CN114644665B - photo/pH dual-response coupled prodrug compound and preparation method and application thereof - Google Patents
photo/pH dual-response coupled prodrug compound and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a light/pH dual response coupled prodrug compound and a preparation method and application thereof, and relates to the technical field of medical materials. The precursor compound has a structure as shown in formula (I). The compound provided by the invention has an o-nitro dithiobenzyl ester structure, the nitro para-position is coupled with the drug molecule doxorubicin through a connecting segment and a Schiff base structure, and the doxorubicin can complex with free copper ions. Under the condition of acidic pH, the compound breaks down Schiff base structure to release drug molecule doxorubicin, breaks down o-nitro dithiobenzyl ester to release diethyl dithiocarbamic acid salt under the condition of illumination, and the released diethyl dithiocarbamic acid salt has stronger complexing ability to copper ions than doxorubicin, so that the compound can effectively rob copper ions complexed by doxorubicin, and simultaneously generates drug molecule doxorubicin Dox and metal drug CuET.
Description
Technical Field
The invention relates to the technical field of medical materials, in particular to a photo/pH dual response coupled prodrug compound and a preparation method and application thereof.
Background
Malignant tumors pose a serious threat to human health. The prodrug molecule is used for responsively releasing the drug active molecules under certain conditions, so that the prodrug is an effective way for improving the anti-tumor effect efficiency of the drug and ensuring the safety. Based on the above, researchers develop molecules or materials having responsiveness to unique exogenous conditions or tumor microenvironment characteristics, and utilize the changes of chemical structure, solubility, stability, charge distribution, size and other properties of prodrug molecules or nanomaterials under the stimulation of different light, sound, heat, force, magnetism, pH value, redox substances, enzymes and the like to realize the controlled release of drugs or active molecules. In addition, through structural design and organic assembly, the time, position and concentration accuracy of the controllable release system can be further improved by drug targeting delivery taking nano materials as carriers.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provides an optical/pH dual response coupled prodrug compound, and a preparation method and application thereof. The precursor compound has an o-nitro dithiobenzyl ester structure, the nitro para-position is coupled with a drug molecule doxorubicin through a connecting segment and a Schiff base structure, and the doxorubicin can complex free copper ions. Under the condition of acidic pH, the compound breaks down Schiff base structure to release drug molecule doxorubicin, breaks down o-nitro dithiobenzyl ester to release diethyl dithiocarbamic acid salt under the condition of illumination, and the released diethyl dithiocarbamic acid salt has stronger complexing ability to copper ions than doxorubicin, so that the compound can effectively rob copper ions complexed by doxorubicin, and simultaneously generates drug molecule doxorubicin Dox and metal drug CuET. Under the conditions of endogenous acidic pH and exogenous illumination, the generated two drug molecules can effectively kill cancer cells, have an effective synergistic cancer treatment effect, have simple preparation process and easy operation, and have wide application prospects in the field of cancer treatment.
A first object of the present invention is to provide an optical/pH dual response coupled prodrug compound having a structure as shown in formula (I),
wherein in formula (i), n=2 to 12.
Preferably, the precursor compound has a photo-responsiveness and/or an acidic pH-responsiveness.
Preferably, the precursor compound is capable of complexing with copper ions and fluorescence quenching after complexing with copper ions.
The second object of the present invention is to provide a method for preparing a photo/pH dual response coupled prodrug compound, comprising the steps of:
s1, under the protection of nitrogen, dissolving a compound of a formula (IV) and p-formylbenzoic acid in anhydrous tetrahydrofuran, stirring for reaction in the presence of 4-dimethylaminopyridine and dicyclohexylcarbodiimide, filtering and removing a solvent to obtain a crude product I, and performing column separation to obtain an intermediate product of the formula (III);
s2, dissolving an intermediate product of the formula (III) and a DTC precursor compound in tetrahydrofuran under the anaerobic condition, stirring for reaction under the catalysis of cuprous iodide and pentamethyldiethylenetriamine, removing a solvent to obtain a crude product II, and performing column separation to obtain the intermediate product of the formula (II);
s3, dissolving doxorubicin hydrochloride in dimethyl sulfoxide and neutralizing with triethylamine, adding dimethyl sulfoxide solution of intermediate product of formula (II), stirring for reaction in dark, extracting and removing solvent to obtain crude product III, washing with acetonitrile and drying to obtain prodrug compound of formula (I);
the structure of the formula (II) is as follows:
the structure of the formula (III) is as follows:
the structure of the formula (IV) is as follows:
wherein in the formulas (II), (III) and (IV), n=2-12;
the structural formula of the DTC precursor compound is as follows:
preferably, in S1, the molar ratio of the compound of the formula (IV) to the p-formylbenzoic acid is 1:1.1-2; the molar ratio of the 4-dimethylaminopyridine to the dicyclohexylcarbodiimide is 0.015:1.5;
the stirring temperature is 25-35 ℃, and the reaction time is 12-24 hours; in the column separation process, the mobile phase is a mixture of petroleum ether and ethyl acetate, and the volume ratio is 15:1-20:1.
Preferably, in S2, the anaerobic condition is that nitrogen is blown into the system twice, and the blowing time is more than or equal to 30min each time.
More preferably, the molar ratio of the DTC precursor compound to the intermediate of formula (III) is 1:1.1-1.5, and the mass ratio of the catalyst cuprous iodide to pentamethyldiethylenetriamine is 1:1.
More preferably, in S2, the reaction temperature is 20-30 ℃ and the reaction time is 24 hours; in the column separation process, petroleum ether is used as the following components: the volume ratio of ethyl acetate is 5:1 to 2:1, and the mobile phase gradient elution is used for column separation.
Preferably, in S3, the molar ratio of the doxorubicin hydrochloride, the triethylamine and the intermediate product of the formula (II) is 1:1.2:0.8-1; the temperature of the stirring reaction is 20-30 ℃; during the purification, 5mL of acetonitrile was added to the solid crude product.
The third object of the invention is to provide an application of the light/pH dual response coupled prodrug compound in preparing anticancer drugs.
Compared with the prior art, the invention has the beneficial effects that:
the photo/pH dual-response coupled prodrug compound provided by the invention has photo-responsiveness and acidic pH responsiveness, and simultaneously has complexation with copper ions and fluorescence quenching after complexation with copper ions. The diethyl dithiocarbamate which can be effectively released under the exogenous 365nm ultraviolet irradiation condition releases the drug molecule doxorubicin under the endogenous acidic pH condition, has excellent controllability, and the diethyl dithiocarbamate which can be effectively released under the ultraviolet irradiation condition can be effectively combined with free copper ions, so that the generated metal drug CuET has the anti-tumor effect.
According to the preparation method of the photo/pH dual-response coupled prodrug compound, the photo-response DTC prodrug compound and doxorubicin molecules are subjected to coupling reaction through the connecting fragments respectively in the structures of 1,2, 3-triazole and Schiff base, so that the yield is considerable, the preparation process is simple and easy to operate, and the practicability is high.
Drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of an intermediate of formula (III);
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of an intermediate of formula (II);
FIG. 3 is a nuclear magnetic resonance hydrogen spectrum of doxorubicin Dox;
FIG. 4 is a nuclear magnetic resonance hydrogen spectrum of the prodrug compound provided in example 1;
FIG. 5 is a hydrogen nuclear magnetic resonance spectrum of the prodrug compound provided in example 1 after acid response;
FIG. 6 is a graph of the ultraviolet absorption spectrum of doxorubicin;
FIG. 7 shows doxorubicin and CuCl 2 A UV absorption spectrum after complexation;
FIG. 8 is an ultraviolet absorption spectrum of the prodrug compound provided in example 1;
FIG. 9 shows the prodrug compound and CuCl provided in example 1 2 A UV absorption spectrum after complexation;
FIG. 10 is a fluorescence emission spectrum of doxorubicin;
FIG. 11 shows doxorubicin and CuCl 2 A fluorescence emission spectrum after complexation;
FIG. 12 is a graph showing fluorescence emission spectra of the prodrug compound provided in example 1;
FIG. 13 shows the prodrug compound and CuCl provided in example 1 2 A fluorescence emission spectrum after complexation;
FIG. 14 shows doxorubicin and CuCl 2 Complexing and illuminating for 5min to obtain a fluorescence emission spectrum;
FIG. 15 shows doxorubicin and CuCl 2 Complexing and illuminating for 10min to obtain a fluorescence emission spectrum;
FIG. 16 shows the prodrug compound and CuCl provided in example 1 2 Complexing and illuminating for 5min to obtain a fluorescence emission spectrum;
FIG. 17 shows the prodrug compound and CuCl provided in example 1 2 Complexing and illuminating for 10min to obtain a fluorescence emission spectrum;
FIG. 18 is a cytogram of doxorubicin;
FIG. 19 is a cytogram of a prodrug compound provided in example 1;
FIG. 20 is a diagram of the process steps in response to cytotoxicity;
FIG. 21 is a cytotoxicity profile of doxorubicin;
figure 22 is a plot of the cytotoxicity response of the prodrug compounds provided in example 1.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the data in the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It should be noted that the technical terms used in the present invention are only for describing specific embodiments, and are not intended to limit the scope of the present invention, and various raw materials, reagents, instruments and equipment used in the following embodiments of the present invention may be purchased commercially or prepared by existing methods unless otherwise specifically described.
The photoresponsive DTC precursor compounds employed in the examples below have the formula:
example 1
An optical/pH dual response coupled prodrug compound comprising the steps of:
s1, 6-azido-1-hexanol (0.71 g,5 mmol), p-formylbenzoic acid (0.83 g,5.5 mmol) and 4-dimethylaminopyridine DMAP (0.12 g,0.075 mmol) of formula (IV) were weighed separately in N 2 Dissolved in 25mL of ultra-dry Tetrahydrofuran (THF) under protection, dicyclohexylcarbodiimide DCC (1.59 g,7.5 mmol) was then added to the solution and the reaction was stirred at 30℃overnight. After the reaction was completed, the solid was removed by suction filtration, and the solid was washed with THF 2 times. The solvent was removed from the resulting solution by rotary evaporation, followed by petroleum ether: ethyl acetate (v/v) =20:1 as mobile phase to give 1.12g of intermediate of formula (iii) as pale yellow oil in 81% yield. 10mg of intermediate of formula (III) is taken and dissolved, and the structure is characterized by nuclear magnetic resonance hydrogen spectrum, as shown in figure 1, 1 H NMR(400M Hz,CDCl 3 ),δ(ppm):10.11(s,1H),8.21/.19(d,2H),7.97/.95(d,2H),4.39/.37/.35(t,2H),3.31/.29/.27(t,2H),1.48～1.85(m,8H).
s2, weighing a photoresponsive DTC precursor compound (1.01 g,3 mmol), an intermediate product (0.91 g,3.3 mmol) of a formula (III) and 16mg of N, N ', N, ' N ' -Pentamethyldiethylenetriamine (PMDETA) to dissolve in 20mLTHF, and blowing nitrogen into the solution to remove oxygen in the system, and blowing for 30min; then weighing 16mg of cuprous iodide (CuI), adding the solution, and continuously blowing nitrogen to discharge oxygen for 30min; the reaction flask was purged with nitrogen and closed, and the reaction was stirred at room temperature for 24 hours. After the reaction, the solvent is removed by rotary evaporation, and the petroleum ether and ethyl acetate are sequentially eluted by taking a mobile phase gradient as 5:1, 4:1, 3:1 and 2:1 according to the volume ratio, so as to carry out column separation, thereby obtaining 1.92g of brown yellow mucus or blocky solid intermediate product (II) with the yield of 94 percent. After 10mg of intermediate of formula (III) is dissolved, the structure is characterized by nuclear magnetic resonance hydrogen spectrum, as shown in figure 2, 1 HNMR(400M Hz,CDCl 3 ),δ(ppm):10.11(s,1H),8.20/.18(d,2H),8.11/.09(d,1H),7.97/.95(d,2H),7.67(s,1H),7.50(s,1H),6.99/.96(d,1H),5.28(s,2H),5.04(s,2H),4.39～4.35(2×t,4H),4.00～3.70(2×q,4H),2.00～1.40(2×m,8H),1.28/.27/.25(t,6H).
s3, weighing doxorubicin Dox (58 mg,0.1 mmol) and dissolving in 2mL of dimethyl sulfoxide DMSO, adding 24 mu L of triethylamine into the solution, and stirring at room temperature in a dark place for 2h; weighing and weighingIntermediate of formula (II) (49 mg,0.08 mmol) was dissolved in 2mL of MSO, added to the above reaction solution, and reacted at room temperature under stirring at a dark place for 48 hours. After the reaction was completed, 75mL of methylene chloride was added to the reaction solution to dilute it, and then an equal volume of deionized water was added to wash it 3 times, and the organic phase was washed with anhydrous Na 2 SO 4 Drying, filtering, and removing the organic phase solvent by rotary evaporation to obtain a crude product. And 5mL of acetonitrile is added into the solid crude product for 3 times, liquid is removed, and then the product is dried in vacuum to obtain a dark red solid product, thus obtaining the prodrug compound of the formula (I) with the yield of 65%. After dissolving 10mg of doxorubicin and the prodrug compound of formula (I), the structure was characterized by nuclear magnetic resonance hydrogen spectrum, as shown in FIG. 3 and FIG. 4. Comparing FIG. 4 with FIG. 2 and FIG. 3, the result shows that the characteristic absorption peak of the aromatic aldehyde (-CHO) at 10.11ppm in FIG. 2 disappears in FIG. 4, and the characteristic absorption peak of-CH=N-in the Schiff base with a chemical shift value of 8.41ppm appears in FIG. 4, namely, doxorubicin is coupled with the intermediate product of formula (II) through the Schiff base bond to obtain the prodrug compound of formula (I);
the preparation route is as follows:
example 2
An optical/pH dual response coupled prodrug compound comprising the steps of:
s1, 2-azido-1-ethanol (0.44 g,5 mmol), p-formylbenzoic acid (1.13 g,7.5 mmol) and 4-dimethylaminopyridine DMAP (0.12 g,0.075 mmol) of formula (IV) were weighed separately in N 2 Dissolved in 25mL of ultra-dry Tetrahydrofuran (THF) under protection, dicyclohexylcarbodiimide DCC (1.59 g,7.5 mmol) was then added to the solution and the reaction was stirred at 30℃overnight. After the reaction was completed, the solid was removed by suction filtration, and the solid was washed with THF 2 times. The solvent was removed from the resulting solution by rotary evaporation, and column separation was carried out by gradient elution with petroleum ether and ethyl acetate in a volume ratio of 5:1, 4:1, 3:1, 2:1 as mobile phase, to give 1.00g of intermediate of formula (III) as a colourless oil, in 91% yield.
S2, weighing a photoresponsive DTC precursor compound (1.01 g,3 mmol), an intermediate product (0.99 g,3.6 mmol) of a formula (III) and 16mg of N, N ', N, ' N ' -Pentamethyldiethylenetriamine (PMDETA) to dissolve in 20mLTHF, and blowing nitrogen into the solution to remove oxygen in the system, wherein the blowing is carried out for 35min; then weighing 16mg of cuprous iodide (CuI), adding the solution, and continuously blowing nitrogen to discharge oxygen for 35min; the reaction flask was purged with nitrogen and closed, and the reaction was stirred at room temperature for 24 hours. After the reaction, removing the solvent by rotary evaporation, and performing column separation by taking petroleum ether and ethyl acetate with the volume ratio of 5:1, 4:1, 3:1 and 2:1 as mobile phase gradient elution to obtain 1.59g of brown yellow mucus as an intermediate product of the formula (II), wherein the yield is 95%.
S3, weighing doxorubicin Dox (58 mg,0.1 mmol) and dissolving in 2mL of dimethyl sulfoxide DMSO, adding 24 mu L of triethylamine into the solution, and stirring at room temperature in a dark place for 2h; the intermediate of formula (II) (56 mg,0.1 mmol) was weighed and dissolved in 2mL of DMSO, and added to the reaction solution, followed by stirring at room temperature for 48 hours under dark conditions. After the reaction was completed, 75mL of methylene chloride was added to the reaction solution to dilute it, and then an equal volume of deionized water was added to wash it 3 times, and the organic phase was washed with anhydrous Na 2 SO 4 Drying, filtering, and removing the organic phase solvent by rotary evaporation to obtain a crude product. Adding 5mL of acetonitrile into the solid crude product, washing for 3 times, pouring out liquid, and then drying in vacuum to obtain a dark red solid product, namely the prodrug compound of the formula (I), wherein the yield is 60%;
the preparation route is as follows:
example 3
An optical/pH dual response coupled prodrug compound comprising the steps of:
s1, respectively weighing 12-azido-1-dodecanol (1.14 g,5 mmol), p-formylbenzoic acid (1.51 g,10.0 mmol) and 4-dimethylaminopyridine DMAP (0.12 g,0.075 mmol) of formula (IV) in N 2 Dissolved in 25mL of ultra-dry Tetrahydrofuran (THF) under protection, dicyclohexylcarbodiimide DCC (1.59 g,7.5 mmol) was then added to the solution and the reaction was stirred at 30℃overnight. After the reaction was completed, the solid was removed by suction filtration, and the solid was washed with THF 2 times.The solvent was removed from the resulting solution by rotary evaporation, followed by petroleum ether: ethyl acetate (v/v) =20:1 as mobile phase to give 1.54g of intermediate of formula (iii) as pale yellow oil in 86% yield.
S2, weighing a photoresponsive DTC precursor compound (1.01 g,3 mmol), an intermediate product (1.62 g,4.5 mmol) of a formula (III) and 16mg of N, N ', N, ' N ' -Pentamethyldiethylenetriamine (PMDETA) to dissolve in 20mLTHF, and blowing nitrogen into the solution to remove oxygen in the system, and blowing for 30min; then weighing 16mg of cuprous iodide (CuI), adding the solution, and continuously blowing nitrogen to discharge oxygen for 40min; the reaction flask was purged with nitrogen and closed, and the reaction was stirred at room temperature for 24 hours. After the reaction is finished, the solvent is removed by rotary evaporation, and the petroleum ether and ethyl acetate are sequentially eluted in a gradient way by taking the petroleum ether and ethyl acetate as mobile phases in the volume ratio of 5:1, 4:1, 3:1 and 2:1 for column separation, so that 1.99g of brown yellow mucus or blocky solid intermediate product with the yield of 95 percent is obtained.
S3, weighing doxorubicin Dox (58 mg,0.1 mmol) and dissolving in 2mL of dimethyl sulfoxide DMSO, adding 24 mu L of triethylamine into the solution, and stirring at room temperature in a dark place for 2h; the intermediate of formula (II) (63 mg,0.09 mmol) was weighed and dissolved in 2mL of DMSO, and added to the reaction solution, followed by stirring at room temperature for 48 hours under dark conditions. After the reaction was completed, 75mL of methylene chloride was added to the reaction solution to dilute it, and then an equal volume of deionized water was added to wash it 3 times, and the organic phase was washed with anhydrous Na 2 SO 4 Drying, filtering, and removing the organic phase solvent by rotary evaporation to obtain a crude product. Adding 5mL of acetonitrile into the solid crude product, washing for 3 times, pouring out liquid, and then drying in vacuum to obtain a dark red solid product, namely the prodrug compound of the formula (I), wherein the yield is 62%;
the preparation route is as follows:
to illustrate the relevant properties of the prodrug compounds provided by the present invention, since the properties of the prodrug compounds provided in examples 1-3 are similar, only the prodrug compounds provided in example 1 were tested for relevant properties;
the solution of the prodrug compound formulation provided in example 1 is labeled as i; adding copper ions to the solution prepared by the prodrug compound provided in the example 1 to mark Cu-I; labeling a solution of doxorubicin (Dox) formulation as Dox; a solution of doxorubicin (Dox) configuration was added to the copper ion labeled Cu-Dox.
To investigate the acidic pH response of the prodrug compound provided by the invention, 10mg of the prodrug compound (I) prepared in example 1 was dissolved in 0.5mL d 6 After addition of 5 μl of deuterated trifluoroacetic acid to the DMSO, the mixture was homogenized and after 1h was subjected to nmr hydrogen spectroscopy characterization as shown in fig. 5. Comparing the results of fig. 5 with those of fig. 2 and fig. 4, it can be seen that the prodrug compound has a nuclear magnetic resonance peak of hydrogen in the Schiff base structure at a chemical shift of 8.41ppm, and the peak completely disappears under the acidic condition of deuterated trifluoroacetic acid, and is converted into an aldehyde hydrogen nuclear magnetic resonance peak with a chemical shift of 10.11ppm, which indicates that the complete responsive cleavage of the Schiff base carbon-nitrogen double bond occurs in the prodrug compound under the acidic condition.
To characterize the binding properties of the prodrug compounds provided in drug example 1 to copper ions, the test was performed by ultraviolet visible absorption (UV-Vis) spectroscopy.
A Dox solution at a concentration of 50. Mu.M was prepared and tested for UV-Vis spectra as shown in FIG. 6, which shows that the Dox has a maximum absorption wavelength of 494nm; cuCl was added to the configured Dox solution at a concentration of 50. Mu.M 2 Obtaining Cu-Dox with copper ion concentration of 50 μm, and its UV-Vis spectrum as shown in FIG. 7, maximum absorption wavelength of Cu-Dox red shifted to 515nm, and Dox and CuCl 2 The color of the solution changed from orange to purple after complexation, indicating that there was complexation between Dox and copper ions.
Likewise, the UV-Vis spectrum of the prodrug compound solution provided in example 1 at a concentration of 50. Mu.M was measured and shown in FIG. 8, which shows that the absorption intensity of the prodrug compound was reduced as compared with Dox by the maximum absorption wavelength of 494nm; adding CuCl to a solution of the prodrug compound provided in example 1 configured at a concentration of 50. Mu.M 2 (copper ion concentration: 50. Mu.M) to obtain Cu-I, the UV-Vis spectrum of which is shown in FIG. 9, the maximum absorption wave of Cu-ILong red shift to about 504nm, and the prodrug compound (I) and CuCl 2 The color of the solution deepens after complexation, indicating that there is also a certain complexation between the prodrug compound and copper ions.
Since doxorubicin (Dox) has fluorescent properties, the fluorescent properties of the prodrug compounds and the copper ion complexation can be tested by fluorescence emission spectroscopy. The fluorescent emission spectrum test conditions are as follows: excitation wavelength 494nm, emission wavelength 500-800 nm.
The fluorescence emission spectrum of the Dox solution with the concentration of 10 mu M is shown in FIG. 10, the maximum emission wavelength is 599nm, and the fluorescence intensity is 5905; copper ions with a concentration of 10 mu M are added into a Dox solution with a concentration of 10 mu M to obtain Cu-Dox, the fluorescence emission spectrum is shown in FIG. 11, the maximum absorption wavelength is still about 599nm, but the fluorescence intensity is only 592; the above results indicate the complexing nature of Dox with copper ions and quenching of Dox fluorescence after copper ion complexation.
Similarly, a prodrug compound solution having a concentration of 10. Mu.M was prepared, and the fluorescence emission spectrum obtained by the test was as shown in FIG. 12, and the maximum emission wavelength was 598nm and the fluorescence intensity was 3086; copper ions at a concentration of 10. Mu.M were added to a prodrug compound solution prepared at a concentration of 10. Mu.M to give Cu-I, the fluorescence emission spectrum of which is shown in FIG. 13, the image was substantially free of peaks, and the fluorescence intensity at 598nm was only 375; the above results indicate that the prodrug compound still has Dox-like copper ion complexing properties and that fluorescence of the prodrug compound is quenched after copper ion complexing.
To test the photoresponsive properties of the prodrug compound, cu-Dox obtained by adding copper ions at a concentration of 10. Mu.M to a Dox solution at a concentration of 10. Mu.M and Cu-I obtained by adding copper ions at a concentration of 10. Mu.M to a prodrug compound solution at a concentration of 10. Mu.M were prepared at an optical power density of 12mW/cm 2 After illumination under 365nm ultraviolet light, fluorescence emission spectroscopy analysis was performed.
As shown in FIG. 14 and FIG. 15, the fluorescence emission spectra obtained after 5min or 10min of Cu-Dox illumination respectively show that the emission intensity at 599nm is further reduced to 259 and 217 respectively, i.e. the illumination has no influence on the Cu-Dox fluorescence emission basically; the fluorescence emission spectra after 5min or 10min of illumination of the Cu-I solution were similarly tested as shown in FIGS. 16 and 17, with the emission intensity at 599nm rising to 1531 and 1954, respectively, and the peak shape consistent with Dox. The above results indicate that the prodrug compound has photo-responsiveness, and that the photo-responsive product DTC can effectively abstract copper ions complexed with the molecule Dox to form CuET while allowing Dox to resume fluorescence.
To test the uptake of the prodrug molecule by cells, their distribution in the cells was characterized by confocal laser cell imaging, according to Dox and the fluorescent properties of the prodrug compound (i) provided in example 1. HeLa cells were seeded into petri dishes (Φ=40 mm) at a density of 5X 10 per well 4 Individual cells and in complete medium containing 10% fetal bovine serum, 5% CO 2 Incubate for 24h at 37 ℃. HeLa cells were incubated with doxorubicin (Dox), cu-Dox, the prodrug compound (I) provided in example 1, and Cu-I, respectively, at a concentration of 5. Mu.M for 12h. Wherein Cu-Dox is Cu-Dox obtained by adding copper ions with a concentration of 5 mu M into a Dox solution with a configuration concentration of 5 mu M; cu-I is Cu-I obtained by adding copper ions at a concentration of 5. Mu.M to a solution of a prodrug compound at a concentration of 5. Mu.M. The imaging excitation wavelength is 490nm, and the emission wavelength range is 500-600 nm. The imaging results of Dox and Cu-Dox are shown in FIG. 18, which shows that Dox enters primarily the nuclei of cancer cells; the results of I and Cu-I imaging are shown in FIG. 19, which shows that the prodrug molecules are also able to enter cancer cells; in cells, however, due to the acidic microenvironment of the cancer tumor cells or lysosomes upon endocytosis, the pro-drug compound may respond to the release site Dox, while copper ions have substantially no effect on the fluorescence of Dox or the pro-drug compound.
In order to test the cytotoxicity of HeLa using doxorubicin (Dox) and the prodrug compound (i) provided in example 1, respectively, the cytotoxicity levels of Dox and the prodrug compound provided in example 1 and their corresponding copper complexes before and after light irradiation were detected by thiazole blue (MTT), in order to find application in the field of cancer treatment. Methods of responding to cytotoxicity of the prodrug compounds provided in example 1The steps are shown in fig. 20. The MTT method is a method commonly used in laboratories to detect cell viability. The detection principle is that succinate dehydrogenase in the mitochondria of living cells can reduce MTT to water-insoluble blue-violet crystalline formazan and deposit in cells, while dead cells have no function. HeLa cells were seeded into 96-well plates (2X 10 per well) 4 And in complete medium containing 10% fetal bovine serum, 5% CO 2 Incubate for 24h at 37 ℃. HeLa cells were incubated with different concentrations of doxorubicin (Dox), cu-Dox, I and Cu-I, respectively; after incubation of cells in the light group for 24h, the cells were incubated with light at an optical power density of 12mW/cm 2 And (3) irradiating 365nm ultraviolet light for 5min, culturing in a non-illumination group in a dark condition, and continuously incubating the cells for 24h after illumination is finished. After discarding the medium and washing 2 times with PBS buffer, fresh medium containing MTT (100. Mu.L, 0.5 mg/mL) was added to each well and incubated for 4h under culture conditions. The supernatant was discarded, washed with PBS buffer, and DMSO (100. Mu.L) was added to solubilize formazan, and UV absorbance at 490nm, 560nm and 720nm was detected with a microplate reader, and the level of viable cell force was indirectly reflected by ratio calculation.
Wherein, the test solutions with different concentrations of doxorubicin (Dox), cu-Dox, I and Cu-I are prepared according to the following methods:
dox: dissolving Dox in DMSO to obtain a concentrated solution with a concentration of 10mM, and diluting the concentrated solution with a complete culture medium to a concentration of 1,2, 4, 6, 8, 10 and 12 mu M respectively, wherein the complete culture medium is used as a blank group;
Cu-Dox: dissolving Dox in DMSO to obtain a Dox solution with the concentration of 10mM, adding copper gluconate to enable the copper ion concentration to be 10mM to obtain a Cu-Dox concentrated solution, diluting the concentrated solution to the concentration of 1,2, 4, 6, 8, 10 and 12 mu M respectively by using a complete culture medium, and taking the complete culture medium as a blank control group;
i: dissolving a prodrug compound I in DMSO to obtain a concentrated solution with the concentration of 10mM, and diluting the concentrated solution with a complete culture medium to the concentration of 1,2, 4, 6, 8, 10 and 12 mu M respectively, wherein the complete culture medium is used as a blank control group;
Cu-I: dissolving a prodrug compound I in DMSO to obtain a solution with the concentration of 10mM, adding copper gluconate to enable the concentration of copper ions to be 10mM to obtain a Cu-I concentrated solution, diluting the concentrated solution with a complete culture medium to the concentrations of 1,2, 4, 6, 8, 10 and 12 mu M respectively, and taking the complete culture medium as a blank control group.
HeLa cytotoxicity test, the results of which are shown in fig. 21 and 22; the results show that: with the increase of the concentration of the prodrug compound, the cell survival rate after incubation treatment is slightly reduced, which indicates that the cytotoxicity of the prodrug compound is weak, as shown in tables 1 and 2; doxorubicin Dox exhibits high cell killing ability in the presence of copper ions, whereas prodrug compounds at the same concentration remain less cytotoxic in the presence of copper ions; under the condition of no copper ion illumination, the cytotoxicity of the doxorubicin (Dox) and the prodrug compound (I) is weak, namely the illumination in a short time does not have excessive influence on the cell state; under the non-illumination condition of copper ions, doxorubicin Dox shows a strong cell killing function in cells, namely, the doxorubicin Dox can release DTCs in test tubes only by illumination, and the influence of nitroreductase and the like in the cells on nitro structures possibly enables the doxorubicin Dox to be broken in the cells to generate DTCs; the prodrug compound can show strong cell killing capability only by adding light under the condition of copper ions, namely the prodrug compound has ultraviolet light irradiation controllability, and the controllable release product can be effectively combined with copper ions in cells to generate metal medicines, and can generate stronger anti-tumor effect by synergistic action with doxorubicin.
TABLE 1 Dox cell viability data for doxorubicin
TABLE 2 cell viability data for prodrug compounds
It should be noted that, when numerical ranges are referred to in the present invention, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and because the adopted step method is the same as the embodiment, in order to prevent redundancy, the present invention describes a preferred embodiment. While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A light/pH dual response coupled prodrug compound is characterized in that the prodrug compound has a structure shown as a formula (I),
wherein in formula (i), n=2 to 12.
2. The dual light/pH responsive coupled prodrug compound of claim 1, wherein the prodrug compound is photo-responsive and/or acidic pH responsive.
3. The dual light/pH responsive coupled prodrug compound of claim 1, wherein the prodrug compound is capable of complexing with copper ions and fluorescence quenching after complexing with copper ions.
4. A method of preparing a light/pH dual response coupled prodrug compound according to claim 1, comprising the steps of:
s1, under the protection of nitrogen, dissolving a compound of a formula (IV) and p-formylbenzoic acid in anhydrous tetrahydrofuran, stirring for reaction in the presence of 4-dimethylaminopyridine and dicyclohexylcarbodiimide, filtering and removing a solvent to obtain a crude product I, and performing column separation to obtain an intermediate product of the formula (III);
s2, dissolving an intermediate product of the formula (III) and a DTC precursor compound in tetrahydrofuran under the anaerobic condition, stirring for reaction under the catalysis of cuprous iodide and pentamethyldiethylenetriamine, removing a solvent to obtain a crude product II, and performing column separation to obtain the intermediate product of the formula (II);
s3, dissolving doxorubicin hydrochloride in dimethyl sulfoxide and neutralizing with triethylamine, adding dimethyl sulfoxide solution of intermediate product of formula (II), stirring for reaction in dark, extracting and removing solvent to obtain crude product III, washing with acetonitrile and drying to obtain prodrug compound of formula (I);
the structure of the formula (II) is as follows:
the structure of the formula (III) is as follows:
the structure of the formula (IV) is as follows:
wherein in the formulas (II), (III) and (IV), n=2-12;
the structure of the DTC precursor compound is as follows:
5. the method of preparing a dual light/pH responsive coupled prodrug compound of claim 4, wherein in S1, the molar ratio of the compound of formula (iv) to p-formylbenzoic acid is 1:1.1-2; the molar ratio of the 4-dimethylaminopyridine to the dicyclohexylcarbodiimide is 0.015:1.5;
the stirring temperature is 25-35 ℃, and the reaction time is 12-24 hours; in the column separation process, the mobile phase is a mixture of petroleum ether and ethyl acetate, and the volume ratio is 20:1.
6. The method for preparing a photo/pH dual response coupled prodrug compound according to claim 4, wherein in S2, the anaerobic condition is that nitrogen is blown into the system twice, and the blowing time is not less than 30min each time.
7. The method for preparing the light/pH dual response coupled prodrug compound according to claim 6, wherein the molar ratio of the DTC precursor compound to the intermediate of formula (III) is 1:1.1-1.5, and the mass ratio of the catalyst cuprous iodide to pentamethyldiethylenetriamine is 1:1.
8. The method for preparing a photo/pH dual response coupled prodrug compound according to claim 6, wherein in S2, the reaction temperature is 20-30 ℃ and the reaction time is 24h; in the column separation process, petroleum ether is used as the following components: the volume ratio of ethyl acetate is 5:1 to 2:1, and the mobile phase gradient elution is used for column separation.
9. The method of preparing a dual light/pH responsive coupled prodrug compound of claim 4, wherein in S3, the molar ratio of doxorubicin hydrochloride, triethylamine, and intermediate of formula (ii) is 1:1.2:0.8-1; the temperature of the stirring reaction is 20-30 ℃; during the purification, 5mL of acetonitrile was added to the solid crude product.
10. Use of the light/pH dual response coupled prodrug compound of claim 1 in the preparation of an anticancer drug.
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