CN115806566A - Preparation method and application of nitroreductase activated multifunctional molecular prodrug for overcoming heterogeneous distribution of tumor oxygen - Google Patents
Preparation method and application of nitroreductase activated multifunctional molecular prodrug for overcoming heterogeneous distribution of tumor oxygen Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a nitroreductase activated multifunctional molecular prodrug for overcoming tumor oxygen heterogeneity distribution. The invention discloses a BODIPY-nitrobenzene-camptothecin conjugate, which is used for overcoming the problem of heterogeneity of oxygen distribution of tumors and achieving the aim of whole tumor treatment. The synthesis method is simple, the raw materials are easy to obtain, the cost is low, the side reaction is less, the yield is high, the purification is easy, and the industrial production is facilitated.
Description
Technical Field
The invention belongs to the field of design and synthesis of anti-cancer drugs, and particularly relates to a preparation method and application of a nitroreductase activated multifunctional molecular prodrug for overcoming tumor oxygen heterogeneity distribution.
Background
Tumor hypoxia is caused by the uncontrolled proliferation of tumor cells and is one of the important features shared by the tumor microenvironment. This is manifested in the form of a significantly lower oxygen level in the intratumoral tissue cells than in normal tissue cells. In view of this phenomenon, scientists have developed hypoxia-responsive prodrugs by chemically modifying active drugs (e.g., paclitaxel, camptothecin, doxorubicin, etc.) with hypoxia-responsive functional groups (e.g., nitroaromatic derivatives, azobenzene, quinone derivatives). The prodrug protects the active site of an active drug and enables the active site to be functionalized, thereby improving the specificity of the drug for treating tumor tissues and reducing the systemic toxic and side effects of the drug. However, the distribution of oxygen in solid tumors is heterogeneous, and the cells of tissues near the blood vessels are still in a normal oxygen state, and the tumor regions far away from the blood vessels are in an anaerobic state, so that the oxygen content of the whole tumor tissues is obviously different. Therefore, hypoxic responsive prodrugs have great application limitations, which only treat hypoxic regions of tumors, but the residual tumor tissue cells still cause cancer recurrence.
Recently, the combined treatment mode of photodynamic therapy and chemotherapy is widely researched, and the combined treatment method can obviously improve the cancer treatment efficiency, reduce the dosage of the medicament and reduce the drug resistance of the lesion tissues to the medicament. Photodynamic therapy (PDT) is an early-developed minimally invasive phototherapy, and has been gradually applied in recent years for the diagnosis and treatment of clinical cancer. Compared with traditional treatment modes such as operation treatment, radiotherapy, chemotherapy and the like, PDT has the advantages of being minimally invasive, high in selectivity, low in side effect, free of drug resistance, wide in tumor treatment spectrum and the like. In PDT, light of specific wavelength locally irradiates tumor part to excite photosensitizer enriched in tumor tissueThe excited photosensitizer is in energy transfer with molecular oxygen in the tumor tissue, producing Singlet oxygen (Singlet oxygen, 1 O 2 )。 1 O 2 has strong oxidizing property, and can induce apoptosis or necrosis. In addition, PDT is being generated as an accurate light-controlled treatment 1 O 2 Meanwhile, oxygen in tumor tissues can be rapidly consumed and severe hypoxia is caused. Therefore, the photosensitizer and the hypoxia response prodrug are integrated, the maximum effects of photodynamic therapy and hypoxia response chemotherapy can be exerted, the purpose of cooperative therapy is achieved, the treatment difficulty caused by the heterogeneity of oxygen distribution of tumor tissues can be overcome, and the whole tumor treatment is realized.
Disclosure of Invention
The invention aims to provide a preparation method and application of a nitroreductase activated multifunctional molecular prodrug for overcoming tumor oxygen heterogeneity distribution. The invention discloses a monomolecular prodrug integrating photodynamic therapy and chemotherapy, which is prepared by covalently connecting chemotherapeutic drugs Camptothecin (CPT) and a BODIPY photosensitizer by utilizing an anaerobic-responsive nitrobenzene connecting arm. Since the hydroxyl active site of CPT is modified, resulting in suppression of CPT cytotoxicity, the entire molecular prodrug has very low dark toxicity. The prodrug synthesized by the invention has the advantages of simple synthesis method, easy purification and separation, less side reaction, high yield, easily obtained raw materials, low cost and contribution to industrial production. The prodrug is enriched to the tumor site by tail vein injection. When the tumor tissue is locally illuminated, the medicine can effectively carry out photodynamic therapy in a tumor normal oxygen region, simultaneously consumes oxygen to accelerate tumor hypoxia, promotes the overexpression of nitrobenzene reductase in tumor cells, reduces nitrobenzene groups in the prodrug into aniline, and further generates intramolecular transformation reaction to release chemotherapeutic medicine CPT; the medicine enriched in the tumor hypoxia area can be reduced by the overexpressed nitroreductase, and the chemotherapeutic medicine CPT is released, so that the defect of insufficient treatment of photodynamic therapy in the tumor hypoxia area is overcome. Therefore, the prodrug is expected to overcome the treatment difficulty caused by the heterogeneity of the oxygen distribution of the tumor and achieve the aim of realizing the treatment of the whole tumor.
In order to realize the purpose, the invention adopts the following technical scheme:
a nitroreductase-activated multifunctional molecular prodrug for overcoming tumor oxygen heterogeneity distribution, which is a BODIPY-Nitrobenzene-camptothecin conjugate having the chemical structural formula:
the preparation method of the compound comprises the following steps: with a compound
As a starting material, the BODIPY-NITROBENZENE-CAMPTOCETIC CONJUGATE is synthesized
(BDP-Nitro-CPT)。
The method comprises the following specific steps: the compound is
Dissolving the mixture in dichloromethane according to a molar ratio of 1:2.2, adding 2-3 equivalents of 4-Dimethylaminopyridine (DMAP), 3 drops of N, N-Diisopropylethylamine (DIPEA) and triphosgene, and vigorously stirring the reaction solution at room temperature for 18-36h; after the reaction is finished, dichloromethane and water are used for extraction, and the organic phase is subjected to anhydrous Na 2 SO 4 Drying and removing the solvent under reduced pressure; and finally, performing silica gel column chromatography separation by using dichloromethane-methanol (volume ratio is 50
(BDP-Nitro-CPT)；
Said 2-3 equivalents of DMAP, to
Based on the molar amount of (a).
The preparation method of the compound 2a comprises the following steps:
(1) To be provided with
Phosphorus tribromide as initial material and synthesis
(2) With the compound synthesized in step (1)
As starting materials, compounds were synthesized
The method comprises the following specific steps:
(1) Will be provided with
Phosphorus tribromide is prepared by mixing the following components in a molar ratio of 2:1, mixing, dissolving in acetonitrile, and reacting for 2h under the ice bath condition; after the reaction is finished, removing the solvent, taking dichloromethane as eluent, and separating by silica gel column chromatography to obtain yellow solid
(2) Will be provided with
According to the mol ratio of 1:2 in acetone, then 10 equivalents of potassium carbonate are added, the reaction is refluxed for 1h in an oil bath at 60 ℃, after the end of the reaction, the potassium carbonate is filtered and the solvent is removed, then extraction is carried out with dichloromethane and water, the organic phase is passed over anhydrous Na 2 SO 4 After drying, the solvent was removed under reduced pressure, and then separated by silica gel column chromatography using dichloromethane-methanol (volume ratio 50
The 10 equivalents of potassium carbonate to
Based on the molar amount of (a). The application comprises the following steps: the multifunctional molecular prodrug activated by nitroreductase for overcoming the heterogeneous distribution of tumor oxygen is applied to the preparation of anticancer drugs.
The BODIPY-NITRO-camptothecin (BDP-Nitro-CPT) prodrug is used for synergistic treatment for overcoming heterogeneity of oxygen distribution of tumors.
Alkaloid is a low molecular weight biomolecule with a nitrogen content of 20%. Some alkaloids have strong biological activity and are often used for treating diseases. The alkaloid anticancer drugs used clinically at present include camptothecin and the like, which is a type I DNA topoisomerase (Top I) inhibitor and has certain curative effect on esophageal cancer, colon cancer, liver cancer and lung cancer. However, camptothecin also has some disadvantages: such as poor water solubility, unstable structure, large systemic toxic and side effects, etc. The photosensitizer is a core element of photodynamic therapy, the BODIPY derivative is one of fluorescent dyes which are widely researched at present, and the photosensitizer meets a series of advantages of an ideal photosensitizer: such as definite chemical structure, strong light absorption, high light stability, low dark toxicity, high singlet oxygen quantum yield after heavy atom modification and the like.
Based on the method, the hypoxia-responsive nitrobenzene group is firstly connected to the BODIPY photosensitizer, and then is covalently connected to the chemotherapeutic drug camptothecin to form the multifunctional molecular prodrug BDP-Nitro-CPT. When the tumor part is irradiated by red light, the medicine enriched in the normal oxygen area of the tumor is excited by the light to generate photodynamic activity, so that the tumor part is hypoxic, the chemotherapeutic medicine camptothecin is induced to be activated, and the photodynamic-chemotherapeutic synergistic treatment effect is achieved. The molecular prodrug in the tumor hypoxia area can directly activate chemotherapeutic camptothecin and make up the defect of poor effect of photodynamic therapy in the tumor hypoxia area, so that the BDP-Nitro-CPT prodrug is expected to overcome the treatment difficulty caused by heterogeneity of tumor oxygen distribution and realize the purpose of whole tumor treatment.
The invention has the remarkable advantages that:
(1) Camptothecin is an alkaloid capable of directly damaging a DNA structure, and when covalently linked to a BODIPY photosensitizer by a nitrobenzene linking arm, the camptothecin can improve the stability of the camptothecin in the blood circulation process and effectively reduce the systemic toxicity of the drug;
(2) During hypoxia condition or photodynamic therapy of the BDP-Nitro-CPT prodrug, the nitrobenzene group can be directly or indirectly reduced into aniline, and further generates intramolecular transformation reaction to release chemotherapeutic drug camptothecin, so that the synergetic treatment effect of photodynamic therapy and chemotherapy is achieved;
(3) The absorption spectrum and the emission spectrum of the prodrug are both positioned in a red light area, the tissue penetration capability of red light is strong, skin phototoxicity is not easy to cause during photodynamic therapy, and the prodrug is low in dark toxicity and is an ideal prodrug;
(4) The target prodrug has a single structure, is simple to synthesize, and is easy to purify;
(5) From cytotoxicity experiments of cancer cells 4T1 (mouse breast cancer cells), hepG2 (human liver cancer cells) and HeLa (human cervical cancer cells), it can be seen that hypoxic cancer cells can induce nitrobenzene groups in BDP-Nitro-CPT to be reduced and then release chemotherapeutic drugs camptothecin.
Drawings
FIG. 1 shows illumination (660nm, 20mW/cm) 2 5 min) and no light, the cytotoxicity of BDP-Nitro-CPT and 2a on 4T1, hepG2 and HeLa cells cultured under different oxygen concentrations;
FIG. 2 shows the antitumor activity of different compounds in the absence of light; (a) The change of tumor volume of different groups of mice in the treatment process; (b) tumor mass and (c) morphology in mice after 15 days; (d) weight change in mice over 15 days; (e) Morphological analysis of tumor tissues after H & E staining of mice of different groups after treatment for 15;
FIG. 3 is a morphological diagram of a tissue section of different groups of mice after H & E staining of organs;
FIG. 4 is a diagram showing the mechanism of action of a BODIPY-Nitro-CPT prodrug;
FIG. 5 shows the reaction of Compound 1a in CDCl 3 Is/are as follows 1 H NMR spectrum;
FIG. 6 shows the reaction of Compound 1a in CDCl 3 Is/are as follows 13 C NMR spectrum;
FIG. 7 is a High Resolution Mass Spectrometry (HRMS) plot of Compound 1 a;
FIG. 8 shows the reaction of Compound 2a in CDCl 3 Is/are as follows 1 H NMR spectrum;
FIG. 9 shows the reaction of Compound 2a in CDCl 3 Is/are as follows 13 C NMR spectrogram;
FIG. 10 is a HRMS spectrum of Compound 2 a;
FIG. 11 shows the reaction of BDP-Nitro-CPT compound in CDCl 3 Is/are as follows 1 H NMR spectrum;
FIG. 12 shows the reaction of BDP-Nitro-CPT compound in CDCl 3 Is/are as follows 13 C NMR spectrum;
FIG. 13 is a HRMS spectrum of BDP-Nitro-CPT compound.
Detailed Description
In order to make the content of the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
The specific preparation process of multifunctional molecular prodrug fluorodipyrrole-nitrobenzene-camptothecin conjugate activated by nitroreductase for overcoming tumor oxygen heterogeneous distribution comprises the following steps:
(1) Will be provided with
Adding the mixture into a 100mL round-bottom flask, dissolving the mixture in acetonitrile, weighing 0.5 equivalent of phosphorus tribromide, dissolving the phosphorus tribromide in acetonitrile, dropwise adding the phosphorus tribromide into a reaction bottle by using a constant-pressure dropping funnel under ice-bath stirring, and reacting for 2 hours; after the reaction is finished, removing the solvent acetonitrile, extracting residues with dichloromethane and water for three times, collecting an organic phase, and removing the dichloromethane by rotary evaporation. Separating with silica gel column chromatography using dichloromethane as eluent, and rotary evaporating to obtain yellow solid compound
The yield is 35-40%;
(2) Will be provided with
According to a mol ratio of 1:2 dissolving in acetone, adding 10 equivalents of potassium carbonate, refluxing the reaction solution at 60 deg.C in oil bath for 1h, filtering potassium carbonate after reaction, removing solvent, extracting with dichloromethane and water, and passing the organic phase through anhydrous Na 2 SO 4 After drying, the solvent was removed under reduced pressure, and then the residue was separated by silica gel column chromatography using dichloromethane-methanol (volume ratio 50
The yield is 52-58%.
(3) Will be provided with
According to a molar ratio of 1:2.2 dissolving in dichloromethane, then adding 2-3 equivalent of 4-Dimethylaminopyridine (DMAP), 3 drops of N, N-Diisopropylethylamine (DIPEA) and triphosgene, and violently stirring the reaction liquid for 18-36h at room temperature; after the reaction is finished, dichloromethane and water are used for extraction, and the organic phase is subjected to anhydrous Na 2 SO 4 Drying and removing the solvent under reduced pressure; and finally, taking dichloromethane-methanol (volume ratio is 50
(BDP-Nitro-CPT) in 69% -74% yield.
Example 1
(1) Will be provided with
(0.30g, 1.00mmol) was added to a 100mL round bottom flask and dissolved with 15mL acetonitrile; then weighing phosphorus tribromide (0.14g, 0.50mmol) and dissolving in 10mL acetonitrile, then dropwise adding the phosphorus tribromide into a reaction bottle by using a constant-pressure dropping funnel under ice-bath stirring, and reacting for 2h; reaction ofAfter completion, the solvent acetonitrile was removed, the residue was extracted three times with dichloromethane and water, the organic phase was collected, and dichloromethane was removed by rotary evaporation. Separating with silica gel column chromatography using dichloromethane as eluent, and rotary evaporating to obtain yellow solid compound
(0.13g,37％)。 1 H NMR(500MHz,CDCl 3 ):δ(ppm)＝8.28(d,2H,J＝9.0Hz,ArH),7.69(d,2H,J＝9.0Hz,ArH),7.21(s,2H,ArH),5.20(s,2H,OCH 2 ),4.69(s,2H,OCH 2 ),4.53(s,2H,CH 2 Br),2.34(s,3H,CH 3 ). 13 C NMR(150MHz,CDCl 3 ) Delta (ppm) =152.65,147.68,144.41,135.19,134.08,131.84,131.21,127.84,123.88,74.85,60.79,27.96,20.76 HRMS (ESI) theoretical calculation (M/z [ M + Na ] M + Na] + ) 388.0160; the actual test value was 388.0145.
(2) Will be provided with
(0.10g,0.10mmol)、
(0.074g, 0.20mmol) was dissolved in acetone, followed by addition of potassium carbonate (0.14g, 1.0mmol), the reaction solution was refluxed in an oil bath at 60 ℃ for 1 hour, after completion of the reaction, the potassium carbonate was filtered, the solvent was removed, followed by extraction with dichloromethane and water, and the organic phase was extracted with anhydrous Na 2 SO 4 After drying, the solvent was removed under reduced pressure, and then the residue was separated by silica gel column chromatography using dichloromethane-methanol (volume ratio 50
(0.071g,53％)。 1 H NMR(400MHz,CDCl 3 ):δ(ppm)＝8.23(d,J＝8.4Hz,2H,ArH),8.10(d,J＝16.4Hz,2H,CH＝CH),7.59-7.63(m,8H,ArH and CH＝CH),7.31(s,1H,ArH),7.30(s,1H,ArH),7.18(d,J＝8.4Hz,2H,ArH),7.08(d,J＝8.8Hz,2H,ArH),6.97(d,J＝8.8Hz,4H,ArH),5.15(s,2H,OCH 2 ),5.13(s,2H,OCH 2 ),4.76(s,2H,OCH 2 ),4.20(t,J＝4.8Hz,4H,OCH 2 ),3.90(t,J＝4.8Hz,4H,OCH 2 ),3.78-3.75(m,4H,OCH 2 ),3.72-3.66(m,8H,OCH 2 ),3.58-3.55(m,4H,OCH 2 ),3.39(s,6H,OCH 3 ),2.39(s,3H,CH 3 ),1.42(s,6H,CH 3 )； 13 C NMR(150MHz,CDCl 3 ):δ(ppm)＝160.01,159.38,153.10,148.41,147.64,144.45,140.78,138.85,134.98,133.97,132.33,131.04,130.94,129.87,129.84,129.27,127.78,127.46,123.83,116.10,115.43,114.97,110.17,75.79,71.94,70.89,70.68,70.59,69.69,67.55,65.75,60.85,59.07,20.89,13.82.HRMS(ESI):C 63 H 68 BBr 2 F 2 N 3 O 13 Theoretical calculation of (M/z [ M + H ]] + ) 1306.3052; the actual test value is 1306.3081.
(3) Will be provided with
(0.03g, 0.086 mmol) was dissolved in dichloromethane, 4-Dimethylaminopyridine (DMAP) (0.03g, 0.246 mmol) and 3 drops of N, N-Diisopropylethylamine (DIPEA) were added and the reaction was stirred at room temperature for 10 minutes, then triphosgene in dichloromethane was added dropwise until the reaction was clear, and 2a was added
(0.05g, 0.039mmol), and vigorously stirring the reaction solution at room temperature for 18 hours; after the reaction is finished, dichloromethane and water are used for extraction, and the organic phase is subjected to anhydrous Na 2 SO 4 Drying and removing the solvent under reduced pressure; and finally, using dichloromethane-methanol (volume ratio is 50: 1) as an eluent, and performing silica gel column chromatography separation to obtain the BODIPY-nitrobenzene-camptothecin (BDP-Nitro-CPT) conjugate
(0.022g,70％)。 1 H NMR(500MHz,CDCl 3 ):δ(ppm)＝8.38(s,1H,ArH),8.14(d,J＝8.5Hz,1H,ArH),8.12(d,J＝16.5Hz,2H,CH＝CH),7.96-7.94(m,3H,ArH),7.83(t,J＝7.5Hz,1H,ArH),7.67(t,J＝7.5Hz,ArH),7.63-7.60(m,6H,ArH and CH＝CH),7.42(d,J＝7.5Hz,2H,ArH),7.32(s,1H,ArH),7.31(s,1H,ArH),7.29(s,1H,ArH),7.15(d,J＝8.5Hz,2H,ArH),7.04(d,J＝8.5Hz,2H,ArH),6.96(d,J＝8.5Hz,4H,ArH),5.72(d,J＝17.0Hz,1H,OCH 2 ),5.41(d,J＝17.0Hz,1H,OCH 2 ),5.30-5.20(m,4H,OCH 2 ),5.07-5.03(m,4H,NCH 2 and OCH 2 ),4.20(t,J＝4.0Hz,4H,OCH 2 ),3.90(t,J＝4.5Hz,4H,OCH 2 ),3.77(t,J＝4.0Hz,4H,OCH 2 ),3.72-3.67(m,8H,OCH 2 ),3.57(m,4H,OCH 2 ),3.39(s,6H,OCH 3 ),2.33(s,3H,CH 3 ),2.30-2.13(m,2H,CH 2 ),1.39(s,6H,CH 3 ),0.88(t,J＝6.5Hz,3H,CH 3 )； 13 C NMR(150MHz,CDCl 3 ):δ(ppm)＝160.01,160.09，159.33,157.29，153.78，153.67，152.22，148.84，148.47，147.55，146.51，145.60，143.89，140.79，138.94，138.43，135.18，131.59，132.50，132.36，131.28，130.86，129.93，129.50，129.34，128.48，128.43，128.29，128.26，127.94，127.56，123.54，120.42，116.14，115.46，115.05，110.23，95.78，78.13，76.37，72.01，70.95，70.74，70.66，69.75，67.62，67.22，65.83，50.05，32.09，20.85，13.89，7.69.HRMS(ESI):C 84 H 82 BBr 2 F 2 N 5 O 18 Theoretical calculation of (M/z [ M + H ]]+) is 1680.3954; the actual measurement value was 1680.4090.
Application example 1
The research on the in vitro anticancer activity of the molecular prodrug BDP-Nitro-CPT can verify the hypoxic response mechanism and the synergistic treatment mechanism of the drug and provide experimental basis for in vivo experiments. Cell activity was measured by the MTT method. MTT (3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazole bromide) is a yellow solid, and an aqueous solution of the MTT can effectively identify living cells in the cancer cell inhibition process. The detection principle is that the applied MTT is reduced to purple crystal formazan insoluble in water by the dehydrogenase succinate in the mitochondria of the living cells and deposited in the cells, and the applied MTT can not be reduced by dead cells because the cell structures of the dead cells are destroyed. After formazan is produced by living cells treated with MTT solution, formazan is dissolved by adding Dimethylsulfoxide (DMSO) to the cells, and the absorbance (OD value) at 570nm is measured with a microplate reader. Cell viability is plotted against log concentration of drugAnd calculating a median inhibitory concentration value (IC) 50 A value). Cell viability calculation formula: cell bioavailability% = (base:Sub>A-base:Sub>A) 0 )/(A 1 -A 0 ) X is 100%; wherein A is the OD value of the experimental group, A 0 Blank OD value, A 1 The OD value is the OD value of the cell control group.
MTT test: normally growing 4T1 cells (mouse breast cancer cells), hepG2 cells (human liver cancer cells) and HeLa cells (human cervical cancer cells) are selected as cell strains for in vitro activity study. The well of each 96-well plate is uniformly paved with about 7X 10 3 The cancer cells were incubated in a cell incubator at 37 ℃ for 24 hours. After incubation, the old medium was replaced with fresh DMEM medium containing different concentrations of BDP-Nitro-CPT (or 2 a), and cells were incubated for 8h under normoxic (21% oxygen concentration), mildly hypoxic (6% oxygen concentration) or hypoxic (0.1% oxygen concentration) conditions. After incubation, cells were washed three times with PBS and fresh DMEM medium was added. Cells were treated with/without light under normoxic, mildly hypoxic, hypoxic conditions (660nm, 20mW/cm) 2 5 min). After the light irradiation is finished, the cells are continuously incubated for 24h. Then 10. Mu.L of MTT (5 mg/mL) solution was added to the 96-well plate and incubation was continued for 4h. After incubation was complete, medium was removed from the 96-well plates using a pipette, and 100 μ L of DMSO was added to sufficiently dissolve formazan in the cells. OD was measured at 490nm using a microplate reader.
We determined the molecular prodrug BDP-Nitro-CPT prepared in the examples and the control drug 2a by MTT method at normal oxygen (21% O) 2 ) Mild hypoxia (6% o) 2 ) And hypoxic (0.1% 2 ) Cytotoxicity to 4T1 cells, hepG2 cells, heLa cells under the conditions. As can be seen from fig. 1: under normal oxygen conditions (c, f and i in figure 1), BDP-Nitro-CPT and 2a have no obvious killing effect on three kinds of cancer cells in the drug concentration range of 0.01-10 mu M, which indicates that the drug has good biocompatibility. When the cells are in hypoxic condition (a, d and g in figure 1), BDP-Nitro-CPT has certain killing effect on 4T1, hepG2 and HeLa cells, and 2a has no obvious cytotoxicity on three cancer cells treated in hypoxic condition. This result indicates that under hypoxic conditions, BDP-Nitro-CPT prodrugs are presentThe nitrobenzene group can be reduced by nitroreductase over-expressed in hypoxic cells, and then chemotherapeutic camptothecin is released to induce cancer cell death. Under the condition of illumination (660nm, 20mW/cm) 2 5 min), BDP-Nitro-CPT has higher cytotoxicity to three cancer cells cultured under normal oxygen and warm and hypoxic conditions, and the IC of the BDP-Nitro-CPT has higher cytotoxicity to 4T1, hepG2 and HeLa cells cultured under normal oxygen 50 IC values of 0.66. Mu.M, 0.67. Mu.M, 0.73. Mu.M for 4T1, hepG2, heLa cells cultured under mild hypoxic conditions 50 The values were 0.73. Mu.M, 0.66. Mu.M, 0.77. Mu.M, and the cytotoxicity was higher than that of the control group 2a (FIG. 1, table 1). The BDP-Nitro-CPT can consume oxygen and promote the release of camptothecin in the process of photodynamic therapy, and realizes the synergistic effect of photodynamic therapy and chemotherapy. In addition, IC of BDP-Nitro-CPT on hypoxic cells 50 Value ratio of IC to 2a for hypoxic cells treated 50 The higher value indicates that camptothecin released by the BDP-Nitro-CPT prodrug under the hypoxic condition can supplement photodynamic therapy.
TABLE 1 IC of BDP-Nitro-CPT and 2a on 4T1, hepG2, heLa cells 50 Value of
The research on the in vivo anticancer activity of the molecular prodrug BDP-Nitro-CPT and the control photosensitizer 2a can further verify the hypoxia response mechanism and the synergistic treatment mechanism of the BDP-Nitro-CPT prodrug. Balb/c mice were used in this experiment for 15-day detection of anti-tumor effect. The tumor inhibiting effect is judged by observing the change of the tumor size during the treatment period, the tumor quality of each group of mice after the treatment and H & E staining of tumor tissue sections, and the safety of the medicine is evaluated.
The experimental method comprises the following specific steps:
(1) Establishing a Balb/c mouse model: 30 female Balb/c mice with the weight of about 20g and the age of 5 weeks are selected, and 1 multiplied by 10 mice are planted on the back of each mouse subcutaneously 6 4T1 mouse breast cancer cells.
(2) Grouping and administration: when the average tumor size of the mice grows to 60mm 3 Mice were randomly divided into six groups (BDP-Nitro-CPT + Light group, 2a + Light group, BDP-Nitro-CPT group, 2a group, saline + Light group, saline group), each group having five mice in parallel, each group being given the corresponding drug (100. Mu.L, 100. Mu.M) by tail vein injection.
(3) Light irradiation treatment: the first administration is day 0, the light group is irradiated 24h after administration, and the tumor part of the mouse is irradiated with 660nm laser for 10min (power density of 300 mW/cm) 2 )。
(4) Data measurement and recording: after the light treatment, the mice were weighed regularly each day and the tumor volume was measured, and repeated for 15 days.
(5) H & E staining: after treatment, one mouse is randomly selected from each group, sacrificed by cervical dislocation, taken out of tumor, heart, liver, spleen, lung and kidney, respectively, soaked in 4% paraformaldehyde solution for 24H for fixation, wrapped with paraffin, sliced, and stained with H & E. Histological morphology of each tissue section was observed and photographed under an optical microscope.
The anti-tumor results of the experimental group and the control group are summarized in FIG. 2. As can be seen from fig. 2: compared with a Saline control group, mice in the Saline + Light group and the group injected with the BDP photosensitizer 2a without Light have no obvious tumor inhibition effect, which indicates that the mice injected with the BDP photosensitizer 2a alone and given Light have no anti-tumor effect. However, 2a +, light and BDP-Nitro-CPT have significant tumor suppression effect in the matt group, due to photodynamic therapy and hypoxia-activated chemotherapy, respectively. In contrast, the antitumor effect was most significant after the mouse was treated with BDP-Nitro-CPT by tail vein injection and light irradiation, and the size and mass of the tumor was relatively small in the dissected mice (c and b in FIG. 2), and even four mice had their tumors completely eliminated without recurrence (c in FIG. 2). Furthermore, H & E staining in tumor histology indicated that the BDP-Nitro-CPT + Light group induced massive necrosis and apoptosis in tumor cells (E in fig. 2). The mice had negligible weight fluctuation throughout the treatment period (d in fig. 2). In addition, it was found from the tissue sections of the respective organs that 2a and BDP-Nitro-CPT did not cause damage to normal tissues even under light (FIG. 3). The experimental results show that: the BDP-Nitro-CPT light-enriched in the tumor region has a photodynamic-chemotherapy synergistic treatment effect, and the BDP-Nitro-CPT shows good biological safety while enhancing tumor inhibition.
The above description is only a preferred embodiment of the present invention, and all the equivalent changes and modifications made according to the claims of the present invention should be covered by the present invention.
Claims (8)
1. A nitroreductase-activated multifunctional molecular prodrug for overcoming tumor oxygen heterogeneity distribution, characterized by: the prodrug is a BODIPY-nitrobenzene-camptothecin conjugate, and the chemical structural formula is as follows:
2. a method of preparing nitroreductase activated multifunctional molecular prodrugs for overcoming tumor oxygen heterogeneity distribution as claimed in claim 1 comprising: the preparation method comprises the following steps: to be provided with
、
As a starting material, the BODIPY-Nitrobenzene-camptothecin conjugates were synthesized.
3. The preparation method according to claim 2, wherein the preparation method specifically comprises the following steps: will be provided with
、
Dissolved in dichloromethane in a molar ratio of 1:2.2, then 2-3 equivalents of 4-dimethylaminopyridine, DMAP, 3 drops of N are addedN-diisopropylethylamine DIPEA and triphosgene, and violently stirring the reaction liquid for 18-36h at room temperature; after the reaction is finished, dichloromethane and water are used for extraction, and the organic phase is subjected to anhydrous Na 2 SO 4 Drying and removing the solvent under reduced pressure; and finally, mixing the raw materials in a volume ratio of 50:1, taking dichloromethane-methanol as eluent, and separating by silica gel column chromatography to obtain the BODIPY-nitrobenzene-camptothecin conjugate.
4. The method of claim 3, wherein the 2-3 equivalents of DMAP are added to
Based on the molar amount of (a).
5. The method according to claim 2 or 3, wherein the method comprises
The preparation method comprises the following steps:
(1) To be provided with
Phosphorus tribromide as initial raw material and synthesizing
；
(2) Synthesized in step (1)
、
As a starting material, synthesized
。
6. According to the claimsThe method according to claim 5, wherein the step of preparing the composition is carried out in the presence of a catalyst
The preparation method specifically comprises the following steps:
(1) Will be provided with
And phosphorus tribromide is prepared by mixing the following components in a molar ratio of (2): 1, mixing, dissolving in acetonitrile, and reacting for 2h under the ice bath condition; after the reaction is finished, removing the solvent, and separating by silica gel column chromatography by using dichloromethane as an eluent to obtain yellow solid
；
(2) Will be provided with
、
According to a mol ratio of 1:2 in acetone, then adding 10 equivalents of potassium carbonate, reacting under reflux for 1h at 60 ℃ in an oil bath, after the reaction is finished, filtering the potassium carbonate, removing the solvent, then extracting with dichloromethane and water, and passing the organic phase through anhydrous Na 2 SO 4 After drying, the solvent was removed under reduced pressure and then mixed in a volume ratio of 50:1, taking dichloromethane-methanol as eluent, and separating by silica gel column chromatography to obtain green solid
。
7. The process according to claim 6, characterized in that the 10 equivalents of potassium carbonate are used in order to obtain
Is calculated.
8. Use of the nitroreductase-activated multifunctional molecular prodrug as claimed in claim 1 for overcoming tumor oxygen heterogeneity distribution in the preparation of an anticancer drug.
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| CN112920210A (en) * | 2021-02-01 | 2021-06-08 | 福州大学 | Red light activatable photodynamic therapy-chemotherapy combined prodrug and preparation and application thereof |
| CN114409687A (en) * | 2022-03-02 | 2022-04-29 | 福州大学 | Intelligent photosensitive medicine capable of switching light treatment modes in tumors and preparation method and application thereof |
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