A sulfonamide-naphthalimide-TB derivative, its synthesis method and application

Abstract

This invention provides a sulfonamide-naphthalimide-TB derivative, its synthesis method, and its application. It is synthesized from p-bromoaniline, paraformaldehyde, p-toluenesulfonyl chloride, ethylenediamine, 4-bromo-1,8-naphthalimide, etc., through a multi-step reaction. Its structural formulas are shown in the first derivative (10) and the second derivative (11) below: The products have large Stokes shifts (both greater than 100 nm) in both solution and solid; they have significant AIE properties; they have a wide pH range and can be applied in the human physiological environment; compared with pure methanol, the fluorescence is enhanced by 1.6 times and 4.3 times at methanol / glycerol = 1 / 9 (v / v), respectively, and both have good responsiveness to viscosity; they can easily enter living A549 cells and have strong targeting ability to their endoplasmic reticulum (Pearson coefficients Pr are 0.81 and 0.71, respectively); the phototoxicity and dark toxicity to A549 cells and HpeG2 cells are both low, indicating that they have high biocompatibility and potential as viscosity probes and endoplasmic reticulum probes for tumor cells.

Description

Technical Field

[0001] This invention belongs to the field of chemical synthesis, specifically relating to sulfonamide-naphthalimide- Base derivatives, their synthesis methods, and their applications in viscosity response and endoplasmic reticulum targeting. Background Technology

[0002] The endoplasmic reticulum (ER) is an important organelle in eukaryotic cells, primarily responsible for lipid synthesis, protein synthesis, processing and modification, and the folding, assembly, and transport of nascent polypeptide chains. Its state and structure are dynamic and susceptible to environmental factors. External triggers such as hypoxia, viral infection, redox reactions, and glucose deficiency can disrupt ER homeostasis, leading to misalignment within the ER lumen, accumulation of unfolded proteins, and calcium ion imbalance, thus causing ER stress (ERS). Following ERS, the cell initiates a protective response, the unfolded protein response (UPR), to restore ER homeostasis. However, excessively strong or prolonged stress can lead to programmed necrosis or apoptosis of the ER, and promote interactions between the ER and other organelles. Therefore, severe ERS can lead to diseases such as heart disease, diabetes, neurodegenerative diseases, and cancer.

[0003] Although there have been reports of ER-targeting fluorescent probes, two problems remain: their underlying molecular mechanisms are unclear, leading to a limited variety; and they exhibit a strong aggregation fluorescence quenching (ACQ) effect in aqueous media, resulting in poor imaging performance. Therefore, developing diverse, highly selective ER fluorescent probes with weak or no ACQ effect, and high efficiency and low toxicity is both a challenge and a hot topic in this field. To date, no probes based on... Reports on ER fluorescent probes with a base (TB) framework.

[0004] 1,8-Naphthalimide is an important luminescent group, exhibiting good photostability, a large Stokes shift, high fluorescence quantum yield, and two-photon emission. Molecules containing 1,8-naphthalimide (NI) are a rapidly developing research hotspot in organic photonics and electronics, with wide applications in fluorescent dyes, laser dyes, metal sensors, pH sensors, bioimaging, and organic light-emitting diodes. The photophysical and electrochemical properties of NI depend on the substitution mode and the nature of the donor unit. Modifying the imine site of the NI unit can not only produce a large number of NI derivatives but also adjust their photophysical and electronic properties.

[0005] Therefore, this invention designs and synthesizes a sulfonamide-naphthalimide with endoplasmic reticulum targeting function by sequentially introducing 1,8-naphthalimide and the ER-directing group p-toluenesulfonamide fragment onto the TB backbone. base derivatives. TB and its derivatives have a unique V-shaped framework and a long conjugated structure. Under photon excitation, they exhibit multiple transition modes (π-π*, n-π*, and spatial transitions), and theoretically have a large molar absorption coefficient, making them an excellent basic framework for ultraviolet light absorption materials. Summary of the Invention

[0006] Technical Problem: The purpose of this invention is to provide a sulfonamide-naphthalimide-TB derivative, its synthesis method, and its application. Using p-bromoaniline, paraformaldehyde, p-toluenesulfonyl chloride, ethylenediamine, 4-bromo-1,8-naphthalimide, etc., as raw materials, sulfonamide-naphthalimide is synthesized through a multi-step reaction process. Base derivatives, and apply them to fields such as viscosity identification and ER localization.

[0007] Technical solution: The structural formula of a class of sulfonamide-naphthalimide-TB derivatives of the present invention is shown in the first and second derivatives below:

[0008]

[0009] The method for synthesizing the sulfonamide-naphthalimide-TB derivative of the present invention includes the following steps:

[0010] Step 1: 4-Bromoaniline 1 reacts with paraformaldehyde 2 to give the first intermediate 3, as shown in the following reaction formula:

[0011]

[0012] Step 2: The first intermediate reacts with n-butyllithium to obtain the second intermediate, as shown in the following reaction formula:

[0013]

[0014] Step 3: p-Toluenesulfonyl chloride reacts with ethylenediamine via a coupling reaction to obtain the third intermediate, as shown in the following reaction formula:

[0015]

[0016] Step 4: The third intermediate is coupled with 4-bromo-1,8-naphthylimide to obtain the fourth intermediate, as shown in the following reaction formula:

[0017]

[0018] Step 5: The second intermediate and the fourth intermediate undergo a coupling reaction to yield the first derivative and the second derivative, as shown in the following reaction formula:

[0019]

[0020] The application of the sulfonamide-naphthalimide-TB derivative of the present invention in the preparation of viscosity probes.

[0021] The application of the sulfonamide-naphthalimide-TB derivative of the present invention in the preparation of endoplasmic reticulum targeting probes.

[0022] The application of the endoplasmic reticulum targeting probe is for the localization of the endoplasmic reticulum in human lung cancer A549 cells.

[0023] Beneficial effects:

[0024] 1. The synthesis method is simple and the post-processing is convenient.

[0025] 2. The product has excellent luminescent properties: it has a large Stokes shift and significant AIE properties.

[0026] 3. The product has a wide pH range and can be used in human physiological environments.

[0027] 4. The product easily enters living A549 cells and has a strong targeting ability to the endoplasmic reticulum (Pearson coefficients Pr are 0.81 and 0.71, respectively).

[0028] 5. The product not only has endoplasmic reticulum targeting ability but also responds well to viscosity, making it of great value for further research.

[0029] 6. The product has high biocompatibility with A549 cells and HpeG2 cells, and has the potential to be used as a viscosity probe and endoplasmic reticulum probe for tumor cells. Attached Figure Description

[0030] Figure 1 It is the first derivative 10 in the embodiment. 1 HNMR spectrum;

[0031] Figure 2 It is the first derivative 10 in the embodiment. 13 CNMR spectrum;

[0032] Figure 3 It is the second derivative 11 in the embodiment. 1 HNMR spectrum;

[0033] Figure 4 It is the second derivative 11 in the embodiment. 13 CNMR spectrum;

[0034] Figure 5 shows the (a) UV absorption spectrum and (b) fluorescence emission spectrum of the first derivative 10 in different solvents in the examples;

[0035] Figure 6 shows the (a) UV absorption spectrum and (b) fluorescence emission spectrum of the second derivative 11 in different solvents in the examples;

[0036] Figure 7 shows the (a) fluorescence emission spectrum and (b) line graph of the first derivative 10 in different ratios of THF / H2O (v / v) and the (c) fluorescence emission spectrum and (d) line graph of the second derivative 11 in different ratios of THF / H2O (v / v).

[0037] Figure 8 shows the SEM images of the first derivative 10 at different THF / H2O (v / v) ratios: (a) THF / H2O = 2 / 8 (v / v) and (b) THF / H2O = 1 / 99 (v / v).

[0038] Figure 9 shows the fluorescence emission spectrum (a) and (b) line graph of the first derivative 10 at different viscosities, and the fluorescence emission spectrum (c) and (d) line graph of compound 11 at different viscosities.

[0039] Figure 10 It is the fluorescence intensity of the first derivative 10 under the interaction of different ions and molecules;

[0040] Figure 11 It is the fluorescence intensity of the second derivative 11 under the interaction of different ions and molecules;

[0041] Figure 12 shows the fluorescence changes of the first derivative 10 at different temperatures, (b) the fluorescence changes after binding with egg white, and (c) the fluorescence changes of the second derivative 11 at different temperatures and (d) the fluorescence changes after binding with egg white.

[0042] Figure 13 Co-localization fluorescence imaging of HeLa cells treated with the first derivative 10 and ER-TrackerGreen;

[0043] Figure 14 Co-localization fluorescence imaging of HeLa cells treated with the second derivative 11 and ER-TrackerGreen;

[0044] Figure 15 shows the phototoxicity and dark toxicity tests of 10 pairs of (a) A549 cells and (b) HpEG2 cells of the first derivative, and the phototoxicity and dark toxicity tests of 11 pairs of (c) A549 cells and (d) HpEG2 cells of the second derivative. Detailed Implementation

[0045] The present invention will be further described below with reference to the embodiments.

[0046] 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.

[0047] The following are: 4-bromoaniline 1, paraformaldehyde 2, first intermediate 3, second intermediate 4, toluenesulfonyl chloride 5, ethylenediamine 6, third intermediate 7, 4-bromo-1,8-naphthylimide 8, fourth intermediate 9, first derivative 10, and second derivative 11.

[0048] sulfonamide-naphthalimide The derivatives, whose structural formulas are shown in the table below:

[0049] Table 1. Structural formulas of first derivative 10 and second derivative 11

[0050]

[0051] The present invention also provides the above-mentioned novel sulfonamide-naphthalimide. Methods for preparing base derivatives include:

[0052] In this embodiment, the product is prepared through a multi-step reaction using p-bromoaniline, paraformaldehyde, n-butyllithium, 4-bromo-1,8-naphthalimide, and ethylenediamine as raw materials. The steps include:

[0053] 4-Bromoaniline reacts with paraformaldehyde to give a first intermediate 3. The first intermediate 3 reacts with n-butyllithium to give a second intermediate 4. The second intermediate 4 and the fourth intermediate 9 undergo a coupling reaction to give a first derivative 10 and a second derivative 11.

[0054] The first derivative 10 and the second derivative 11 in the above-described synthesis method were prepared as follows:

[0055] 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.

[0056]

[0057] Synthesis of intermediate 3 in Equation 1

[0058] (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, and then 0.6 mL of trimethyl borate was added dropwise. 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%).

[0059]

[0060] Equation 2 Intermediate 4 Synthesis

[0061] (4) Dissolve p-toluenesulfonyl chloride (1.0 mmol) in dichloromethane (5 mL) and slowly add it to a solution of ethylenediamine (10 mmol) dissolved in dichloromethane. Stir at 40 °C for 15 minutes, then wash the mixture twice with water (25 mL) and dry it with Na2SO4. Remove the solvent under vacuum to obtain the third intermediate 7, which is a white solid product (83%).

[0062]

[0063] Synthesis of the third intermediate 7 in Equation 3

[0064] (5) The third intermediate 7 (1.0 mmol) and 4-bromo-1,8-naphthylimide (0.28 g, 1.0 mmol) were dissolved in ethanol (20 mL). The mixture was stirred at 80 °C for 4 hours under argon protection. After the reaction was complete (TLC monitoring), the mixture was quenched with water and filtered. The filter cake was purified by column chromatography using CH2Cl2 / CH3OH = 25 / 1 (v / v) as the eluent to obtain the fourth intermediate 9 (72%).

[0065]

[0066] Synthesis of intermediate 9 in Equation 4

[0067] (5) First intermediate 3 (1.0 mmol), fourth intermediate 9 (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), dried over Na2SO4, and the crude product obtained by rotary evaporation was purified by column chromatography (V 石油醚 :V 乙酸乙酯 =7:1), yielding the first derivative 10 (38%) and the second derivative 11 (33%).

[0068]

[0069] Synthesis of the first derivative 10 and the second derivative 11 of Equation 5

[0070] (8-(2-(2-((4-methylphenyl)sulfinylamino)ethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-6-yl)-6H,12H-5,11-dibenzo[b,f][1,5]diaza-octyl-2-yl)boronic acid (10)

[0071] 1 H NMR (400MHz, CDCl3) δ8.51-8.44(m,2H,Ar-H),8.26(d,J=8.5Hz,1H,Ar-H),7.64(t,J=7.8Hz, 1H),7.58-7.51(m,3H,Ar-H),7.30(t,J=7.0Hz,2H),7.19(t,J=7.9Hz,2H),7.07-6.96(m,3H, Ar-H),6.74(d,J=7.8Hz,2H,Ar-H),5.30(d,J=9.3Hz,1H,Ar-H),4.86-4.75(m,2H,-CH2-brid ge), 4.39 (s, 2H), 4.32-4.22 (m, 4H, -CH2-bridge), 3.45 (d, J = 5.2Hz, 2H, Ar-H), 1.91 (s, 3H). 13 C NMR (100MHz, CDCl3) δ164.8,164.5,146.9,142.7,137.0,134.3,133.2,131.5,131.1,129.8,129 .3,129.0,128.7,128.4,127.8,127.6,127.1,126.9,126.8,66.8,58.8,58.7,42.6,39.2,21.3.

[0072] N,N'-(((6H,12H-5,11-dibenzo[b,f][1,5]diaza-2,8-diyl)bis(1,3-dioxo-1H-benzo[de]isoquinoline-6,2(3H)-diyl))bis(ethyl-2,1-diyl))bis(4-methylbenzenesulfonamide)(11)

[0073] 1 H NMR (400MHz, CDCl3) δ8.53(t,J=8.2Hz,4H),8.32(d,J=8.5Hz,2H),7.70(t,J=7.9Hz,2H),7.64(d,J=7.6Hz,2H),7.56(d,J=7 .9Hz,4H),7.37(s,4H),7.15(s,2H),6.80(s,4H),5.25(s,2H),4.92(d,J=16.9Hz,2H,-CH2-bridge),4.50-4.37(m,4H,TB-CH 2* 2),4.29(s,4H),3.47(s,4H),1.99(s,6H). 13 C NMR (100MHz, CDCl3) δ164.8,164.5,146.6,142.8,137.0,133.0,131.5,131.1,129.9,1 29.3,128.8,128.5,127.9,127.0,126.8,125.6,122.5,121.2,58.7,42.6,39.2,21.30.

[0074] Optical performance testing

[0075] The solvation effect of the compounds of this invention was tested, and the specific experimental scheme is as follows:

[0076] Derivative 10 and derivative 11 were respectively 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. -5 mol·L -1 The working solution was tested, and its ultraviolet absorption spectrum and fluorescence emission spectrum were measured. For example... Figures 7a-7d As shown.

[0077] from Figures 7a-7d It can be seen that the ultraviolet absorption wavelengths of the first derivative 10 and the second derivative 11 increase with increasing solvent polarity. emA significant blue shift occurs, attributed to the R-band absorption of heteroatoms. With increasing polarity, the n-π* transitions of the first derivative 10 and the second derivative 11 are enhanced. The ground state is more polar than the excited state, thus enabling the ground state to form stronger hydrogen bonds with the polar solvent, resulting in a larger energy decrease, while the excited state energy decreases less. Consequently, the transition energy increases, leading to a blue shift in the fluorescence emission wavelength.

[0078] The UV absorption and fluorescence emission spectra of the first derivative 10 and the second derivative 11 in THF solution, as well as their solid-state fluorescence emission spectra, were tested. The specific experimental protocol is as follows:

[0079] Weigh out 10 -5 mol of 4-bromoaniline, second intermediate 4, first derivative 10, and second derivative 11 were diluted with THF solution to a concentration of 1 × 10⁻⁶. -5 The concentration was mol / L, and its ultraviolet absorption, fluorescence emission, and solid-state fluorescence emission spectra were measured.

[0080] The spectral data of 4-bromoaniline, second intermediate 4, first derivative 10 and second derivative 11 are shown in Table 2.

[0081] As shown in Table 2, compared with 4-bromoaniline and the second intermediate 4, the fluorescence properties of the first derivative 10 and the second derivative 11 have the following changes:

[0082] (1) Solution and solid λ of the first derivative 10 em Both showed a significant red shift, with the Stokes shifts in solution and solid (103 and 182 nm, respectively) increasing significantly.

[0083] (2) Solution and solid λ of the second derivative 11 em Both showed a significant red shift, with the Stokes shifts in solution and solid (125 and 173 nm, respectively) increasing significantly.

[0084] (3) Compared with the first derivative 10, the solution and solid fluorescence intensity of the second derivative 11 are increased. This may be because the fluorescence quantum yield is increased when the number of naphthalimide increases, and the luminescence intensity is enhanced. Therefore, the fluorescence emission intensity of the second derivative 11 is greater than that of the first derivative 10.

[0085] The results above demonstrate that combining the TB backbone with the 1,8-naphthalimide fragment can amplify the advantages of both in terms of luminescence performance, providing a new approach to obtaining products with excellent luminescence properties.

[0086] Table 2. Spectral data (THF) of compounds 1, 4, 10, and 11.

[0087]

[0088] 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.

[0089] pH response

[0090] A suitable pH response range is one of the key factors for the successful application of probes in biological systems; therefore, we explored the pH range of the first derivative 10. Using THF as a solvent, 6 was prepared into a concentration of 1×10⁶. -4 mol·L -1 For the working solution, 1.0 mL of the first derivative 10 working solution was measured into nine 10.0 mL volumetric flasks, and then 1.0 mL of a buffer solution with a pH 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 for each flask. -5 molL -1 Its fluorescence emission spectrum (λ) was measured. ex =370nm, slit: 5 / 5nm).

[0091] The reagent preparation method for the second derivative 11 is the same as that for the first derivative 10. The fluorescence intensity of the first derivative 10 and the second derivative 11 remains almost constant in the pH range of 2.2-10.0, indicating that the first derivative 10 and the second derivative 11 have a wide pH range of applicability.

[0092] AIE features

[0093] Since the first derivative 10 is readily soluble in THF but poorly soluble in water, 10 was prepared into a concentration of 1×10⁻⁶. -4 mol·L -1For 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 the first derivative 10 is prepared to 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 molL -1 This results in THF / H₂O = 1 / 99 (v / v). The measured fluorescence emission spectra are shown in Figures 7a-7b (λ). ex =370nm, slit: 5 / 5nm).

[0094] The preparation method of the reagents for the second derivative 11 is the same as that for the first derivative 10 (λ). ex =380nm, slit: 5 / 5nm, Figures 7c-7d ).

[0095] As shown in 7a-7d, the fluorescence of the first derivative 10 gradually decreases when the water content is between 10% and 80%. When the water content continues to increase from 80% to 99%, the fluorescence continuously increases due to restricted intramolecular motion, reaching a peak intensity at 99% water content, and exhibiting a redshift into the red light region, which is beneficial for cell imaging. The fluorescence of the second derivative 11 decreases with increasing solution polarity, exhibiting the ACQ phenomenon. This may be due to the presence of two long fatty acid chains in the second derivative 11, which reduces intramolecular rigidity.

[0096] To investigate the formation mechanism of the AIE properties of the first derivative 10, we used scanning electron microscopy (SEM) to observe the morphological characteristics of the first derivative 10 at THF / H2O = 2 / 8 (v / v) and THF / H2O = 1 / 99 (v / v), such as... Figures 8a-8b As shown.

[0097] Depend on Figures 8a-8b It can be seen that the first derivative 10 exhibits an amorphous state when THF / H2O = 2 / 8 (v / v), while it shows a uniform sheet-like aggregation when THF / H2O = 1 / 99 (v / v), with a sharp increase in fluorescence, demonstrating AIE properties. This nano-aggregate may be the reason for its AIE performance. The average diameter of the first derivative 10 molecule at THF / H2O = 1 / 99 (v / v) is 2 μm.

[0098] Viscosity response

[0099] The viscosity response of the first derivative 10 and the second derivative 11 was tested: 10 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 9a-9b (λ). ex =370nm, slit: 5 / 5nm).

[0100] The preparation method of the reagents for derivative 11 is the same as that for 10 (λ). ex =430nm, slit width: 5 / 10nm Figures 7c-7d ).

[0101] As shown in Figure 9, the fluorescence intensity of the first derivative 10 and the second derivative 11 increases with increasing viscosity. This may be because the increased viscosity leads to impaired intramolecular motion, indicating that the first derivative 10 and the second derivative 11 may produce a high-level response to changes in viscosity in the endoplasmic reticulum and can be further studied as multifunctional fluorescent probes for the endoplasmic reticulum.

[0102] Interference experiment

[0103] Common cation Fe was examined 3+ Al 3+ Na + Ca 2+ Cu 2+ Cr 3+ and K + CO3 anion 2- HCO3 - CH3COO - PO4 2- 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 the first derivative 10, are shown below. Figure 10 As shown (λ) ex =370nm, slit width: 5 / 10nm).

[0104] Similarly, the effects of different ions and molecules on the second derivative 11 were tested (λ). ex=380nm, slit width: 5 / 10nm, Figure 11 ).

[0105] The results show that the fluorescence intensity of the first derivative 10 and the second derivative 11 remained essentially unchanged after the addition of various anions, cations, and biothiols, and differed significantly from the fluorescence intensity of the first derivative 10 and the second derivative 11 in 90% glycerol solvent. Therefore, both the first derivative 10 and the second derivative 11 exhibit specific responses to viscosity, enabling specific detection of viscosity in complex biological environments.

[0106] Protein aggregation experiment

[0107] The dense polypeptide chains formed during protein misfolding and aggregation can lead to changes in viscosity.

[74] 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 10, observing its fluorescence changes.

[0108] First, the first derivative 10 was tested. Figure 12a , λ ex =370nm, slit: 5 / 10nm) and ( Figure 12b , λ ex The fluorescence changes of the first derivative 10 and the second derivative 11 from 0 to 100 °C (λ = 380 nm, slit width: 5 / 10 nm) were analyzed. The results showed that the fluorescence intensity of the first derivative 10 and the second derivative 11 did not change significantly from 0 to 100 °C, indicating that they have good thermal stability (λ). ex =430nm, slit width: 5 / 10nm).

[0109] When the first derivative 10 and the second derivative 11 were mixed with an appropriate amount of egg white, the proteins gradually aggregated and their viscosity increased as the temperature rose. The fluorescence of the first derivative 10 and the second derivative 11 gradually increased accordingly, indicating that they can be used to monitor viscosity changes during protein aggregation and have the potential to become viscosity-responsive probes for protein aggregation. Figures 12c-12d ).

[0110] ER localization experiment

[0111] The ER, an important organelle in cells, organically connects the nucleus, cytoplasm, and cell membrane into a unified whole, and is responsible for the transport of substances within the cell. It is also the site of protein and lipid synthesis. Since cyano groups are highly lipid-soluble, they can theoretically be localized to the ER. Therefore, a reasonable co-localization experiment was designed to verify this.

[0112] First, the commercial ER probe ER-Tracker Green was co-incubated with HeLa cells for 30 minutes under suitable conditions with the first derivative 10 and the second derivative 11, respectively. After washing twice with phosphate-buffered saline (PBS), confocal imaging was performed using PBS. The results are as follows: Figure 13-14 As shown.

[0113] Both the first derivative 10 and the second derivative 11 can easily enter living HeLa cells. The Pr of the first derivative 10 is 0.81 and the Pr of the second derivative 11 is 0.71, indicating that both the first derivative 10 and the second derivative 11 can accurately locate the ER and perform fluorescence imaging on the ER, and have strong ER targeting ability.

[0114] Extracorporeal photodynamic therapy

[0115] Using human non-small cell lung cancer (A549) cells and human hepatocellular carcinoma (HepG2) cells as models, the cytotoxicity of the first derivative 10 and the second derivative 11 of compounds on A549 and HepG2 cells was detected by the MTT assay. A549 and HepG2 cells were seeded in 96-well microplates (1×10⁻⁶ cells / wells). -5 Cells were incubated in a CO2 incubator at 37°C for 24 hours. Then, different concentrations of the third intermediate 7 were added to the inoculated cells and incubated for another 24 hours. The microplate was then washed three times with PBS buffer, and 10 μL of MTT solution was added to each well for 4 hours of further incubation. 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 slowly for 5-7 minutes to ensure complete dissolution of the crystals. Finally, the absorbance values ​​at 560 nm and 670 nm were measured using an ELISA reader. Cytotoxicity was calculated using the following formula:

[0116] %viability=[∑(A i / A0×100) / n]

[0117] 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.

[0118] The dark toxicity and phototoxicity of derivative 10 and derivative 11 on 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 at 560 nm and 670 nm were measured using an enzyme-linked immunosorbent assay (ELISA). The dark toxicity and phototoxicity of derivative 10 and derivative 11 on A549 and HpeG2 cells are shown below. Figures 15a-15d As shown.

[0119] Table 3 shows the half-maximal inhibitory rates of the first derivative 10 and the second derivative 11 on HepG2 and A549 cells. The results indicate that the first derivative 10 and the second derivative 11 have low phototoxicity and dark toxicity to both A549 and HpeG2 cells, indicating that they have high biocompatibility with these two cell types and are suitable for monitoring viscosity changes and localizing endoplasmic reticulum in cancer cells.

[0120] Table 3. Half-maximal inhibitory rates (IC50) of derivative 10 and derivative 11 against two cell types. 50 )

[0121]

[0122] 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. A class of sulfonamide-naphthalimide-TB derivatives, characterized in that, Its structural formulas are shown in the first derivative (10) and the second derivative (11) below:

2. A method for synthesizing the sulfonamide-naphthalimide-TB derivative as described in claim 1, characterized in that, 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: p-Toluenesulfonyl chloride (5) and ethylenediamine (6) undergo a coupling reaction to obtain the third intermediate (7), as shown in the following reaction formula: Step 4: The third intermediate 7 and 4-bromo-1,8-naphthalimide (8) undergo a coupling reaction to obtain the fourth intermediate (9), as shown in the following reaction formula: Step 5: The second intermediate (4) and the fourth intermediate (9) undergo a coupling reaction to obtain the first derivative (10) and the second derivative (11), as shown in the following reaction formula:

3. The application of the sulfonamide-naphthalimide-TB derivative as described in claim 1 in the preparation of a viscosity probe.

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