A TB-thiophene methylene malononitrile derivative and its preparation and application

Abstract

This invention provides a TB-thiophene methylene malononitrile derivative, its preparation, and its applications. It is synthesized from p-bromoaniline, paraformaldehyde, 5-bromothiophene-2-carboxaldehyde, and malononitrile via a multi-step reaction. The product exhibits large Stokes shifts (147 nm and 213 nm, respectively) in both solution and solid states, demonstrating excellent solid-state luminescence. It forms uniform petal-like nanoaggregates in a THF / H₂O = 1 / 99 (v / v) solution, exhibiting significant AIE properties. It has a wide pH range, making it suitable for use in human physiological environments. Compared to pure methanol, the fluorescence is enhanced by 6.1 times at a methanol / glycerol = 1 / 9 (v / v) ratio, showing good responsiveness to viscosity. Under dark conditions, the IC50 of HpeG2 and A549 cells... 50 71.7 and 100 μmol·L, respectively ‑1 Under illumination (428 nm), the inhibition rates against HpeG2 cells and A549 cells were 9.2 and 19.9 μmol·L⁻¹, respectively. ‑1 It exhibits excellent photodynamic anticancer activity.

Description

Technical Field

[0001] This invention belongs to the field of chemical synthesis, specifically involving Base-thiophene methylene malononitrile derivatives, their synthesis methods, and photodynamic anticancer activity. Background Technology

[0002] Photodynamic therapy (PDT) is a novel approach to treating tumors and other diseases using photosensitizing drugs and laser activation. Compared to traditional methods, it offers advantages such as fewer side effects, minimal invasiveness, high safety, no drug resistance, high selectivity in tumor destruction, and ease of combination with other therapies. It demonstrates good spatiotemporal precision and effectiveness in clinical research and treatment, and has become an important emerging tool for precision oncology.

[0003] The efficacy of photosensitizers is a core factor determining the effectiveness of phototherapy (PDT). With the rapid development of PDT, there are increasingly higher demands for the diversity and efficiency of photosensitizers. Addressing the issues of visible absorption and poor penetration inherent in traditional PDT photosensitizers, a growing number of novel, highly efficient photosensitizers have been developed. However, based on… Photosensitizers with a base (TB) framework are still in the early stages, with only one related report.

[0004] Thiophene is an electron-rich aromatic heterocyclic compound with good modifiability. Due to its high electron mobility, low electrode potential, and good conductivity, thiophene derivatives exhibit excellent photoelectric properties and are widely used in the design of biological diagnostic reagents, pharmaceuticals, electronic and optoelectronic devices, as well as conductive and electroluminescent materials.

[0005] The malononitrile molecule contains two strongly electron-withdrawing cyano groups, which make its methylene group readily form a carbanion, resulting in high reactivity and diverse reaction types. The aromatic methylene malononitrile building blocks prepared by the Knoevenagal reaction of malononitrile with aldehydes possess a long conjugated system and strong electron-withdrawing ability; their introduction into the molecule helps to improve its light absorption and emission capabilities.

[0006] Therefore, this invention designs and synthesizes a D-π-A structure by introducing an electron-withdrawing thiophene methylene malononitrile fragment onto the TB backbone. base-thiophene methylene malononitrile compound. Summary of the Invention

[0007] Technical Problem: The purpose of this invention is to provide a TB-thiophene methylene malononitrile derivative, its preparation and application, which is synthesized from p-bromoaniline, paraformaldehyde, 5-bromothiophene-2-carboxaldehyde, and malononitrile through a multi-step reaction. Base-thiophene methylene malononitrile compounds have been applied to fields such as viscosity identification and PDT therapy.

[0008] Technical solution: The present invention provides a TB-thiophene methylene malononitrile derivative, the structural formula of which is as follows:

[0009]

[0010] The preparation method of a TB-thiophene methylene malononitrile derivative according to the present invention includes the following steps:

[0011] Step 1: 4-Bromoaniline reacts with paraformaldehyde to give the first intermediate, as shown in the following reaction formula:

[0012]

[0013] Step 2: The first intermediate reacts with n-butyllithium to obtain the second intermediate, as shown in the following reaction formula:

[0014]

[0015] Step 3: The second intermediate reacts with 5-bromothiophene-2-carboxaldehyde via a coupling reaction to obtain the third intermediate, as shown in the following reaction formula:

[0016]

[0017] Step 4: The third intermediate reacts with malononitrile to obtain the derivative, as shown in the following reaction formula:

[0018]

[0019] The present invention relates to the application of a TB-thiophene methylene malononitrile derivative in the preparation of a viscosity probe.

[0020] The present invention relates to the application of a TB-thiophene methylene malononitrile derivative in the preparation of a photodynamic therapy drug for cancer.

[0021] The aforementioned photodynamic therapy drug for cancer is used to inhibit human liver cancer HpeG2 cells and human lung cancer A549 cells.

[0022] Beneficial effects:

[0023] 1. The synthesis method is simple and the post-processing is convenient.

[0024] 2. It exhibits a large Stokes shift, excellent solid-state luminescence, and significant AIE properties; it also has a wide pH range, making it suitable for use in human physiological environments.

[0025] 3. The product has a good response to viscosity and has the potential to become a viscosity-responsive fluorescent probe;

[0026] 5. IC50 of HpeG2 and A549 cells under dark conditions 50 71.7 and 100 μmol·L, respectively -1 Under illumination (428 nm), the inhibition rates against HpeG2 cells and A549 cells were 9.2 and 19.9 μmol·L⁻¹, respectively. -1 It exhibits excellent photodynamic anticancer activity and has the potential to be developed into a novel photodynamic antitumor photosensitized drug. Attached Figure Description

[0027] Figure 1 It is a derivative of the product in the example. 1 HNMR spectrum;

[0028] Figure 2 It is the product derivative 7 in the example 13 C NMR spectrum;

[0029] Figure 3a These are the UV absorption spectra of the third intermediate 6 in different solvents;

[0030] Figure 3b These are the fluorescence emission spectra of the third intermediate 6 in different solvents;

[0031] Figure 3c These are the ultraviolet absorption spectra of derivative 7 in different solvents;

[0032] Figure 3d The fluorescence emission spectra of derivative 7 in different solvents;

[0033] Figure 4 shows the (a) fluorescence emission spectrum and (b) line graph of the third intermediate 6 at different pH values, and the (c) fluorescence emission spectrum and (d) line graph of compound 7 at different pH values.

[0034] Figure 5 shows the (a) fluorescence emission spectrum and (b) line graph of the third intermediate 6 in different ratios of THF / H2O (v / v) and the (c) fluorescence emission spectrum and (d) line graph of the derivative 7 in different ratios of THF / H2O (v / v).

[0035] Figure 6 shows the SEM images of derivative 7 at different THF / H2O (v / v) ratios: (a) THF / H2O = 1 / 9 (v / v) and (b) THF / H2O = 1 / 99 (v / v).

[0036] Figure 7 shows the (a) fluorescence emission spectrum and (b) line graph of the third intermediate 6 at different viscosities, and the (c) fluorescence emission spectrum and (d) line graph of compound 7 at different viscosities.

[0037] Figure 8It is the fluorescence intensity of derivative 7 under the interaction of different ions and molecules;

[0038] Figure 9 shows the fluorescence emission spectrum and (b) line graph of derivative 7 at different temperatures; and the fluorescence emission spectrum and (d) line graph after binding with egg white.

[0039] Figure 10 shows the phototoxicity and dark toxicity of derivative 7 on (a) A549 cells and (b) HpEG2 cells. Detailed Implementation

[0040] The present invention will be further described below with reference to the embodiments.

[0041] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0042] The following are: 4-bromoaniline 1, paraformaldehyde 2, first intermediate 3, second intermediate 4, 5-bromothiophene-2-carboxaldehyde 5, third intermediate 6, and derivative 7.

[0043] The structural formula of the base-thiophene methylene malononitrile compound is shown in the table below:

[0044] Table 1. Structural formulas of derivative 7

[0045]

[0046] The present invention also provides the above-mentioned novel Methods for preparing base-thiophene methylene malononitrile compounds include:

[0047] In this embodiment, the product is prepared via a coupling reaction using p-bromoaniline, paraformaldehyde, n-butyllithium, 5-bromo-thiophene-2-carboxaldehyde, and malononitrile as raw materials. The process includes the following steps:

[0048] 4-Bromoaniline 1 reacts with paraformaldehyde 2 to give a first intermediate 3. The first intermediate 3 reacts with n-butyllithium to give a second intermediate 4. The second intermediate 4 reacts with 5-bromo-thiophene-2-carboxaldehyde to give a third intermediate 6. The third intermediate 6 reacts with malononitrile via a coupling reaction to give a derivative 7.

[0049] The first intermediate 3 in the above-described synthesis method was prepared as follows:

[0050] 4-Bromoaniline (50.0 mmol) and paraformaldehyde (100.0 mmol) were added sequentially to a 200.0 mL round-bottom flask, which was then placed in a cryogenic bath and heated to -15 °C. Trifluoroacetic acid (100.0 mL, added over approximately 30 min) was slowly added dropwise with stirring, and the mixture was allowed to react at room temperature for 7 days. After the reaction was complete (tracked by TLC), the mixture was poured into ice water, the pH was adjusted to 9-10 with ammonia, and the mixture was cooled to room temperature. The mixture was extracted with dichloromethane (50.0 mL × 3), and the extract was evaporated to dryness to obtain the crude product. Acetone was added, and the mixture was heated until the crude product was completely dissolved. The product was recrystallized at room temperature, filtered, and washed with acetone to obtain the first intermediate 3.

[0051]

[0052] (3) First intermediate 3 (5.0 mmol) was added to a 100 mL round-bottom flask. After three evacuations, the flask was placed in a cryogenic bath and the temperature was adjusted to -78 °C. With stirring, 20.0 mL of anhydrous tetrahydrofuran was added, followed by 2.5 mL of n-butyllithium. The reaction was carried out under argon protection for 1 h, followed by the addition of 0.6 mL of trimethyl borate. The reaction was then carried out at room temperature for 4 h. TLC was used to monitor the reaction until complete. The mixture was extracted with dichloromethane (30.0 mL × 3) and evaporated to dryness to obtain the crude product. The crude product was purified by column chromatography (V... PE :V EA =5:1) yields the second intermediate 4 (65%).

[0053]

[0054] (4) Second intermediate 4 (1.0 mmol), 5-bromothiophene-2-carboxaldehyde (1.2 mmol), tetrakis(triphenylphosphine)palladium (20% mmol, 0.03 g), and K2CO3 (0.2 mmol) were added sequentially to a 100 mL round-bottom flask. Under argon protection, 20 mL of anhydrous toluene was added, and the reaction was carried out at 108 °C for 24 h. After the reaction was complete (TLC monitoring), the mixture was quenched with water, extracted with dichloromethane (10.0 mL × 3), and the organic phase was dried over Na2SO4 and then evaporated to dryness. The crude product was purified by column chromatography (V 石油醚 :V 乙酸乙酯 =6:1) yields the third intermediate 6 (65%).

[0055]

[0056] (5) Add the third intermediate 6 (1.0 mmol), malononitrile (1.2 mmol), and 10.0 mL of anhydrous ethanol sequentially to a 100 mL round-bottom flask, and react at 70 °C for 6 h under argon protection. After the reaction is complete (TLC monitoring), quench with water, extract with dichloromethane (10.0 mL × 3), and the crude product after drying the organic phase with Na2SO4 is purified by column chromatography (V 石油醚 :V乙酸乙酯 =3:1) yielded derivative 7 (77%).

[0057]

[0058] Derivative 7 has the molecular formula: C 23 H 17 BN4O2S

[0059] The Chinese name is: (8-(5-(2,2-dicyanovinyl)thiophen-2-yl)-6H,12H-5,11-methyldibenzo[b,f][1,5]diazoindene-2-yl)boronic acid

[0060] The English name is:

[0061] (8-(5-(2,2-dicyanovinyl)thiophen-2-yl)-6H,12H-5,11-methanodibenzo[b,f][1,5]diazocin-2-yl)boronic acid

[0062] Appearance: Red solid

[0063] Melting point: 184.2-184.8℃

[0064] 1H NMR spectrum: 1 H NMR (400MHz, CDCl3) δ7.73(s,1H),7.64(d,J=4.0Hz,1H),7.49(d,J=8.1Hz,1H,Ar-H),7.39-7.38(m,1H,-OH),7.30(d,J=4.0Hz,1H),7.24(s,1H,-OH ),7.21-7.15(m,3H,Ar-H),7.00(d,J=7.2Hz,1H,Ar-H),6.93(d,J=7.5Hz,1H,Ar-H),4.76-4.72(m,2H,-CH2-bridge),4.38-4.20(m,4H,TB-CH2*2).

[0065] Carbon NMR spectrum: 13 C NMR (100MHz, DMSO-d6) δ155.06,144.55,131.35,120.50,109.10,107.78,66.06,56.83,56.63,40.49,40.28,40.07,39.96,39.65,39.44,39.23.

[0066] Mass spectrometry: HRMS(ESI) m / z calcd for C 23 H17 BN4O2S[M+H] + :425.1242; found,425.1268.

[0067] Optical performance

[0068] The solvation effect of the compounds of this invention was tested, and the specific experimental scheme is as follows:

[0069] Compound 6 and derivative 7 were prepared into concentrations of 1×10⁻⁶ using methanol, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), tetrahydrofuran (THF), ethyl acetate, chloroform (CHCl₃), toluene, and n-hexane, respectively. -5 mol·L -1 The working solution was tested, and its ultraviolet absorption spectrum and fluorescence emission spectrum were measured. For example... Figures 3a-3d As shown.

[0070] from Figures 3a-3d It can be seen that the UV absorption spectra of the third intermediate 6 and derivative 7 are not significantly different in various solvents; however, the relative fluorescence intensity of the third intermediate 6 and derivative 7 is stronger in medium and low polar solvents due to their higher solubility.

[0071] The UV absorption and fluorescence emission spectra of the third intermediate 6 and derivative 7 in THF solution, as well as their solid-state fluorescence emission spectra, were tested. The specific experimental protocol is as follows:

[0072] Weigh out 10 -5 The second intermediate 4, the third intermediate 6, and the derivative 7 were diluted with THF solution to a concentration of 1 × 10⁻⁶. -5 The concentration was measured to mol / L, and its ultraviolet absorption, fluorescence emission, and solid-state fluorescence emission spectra were tested. The spectral data of the second intermediate 4, the third intermediate 6, and the derivative 7 are shown in Table 2.

[0073] Table 2. Spectral data (THF) of the second intermediate 4, the third intermediate 6, and the derivative 7.

[0074]

[0075] a Ultraviolet absorption wavelength in solution (slit width 2.5 / 5 nm); b The molar extinction coefficient ε = A / bC, with units of 1 × 10⁻⁶. 5 L·mol -1 ·cm -1 ; c Fluorescence emission wavelength in solution; d Stokes displacement in solution;e Relative fluorescence quantum yield (reference: quinine sulfate); f Fluorescence intensity, in L·mol -1 ·cm -1 ; g Solid-state excitation wavelength (5 / 5 nm slit); h Solid-state fluorescence emission wavelength; i Solid-state Stokes displacement.

[0076] As shown in Table 2, compared with the second intermediate 4 and the third intermediate 6, the fluorescence properties of derivative 7 have the following changes:

[0077] (1) Solution and solid λ of derivative 7 em Both showed a significant red shift, with the Stokes shifts in solution and solid (147 and 213 nm, respectively) increasing significantly.

[0078] (2) The relative fluorescence quantum yield of derivative 7 solution increased significantly.

[0079] This may be because the electron-withdrawing group thiophene methylene malononitrile promotes the flow of electrons within its molecule, lowers the overall energy of the molecule, makes it easier to emit fluorescence, has a longer maximum fluorescence emission wavelength, and increases the Stokes shift;

[0080] (3) The combined effect of C=C and two -C≡N restricts intramolecular rotation, giving the entire molecule a highly twisted structure, which in turn enhances the intensity of solid-state luminescence.

[0081] The above results demonstrate that combining the TB backbone with thiophene and methylene malononitrile fragments can amplify the advantages of both in terms of luminescence performance, providing a new approach to obtaining products with excellent luminescence properties.

[0082] pH response

[0083] The third intermediate 6 was prepared with THF as solvent to a concentration of 1×10⁻⁶. -4 mol·L -1 For the working solution, 1.0 mL of the working solution of the third intermediate 6 was measured into nine 10.0 mL volumetric flasks. Then, 1.0 mL of a buffer solution with a pH range of 2.2-10.0 was added to each flask (citric acid / disodium hydrogen phosphate system for pH 2.2-8.0, and sodium bicarbonate / sodium carbonate system for pH 9.0-10.0). The solution was then diluted to volume with THF to a concentration of 1×10⁻⁶. -5 mol L -1 Its fluorescence emission spectrum (λ) was measured. ex =380nm, slit: 5 / 5nm, Figures 4a-4b The reagent preparation method for derivative 7 is the same as that for the third intermediate 6 (λ).ex =430nm, slit width: 5 / 5nm, Figures 4c-4d ).

[0084] Depend on Figures 4a-4d It can be seen that the fluorescence intensity of the third intermediate 6 and derivative 7 remains almost constant in the pH range of 2.2-10.0, indicating that the third intermediate 6 and derivative 7 have a wide pH range of applicability.

[0085] AIE features

[0086] Since the third intermediate 6 is readily soluble in THF but poorly soluble in water, the third intermediate 6 was formulated to a concentration of 1×10⁻⁶. - 4 mol·L -1 For the working solution, measure 1.0 mL of the working solution into ten 10.0 mL volumetric flasks. Then, add 0.0–9.0 mL of double-distilled water to each of the ten 10.0 mL volumetric flasks, and dilute to volume with THF to make the concentration of each flask 1 × 10⁻⁶. -5 mol·L -1 (THF / H2O(v / v) is 1 / 9-9 / 1 respectively), and 6 is prepared into a solution with a concentration of 1×10 -3 mol·L -1 For the working solution, measure 100.0 μL of the working solution into a 10.0 mL volumetric flask, then add 9.9 mL of double-distilled water and THF to bring the volume to 1 × 10⁻⁶. - 5 mol L -1 This results in THF / H₂O = 1 / 99 (v / v). The measured fluorescence emission spectra are shown in 5a-5b (λ). ex =365nm, slit width: 5 / 10nm).

[0087] The reagent preparation method for derivative 7 is the same as that for the third intermediate 6 (λ). ex =430nm, slit width: 5 / 5nm, Figures 5c-5d ).

[0088] As shown in 5a-5d, the fluorescence of the third intermediate 6 decreases with increasing solution polarity, exhibiting the ACQ phenomenon. Derivative 7 shows a continuous decrease in fluorescence due to the TICT effect when the water content is between 10% and 90%. With further increases in water content, due to the restriction of intramolecular vibration and rotation in the aggregated state, derivative 7 aggregates at a water content of 99%, reaching its peak fluorescence intensity. This indicates that it has good AIE performance and is suitable for stable imaging in the human body environment.

[0089] The morphological characteristics of compound 7 at THF / H2O = 1 / 9 (v / v) and THF / H2O = 1 / 99 (v / v) were observed using scanning electron microscopy (SEM), such as... Figures 6a-6b As shown.

[0090] Depend on Figures 6a-6b It can be seen that when THF / H2O = 1 / 9 (v / v), the molecules of compound 7 are in an amorphous state, while when THF / H2O = 1 / 99 (v / v), the molecules rapidly aggregate and become uniform petal-shaped nano-aggregates with an average diameter of 10 μm.

[0091] Viscosity response

[0092] The viscosity response of the third intermediate 6 and derivative 7 was tested: 6 was prepared into a concentration of 1×10⁻⁶ using methanol as a solvent. -4 mol·L -1 The working solution. Take ten 10.0 mL volumetric flasks, add 0.0–9.0 mL of glycerol to each flask, measure 1.0 mL of the working solution into each flask, and dilute to volume with methanol to make the concentration of each flask 1 × 10⁻⁶. -5 mol·L -1 The fluorescence emission spectra were measured (the volume ratios of methanol / glycerol were 1 / 9-10 / 0), as shown in Figures 7a-7b (λ). ex =365nm, slit width: 5 / 10nm).

[0093] The reagent preparation method for compound 7 is the same as that for the third intermediate 6 (λ). ex =430nm, slit width: 5 / 10nm Figures 7c-7d ).

[0094] As shown in Figure 7, the fluorescence intensity of both the third intermediate 6 and derivative 7 increases with increasing viscosity. This may be because the increased viscosity hinders the intramolecular motion of the third intermediate 6. However, the aromatic methylmalononitrile fragment in derivative 7 further restricts intramolecular motion, making it more responsive to viscosity. This suggests that derivative 7 has the potential to become a viscosity-responsive fluorescent probe.

[0095] Interference experiment

[0096] Common cation Fe was examined 3+ Al 3+ Na + Ca 2+ Cu 2+ Cr 3+ and K + CO3 anion 2- HCO3 - CH3COO - PO42- SO4 2- SCN - and HS - The effects of biothiols Cys, Hcy, and GSH, as well as 90% glycerol (from left to right 2-18, 1 being the blank control) on the fluorescence emission spectrum of derivative 7, such as... Figure 8 As shown (λ) ex =430nm, slit: 10 / 10nm).

[0097] The results show that the fluorescence intensity of derivative 7 remained largely unchanged after the addition of various anions, cations, and biothiols. However, the fluorescence intensity of derivative 7 in 90% glycerol was significantly enhanced, indicating that derivative 7 can achieve specific detection of viscosity in complex biological environments.

[0098] Protein aggregation experiment

[0099] The formation of dense polypeptide chains during protein misfolding and aggregation leads to changes in viscosity. Furthermore, protein denaturation alters its physicochemical properties and exposes hydrophobic groups within the molecule, accelerating aggregation and causing precipitation from aqueous solutions, thus increasing viscosity. Therefore, we simulated the viscosity changes during protein aggregation using the thermal denaturation process of egg white and monitored these changes with Derivative 7, observing alterations in its fluorescence properties.

[0100] First, the fluorescence change of derivative 7 was tested from 0 to 100 °C. Figures 9a-9b The results showed that derivative 7 exhibited no significant change in fluorescence intensity from 0 to 100 °C, indicating that it possesses good thermal stability (λ). ex =430nm, slit width: 5 / 10nm).

[0101] When derivative 7 was mixed with an appropriate amount of egg white, the protein gradually aggregated and its viscosity increased as the temperature rose. The fluorescence of derivative 7 also gradually increased, indicating that it can be used to monitor viscosity changes during protein aggregation and has the potential to become a viscosity-responsive probe for protein aggregation. Figures 9c-9d ).

[0102] Extracorporeal photodynamic therapy

[0103] Using human non-small cell lung cancer (A549) cells and human hepatocellular carcinoma (HepG2) cells as models, the cytotoxicity of derivative 7 on A549 and HepG2 cells was detected by the MTT assay. A549 and HepG2 cells were seeded in 96-well microplates (1×10⁻⁶). -5Cells were inoculated with 100 μL of culture medium per well (cells / mL). After incubation at 37°C in a CO2 incubator for 24 h, different concentrations of derivative 7 were added to the inoculated cells and incubated for another 24 h. The microplate was then washed three times with PBS buffer, and 10 μL of MTT solution was added to each well for further incubation for 4 h. The culture medium was removed from the wells, and 150 μL of DMSO was added to each well to dissolve the blue-purple formazam crystals within the cells. The wells were then placed on a shaker and shaken at low speed for 5-7 min to ensure complete dissolution of the crystals. Finally, the absorbance values ​​of each well at 560 nm and 670 nm were measured using an ELISA reader. Cytotoxicity was calculated using the following formula:

[0104] Viability% = [∑(A i / A0×100) / n]

[0105] In the formula A i A1 represents the absorbance values ​​of different concentrations of the compound; A0 represents the average absorbance value of the control well without added compound; n (=3) represents three parallel experiments.

[0106] The dark toxicity and phototoxicity of derivative 7 to HpeG2 and A549 cells were detected using the MTT assay. The light source was 430 nm, and the control group received no light treatment. The absorbance values ​​of each well were measured at 560 nm and 670 nm using an enzyme-linked immunosorbent assay (ELISA). The dark toxicity and phototoxicity of derivative 7 to A549 and HpeG2 cells are shown below. Figures 10a-10b As shown.

[0107] Table 5. Half-maximal inhibitory effect (IC50) of derivative 7 on two cell types. 50 )

[0108]

[0109] The half-maximal inhibitory concentrations (IC50) of derivative 7 against HepG2 and A549 cells are shown in Table 5. The results indicate that derivative 7 exhibits low dark toxicity and high phototoxicity against both HepG2 and A549 cells, demonstrating its excellent phototoxicity response (PDT). Particularly noteworthy is the very low dark toxicity (IC50) of derivative 7 against A549 cells. 50 >100.0 μmol·L -1 ), and has high phototoxicity (IC). 50 =19.9 μmol·L -1 This indicates that it has the potential to be developed into an anti-liver cancer drug.

[0110] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a TB-thiophene methylene malononitrile derivative in the preparation of a viscosity probe, characterized in that, The structural formula of the TB-thiophene methylene malononitrile derivative is: The preparation method of TB-thiophene methylene malononitrile derivative includes the following steps: Step 1: 4-Bromoaniline (1) reacts with paraformaldehyde (2) to obtain the first intermediate (3), as shown in the following reaction formula: Step 2: The first intermediate (3) reacts with n-butyllithium to obtain the second intermediate (4), as shown in the following reaction formula: Step 3: The second intermediate (4) and 5-bromothiophene-2-carboxaldehyde (5) undergo a coupling reaction to obtain the third intermediate (6), as shown in the following reaction formula: Step 4: The third intermediate (6) reacts with malononitrile via a coupling reaction to obtain derivative (7), as shown in the following reaction formula: 。 2. The application of a TB-thiophene methylene malononitrile derivative in the preparation of cancer photodynamic therapy drugs, characterized in that, The structural formula of the TB-thiophene methylene malononitrile derivative is: The application described is aimed at inhibiting human liver cancer HepG2 cells and human lung cancer A549 cells.

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