CN112341472B - Tyrosinase activated double-quenching diagnosis and treatment prodrug and preparation thereof - Google Patents

Tyrosinase activated double-quenching diagnosis and treatment prodrug and preparation thereof Download PDF

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CN112341472B
CN112341472B CN202011314583.7A CN202011314583A CN112341472B CN 112341472 B CN112341472 B CN 112341472B CN 202011314583 A CN202011314583 A CN 202011314583A CN 112341472 B CN112341472 B CN 112341472B
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tyrosinase
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颜梅
卫先哲
张晶
魏全勇
郝梦娇
王妍
马云飞
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University of Jinan
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Abstract

The invention discloses a tyrosinase activated double-quenching diagnosis and treatment prodrug and a preparation method thereof, wherein the structure of a diagnosis and treatment prodrug compound is shown as a formula I. The prodrug molecule simultaneously introduces tyrosinase activated 3-hydroxy benzyloxy and an anticancer drug melphalan as fluorescence quenching groups to reduce background fluorescence, and utilizes hydroxylation of enzyme to self-eliminate double quenching groups so as to form lactam, so that the fluorescent groups are activated, and simultaneously, the active melphalan is released for treating cancers. The double-quenched fluorescent prodrug has the advantages that background fluorescence is reduced, the double-quenched fluorescent prodrug has quick-start fluorescence response, and the prodrug is successfully applied to tracking of in-vivo drug release and targeted therapy of a mouse model of tumor with abnormal enzyme activity expression after being modified by lysosome targeting groups.

Description

Tyrosinase activated double-quenching diagnosis and treatment prodrug and preparation thereof
Technical Field
The invention relates to an enzyme-activated double-quenching fluorescent diagnosis prodrug compound in tumor cells, in particular to a double-quenching fluorescent prodrug molecule for releasing an anticancer drug melphalan after a target lysosome is activated by tyrosinase, and a prodrug compound for matching tumor diagnosis and treatment by tracking drug release by utilizing fluorescence imaging and preparation, belonging to the field of pharmaceutical analytical chemistry.
Background
Cancer is the most common life-threatening disease in the world, and although various methods for treating cancer have been developed, there is still a shortage of research on how to improve the effectiveness of treatment and reduce side effects. Generally, one of the major causes of death from cancer is metastasis of the tumor, and cancer cells are generated by genetic differentiation of normal cells, which allow cancer cells to migrate and invade adjacent tissues or organs. In addition, tyrosinase is a copper-containing enzyme that catalyzes the further oxidation of phenolic hydroxyl groups to o-quinones in the presence of molecular oxygen. This enzyme is widely present in plants, animals and microorganisms, and tyrosinase is considered to be an important biomarker in melanoma cells. In addition, the abnormal expression of tyrosinase may cause related diseases such as vitiligo and parkinsonism, so that the detection of tyrosinase in living organisms has important significance for clinical research.
The prodrug based on the probe molecule is an emerging technology in the biomedical field, and can simultaneously achieve the dual purposes of improving the early diagnosis and treatment of cancer. Taking advantage of this advantage, various types of prodrug systems have been developed, including anticancer drugs, fluorophores, and recognition groups. A number of strategies for detecting tyrosinase have been explored, with fluorescent probe-based methods showing higher specificity and sensitivity in detecting endogenous tyrosinase in living cells. The fluorescence of the tyrosinase probe based on the rhodamine fluorescent group is masked by a tyrosinase recognition group 3-hydroxy benzyloxy, 1, 6-rearrangement elimination is initiated through hydroxylation of tyrosinase, and closing of carboxyl is effectively promoted, so that a fluorescence signal can be accumulated more quickly. However, monofluorescence quenchers with fluorescence elimination usually depend on the type of fluorophore, resulting in inefficient fluorescence masking or incomplete shift of the equilibrium to the blocked lactone form, and therefore, most probes have only moderate fluorescence turn-on responses.
The invention discloses a tyrosinase activated double-quenching diagnosis and treatment prodrug and a preparation method thereof. The prodrug molecule simultaneously introduces tyrosinase activated 3-hydroxybenzyloxy and an anticancer drug melphalan as fluorescence quenching groups to reduce background fluorescence, and utilizes hydroxylation of enzyme to self-eliminate double quenching parts so as to form lactam, so that the fluorescent groups are activated, and simultaneously, the active melphalan is released for treating cancers. The double-quenched fluorescent prodrug has the advantages that background fluorescence is reduced, the double-quenched fluorescent prodrug has quick-start fluorescence response, and the prodrug is successfully applied to tracking of in-vivo drug release and targeted therapy of a mouse model of tumor with abnormal enzyme activity expression after being modified by lysosome targeting groups.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: the prodrug compound has the advantages of carrying out fluorescence imaging diagnosis and releasing drug therapy on cancer at the same time. And secondly, a fluorescence diagnostic reagent is provided for effectively, accurately and in-situ detecting the lysosome tyrosinase. Thirdly, a prodrug compound is provided, which has the advantage of high-efficiency drug release after tyrosinase activation. And fourthly, the application of tracking the drug release in vivo and treating in a tumor mouse model with abnormally expressed enzyme activity is provided.
In order to solve the technical problems, the technical scheme is as follows:
the invention provides a tyrosinase activated double-quenching diagnosis and treatment prodrug, which has the following molecular structural formula:
Figure DEST_PATH_IMAGE001
compound lyso-MT
The invention also provides a preparation method of the tyrosinase activated double-quenching diagnosis and treatment prodrug, which comprises the following steps:
(1) (a) preparation of Compound A from phthalic anhydride and 3-diethylaminophenol, dissolving Compound A (1 eq) and 2, 4-dihydroxybenzaldehyde (1-1.2 eq) in trifluoroacetic acid solution, stirring at 80 deg.C for 15-20 h, cooling, and adding saturated Na2CO3Neutralizing the aqueous solution to pH (7-7.5), filtering, and purifying with column (dichloromethane/ethanol) to obtain red powder of compound 1; (b) mixing compound 1 (1 eq), methyl 3- (bromomethyl) benzoate (1.5-1.8 eq) and Cs2CO3(1.5-1.8 eq) in DMF solution, stirring at 75 ℃ for 10-15 hours, cooling to room temperature, and column purification (dichloromethane/ethanol) to obtain solid compound 2;
Figure 247248DEST_PATH_IMAGE002
(2) (c) reacting 2 (1 eq) with NaBH4(1-1.2 eq) in ethanol solution, stirring under nitrogen for 5-8 hours, and column purifying (dichloromethane/ethanol) to obtain solid compound 3; (d) dissolving a mixture of the compound 3 (1 eq) and melphalan (1-1.4 eq) in a dichloromethane solution, adding DCC (1-1.3 eq) and DMAP (1-1.4 eq) to the solution, stirring the reaction mixture at 80 ℃ for 8-12 hours, cooling to room temperature, and performing column purification (ethyl acetate/petroleum ether) to obtain a solid compound 4;
Figure DEST_PATH_IMAGE003
(3) (e) 4- (2-aminoethyl) morpholine (2-2.5 eq) was added dropwise to a solution of compound 4 (1 eq) in ethanol, refluxed for 4-7 hours, cooled and then the solvent was removed under reduced pressure, the solid compound and potassium carbonate (2-2.4 eq) were dissolved in an aqueous solution of methanol, stirred at room temperature for 10-12 hours, and column-purified (dichloromethane/ethanol) to obtain lyso-MT as a product.
Figure 508465DEST_PATH_IMAGE004
The invention has the advantages that:
the prodrug molecule has the advantages of low background fluorescence and high response rate.
The prodrug molecules of the invention have the advantages of monitoring the biodistribution and the treatment effect of chemical drugs.
The prodrug molecules of the present invention, comprising the anticancer drugs melphalan (Mel) and rhodamine fluorophore, can be used to monitor lysosomal tyrosinase activity and anti-tumor therapeutic efficacy in situ.
The prodrug molecule of the invention shows quite high activity and has obvious inhibition effect on tumor growth.
The prodrug molecule of the invention integrates the advantages of auxiliary cancer diagnosis by fluorescence imaging, high drug loading capacity, controllable drug release and the like.
Therefore, the invention is a non-invasive diagnosis and treatment integrated tool which realizes the sustained release of melphalan in the tumor cells with the overexpression of tyrosinase, is accompanied with continuous fluorescence emission and can effectively inhibit the growth of tumors. Has wide application prospect in the field of pharmaceutical chemistry analysis and detection.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
Example 1
A preparation method of a tyrosinase activated double-quenching diagnosis and treatment prodrug comprises the following steps:
1) synthesis of Compound 1:
preparation of Compound A from phthalic anhydride and 3-diethylaminophenol Compound A (3.15 g, 10 mmol) and 2, 4-dihydroxybenzaldehyde (2 g, 12 mmol) were dissolved in trifluoroacetic acid solution (25 mL), stirred at 80 ℃ for 18 h, the cooled mixture was poured into 25 mL ice water, saturated Na was added thereto2CO3The aqueous solution was neutralized to pH 7.5, the solvent was evaporated under reduced pressure and column purified (dichloromethane/ethanol = 20/1) to give compound 1 as a red powder with 50% yield.
Synthesis of Compound 2:
compound 1 (0.75 g, 1.8 mmol), methyl 3- (bromomethyl) benzoate (0.62 g, 2.7 mmol) and Cs2CO3(1.05 g, 3.24 mmol) was dissolved in DMF solution (15 mL), stirred at 75 deg.C for 15 h, cooled to room temperature, added to the mixture 25 mL water and added with CH2Cl2(10 mL. times.3), the organic phases were combined and extracted with anhydrous Na2SO4Drying, evaporation of the solvent under reduced pressure and column purification (dichloromethane/ethanol = 10/1) gave compound 2 as a solid in 60% yield.
2) Synthesis of Compound 3:
compound 2 (0.4 g, 0.7 mmol) was reacted with NaBH4(0.03 g, 0.85 mmol) in ethanolTo the solution (35 mL), stirred under nitrogen for 8 hours, evaporated under reduced pressure to remove the solvent, and column purified (dichloromethane/ethanol = 25/1) to give compound 3 as a solid in 40% yield.
Synthesis of Compound 4:
a mixture of compound 3 (0.23 g, 0.4 mmol) and melphalan (0.17 g, 0.56 mmol) was dissolved in dichloromethane solution (20 mL), DCC (0.11 g, 0.52 mmol) and DMAP (0.06 g, 0.48 mmol) were added to the solution, the reaction mixture was stirred at 80 ℃ for 8 hours, cooled to room temperature, the solvent was evaporated under reduced pressure and column purified (ethyl acetate/petroleum ether = 6/5) to give compound 4 as a solid in 35% yield.
3) Synthesis of the Compound lyso-MT:
4- (2-aminoethyl) morpholine (0.35 mL, 2.64 mmol) was added dropwise to a solution of Compound 4 (1.02 g, 1.2 mmol) in ethanol (20 mL) under reflux for 5 hours, cooled and the solvent removed under reduced pressure, the solid compound and potassium carbonate (0.34 g, 2.4 mmol) were dissolved in an aqueous solution of methanol, stirred at room temperature for 10 hours and then treated with CH2Cl2(25 mL. times.3), the organic phases were combined and extracted with anhydrous MgSO4Drying, evaporation of the solvent under reduced pressure and column purification (dichloromethane/ethanol = 25/1) gave the product lyso-MT in 75% yield.
Example 2
In vitro detection method for reaction of prodrug molecule lyso-MT and tyrosinase
In a test tube, a stock solution was prepared by thoroughly mixing the prodrug molecule lyso-MT (50. mu.M) synthesized in example 1 in 4 mL of phosphate buffer and DMSO, followed by addition of tyrosinase solution, and the volume was adjusted to 5 mL with phosphate buffer. After 30 minutes of reaction at 37 ℃ in an incubator, 3 mL of the reaction solution was transferred to a 1 cm quartz cell at lambdaex/ emWavelength of = 470/528 nm. Meanwhile, a solution without tyrosinase was prepared as a control, and comparison was performed under the same conditions.
Example 3
Fluorescent response of prodrug molecule lyso-MT to tyrosinase
Taking a solution of the prodrug compound of example 2 (10 μ M), an increase in fluorescence of about 55-fold was observed at about 528 nm after tyrosinase was added, increasing the fluorescence quantum yield from less than 0.01 to 0.14. lyso-MT showed good linear fluorescence response to tyrosinase at concentrations ranging from 0.5 to 60U/mL, with a detection limit determined to be 0.7U/L, indicating that the prodrug had a sensitive and rapid response to tyrosinase.
Example 4
Dependence of the reaction of the prodrug molecule lyso-MT with tyrosinase on pH and temperature
A solution of the prodrug compound of example 2 (10. mu.M) was taken for fluorometric assay and showed substantially no fluorescence in the pH and temperature ranges of 4.0-8.0 and 23-40 ℃ respectively, indicating that lyso-MT had high stability. After reaction with tyrosinase, a significant fluorescence response occurred throughout the pH range tested, with the strongest fluorescence occurring at pH in the range of 6.0-7.4. Furthermore, the fluorescence of the reaction solution rapidly increases at about 37 ℃, consistent with the fact that enzymes generally have maximal activity at 37 ℃. It was shown that lyso-MT has a stable fluorescence signal under normal physiological conditions.
Example 5
Fluorescent detection of tyrosinase-induced prodrug molecule lyso-MT drug release
The prodrug compound stock solution (5. mu.M) of example 2 was taken, transferred to a dialysis membrane (MWCO 1000), and then the dialysis membrane was immersed in 50 mL of the corresponding buffer solution at 37oAnd C, incubating, and monitoring whether the tyrosinase is added in vitro drug release and fluorescence change by using a fluorescence spectrometer at fixed time intervals (1 min). lyso-MT, after reaction with tyrosinase, released the anticancer drug melphalan significantly, and within 12 hours, released approximately 95% of the melphalan, whereas the prodrug, after pretreatment with tyrosinase inhibitor, released essentially no melphalan. These results indicate that lyso-MT ensures melphalan stability under extracellular conditions, but that tyrosinase will induce a rapid release of melphalan.
Example 6
Real-time monitoring capability of prodrug molecule lyso-MT in cancer cells based on fluorescence imaging
Tyrosinase-overexpressing B16 cells were incubated in DMEM containing 10% (v/v) bovine serum 1% (v/v) penicillin-streptomycin at 5% CO2And cultured at 37 ℃ for 2 hours. Subsequently, B16 cells were stained with a commercially available lysosomal tracer for 15 minutes in the dark, the medium was removed and washed three times with PBS, and cell images were taken by a fluorimeter after pretreatment with kojic acid (tyrosinase inhibitor) and further incubation for 0, 5 and 12 hours before addition of lyso-MT or addition of prodrug molecules, after allowing lyso-MT to react well within the cells, removing the medium and washing twice with PBS. The fluorescence intensity of the lyso-MT-treated cells gradually increased with the increase of the incubation time, whereas the fluorescence intensity of B16 cells pretreated with kojic acid did not change significantly. These results are consistent with in vitro fluorescence experiments, indicating that lyso-MT can monitor drug release in real time.
The foregoing is only a preferred embodiment of this invention and is not intended to limit the invention in any way, so that any person skilled in the art may, using the teachings disclosed above, modify or adapt for various equivalent embodiments with equivalent modifications. The design concept of the present invention is not limited thereto, and any insubstantial modifications made to the present invention using this concept shall fall within the scope of infringing upon the present invention.

Claims (4)

1. A preparation method of a tyrosinase-activated double-quenching diagnosis and treatment prodrug is characterized in that the structure of the compound is shown as lyso-MT:
Figure DEST_PATH_IMAGE002
lyso-MT；
the preparation method comprises the following steps:
(1) (a) preparation of Compound A from phthalic anhydride and 3-diethylaminophenol by dissolving Compound A and 2, 4-dihydroxybenzaldehyde in trifluoroacetic acid solution, stirring at 80 deg.C for 15-20 hr, cooling, and adding saturated Na2CO3Neutralizing the aqueous solution, filtering and purifying the solution to obtainRed powder compound 1; (b) the compound 1, 3- (bromomethyl) acetic acid phenyl ester and Cs2CO3Dissolving in DMF solution, stirring at 75 deg.C for 10-15 hr, cooling to room temperature, and column purifying to obtain solid compound 2;
Figure DEST_PATH_IMAGE004
(2) (c) reacting Compound 2 with NaBH4Dissolving in ethanol solution, stirring under nitrogen for 5-8 hr, and purifying with column to obtain solid compound 3; (d) dissolving a mixture of the compound 3 and melphalan in a dichloromethane solution, adding DCC and DMAP into the solution, stirring the reaction mixture at 80 ℃ for 8-12 hours, cooling to room temperature, and performing column purification to obtain a solid compound 4;
Figure DEST_PATH_IMAGE006
(3) (e) adding 4- (2-aminoethyl) morpholine dropwise to the ethanol solution of compound 4, refluxing for 4-7 hours, cooling, removing the solvent under reduced pressure, dissolving the solid compound and potassium carbonate in the aqueous solution of methanol, stirring at room temperature for 10-12 hours, and purifying by column to obtain lyso-MT
Figure DEST_PATH_IMAGE008
。
2. The method for preparing the tyrosinase-activated double-quenching prodrug for diagnosis and treatment according to claim 1, wherein the method comprises the following steps: the molar ratio of the compound A and the 2, 4-dihydroxy benzaldehyde in the step (1) (a) is 1: (1-1.2); the molar concentration range of the compound A dissolved in trifluoroacetic acid solution is 0.2-0.5 mol.L-1(ii) a The pH after neutralization ranges from 7 to 7.5; the volume ratio of the dichloromethane to the ethanol in the column purification is (30-15): 1; (b) the compounds 1, 3- (bromomethyl) phenyl acetate and Cs2CO3The molar ratio ranges from 1: (1.5-1.8): (1.5E &1.8); the molar concentration range of the compound 1 dissolved in a DMF solution is 0.1-0.15 mol.L-1(ii) a The volume ratio of the dichloromethane to the ethanol in the column purification is (20-10): 1.
3. the method for preparing the tyrosinase-activated double-quenching prodrug for diagnosis and treatment according to claim 1, wherein the method comprises the following steps: the compound 2 and NaBH in the step (2) (c)4The molar ratio ranges from 1: (1-1.2); the molar concentration range of the compound 2 dissolved in ethanol is 0.015-0.02 mol.L-1(ii) a The volume ratio of the dichloromethane to the ethanol in the column purification is (50-20): 1; (d) the molar ratio of the compound 3 to the melphalan, DCC and DMAP is 1: (1-1.4): (1-1.3): (1-1.2); the molar concentration range of the compound 3 dissolved in dichloromethane is 0.02-0.05 mol.L-1(ii) a The volume ratio of ethyl acetate to petroleum ether in column purification is (1.5-1): 1.
4. the method for preparing the tyrosinase-activated double-quenching prodrug for diagnosis and treatment according to claim 1, wherein the method comprises the following steps: the molar ratio of the compound 4 and the 4- (2-aminoethyl) morpholine to the potassium carbonate in the step (3) (e) is 1: (2-2.5): (2-2.4); the molar concentration range of the compound 4 dissolved in ethanol is 0.05-0.1 mol.L-1(ii) a The volume ratio of the dichloromethane to the ethanol in the column purification is (40-20): 1.
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CN105567216A (en) * 2015-10-27 2016-05-11 西北大学 Lysosome targeted fluorescent probe and preparation method and application thereof
CN106420678B (en) * 2016-09-30 2019-04-23 广东轻工职业技术学院 Aryl compound containing phenolic hydroxyl group is used as tyrosinase activator and its application
CN106674284A (en) * 2016-12-26 2017-05-17 南京工业大学 Fluorescent probe for accurately detecting copper ions in grains through light control and preparation method and application thereof
CN107286173B (en) * 2017-05-17 2020-01-07 扶风慈缘生物科技有限公司 Rhodol derivative and preparation method and application thereof
WO2019236522A1 (en) * 2018-06-04 2019-12-12 Dana-Farber Cancer Institute, Inc. Compositions and methods for targeted immunotherapy
CN109232505A (en) * 2018-10-12 2019-01-18 陕西工业职业技术学院 The fluorescence probe and its synthetic method of a kind of quick detection cysteine and application
CN109608474B (en) * 2018-12-13 2021-02-26 湖北大学 Compound for detecting tyrosinase and preparation method and application thereof
CN109970618A (en) * 2019-03-28 2019-07-05 青岛科技大学 A kind of Ratio-type near infrared fluorescent probe and the preparation method and application thereof
CN110746965A (en) * 2019-11-25 2020-02-04 中国石油大学(华东) Tyrosinase detection probe constructed based on carbon quantum dots, and preparation method and application thereof
CN111138429A (en) * 2019-12-31 2020-05-12 西安邮电大学 Synthesis method of 1,2, 4-triazole pyridine derivatives
CN111678900B (en) * 2020-06-30 2023-03-21 东南大学 Cell membrane and cell wall fluorescence labeling method based on tyrosinase catalysis and application

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