Photosensitive probe for breast tumor cells

Abstract

The invention provides a photosensitive probe for breast tumor cells, and belongs to the technical field of compound synthesis. By utilizing good biocompatibility and high fluorescence quantum yield of 2-benzothiazole-2-yl phenol (HBT) mother nucleus, a blue fluorescence signal which can be shown in breast cancer tumor cells, is firstly excited by reactive oxygen species (ROS) and is bright green in fluorescence under the condition that the blue fluorescence signal is irradiated by light (lambda is greater than or equal to 410 nm) after being catalyzed by tyrosinase is developed, and the fluorescence quantum yield is high. The drug delivery system for releasing the anti-cancer drug (bendamustine) reduces the toxicity of bendamustine to normal cells and tissues on one hand, and also avoids serious side effects in the cancer treatment process of bendamustine.

Description

Technical Field

[0001] The invention belongs to the technical field of compound synthesis and relates to a photosensitive probe for breast tumor cells. Background Art

[0002] Many fluorescent probes are susceptible to photobleaching under continuous illumination, which means that their fluorescence intensity will gradually weaken over time, limiting the possibility of long-term imaging; fluorophores with low quantum yield may result in insufficient signal intensity and affect detection sensitivity; most are single-target types, resulting in poor control and diagnosis and treatment accuracy of cancer cells. Summary of the invention

[0003] The purpose of the present invention is to provide a photosensitive probe for breast tumor cells in view of the above problems existing in the prior art. The technical problem to be solved by the present invention is to prepare a compound for photosensitizing breast tumor cells.

[0004] The purpose of the present invention can be achieved by the following technical scheme: A photosensitive probe for breast tumor cells, comprising the following steps:

[0005] 1. Preparation of Compound I: 5-(2-bromoacetyl)-2-hydroxybenzaldehyde is used as a reaction raw material, mixed with bendamustine hydrochloride, and then added with dry potassium carbonate in N,N-dimethylformamide to obtain a mixed product containing Compound I after reaction. After purification and separation, a brown solid Compound I is obtained. The reaction expression is as follows:

[0006]

[0007] 2. Preparation of Compound II: Compound I was used as a raw material and dissolved in dimethyl sulfoxide. 2-aminothiophenol was then added to the mixture and refluxed at 110 degrees Celsius for 1 hour. After the reaction was completed, ice water and ethyl acetate were quickly poured into the reaction mixture. The organic layer was collected to obtain a mixture containing Compound II. After purification, a light yellow solid Compound II was obtained. The reaction expression is as follows:

[0008]

[0009] 3. Prepare compound III: Add acetonitrile, acetone or a small amount of N,N-dimethylformamide to the pure compound II as the reaction solvent, add dry potassium carbonate, and then add dry potassium iodide to m-hydroxybenzyl bromide to prepare a drop solution, slowly add the drop solution to the reaction solvent and react for 5-8 hours. After the reaction is completed, purify to obtain compound III, and the reaction expression is as follows:

[0010]

[0011] 4. Prepare compound IV: Add compound III into a mixture of DMF, K2CO3, and 2-(3-(bromomethyl)phenyl)-4,4,5-5-tetramethyl-1,3,2-dioxaborane, stir at room temperature overnight, dilute with ethyl acetate, and then wash with saturated NaCl solution. Recover the organic phase and concentrate in vacuo to obtain a crude product containing compound IV. The crude product is purified to obtain an orange solid compound IV, i.e., the target product. The reaction expression is as follows:

[0012]

[0013] The present invention utilizes the good biocompatibility and high fluorescence quantum yield of the 2-benzothiazol-2-ylphenol (HBT) mother nucleus itself to develop a drug delivery system that can show a blue fluorescence signal in breast cancer tumor cells. The system first excites reactive oxygen species (ROS), and then after tyrosinase catalysis, the fluorescence shows bright green under light irradiation conditions (λ≥410nm), and releases an anticancer drug (bendamustine). On the one hand, the toxicity of bendamustine to normal cells and tissues is reduced, and the serious side effects of bendamustine in the treatment of cancer are avoided, so that the tumor is clearer during diagnosis and treatment.

[0014] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: using HBT as a carrier, combining an active oxygen detection group and a drug delivery system of bendamustine.

[0015] Bendamustine is a bifunctional alkylating agent that has a good inhibitory effect on malignant tumors such as breast cancer. However, bendamustine is toxic to both cancer cells and normal cells during cancer treatment. The fluorescent dye HBT itself has good biocompatibility and high fluorescence quantum yield, and can spontaneously locate the mitochondria of cancer cells with excessive reactive oxygen species (ROS). The triggering probe releases the drug through a tyrosinase-catalyzed reaction, avoiding the side effects of bendamustine in the treatment of cancer. At the same time, it has the advantages of HBT, which can release the anticancer drug bendamustine under (λ≥410nm) irradiation. Therefore, the developed drug dual-corresponding delivery system may play a pivotal role in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the H-NMR spectrum of the photosensitive probe 1 (MUS);

[0017] Figure 2 This is the NMR carbon spectrum of the photosensitive probe 1 (MUS);

[0018] Figure 3 This is the mass spectrometry characterization result of the photosensitive probe 2 (MUS-OH);

[0019] Figure 4 This is the H-NMR spectrum of the photosensitive probe 2 (MUS-OH);

[0020] Figure 5 This is the NMR carbon spectrum of the photosensitive probe 3 (MUS-AOH);

[0021] Figure 6 This is the mass spectrometry characterization result of the photosensitive probe 3 (MUS-AOH);

[0022] Figure 7 This is the H-NMR spectrum of the photosensitive probe 4 (MUS-AOR);

[0023] Figure 8 This is the NMR carbon spectrum of the photosensitive probe 4 (MUS-AOR);

[0024] Fig. 9 This is the characterization result diagram of the blue-green fluorescence conversion of the photosensitive probe;

[0025] Fig.10 The figures are the results of MTT assay to evaluate the survival rate and inhibition rate of different concentrations of MUS-AOR in normal cells and BC cells, wherein (a) is the MTT assay of the survival rate of NCM460 cells (bar graph) and BC cells (line graph) treated with different concentrations of AOR (from 0 to 64 μM) for 24 h; (b) is the MTT assay of the inhibition rate of BC cells treated with different concentrations of AOR (from 0 to 64 μM) for 24 h.

[0026] Fig.11 is the fluorescence intensity change fitted by enzyme kinetic study;

[0027] Fig.12 This is the plotting result of the phototoxicity test of MUS-AOR on normal cells and breast cancer cells under different conditions;

[0028] Fig.13 The photoreaction lifetime decay of this compound fluorescent probe was fitted for the photosensitive probe. DETAILED DESCRIPTION

[0029] The following are specific embodiments of the present invention and the accompanying drawings to further describe the technical solution of the present invention, but the present invention is not limited to these embodiments.

[0030] Preferred selection of fluorescent nucleosome drugs

[0031] 2-Benzothiazol-2-ylphenol (HBT) derivatives have shown great potential in the field of bioactivity detection due to their excellent fluorescence properties, especially in monitoring the levels of reactive oxygen species and esterases in vivo. These derivatives have a series of significant advantages, including their large conjugated plane and extensive π electron delocalization, which endow them with excellent optical properties. In addition, they have good biocompatibility, anti-fluorescence interference ability, simple synthesis, significant Stokes shift, and tunable fluorescence emission characteristics, which have made them of great interest in biomedical research. It is particularly noteworthy that HBT derivatives emit green fluorescence in the keto form after photoexcitation, and their fluorescence emission gradually changes from weak enol form to blue during the process of electrophilic substitution of hydrogen atoms. This color change provides an intuitive visualization effect for the diagnostic and therapeutic process of the probe, making the HBT structure an ideal choice for the development of new fluorescent probes. Therefore, the selection of HBT-based fluorescent labeling molecules as the core of the study is of great significance for the design of bioactivity detection tools with high specificity and sensitivity.

[0032] A new type of fluorescent probe MUS-AOR was synthesized. The specific process is as follows:

[0033] (1) MUS synthesis: 5-(2-bromoacetyl)-2-hydroxybenzaldehyde (0.183 g, 0.76 mmol) was mixed with bendamustine hydrochloride (0.3 g, 0.76 mmol) as the reaction material, and then dried potassium carbonate (0.258 g) was added to N, N-dimethylformamide (2 ml) to obtain a brown solid after 6-10 hours at room temperature. The crude reaction mixture of compound I was purified by column chromatography using petroleum ether and ethyl acetate (2:1 / 1:2) and dichloromethane.

[0034] The caged conjugate was separated by chromatography using alkane and methanol (20:1) to obtain a brown solid with a yield of 62% and a melting point of 78°C (attached Figure 1 , 2).

[0035] (2) Synthesis of MUS-OH: Compound I (113 mg, 0.217 mmol) was used as the raw material and dissolved in dimethyl sulfoxide (9 ml). Then 2-aminothiophenol (20 μl) was added to the mixture and refluxed at 110 °C for 1 hour. After the reaction was completed, ice water and ethyl acetate were quickly poured into the reaction mixture and the organic layer was collected. The crude product of compound II was purified using petroleum ether and ethyl acetate (1:2), dichloromethane and methanol (40:1) to obtain a pure product with a yield of 64.6%, a light yellow solid (attached Figure 3 , 4).

[0036]

[0037] (3) Synthesis of MUS-AOH: Add acetonitrile, acetone or a small amount of N,N-dimethylformamide to the pure compound II as the reaction solvent, add dry potassium carbonate (42.1 mg, 0.3046 mmol), and then add dry potassium iodide (0.0758 g, 0.4569 mmol) to m-hydroxybenzyl bromide. Then, slowly add m-hydroxybenzyl bromide (85.5 g, 0.4569 mmol) dropwise to the reaction solvent and react for 5-8 hours. After the reaction, purify the crude product compound III by first extracting it, separating it with ethyl acetate and saturated brine, and then separating it with silica gel column using dichloromethane and methanol (40:1) to obtain the pure compound (attached Figure 5 , 6).

[0038]

[0039] (4) MUS-AOR synthesis: Compound III (23.4 mg, 0.032 mmol) was dissolved in DMF (10 ml), K2CO3 (14 mg, 0.096 mmol) and 2-(3-(bromomethyl)phenyl)-4,4,5-5-tetramethyl-1,3,2-dioxaborane (12 mg, 0.04 mmol). After stirring overnight at room temperature, the mixture was diluted with ethyl acetate (50 mL) and then washed with saturated NaCl. The organic phase was collected and concentrated under vacuum. The crude product was purified by using dichloromethane / methanol (40 / 1, v / v) as the eluent. An orange solid (attached) was obtained in a yield of 58%. Figure 7 , 8).

[0040]

[0041] This new fluorescent probe has good imaging effect and stability in living cells.

[0042] The cells were divided into a normal cell group and a BC cell group, and two groups of experiments were performed under dark or irradiation conditions, respectively. Regardless of whether it was irradiated or not, in the normal cell group, we could only observe that the cells exhibited blue fluorescence, indicating that there was no release of MUS-OH (the product of MUS-AOR after induction by reactive oxygen species and enzyme catalysis) in the cell clusters. In contrast, in the BC cell group, clear bright green fluorescence was observed in the cells under dark conditions, and blue fluorescence was shown again after a period of irradiation. These results indicate that tyrosinase hydrolyzes the ether bond of MUS-AOH (the product of MUS-AOR after induction by reactive oxygen species), releasing the fluorophore MUS-OH, which is then further photolyzed by radiation to release the drug, and the fluorescence changes from green to blue (attached Fig. 9This process also provides real-time information about drug delivery. Based on the above results, a cytotoxicity test was performed to evaluate the survival rate and inhibition rate of MUS-AOR at different concentrations in normal cells and BC cells by MTT assay. The results are shown in (Appendix Fig.10 ); and the enzyme kinetics study of MUS-AOR drugs, the results show (Appendix Fig.11 In addition, MUS-AOR has no obvious dark toxicity to breast cancer cells, but has obvious phototoxicity (Appendix Fig.12 ), relatively speaking, the toxicity of MUS-AOR to normal cells was relatively low, regardless of whether it was irradiated or not. In short, the results of the cell imaging experiment were consistent with those of the cytotoxicity experiment. The above results show that MUS-AOH can accurately track tyrosinase in BC cells and achieve controlled release of antitumor drugs under in vitro light irradiation as needed.

[0043] Reactive oxygen-induced tyrosinase activation, i.e., the application of photosensitivity probes in the diagnosis and treatment of breast cancer:

[0044] By detecting tyrosinase activity by fluorescence, not only the recognition efficiency of cancer cells is improved, but also the killing ability of cancer cells is enhanced. In addition, the experimental design integrates the detection function of hydrogen peroxide, which provides double protection for the diagnosis of cancer. More importantly, bendamustine, an alkylating anti-tumor drug, is introduced into the probe. It inhibits the proliferation of tumor cells by forming cross-links with DNA and interferes with cell signaling in the tumor microenvironment, especially those signaling pathways involving tyrosinase. This design takes advantage of the characteristics of bendamustine and combines it with the fluorescent probe to produce a synergistic effect, which not only enhances the pharmacokinetic properties of the drug, but also improves the targeting and therapeutic effect of the drug in tumor tissue, making the fluorescent probe have the potential for treatment while diagnosing.

[0045] This innovative method allows the direct detection of abnormal levels of the enzyme in living cells and tissues, and enables the imaging of cancer cells with high tyrosinase levels, which provides a powerful tool for diagnosis and treatment. Research progress has shown that fluorescence detection and imaging are important and rapid technologies for early diagnosis of cancer, providing a new strategy for the diagnosis and treatment of breast cancer.

[0046] Sensing performance of photosensitive probes in breast cancer diagnosis and treatment:

[0047] Excited State Intramolecular Proton Transfer (ESIPT) is a new sensing mechanism for fluorescent molecules. Its most significant photophysical property is the large Stokes shift. The large Stokes shift can effectively eliminate the luminescence quenching caused by the self-absorption of the dye, thereby avoiding light loss and reducing the interference of the self-luminescence of living organisms, and improving the sensitivity of fluorescent dyes. First, we fitted the light lifetime decay of the compound fluorescent probe (see Appendix Fig.13 ) and combined with enzyme kinetics research, supported by this theoretical knowledge, we also found that typical ESIPT compounds include 2-(2'-hydroxyphenyl)benzoxazole (HBO) derivatives, 2-(2'-hydroxyphenyl)benzothiazole (HBT) derivatives, and 2-(2'-hydroxyphenyl)benzimidazole (HBI) derivatives, all of which have large Stokes shifts. In order to further understand the sensing mechanism of MUS-OH after induced excitation, the highest occupied and lowest unoccupied molecular orbitals were calculated and compared. The results show that compared with MUS-COOH, MUS-OH has a smaller LUMO-HOMO energy gap, which leads to a red shift in the emission wavelength. It is worth noting that when MUS-OH is excited by light, keto-enol tautomerism occurs within the molecule (excited state intramolecular proton transfer, ESIPT). Since the keto form has a smaller LUMO-HOMO energy gap, it effectively reduces the self-absorption of OH and produces a stronger green fluorescence signal. In contrast, the photolysis product shows a larger LUMO-HOMO energy gap and a blue-shifted fluorescence (blue fluorescence signal) due to the interruption of the coupling between the phenolic hydroxyl group and the carbonyl group.

[0048] The specific embodiments described herein are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

Claims

1. A photosensitive probe for breast tumor cells, characterized in that: The method comprises the following steps: 1) preparing compound I: using 5-(2-bromoacetyl)-2-hydroxybenzaldehyde as a reaction raw material, mixing it with bendamustine hydrochloride, adding dry potassium carbonate to N,N-dimethylformamide, and reacting to obtain a mixed product containing compound I. After purification and separation, a brown solid compound I is obtained. The reaction expression is as follows: 2) Prepare compound II: Compound I is used as a raw material, dissolved in dimethyl sulfoxide, and then 2-aminothiophenol is added to the mixture, refluxed at 110 degrees Celsius for 1 hour, and ice water and ethyl acetate are quickly poured into the reaction mixture after the reaction is completed. The organic layer is collected to obtain a mixture containing compound II, and a light yellow solid compound II is obtained after purification. The reaction expression is as follows: 3) Prepare compound III: Add acetonitrile, acetone or a small amount of N,N-dimethylformamide to the pure compound II as a reaction solvent, add dry potassium carbonate, and then add dry potassium iodide to m-hydroxybenzyl bromide to prepare a drop solution, slowly add the drop solution to the reaction solvent for 5-8 hours, and purify after the reaction to obtain compound III, the reaction expression of which is as follows: 4) Prepare compound IV: Add compound III into a mixture of DMF, K2CO3, and 2-(3-(bromomethyl)phenyl)-4,4,5-5-tetramethyl-1,3,2-dioxaborane, stir overnight at room temperature, dilute with ethyl acetate, and then wash with saturated NaCl solution. Recover the organic phase and concentrate in vacuo to obtain a crude product containing compound IV. The crude product is purified to obtain an orange solid compound IV, i.e., the target product. The reaction expression is as follows:

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