A semi-fluorochrome chemotherapeutic drug and a synthesis method thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HUBEI UNIV OF SCI & TECH
- Filing Date
- 2026-02-04
- Publication Date
- 2026-05-29
AI Technical Summary
Existing traditional chemotherapy drugs have poor selectivity, strong toxic side effects, and are prone to drug resistance. Hemicyanine derivatives have insufficient selectivity in tumor treatment, and their synthesis methods are complex and their mechanisms are unclear.
We designed and synthesized novel hemiflorin salt derivatives, enhanced their selective killing ability against tumor cells through structural optimization, developed an efficient synthetic route, and revealed the mechanism of ROS-mediated mitochondrial membrane potential collapse-induced late apoptosis.
It significantly improves the selective killing ability of tumor cells, and provides novel anti-tumor drug candidate compounds with clear mechanisms of action, exhibiting good biological activity and selectivity.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of drug synthesis technology, and in particular to a hemicyanine chemotherapy drug and its synthesis method. Background Technology
[0002] Cancer, as one of the leading causes of death worldwide, faces significant challenges due to the limited clinical efficacy of traditional chemotherapy drugs, which suffer from poor selectivity, strong toxicity, and easy drug resistance. Developing novel, low-toxicity, and highly effective anti-tumor drugs has become a research focus. In recent years, the mitochondrial apoptosis pathway regulated by reactive oxygen species (ROS) has attracted considerable attention due to its precise targeting of metabolic weaknesses in tumor cells. Tumor cells, with their higher baseline ROS levels, are more sensitive to oxidative stress. Excessive ROS can oxidize mitochondrial membrane proteins, leading to membrane potential collapse, which in turn triggers cytochrome c release and a caspase cascade reaction, ultimately inducing apoptosis. Although hemicyanine derivatives containing conjugated systems are widely used in bioimaging and photodynamic therapy due to their tunable photophysical properties and good biocompatibility, and some derivatives have been reported to induce ROS bursts in tumor cells through non-photodependent pathways, they still suffer from limitations such as insufficient selectivity (significant toxicity to normal hepatocytes such as LO2), unclear mechanisms of action (lack of direct evidence for the causal relationship between elevated ROS and mitochondrial damage), and complex synthesis methods (cumbersome routes and low yields). Based on this, this invention addresses the shortcomings of existing technologies by designing and synthesizing a series of novel hemicyanine derivatives. Through structural optimization, the selective killing ability against tumor cells has been significantly improved. The invention also systematically reveals the clear mechanism by which late apoptosis is induced by ROS-mediated mitochondrial membrane potential collapse, and develops an efficient synthetic route. This provides novel candidate compounds with clear mechanisms of action and theoretical basis for the development of targeted anti-tumor drugs.
[0003] The hemiflorin salt derivative involved in this invention is a novel small molecule chemotherapy drug for tumors, which has the technical advantages of good anti-tumor activity and good selectivity. Summary of the Invention
[0004] To address the shortcomings of existing technologies, this invention proposes a small molecule drug with good anti-tumor activity and selectivity.
[0005] To achieve the above objectives, the present invention adopts the following technical solution: a small molecule drug for tumor chemotherapy, which is hemicyanine salt, appears red in aqueous solution under natural light and exhibits selective therapeutic effects on cancer cells.
[0006] The hemicyanine salt derivative is soluble in organic solvents such as methanol, ethanol, acetone, dichloromethane, petroleum ether, ethyl acetate, acetonitrile, and dimethyl sulfoxide.
[0007] A method for synthesizing hemicyanine salt derivatives, characterized in that 3-ethyl-2-methylbenzothiazole iodide and 4-(bis(4-(pyridin-4-yl)phenyl)amino)benzaldehyde are reacted under reflux at 90°C with anhydrous ethanol as solvent to obtain hemicyanine derivatives.
[0008] Specifically, The general formula of hemicyanine salt derivatives with tumor chemotherapy function is shown in formula (I): The preparation method of the hemiflorum cyanine salt derivative includes the following technical route: Step 1), Preparation of compound 1: 2-Methylbenzothiazole (0.64 mL, 5 mmol) and iodoethane (0.8 mL, 10 mmol) were heated to reflux at 83 °C for 12 hours in 50 mL of acetonitrile solution. The reaction process was monitored by thin-layer chromatography. After the reaction was completed, the mixture was cooled to room temperature and concentrated under reduced pressure. The mixture was then purified by silica gel column chromatography to obtain compound 1 as a white solid with a yield of 80%.
[0009] Step 2), preparation of compound 2: 4-Formyltriphenylamine (1365 mg, 5 mmol) was dissolved in 50 mL of anhydrous tetrahydrofuran. N-Bromosuccinimide (1770 mg, 10 mmol) was added at room temperature. The mixture was heated under reflux at 100 °C for 5 h, cooled to room temperature, and concentrated under reduced pressure. The mixture was then purified by separation with ethyl acetate using a neutral alumina column to give compound 2 as a pale yellow solid in 85% yield.
[0010] Step 3), preparation of compound 3: Compound 2 (2155 mg, 5 mmol) and Pd(dppf)Cl2 (365.85 mg, 0.5 mmol) were dissolved in 50 mL of toluene. 10 mmol of 4-pyridineboronic acid and 12.5 mmol of K2CO3 dissolved in 50 mL of methanol were added. The mixture was refluxed at 120 °C for 16 h, cooled to room temperature, concentrated under reduced pressure, and purified with 200-300 mesh silica gel. The solution was eluted with dichloromethane:methanol = 20:1 to give compound 3 as a pale yellow solid in 60% yield.
[0011] Step 4), preparation of compound 4: Compound 1 (305 mg, 1 mmol) and compound 3 (427 mg, 1 mmol) were dissolved in anhydrous ethanol and refluxed at 90 °C for 24 h. After cooling to room temperature, the mixture was concentrated under reduced pressure and purified with 200-300 mesh silica gel. The mixture was then eluted with dichloromethane:methanol = 10:1 to give compound 4 as a red solid in 50% yield.
[0012] The cytotoxicity of PNSN and TSN was evaluated using the MTT assay. The results showed that PNSN exhibited good biological activity against cancer cells, such as cervical cancer cells (HeLa). IC 50 =5.0 μM, against mouse breast cancer cells (4T1) IC 50 =4.2μM, PNSN on normal human hepatocytes (LO2) cells IC 50 The concentration was much greater than 20 μM, indicating that PNSN exhibited different levels of cytotoxicity against normal and cancer cells, suggesting that PNSN has good selectivity for both. In contrast, TSN showed very low cytotoxicity, with cell viability exceeding 75% in all cases.
[0013] The antitumor activity of two compounds, PNSN and TSN, was detected using a calcein-AM / propidium iodide (PI) double staining kit. The results showed that PNSN exhibited significantly superior antitumor activity compared to TSN, consistent with the findings of the MTT study, thus confirming the chemotherapeutic potential of PNSN.
[0014] Intracellular reactive oxygen species (ROS) levels were detected using the DCFH-DA probe. After co-incubating HeLa cells with 5 μM PNSN for 24 hours, an enhanced green fluorescence signal was observed in the cells, indicating that PNSN can induce intracellular ROS production.
[0015] Using Rhodamine 123 (Rh123) dye probes to observe changes in mitochondrial membrane potential, it was found that after 5 μM PNSN was co-incubated with HeLa cells for 24 hours, the intensity of green fluorescence in the cells decreased, indicating that PNSN can cause the collapse of mitochondrial membrane potential.
[0016] The effect of PNSN on cell apoptosis was detected using the Annexin V-iFluor 488 / PI apoptosis detection kit. Cells were incubated with 5 μM PNSN for 24 hours, and the results showed that some cells exhibited red fluorescence, indicating late apoptosis. This suggests that PNSN has excellent chemotherapeutic potential and can induce apoptosis in cancer cells. Attached Figure Description
[0017] Figure 1 This is the general formula of the molecular structure of the present invention; Figure 2 This is the synthetic route for compounds 1, 2, 3, and 4; Figure 3 This is the NMR spectrum (H1N M spectrum) of compound 1. Figure 4 This is the NMR spectrum (carbon spectrum) of compound 1. Figure 5 This is the NMR spectrum (H1N1 spectrum) of compound 2. Figure 6 This is the NMR spectrum (carbon spectrum) of compound 2. Figure 7 This is the NMR spectrum (H1N1 spectrum) of compound 3. Figure 8 This is the NMR spectrum (carbon spectrum) of compound 3. Figure 9 This is the NMR spectrum (H1N1 spectrum) of the compound TSN. Figure 10 It is the NMR spectrum (carbon spectrum) of the compound TSN. Figure 11 It is the NMR spectrum (H1N1 spectrum) of the compound PNSN. Figure 12 It is the NMR spectrum (carbon spectrum) of the compound PNSN. Figure 13 These are the results of cytotoxicity assays for compounds PNSN and TSN; Figure 14 This is the result of the compound PNSN calcein-AM / propidium iodide (Calcein-AM / PI) double staining kit; Figure 15 The effect of PNSN on intracellular reactive oxygen species (ROS) levels was detected using the DCFH-DA intracellular reactive oxygen species detection kit. Figure 16 The effect of PNSN on mitochondrial membrane potential was detected using a Rhodamine 123 (Rh123) mitochondrial membrane potential assay kit. Figure 17 The effect of PNSN on apoptosis was detected using the Annexin V-iFluor 488 / PI apoptosis detection kit. Detailed Implementation
[0018] The following are specific embodiments of the present invention, which are described in conjunction with the accompanying drawings. However, the present invention is not limited to these embodiments. Example
[0019] like Figure 2 As shown, a method for detecting HSO3 - The preparation method of the fluorescent probe, specifically compound 1, is as follows: 2-methylbenzothiazole (0.64 mL, 5 mmol) and iodoethane (0.8 mL, 10 mmol) were heated to reflux at 83 °C for 12 hours in 50 mL of acetonitrile solution. After cooling to room temperature, the mixture was concentrated under reduced pressure and purified with 200-300 mesh silica gel. Elution was performed using dichloromethane:methanol = 100:1 to obtain compound 1, a white solid with a yield of 80%. The molar ratio of compound 1 to iodoethane was 1:2. Its proton NMR spectrum is shown below. Figure 3 As shown, the carbon spectrum is as follows Figure 4 As shown. Example
[0020] like Figure 2 As shown, a method for detecting HSO3 - The preparation method of the fluorescent probe, compound 2, is as follows: 4-Formyltriphenylamine (1365 mg, 5 mmol) was dissolved in 50 mL of anhydrous tetrahydrofuran. N-bromosuccinimide (1770 mg, 10 mmol) was added at room temperature. The mixture was heated under reflux at 100 °C for 5 h, cooled to room temperature, and concentrated under reduced pressure. The mixture was then purified by separation with ethyl acetate using a neutral alumina column to obtain compound 2, a pale yellow solid, in 85% yield. The molar ratio of 4-formyltriphenylamine to N-bromosuccinimide was 1:2. Its 1H NMR spectrum is shown below. Figure 5 As shown, the carbon spectrum is as follows Figure 6 As shown. Example
[0021] like Figure 2 As shown, a method for detecting HSO3 - The preparation method of the fluorescent probe, compound 3, is as follows: Compound 2 (2155 mg, 5 mmol) and Pd(dppf)Cl2 (365.85 mg, 0.5 mmol) were dissolved in 50 mL toluene. 10 mmol of 4-pyridineboronic acid and 12.5 mmol of K2CO3 dissolved in 50 mL methanol were added. The mixture was refluxed at 120 °C for 16 h, then cooled to room temperature, concentrated under reduced pressure, and purified with 200-300 mesh silica gel. Elution with dichloromethane:methanol = 20:1 yielded compound 3, a pale yellow solid, with a yield of 60%. The molar ratio of compound 2 to 4-pyridineboronic acid was 1:2. Its 1H NMR spectrum is shown below. Figure 7 As shown, the carbon spectrum is as follows Figure 8 As shown. Example
[0022] like Figure 2As shown, a method for detecting HSO3 - The preparation method of the fluorescent probe, specifically compound 4, is as follows: Compound 1 (305 mg, 1 mmol) and compound 3 (427 mg, 1 mmol) were dissolved in anhydrous ethanol and reacted at 90 °C for 24 h. After cooling to room temperature, the mixture was concentrated under reduced pressure and purified with 200-300 mesh silica gel. Elution was performed using dichloromethane:methanol = 10:1 to obtain compound PNSN as a red solid with a yield of 50%. The molar ratio of compound 1 to compound 3 was 1:1. Example
[0023] The MTT assay, or tetramethylazozid salt colorimetric assay, is based on the activity of succinate dehydrogenase in the mitochondria of living cells. This enzyme catalyzes the reduction of exogenous MTT (a yellow compound) to water-insoluble blue-purple formazan crystals, which then deposit within the cells. Dead cells, lacking this enzyme activity, cannot produce this reaction. After the reaction, the formazan crystals are dissolved in dimethyl sulfoxide (DMSO), and the absorbance (OD) value is measured at 490 nm or 570 nm using a microplate reader. Within a certain cell number range, the OD value is directly proportional to the number of living cells, thus indirectly reflecting cell activity or proliferation capacity. This invention uses the MTT assay to test PNSN (its proton spectrum is shown below). Figure 11 As shown, the carbon spectrum is as follows Figure 12 (as shown) and TSN (its proton spectrum is as shown) Figure 9 As shown, the carbon spectrum is as follows Figure 10 As shown in the figure, PNSN exhibits good biological activity against cancer cells, such as cervical cancer cells (HeLa). IC 50 =5.0 μM, against mouse breast cancer cells (4T1) IC 50 = 4.2 μM, PNSN on normal human hepatocytes (LO2) IC 50 The concentration is much greater than 20 μM, indicating that PNSN exhibits different toxicities to normal cells and cancer cells. Figure 13 (a) This indicates that PNSN has very good selectivity for both normal and cancer cells. In contrast, TSN has low cytotoxicity, with cell survival rates exceeding 75% in all cases (a). Figure 13 b). Example
[0024] The cytotoxicity of PNSN and TSN was investigated using a calcein-AM / PI double staining kit. A 5 μM concentration of PNSN induced red fluorescence in some cancer cells, while other cells showed green fluorescence. In contrast, a small number of cells showed red fluorescence with a 5 μM concentration of TSN, with the majority showing green fluorescence. HeLa cell survival was almost unaffected under PBS solution conditions. Figure 14 This is consistent with the results of the MTT study, confirming the chemotherapeutic efficacy of PNSN. Example
[0025] To investigate the cytotoxic mechanism of PNSN, we used the DCFH-DA probe to detect intracellular reactive oxygen species (ROS) levels. The results showed that after co-incubating HeLa cells with 5 μmol of PNSN for 24 hours, the intracellular green fluorescence signal was enhanced, indicating an increase in intracellular ROS levels. Figure 15 ). Example
[0026] Changes in mitochondrial membrane potential were observed using a Rhodamine 123 (Rh123) dye probe. Furthermore, after co-incubating HeLa cells with 5 μmol of PNSN for 24 hours, the intensity of green fluorescence within the cells decreased, while cells under PBS conditions exhibited bright green fluorescence. This indicates a decrease in mitochondrial membrane potential. Figure 16 ). Example
[0027] Cells incubated with 5 μmol PNSN and HeLa cells for 24 hours were analyzed using the Annexin V-iFluor 488 / PI apoptosis detection kit. The results showed that some cells exhibited red fluorescence, indicating late apoptosis. Figure 17 This indicates that PNSN has excellent chemotherapeutic capabilities and can induce apoptosis in cancer cells.
[0028] The specific embodiments described herein are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.
Claims
1. A hemicyanine chemotherapy drug, characterized in that, The general formula of the hemicyanine derivative is shown in formula (I), which has cell selectivity and anti-tumor effect.
2. The hemicyanine chemotherapy drug according to claim 1, characterized in that, Hemicyanine derivatives can induce the production of reactive oxygen species in cells.
3. The hemicyanine chemotherapy drug according to claim 1, characterized in that, The hemicyanine derivative can induce the collapse of mitochondrial membrane potential.
4. The hemicyanine chemotherapy drug according to claim 1, characterized in that, The hemicyanine derivative can induce late apoptosis in cells.
5. The application of the hemicyanine derivative with antitumor activity according to claim 1, characterized in that, The antitumor active drug can be formulated into pharmaceutically acceptable injections, sprays, inhalers, or oral formulations.
6. The application of the hemicyanine derivative with antitumor activity according to claim 1, characterized in that, Using the hemicyanine derivative as the active ingredient, and one or more pharmaceutically acceptable carriers, it is used to prepare an antitumor active drug.
7. A method for synthesizing the hemicyanine chemotherapeutic drug according to any one of claims 1 to 6, characterized in that, It includes the following steps: Step 1), Preparation of compound 1: 2-Methylbenzothiazole (0.64 mL, 5 mmol) and iodoethane (0.8 mL, 10 mmol) were heated to reflux at 83 °C for 12 hours in 50 mL of acetonitrile solution. The reaction process was monitored by thin-layer chromatography. After the reaction was completed, the mixture was cooled to room temperature and concentrated under reduced pressure. The solution was then purified by silica gel column chromatography to give compound 1 as a white solid with a yield of 80%. Step 2), preparation of compound 2: 4-Formyltriphenylamine (1365 mg, 5 mmol) was dissolved in 50 mL of anhydrous tetrahydrofuran. N-Bromosuccinimide (1770 mg, 10 mmol) was added at room temperature; the mixture was heated under reflux at 100 °C for 5 h, cooled to room temperature, and concentrated under reduced pressure. The mixture was then purified by separation with ethyl acetate using a neutral alumina column to give compound 2 as a pale yellow solid in 85% yield. Step 3), preparation of compound 3: Compound 2 (2155 mg, 5 mmol) and Pd(dppf)Cl2 (365.85 mg, 0.5 mmol) were dissolved in 50 mL of toluene. 10 mmol of 4-pyridineboronic acid and 12.5 mmol of K2CO3 dissolved in 50 mL of methanol were added. The mixture was refluxed at 120 °C for 16 h, cooled to room temperature, concentrated under reduced pressure, and purified with 200-300 mesh silica gel. Elution with dichloromethane:methanol = 20:1 gave compound 3 as a pale yellow solid with a yield of 60%. Step 4), preparation of compound 4: Compound 1 (305 mg, 1 mmol) and compound 3 (427 mg, 1 mmol) were dissolved in anhydrous ethanol and refluxed at 90 °C for 24 h. After cooling to room temperature, the mixture was concentrated under reduced pressure and purified with 200-300 mesh silica gel. The mixture was then eluted with dichloromethane:methanol = 10:1 to give compound 4 as a red solid in 50% yield.