(100MHz,CDCl3)δ169.2,145.0,140.3,126.8,124.0,71.9,71.1,70.8-70.4(m),68.4,59.1,27.6,23.5.HRMS(ESI)calcd for C23H38NaO7 +([M+Na]+),449.2510；found,449.2500。
(4) Synthesis of Compound 2d (n ═ 6)
Hexaethyleneglycol monomethyl ether tert-butyl acetate derivative (1.04g,2.44mmol) was dissolved in dichloromethane (30mL), trifluoroacetic acid (4.5mL,112.50mmol) and anisole (0.3mL,5.40mmol) were added, and the mixture was stirred at room temperature for 5h and then drained to give the corresponding acid. Dicyclohexylcarbodiimide (0.60g,2.92mmol) was added to a solution of the acid obtained in the previous step and 4-dimethylaminopyridine (0.15g,1.22mmol) in dichloromethane (50mL) under nitrogen, and after stirring for 10 minutes, propofol (0.65g,3.65mmol) was dissolved in dichloromethane (10mL) and slowly added dropwise to the reaction mixture, after dropwise addition, the reaction mixture was allowed to return to room temperature and stirred for 16 hours. After the reaction was completed, suction filtration was performed, the filtrate was washed twice with dilute hydrochloric acid, the organic phase was spin-dried, and purification by column chromatography gave compound 2d (0.56g, 45%).1H NMR(400MHz,CDCl3)δ7.25-7.14(m,3H),4.49(s,2H),3.87-3.82(m,2H),3.77-3.73(m,2H),3.71-3.63(m,18H),3.57-3.53(m,2H),3.38(s,3H),2.94-2.83(m,2H),1.19(d,J＝6.9Hz,12H).13C NMR(100MHz,CDCl3)δ169.2,145.0,140.3,126.8,124.0,71.9,71.1,70.8-70.4(m),68.4,59.1,27.6,23.2.HRMS(ESI)calcd for C27H50NO9 +([M+NH4]+),532.3480；found,532.3474。
(5) Synthesis of Compound 2e (n ═ 7)
Heptaethyleneglycol monomethyl ether tert-butyl acetate derivative (1.64g,3.61mmol) was dissolved in methylene chloride (30mL), and trifluoroacetic acid (6.9 m)L,90.00mmol) and anisole (0.5ml,4.33mmol), stirred at room temperature for 5h and then drained to give the corresponding acid. Dicyclohexylcarbodiimide (0.90g,4.33mmol) was added to a solution of the acid obtained in the previous step and 4-dimethylaminopyridine (0.22g,1.80mmol) in dichloromethane (50mL) under nitrogen, and after stirring for 10 minutes, propofol (0.97g,5.41mmol) was dissolved in dichloromethane (10mL) and slowly added dropwise to the reaction mixture, after dropwise addition, the reaction mixture was allowed to return to room temperature and stirred for 16 hours. After the reaction was completed, suction filtration was performed, the filtrate was washed twice with dilute hydrochloric acid, the organic phase was spin-dried, and purification by column chromatography gave compound 2e (1.20g, 60%).1H NMR(400MHz,CDCl3)δ7.25-7.15(m,3H),4.49(s,2H),3.86-3.82(m,2H),3.77-3.73(m,2H),3.72-3.61(m,22H),3.57-3.53(m,2H),3.38(s,3H),2.93-2.83(m,2H),1.19(d,J＝6.9Hz,12H).13C NMR(100MHz,CDCl3)δ169.2,145.0,140.3,126.7,124.0,71.9,71.1,70.8-70.3(m),68.4,59.0,27.6,23.3.HRMS(ESI)calcd for C29H54NO10 +([M+NH4]+),576.3742；found,576.3737。
(6) Synthesis of Compound 2f (n ═ 10)
Decaethylene glycol monomethyl ether tert-butyl acetate derivative (2.09g,3.56mmol) was dissolved in dichloromethane (30mL), trifluoroacetic acid (7.3mL,89.00mmol) and anisole (0.5mL,4.27mmol) were added, and the mixture was stirred at room temperature for 5h and then drained to give the corresponding acid. Dicyclohexylcarbodiimide (0.88g,4.27mmol) was added to a solution of the acid obtained in the previous step and 4-dimethylaminopyridine (0.22g,1.78mmol) in dichloromethane (50mL) under nitrogen, and after stirring for 10 minutes, propofol (1.27g,7.12mmol) was dissolved in dichloromethane (10mL) and slowly added dropwise to the reaction mixture, after dropwise addition, the reaction mixture was allowed to return to room temperature and stirred for 16 hours. After the reaction was completed, suction filtration was performed, the filtrate was washed twice with dilute hydrochloric acid, the organic phase was spin-dried, and purification by column chromatography gave compound 2f (1.29g, 44%).1H NMR(400MHz,CDCl3)δ7.25-7.12(m,3H),4.49(s,2H),3.86-3.82(m,2H),3.76-3.72(m,2H),3.70-3.61(m,34H),3.57-3.52(m,2H),3.37(s,3H),2.95-2.78(m,2H),1.19(d,J＝6.9Hz,12H).13C NMR(100MHz,CDCl3)δ169.2,145.0,140.3,126.7,124.0,71.9,71.1,70.8-70.3(m),68.4,59.0,27.6,23.3.HRMS(ESI)calcd for C35H66NO13 +([M+NH4]+),708.4529；found,708.4525。
Example 3 solubility determination experiment
The solubility of compounds 1a-1f and 2a-2f in PBS buffer (pH 7.4) was determined by uv spectrophotometry. Firstly, measuring ultraviolet absorbance of solutions with different concentrations of a compound at a wavelength of 257nm, drawing a standard curve to obtain a regression curve equation, then preparing a saturated solution of the compound, centrifuging the saturated solution, taking a supernatant to dilute to a certain concentration, then measuring the absorbance value of the supernatant at the wavelength of 257nm, substituting the value into the standard curve equation, converting to obtain the concentration of the diluted saturated solution of the compound, and multiplying the concentration by the dilution factor to obtain the solubility of the compound in a PBS (pH 7.4). As shown in Table 1, the solubility of the propofol prodrug was better as the chain length of the monodisperse polyethylene glycol monomethyl ether was longer, and when n.gtoreq.7, the solubility increased sharply, and the solubility of compounds 1f,2e and 2f was very good without an upper limit.
Table 1 solubility of compounds 1a-1f and 2a-2f in PBS buffer (pH 7.4) (unit mg/mL)
| |
2 | 3 | 4 | 6 | 7 | 10 |
| 1a-1f | 0.07 | 0.19 | 0.38 | 0.83 | 2.73 | >314.75 |
| 2a-2f | 0.42 | 0.99 | 1.58 | 2.02 | >216.33 | >371.09 |
Example 4 cell viability assay
(1) Preparation of the solution
Preparing a DMEM high-sugar culture solution: DMEM high-sugar culture medium is purchased, 500mL of DMEM high-sugar culture medium is added into each bottle, 10% of fetal calf serum and 1% of penicillin-streptomycin solution are added into each bottle of DMEM high-sugar culture medium, namely 50mL of fetal calf serum and 5mL of penicillin-streptomycin solution are added into each bottle of DMEM high-sugar culture medium, the DMEM high-sugar culture medium is prepared in a clean bench, and the DMEM high-sugar culture medium is stored in a refrigerator at 4 ℃.
Preparation of PBS buffer: in a 1000mL conical flask, 8g of sodium chloride, 0.2g of potassium chloride, 2.9g of disodium hydrogen phosphate dodecahydrate and 0.2g of potassium dihydrogen phosphate are weighed, 800mL of purified water is added, the mixture is fully stirred and dissolved, the volume is determined to be 1000mL, and the mixture is placed in a refrigerator for storage at 4 ℃ after autoclaving.
Preparation of MTT solution: 0.5g of MTT dry powder was weighed, dissolved in 100mL of PBS buffer, sterilized by filtration through a 0.22. mu.M filter, and stored at-12 ℃ in a refrigerator.
(2) Cell viability assay
Cell viability assay selection of human non-Small cell Lung cancer (A549) and mouse fibroblast (L929)
A: cell survival determination using human non-small cell lung carcinoma (A549)
The culture solution used for A549 cells is DMEM high-glucose culture solution prepared above, and the culture conditions are 37 deg.C and 5% CO2The constant temperature incubator. The method comprises the following specific steps:
1) cells in logarithmic growth phase were taken and diluted 10-fold with DMEM high-glucose medium.
2) 200. mu.L of the diluted cell suspension was added to each well of a 96-well plate and incubated at 37 ℃ for 24 hours in an incubator.
3) The compound to be tested was diluted to 0.05mM with DMEM high-sugar medium, dosed at 100. mu.L/well and incubated in an incubator at 37 ℃ for 24 h.
4) mu.L of MTT at a concentration of 5mg/mL was added and incubated in an incubator at 37 ℃ for 4 h.
5) Cells were lysed by adding 200. mu.L DMSO and OD measured at 490nm using a microplate reader.
6) And processing the data to obtain the cell survival rate.
B: cell viability assay Using mouse fibroblasts (L929)
The culture solution used for L929 cells is DMEM high-glucose culture solution prepared above, and the culture conditions are 37 ℃ and 5% CO2The constant temperature incubator. The method comprises the following specific steps:
1) cells in logarithmic growth phase were taken and diluted 10-fold with DMEM high-glucose medium.
2) 200. mu.L of the diluted cell suspension was added to each well of a 96-well plate and incubated at 37 ℃ for 24 hours in an incubator.
3) The compound to be tested was diluted to 0.05mM with DMEM high-sugar medium, dosed at 100. mu.L/well and incubated in an incubator at 37 ℃ for 24 h.
4) mu.L of MTT at a concentration of 5mg/mL was added and incubated in an incubator at 37 ℃ for 4 h.
5) Cells were lysed by adding 200. mu.L DMSO and OD measured at 490nm using a microplate reader.
6) And processing the data to obtain the cell survival rate.
(3) Cell viability assay results
The results of the assay were all cell viability at a compound concentration of 0.05 mM. FIG. 1 is the cell survival rate of Propofol, Compounds 1a-1f and 2a-2f on A549 cells, FIG. 2 is the cell survival rate of Propofol, Compounds 1a-1f and 2a-2f on L929 cells; it can be seen from FIGS. 1 and 2 that cell viability is greater than 90% at the 0.05mM dose, which is close to the therapeutic dose of propofol, indicating that these propofol prodrugs are safe at therapeutic doses.
Example 5 animal anesthesia experiment
Only propofol prodrugs showing good water solubility as a result of solubility measurements are considered here. 12 healthy SD rats with the body weight of 180-200g were selected and randomly divided into five groups of 3 animals, and were bred under normal conditions before the test and during the observation period. The compounds 1f,2e and 2f with good water solubility are respectively dissolved in physiological saline for injection. A commercially available propofol emulsion was used as a control, and the dosage was measured as an effective propofol dose of 13mg/kg (0.073 mmol/kg). The phenomenon of anesthesia in rats was observed by tail vein injection, and the duration of anesthesia was recorded, and the experimental results are shown in table 2.
TABLE 2 anesthetic Effect of different Compounds on rats
| Compound (I) | Post-injection phenomenon | Duration of anesthesia |
| Reference substance | The appearance of anesthetic reaction | 10.25 |
| 1f | The appearance of anesthetic reaction | 5.56 |
| 2e | The appearance of anesthetic reaction | 10.50 |
| 2f | The appearance of anesthetic reaction | 8.5 minutes |
Claims (5)
1. A monodisperse polyethylene glycol monomethyl ether modified propofol prodrug is characterized in that the structure is shown as formula I:
wherein Y is-OCCH2N is 7 or 10.
2. The method of preparing a propofol prodrug as claimed in claim 1, wherein, in formula I, Y is-OCCH2The synthesis method of the propofol prodrug comprises the following steps:
(1) reacting monodisperse polyethylene glycol monomethyl ether with tert-butyl bromoacetate to obtain a monodisperse polyethylene glycol monomethyl ether tert-butyl acetate derivative;
(2) reacting the obtained monodisperse polyethylene glycol monomethyl ether acetic acid tert-butyl ester derivative with trifluoroacetic acid, and removing tert-butyl to obtain corresponding acid;
(3) the obtained acid reacts with propofol under the action of alkali and a condensing agent to obtain an acetate type propofol prodrug shown in a formula III:
3. the method of claim 2, wherein the base is 4-dimethylaminopyridine, pyridine, triethylamine, N-diisopropylethylamine, sodium carbonate, potassium carbonate.
4. The method of claim 2, wherein the condensing agent comprises dicyclohexylcarbodiimide, diisopropylcarbodiimide, (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, N, N, N ', N' -tetramethylformamidine hexafluorophosphate.
5. Use of a propofol prodrug as claimed in claim 1 for the manufacture of a centrally inhibitory drug for producing sedative hypnotic and/or anesthetic effects for animals or humans by intravenous or intravenous route.
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