CN108395460B - Hypoxia activated adriamycin prodrug and preparation method thereof - Google Patents
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Abstract
The invention discloses a hypoxia activated adriamycin prodrug and a preparation method thereof, wherein an azo bond with hypoxia activation effect and a response functional group of a self-occlusion structure are modified on the amino group at the 3' position of adriamycin to synthesize the adriamycin prodrug, the drug can effectively realize selective activation in a hypoxia environment, and can realize the regulation and control of the growth activity of tumor cells by regulating the distribution of subcellular organelles of the adriamycin in a normoxic and hypoxic environment, so that the toxic and side effects of the drug on normal cells are reduced, the targeting efficiency of tumor treatment is improved, and more new ideas are provided for the development of antitumor drugs. The hypoxic activated adriamycin prodrug provided by the invention has the advantages of simple synthesis method, cheap and easily available raw materials, and easiness in industrial production and clinical transformation.
Description
Technical Field
The invention relates to the fields of medicinal chemistry and biomedicine, in particular to a hypoxia activated adriamycin prodrug and a preparation method thereof.
Background
Cancer, also known as malignant tumor, has become one of the major diseases threatening human health. Published data from the world health organization show that a total of 800 million people die of cancer in 2012 worldwide and it is expected that 1300 million will be reached in 2030. According to the annual report of 2017 Chinese tumor registration published by the national tumor center, about 1 million people in China have diagnosed cancer every day, which means that one person is diagnosed with cancer every 7 minutes on average. By the age of 85, a person has an accumulated risk of cancer of up to 36%, and is on the verge of achieving effective treatment of cancer, moving towards a high morbidity, high mortality, and a trend towards younger age. The treatment modes of chemotherapy, radiotherapy, gene therapy and the like can effectively realize the inhibition of tumor growth, but the low tumor treatment effect and the great toxic and side effect are caused by the poor selectivity of the anti-tumor drugs and the individualized difference of tumor patients. Therefore, it is highly desirable to construct a therapeutic drug with tumor selectivity and specificity to achieve precise tumor treatment.
Tumor cells are abnormally proliferated by genetic and adaptive changes, and the rapid proliferation of tumor cells can cause a special microenvironment in a tumor area. Among them, the hypoxic microenvironment of a tumor region, which is caused by increased oxygen consumption of rapidly proliferating tumor cells in the tumor region and insufficient angiogenesis, is a common feature of all tumor types, including hematological tumors. Tumor hypoxia induces tumor tolerance to, for example, chemotherapy and radiation therapy, and causes problems with tumor invasion and metastasis, which are major factors in tumor patient death.
Doxorubicin (Doxorubicin) is a high-efficiency antitumor drug used in clinic, is commonly used for treating cancers such as ovarian cancer, lung cancer, breast cancer, prostatic cancer and the like, and has a main action mechanism that a compound is formed with DNA and DNA topoisomerase II to further inhibit the growth of tumors. However, because doxorubicin has no selectivity to cytotoxicity generated by normal cells and tumor cells, doxorubicin has significant toxic and side effects in clinical application, particularly cardiotoxicity such as chronic heart disease and congestive heart failure, and side effects such as nausea, alopecia, appetite decrease, diarrhea, cytopenia, oral ulcer and the like can be caused. Generally, researchers construct prodrugs by liposome encapsulation or chemical modification of doxorubicin in order to improve the selectivity of doxorubicin for tumor therapy, for example, conventional doxorubicin prodrugs modulate the intracellular distribution of doxorubicin by chemically modifying the subcellular organelle targeting group of doxorubicin. Nevertheless, doxorubicin prodrugs have been reported to be generally directed against only a certain tumor or class of tumors, and lack versatility in tumor therapy. Nitrogen-containing drugs are tumor cell inhibitors, which inhibit tumor cell proliferation by methylating DNA and then cross-linking DNA-DNA or DNA-protein, unlike doxorubicin. However, because of the lack of targeting of nitrogen-mediated drugs to tumor cells, severe toxic and side effects are caused to the liver, especially in the process of treating tumors. The reported medicines lack the universality and targeting property of tumor treatment and cannot meet the requirements of clinical use, so that a tumor-specific medicine is urgently needed to realize safe and effective treatment of tumor diseases.
Disclosure of Invention
Aiming at the problems in the prior art, the invention constructs an anti-tumor treatment medicament with hypoxic targeting property and hypoxic responsiveness, realizes the universality of tumor treatment, greatly improves the tumor treatment effect and reduces toxic and side effects.
The technical scheme provided by the invention is as follows:
a hypoxia activated doxorubicin prodrug has a structural formula shown in formula (I):
wherein R represents bis (2-chloroethyl) amino
Bis (2-hydroxyethyl) amino
Diethylamino group
Dimethylamino group
Wherein X represents a nitrogen atom or a methine group.
The hypoxia-activated doxorubicin prodrug has a structure represented by the formula (II):
a method for preparing a hypoxia activated doxorubicin prodrug having a structure represented by formula (II), comprising the steps of:
(1) adding a sodium nitrite aqueous solution into a concentrated hydrochloric acid solution of 4-aminobenzol, reacting at room temperature for 20 minutes, adding a reaction solution into an ethanol solution of N, N-bis (2-chloroethyl) aniline, reacting for 2 hours, and purifying the reaction solution to obtain a compound A (raw material 3); the structural formula of the compound A is
(2) Dissolving a compound A and 4-dimethylaminopyridine in anhydrous dichloromethane together, slowly dropwise adding a dichloromethane solution of p-nitrophenyl chloroformate to the mixture under the condition of stirring at 0 ℃, and after dropwise adding, placing the reaction solution at normal temperature for reaction; after the reaction is finished, concentrating and purifying the reaction solution to obtain a compound B (raw material 4); the structural formula of the compound B is
(3) Dissolving doxorubicin hydrochloride and triethylamine in anhydrous DMF together, stirring for 1 hour in the dark, adding a DMF solution of a compound B, continuing to react for 12 hours, dropping the reaction solution into anhydrous ether to terminate the reaction, performing centrifugal separation to obtain a crude product, and purifying the obtained crude product to obtain the hypoxia-activated doxorubicin prodrug with the structure shown in the formula (II).
The invention utilizes aniline structure in nitrogen-mediated drug to link adriamycin and nitrogen-mediated two antitumor drugs through azo bond activated by hypoxia to form adriamycin prodrug. After the adriamycin prodrug enters cells, because a tumor area has a hypoxic microenvironment, azo bonds on the adriamycin prodrug are broken, the adriamycin part is subjected to self-occlusion, the original shape is recovered, the adriamycin is enriched in cell nuclei, and the adriamycin prodrug and DNA topoisomerase in the cell nuclei interact to realize the inhibition of cell growth; and the normal cell area does not have hypoxic microenvironment, and the adriamycin prodrug is mainly distributed in the cytoplasm of the cell, so that the adriamycin prodrug has lower toxicity to the normal cell. The distribution of the adriamycin in cell nucleus and cytoplasm is regulated and controlled by the tumor hypoxia microenvironment, so that the treatment effect of the adriamycin is greatly improved, and the toxic and side effects of the adriamycin on normal cells and tissues are reduced. The design of the prodrug has no relevant report at home and abroad.
Because of the repair mechanism of DNA, the methylation drug is often induced to generate drug resistance in the treatment process, and the drug resistance generated in the repeated administration process depending on a single anti-tumor drug can be effectively solved in a cooperative treatment mode, so that the establishment of the cooperative treatment prodrug of the nitrogen-mediated drug responding to the tumor microenvironment can greatly promote the process of tumor targeted accurate treatment. The invention simultaneously realizes the regulation and control of the adriamycin at the level of subcellular organelles under the condition of hypoxia.
The invention has the following advantages and beneficial effects:
(1) the invention modifies the azo bond and the self-occlusion group of the adriamycin, thereby endowing the small molecule prodrug with the characteristic of drug controlled release under the condition of tumor hypoxia;
(2) the invention reduces the toxic and side effect of the adriamycin on normal cells and enhances the killing effect of the adriamycin on tumor cells under the condition of hypoxia through chemical modification;
(3) the invention links two antitumor drugs of adriamycin and nitrogen medium through the hypoxia response azo bond, thus realizing the effect of cooperative treatment on tumor cells;
(4) the hypoxia-activated adriamycin prodrug provided by the invention can realize the distribution of a drug in a cell on the level of a subcellular organelle, and can greatly enhance the selectivity and specificity of tumor hypoxia targeted therapy;
(5) the hypoxic activated adriamycin prodrug provided by the invention has the advantages of simple synthesis method, cheap and easily available raw materials, and easiness in industrial production and clinical transformation.
Drawings
FIG. 1 is an electrospray mass spectrum of a doxorubicin prodrug represented by formula (II).
FIG. 2 is a confocal microscope of 4T1 endocytosis of doxorubicin prodrug or doxorubicin under normoxic and hypoxic conditions;
wherein, FIG. 2(A1, A2, A3) represents the nuclear fluorescence of 4T1 cells, the doxorubicin fluorescence of 4T1 cells cultured with doxorubicin hydrochloride, and the superimposed fluorescence of both, respectively; FIG. 2(B1, B2, B3) represents the nuclear fluorescence of 4T1 cells under normoxic conditions, the doxorubicin prodrug fluorescence of 4T1 cells cultured with doxorubicin prodrug, and the superimposed fluorescence of both, respectively; FIG. 2(C1, C2, C3) represents the nuclear fluorescence of 4T1 cells under hypoxic conditions, the doxorubicin prodrug fluorescence of 4T1 cells cultured with doxorubicin prodrug, and the superimposed fluorescence of both, respectively.
FIG. 3 is a confocal microscope of B16 endocytosis of doxorubicin prodrug under normoxic and hypoxic conditions;
wherein, fig. 3(a1, a2, A3) respectively represent under normal oxygen conditions: nuclear fluorescence of B16 cells, doxorubicin prodrug fluorescence of B16 cells cultured with doxorubicin prodrug, and the superimposed fluorescence of both; FIG. 3(B1, B2, B3) represents respectively under hypoxic conditions: nuclear fluorescence of B16 cells, doxorubicin prodrug fluorescence of B16 cells cultured with doxorubicin prodrug, and the superimposed fluorescence of both;
FIG. 4 is a confocal microscope showing the endocytosis of doxorubicin prodrug by fibroblasts under normoxic and hypoxic conditions;
wherein, fig. 4(a1, a2, A3) respectively represent under normal oxygen conditions: the nuclear fluorescence of fibroblasts, the adriamycin prodrug fluorescence of fibroblasts cultured by adriamycin prodrug and the superposition fluorescence of the adriamycin prodrug and the adriamycin prodrug; FIG. 4(B1, B2, B3) represents respectively under hypoxic conditions: nuclear fluorescence of fibroblasts, doxorubicin prodrug fluorescence of fibroblasts cultured with doxorubicin prodrugs, and the superimposed fluorescence of both.
FIG. 5 is a confocal microscope of 4T1 endocytosis of doxorubicin prodrug under normoxic and hypoxic induction conditions;
wherein, fig. 5(a1, A3) represents doxorubicin prodrug fluorescence of 4T1 cells cultured with doxorubicin prodrug under normoxic or hypoxic conditions, respectively; fig. 5(a2, a4) represents the nuclear fluorescence of 4T1 cells cultured with doxorubicin prodrug under normoxic and hypoxic conditions and their superimposed fluorescence with doxorubicin prodrug, respectively.
FIG. 6 is a confocal microscope of 4T1 endocytosis of doxorubicin prodrug under normoxic and hypoxic induction conditions;
wherein, fig. 6(a1, a2, A3) represents the nuclear fluorescence of 4T1 cells under normoxic condition, the adriamycin prodrug fluorescence of 4T1 cells cultured with adriamycin prodrug, and the superimposed fluorescence of both, respectively; FIG. 6(A4, A5, A6) represents the nuclear fluorescence of 4T1 cells under hypoxic conditions, the doxorubicin prodrug fluorescence of 4T1 cells cultured with doxorubicin prodrug, and the superimposed fluorescence of both, respectively.
FIG. 7 is a confocal microscope of mitochondrial staining following normoxic culture or hypoxia induction following endocytosis of doxorubicin prodrug by 4T1 cells;
wherein, fig. 7(a1, a2, A3, a4) respectively represents the nuclear fluorescence, mitochondrial fluorescence, doxorubicin prodrug fluorescence and its superimposed fluorescence of 4T1 cells cultured with doxorubicin prodrug under normoxic conditions; FIG. 7(B1, B2, B3, B4) represents nuclear fluorescence, mitochondrial fluorescence, doxorubicin prodrug fluorescence and superimposed fluorescence of 4T1 cells cultured with doxorubicin prodrug under hypoxic conditions, respectively.
FIG. 8 is a confocal microscope of lysosomal staining following normoxic culture or hypoxia induction following endocytosis of doxorubicin prodrug by 4T1 cells;
wherein, fig. 8(a1, a2, A3, a4) respectively represents the nuclear fluorescence, lysosomal fluorescence, doxorubicin prodrug fluorescence and its superimposed fluorescence of 4T1 cells cultured with doxorubicin prodrug under normoxic conditions; FIG. 8(B1, B2, B3, B4) represents the nuclear fluorescence, lysosomal fluorescence, doxorubicin prodrug fluorescence and its superimposed fluorescence, respectively, of 4T1 cells cultured with doxorubicin prodrug under hypoxic conditions.
FIG. 9 shows cell viability under various doxorubicin prodrug concentrations and normoxic and hypoxic conditions;
among them, FIG. 9(A) represents 4T1 cells, and FIG. 9(B) represents B16 cells.
Figure 10 is a synthetic scheme for a hypoxia activated doxorubicin prodrug represented by formula (II).
Detailed Description
The technical solution of the present invention will be described in further detail with reference to examples.
Example 1: synthesis of hypoxia-activated doxorubicin prodrug represented by formula (II)
(1) Synthesis of N, N-bis (2-chloroethyl) aniline:
putting phosphorus oxychloride (5mL) into a round-bottom flask, slowly adding N, N-bis (2-hydroxyethyl) aniline (3.9 g) into the round-bottom flask under the condition of stirring at 0 ℃, after the solid is dissolved, refluxing the reaction solution at 110 ℃ for 1 hour, and performing rotary evaporation and concentration to obtain a crude product; dissolving the obtained crude product in 200mL ethyl acetate, washing with pure water for three times, drying the organic phase with anhydrous magnesium sulfate overnight after liquid separation, carrying out rotary evaporation and concentration on the obtained product, separating by column chromatography to obtain N, N-bis (2-chloroethyl) aniline, and eluting with a methanol-dichloromethane mixed solution (the volume ratio of methanol to dichloromethane is 2: 98).
(2) Synthesizing a raw material 3:
sodium nitrite (5.3 g) was dissolved in 30mL of pure water, and then added to a pure aqueous solution (120mL) containing 18mL of concentrated hydrochloric acid containing 4-aminobenzyl alcohol (8.2 g), and after 20 minutes of reaction at room temperature, the resulting reaction solution was added to an ethanol solution containing N, N-bis (2-chloroethyl) aniline, and after 2 hours of reaction, the resulting reaction solution was diluted with dichloromethane (500mL) and washed twice with water. The organic phase obtained is treated with Na2SO4Dry overnight. And (3) performing rotary evaporation and concentration on the obtained product, and performing column chromatography separation to obtain a raw material 3, wherein the eluent is a methanol-dichloromethane mixed solution (the volume ratio of methanol to dichloromethane is 1: 13).
(3) Synthesizing a raw material 4:
feed 3(0.772 g) and 4-dimethylaminopyridine (DMAP, 0.4 g) were dissolved together in dry dichloromethane (15mL) and stirred at 0 ℃. Then, p-nitrophenyl chloroformate (0.62 g) was dissolved in 8mL of dry methylene chloride and slowly added dropwise to the reaction mixture of starting material 3 and DMAP. After the completion of the dropwise addition, the reaction mixture was left at room temperature to react for 12 hours. After the reaction is finished, concentrating the reaction solution, and separating by column chromatography to obtain a raw material 4, wherein the eluent is a mixed solution of n-hexane and ethyl acetate (the biological volume ratio of n-hexane to ethyl acetate is 3: 1).
(4) Synthesizing a prodrug of doxorubicin represented by the formula (II):
doxorubicin hydrochloride (0.4 g) and triethylamine (0.29mL) were dissolved in 6mL of dry DMF and stirred for 1 hour under the exclusion of light. Then, starting material 4(0.52 g) was dissolved in 4ml of DMF and added to a DMF solution containing doxorubicin and triethylamine. After 12 hours of reaction, the reaction solution was dropped into anhydrous ether to terminate the reaction, and the crude product was obtained by centrifugal separation. And (3) separating the obtained crude product by column chromatography to obtain the adriamycin prodrug shown in the formula (II), wherein the eluent is a methanol-dichloromethane mixed solution (the volume ratio of methanol to dichloromethane is 1: 13).
The molecular structure of the obtained compound was characterized by electrospray mass spectrometry, and as a result, as shown in FIG. 1, the theoretical molecular weight of the doxorubicin prodrug was 920.24, and the obtained [ M-H ] was characterized by electrospray mass spectrometry]-The molecular ion peak was 919.23, which coincides with the theoretical molecular weight, thus demonstrating the successful synthesis of the doxorubicin prodrug represented by formula (II).
Example 2
Mouse breast cancer cells (4T1) at 1X 105The cells/well were seeded at a density and cultured in 1mL of medium at 37 ℃. After 24 hours, doxorubicin hydrochloride and the doxorubicin prodrug were dissolved in the medium, respectively, and 1mL of a solution containing doxorubicin hydrochloride was added to 4T1 cellsMedium of either doxorubicin (5 μmol/l) or doxorubicin prodrug (20 μmol/l). Meanwhile, 4T1 cells added with the adriamycin prodrug are respectively cultured under the condition of normal oxygen or hypoxic conditions. After 24 hours, the cells were washed three times with PBS, and after staining the nucleus with Hoechst 33342, the distribution of doxorubicin inside the cells was observed with a confocal microscope.
As shown in FIG. 2, doxorubicin hydrochloride-cultured 4T1 cells showed that doxorubicin was mainly distributed in the nucleus of 4T1 cells. 4T1 cells cultured with doxorubicin prodrug under normoxic conditions showed that the red fluorescence was distributed mainly in the cytoplasm of the cells. And 4T1 cells cultured by the adriamycin prodrug under hypoxic condition have red fluorescence enriched towards the nucleus. These results indicate that hypoxic conditions stimulate the activation of doxorubicin prodrugs in 4T1 cells.
Example 3
Mouse melanoma cells (B16) were cultured at 1X 105The cells/well were seeded at a density and cultured in 1mL of medium at 37 ℃. After 24 hours, the doxorubicin prodrug was dissolved in the medium and 1mL of the medium containing the doxorubicin prodrug (20 μmol/l) was added to B16 cells. Meanwhile, B16 cells added with the adriamycin prodrug are respectively cultured under the condition of normal oxygen or hypoxic oxygen. After 24 hours, the cells were washed three times with PBS, and after staining the nucleus with Hoechst 33342, the distribution of doxorubicin inside the cells was observed with a confocal microscope.
As shown in FIG. 3, the red fluorescence of B16 cells cultured with doxorubicin prodrug under normoxic conditions was distributed mainly in the cytoplasm of the cells. And the red fluorescence of B16 cells cultured by the adriamycin prodrug under the hypoxic condition is enriched to the nucleus. These results indicate that hypoxic conditions induce doxorubicin activation in B16 cells, which in turn enriches into the nucleus of tumor cells.
Example 4
Fibroblast cells were cultured at 1X 105The cells/well were seeded at a density and cultured in 1mL of medium at 37 ℃. After 24 hours, the doxorubicin prodrug was dissolved in the culture medium and 1mL of the culture medium containing the doxorubicin prodrug (20 micromoles/liter) was added to the fibroblasts. At the same time, will addThe fibroblasts with the doxorubicin prodrug are cultured under normoxic or hypoxic conditions, respectively. After 24 hours, the cells were washed three times with PBS, and after staining the nucleus with Hoechst 33342, the distribution of doxorubicin inside the cells was observed with a confocal microscope.
As shown in FIG. 4, the red fluorescence of fibroblasts cultured with doxorubicin prodrug under normoxic conditions is mainly distributed in the cytoplasm of the cells. And the red fluorescence of the fibroblasts cultured by the adriamycin prodrug under the hypoxic condition is enriched to the cell nucleus. These results indicate that hypoxic conditions induce doxorubicin activation in fibroblasts, which in turn enriches into the nucleus of fibroblasts. As can be demonstrated by fig. 2, fig. 3 and fig. 4, the hypoxic microenvironment can effectively induce the activation of the doxorubicin prodrug, and further induce the enrichment thereof to the nucleus of the cell, and these properties have good versatility.
Example 5
4T1 cells were plated at 1X 105The cells/well were seeded at a density and cultured in 1mL of medium at 37 ℃. After 24 hours, the doxorubicin prodrug was dissolved in the medium and 1mL of the doxorubicin prodrug (20 μmol/l) of the medium was added to 4T1 cells. After 12 hours of endocytosis, half of the cells in the dish were covered with a cover slip to reduce the exposure of the cells under the cover slip to oxygen, and then the cells were incubated at 37 ℃. After 12 hours, the cells were washed three times with PBS, and after staining the nucleus with Hoechst 33342, the distribution of doxorubicin inside the cells was observed with a confocal microscope.
As a result, as shown in FIG. 5, in the cells not covered with the cover glass, the red fluorescence was mainly distributed in the cytoplasm of the cells, while in the cells covered with the cover glass, the red fluorescence was mainly distributed in the nuclei of the cells. These results again demonstrate the responsiveness of the hypoxia activated doxorubicin prodrug to the hypoxic microenvironment and the property of hypoxia to induce nuclear targeted enrichment.
Example 6
4T1 cells were plated at 1X 105The cells/well were seeded at a density and cultured in 1mL of medium at 37 ℃. After 24 hours, the doxorubicin prodrug was dissolved in the medium,to 4T1 cells, 1mL of doxorubicin prodrug (20. mu. mol/L) in medium was added. After 12 hours of endocytosis, the cells were washed three times with PBS, stained with Hoechst 33342 for nucleus, and then distribution of doxorubicin in the cells was observed with a confocal microscope. After the observation, the cells were covered with a cover glass, and after further culturing for 12 hours, the cells were washed three times with PBS, and after staining the nuclei with Hoechst 33342, the distribution of doxorubicin in the cells was observed with a confocal microscope.
As shown in fig. 6, in the 4T1 cells without hypoxic treatment, the red fluorescence was mainly distributed in the cytoplasm of the cells, whereas after incubation in a hypoxic microenvironment induced by coverslips, the red fluorescence was mainly distributed in the nucleus of the cells. These results again demonstrate the responsiveness of the hypoxia activated doxorubicin prodrug to the hypoxic microenvironment, as well as the characteristic hypoxia-induced nuclear-targeted enrichment that the hypoxia activated doxorubicin prodrug possesses.
Example 7
4T1 cells were plated at 1X 105The cells/well were seeded at a density and cultured in 1mL of medium at 37 ℃. After 24 hours, the doxorubicin prodrug was dissolved in the medium and 1mL of the doxorubicin prodrug (20 μmol/l) of the medium was added to 4T1 cells. After 24 hours incubation in normoxic and hypoxic conditions, respectively, the cells were washed three times with PBS, and after staining the mitochondria with mitotracker green, the cells were washed three times with PBS. After the nucleus of the cell is stained by Hoechst 33342, the distribution of adriamycin in the cell is observed by confocal observation.
As a result, as shown in fig. 7, under the normoxic condition, the red fluorescence was distributed mainly in the cytoplasm of the cell and did not overlap with the mitochondria. Under hypoxic conditions, however, red fluorescence is concentrated towards the nucleus of the cell and coincides well with the nucleus. These results indicate that the doxorubicin prodrug is not distributed for mitochondrial targeting properties, and again demonstrate that the hypoxia-activated doxorubicin prodrug is hypoxia-inducible for nuclear targeted enrichment.
Example 8
4T1 cells were plated at 1X 105The cells/well were seeded at a density and cultured in 1mL of medium at 37 ℃. After 24 hours, the adriamycin is addedThe prodrug was dissolved in culture medium and 1mL of doxorubicin prodrug (20 μmol/l) was added to 4T1 cells. After 24 hours incubation in normoxic and hypoxic conditions, respectively, the cells were washed three times with PBS, and after lysosome staining with lysotracergreen, the cells were washed three times with PBS. After the nucleus of the cell is stained by Hoechst 33342, the distribution of adriamycin in the cell is observed by confocal observation.
The results are shown in figure 8, where under normoxic conditions, the red fluorescence is predominantly distributed in the cytoplasm of the cell and coincides with the lysosomal green fluorescent dye, indicating that the pro-drug in the cytoplasm is predominantly distributed in the lysosomes of the cell. Under the hypoxic condition, the red fluorescence is enriched to the cell nucleus of the cell and well overlapped with the cell nucleus. These results indicate that the doxorubicin prodrug may enter the cell by endocytosis, and demonstrate the property that the doxorubicin prodrug in the cytoplasm can be efficiently transferred from the cytoplasm to the nucleus after activation by hypoxia.
Example 9
4T1 cells and B16 cells were seeded at 6000 cells/well in 96-well plates and cultured with 100. mu.l of medium for 24 hours. Then, 100. mu.l of the doxorubicin prodrug-containing solution prepared in the medium at various concentrations was added to each well, respectively. All cells were cultured under normoxic or hypoxic conditions for 24 hours. Subsequently 20 μ l of 5 mg/ml MTT (MTT dissolved in PBS buffer) was added to each well. After 4 hours of co-cultivation, the medium was aspirated and 150. mu.l of dimethyl sulfoxide was added. And measuring the light absorption value at 570 nm in each hole by using a microplate reader, and calculating the cell survival rate according to the light absorption value to further obtain the cytotoxicity of the adriamycin prodrug on 4T1 cells and B16 cells under the hypoxic and normoxic conditions.
The results are shown in figure 9, where 4T1 cells survived 54.3% and 33.6% under normoxic and hypoxic conditions, respectively, and B16 cells survived 43.7% and 33.5% under normoxic and hypoxic conditions, respectively, in cells co-cultured at 20 micromolar prodrug concentration. The doxorubicin prodrug was demonstrated to have concentration-dependent cytotoxicity against both 4T1 cells and B16 cells, and toxicity increased with increasing concentration of the doxorubicin prodrug. More specifically, doxorubicin prodrug is more cytotoxic to both 4T1 cells and B16 cells under hypoxic conditions relative to normoxic conditions, demonstrating the hypoxic-activated cytotoxic properties of the doxorubicin prodrug.
Claims (3)
1. A hypoxia activated doxorubicin prodrug having the structural formula shown in formula (I):
wherein R represents a bis (2-chloroethyl) amino group, and X represents a nitrogen atom.
2. The hypoxia-activated doxorubicin prodrug according to claim 1, having the structure represented by formula (II):
3. a process for preparing the hypoxia-activated doxorubicin prodrug of claim 2, comprising the steps of:
(1) adding a sodium nitrite aqueous solution into a concentrated hydrochloric acid solution of 4-aminobenzol, reacting at room temperature for 20 minutes, adding a reaction solution into an ethanol solution of N, N-bis (2-chloroethyl) aniline, reacting for 2 hours, and purifying the reaction solution to obtain a compound A; the structural formula of the compound A is
(2) Dissolving a compound A and 4-dimethylaminopyridine in anhydrous dichloromethane together, slowly dropwise adding a dichloromethane solution of p-nitrophenyl chloroformate to the mixture under the condition of stirring at 0 ℃, and after dropwise adding, placing the reaction solution at normal temperature for reaction; after the reaction is finished, concentrating and purifying the reaction solution to obtain a compound B; the structural formula of the compound B is
(3) Dissolving doxorubicin hydrochloride and triethylamine together in anhydrous DMF, stirring for 1 hour in the dark, adding a DMF solution of a compound B, continuing to react for 12 hours, dropping the reaction solution into anhydrous diethyl ether to terminate the reaction, performing centrifugal separation to obtain a crude product, and purifying the obtained crude product to obtain the hypoxic-activated doxorubicin prodrug as claimed in claim 2.
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