An indole derivative, a preparation method and application thereof

Abstract

The present application provides an indole derivative and a preparation method and application thereof. The preparation method comprises the following steps: dissolving copper acetate and triethylamine in isopropyl alcohol, stirring until completely dissolved to obtain a reaction medium; adding 5-bromo indigo and indole into the reaction medium, fully stirring at room temperature until the reaction is completely converted; removing the reaction solvent by distillation to obtain a crude product, purifying and recrystallizing to obtain a high-purity solid powder of the indole derivative. The indole derivative has a significant inhibitory effect on tyrosinase, melanin production and transfer; has high antioxidant capacity for scavenging intracellular active oxygen free radicals; through the mechanisms of affecting the membrane potential of mitochondria, activating the related apoptosis signaling pathway and promoting the expression of apoptosis proteins, etc., the A549 cell is prompted to enter the apoptosis program, thereby providing a new potential strategy and research direction for the apoptosis regulation of tumor cells, and having important research value and application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of organic compound synthesis, and in particular to an indole derivative, a preparation method and an application thereof. Background Art

[0002] Tyrosinase (TYR), an oxidase containing divalent copper ions, is widely distributed in natural organisms. It plays the role of a key rate-limiting enzyme in the melanin synthesis process and plays a decisive regulatory role in the rate of melanin production. The production of melanin is a complex biological process, involving key links such as the production of melanosomes in melanocytes and the transport of melanin between cells. An appropriate amount of melanin has an important protective function for the skin and can effectively protect the skin from damage caused by ultraviolet radiation. However, when melanin is overproduced, it will cause a series of related skin diseases such as pigmentation, bringing many troubles and challenges to human skin health.

[0003] Lung cancer is one of the most common malignant tumors in the world. With the increase in the global population and the intensification of the aging trend, its incidence continues to rise, and it has become the type of tumor with the highest mortality rate. Non-small cell lung cancer (NSCLC) is a common malignant tumor of the lungs, accounting for about 80-85% of all lung cancers. It is a tumor with significant heterogeneity and can be further divided into subtypes such as squamous cell carcinoma, adenocarcinoma and large cell carcinoma. Most patients are in the advanced stage when diagnosed. For patients with locally advanced NSCLC who are in good physical condition, although a disease-free survival of 8 months can be achieved after a series of chemotherapy, radiotherapy and / or surgical treatment, the 5-year survival rate is still less than 15%.

[0004] Currently, there are several drugs on the market for the treatment of non-small cell lung cancer. Among them, the first-generation epidermal growth factor tyrosine kinase inhibitors (EGFR TKIs) include gefitinib and erlotinib; the second-generation EGFR TKIs include afatinib and dacomitinib; and the third-generation EGFR TKIs mainly include osimertinib. However, almost all patients treated with these three types of drugs will inevitably develop acquired drug resistance. To prevent patients from falling into the dilemma of having no available drugs after the development of acquired drug resistance, the continuous development of new anti-tumor drugs has become a key issue that needs to be addressed urgently. This is of vital significance for improving the treatment effect and prognosis of NSCLC patients and also provides new direction and motivation for promoting the development of the field of tumor treatment. Summary of the Invention

[0005] In order to solve the problems mentioned in the background technology, the present application provides an indole derivative and a preparation method and application thereof.

[0006] In the first aspect of the present invention, an indole derivative is provided, the chemical structure of which is:

[0007]

[0008] Furthermore, the indole derivative is a light brown solid powder.

[0009] The second aspect of the present invention provides a method for preparing an indole derivative, the method comprising:

[0010] S1, preparing a reaction medium, dissolving copper acetate and triethylamine in isopropyl alcohol, and stirring until completely dissolved to obtain a reaction medium;

[0011] S2, preparing an indole derivative solution, adding 5-bromoisatin and indole to the reaction medium, and stirring thoroughly at room temperature until the reactants are completely converted;

[0012] S3, extracting the indole derivative powder, distilling off the reaction solvent to obtain a crude product, purifying and recrystallizing to obtain a high-purity solid powder of the indole derivative.

[0013] Furthermore, step S1 includes the following:

[0014] S11, weigh 0.4 g (2 mmol) of copper acetate and 0.2 g (2 mmol) of triethylamine and place them in a reaction flask;

[0015] S12, adding 10 ml of isopropanol to the reaction flask and stirring for 10 minutes to completely dissolve the isopropanol to obtain a reaction medium.

[0016] Furthermore, step S2 includes adding 4.52 g, 20 mmol, of 5-bromoisatin and 2.8 g, 24 mmol, of indole into a reaction flask, stirring the mixture sufficiently at room temperature to allow a post-coupling reaction to occur, and monitoring the reaction progress by thin layer chromatography.

[0017] Furthermore, the S3 step includes:

[0018] S31, performing reduced pressure distillation using a rotary evaporator to remove the isopropanol solvent in the reaction system, collecting the residue to obtain a crude product of the reactant;

[0019] S32, adding the crude product to a mixed solvent of petroleum ether and dichloromethane, stirring thoroughly to dissolve, and then recrystallizing to obtain a solid powder of the indole derivative.

[0020] The third aspect of the present invention provides a use of an indole derivative, namely, the use of the indole derivative described in the first aspect or the indole derivative prepared by the method described in the second aspect as a tyrosinase inhibitor.

[0021] The fourth aspect of the present invention provides an application of an indole derivative, wherein the indole derivative described in the first aspect or the indole derivative prepared by the method described in the second aspect is used to inhibit the production and / or transfer of melanin in cells.

[0022] The fifth aspect of the present invention provides a use of an indole derivative, that is, the use of the indole derivative described in the first aspect or the indole derivative prepared by the method described in the second aspect as an antioxidant.

[0023] The sixth aspect of the present invention provides an application of an indole derivative, namely, the application of the indole derivative described in the first aspect or the indole derivative prepared by the method described in the second aspect in the treatment of lung cancer.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] (1) The present invention provides a novel indole derivative.

[0026] (2) The method for preparing the indole derivatives provided by the present invention is simple to operate and has high feasibility and repeatability, making it possible to prepare the indole derivatives on a large scale, laying the foundation for their industrial production and wide application, and ensuring the stability and consistency of product quality.

[0027] (3) When used as tyrosinase inhibitors, the indole derivatives provided by the present invention can effectively inhibit the activity of tyrosinase. This has important implications in the fields of medicine and cosmetics. For example, in cosmetics, they can reduce the production of melanin and whiten the skin. In the medical field, they may be used to treat diseases associated with abnormal tyrosinase activity, such as pigmentation disorders.

[0028] (4) The indole derivatives provided by the present invention have a regulatory effect on the production and transfer of melanin in cells. They can inhibit the excessive production of melanin or promote its rational distribution. This has potential application value in the treatment of skin diseases, the development of whitening products, and pigment-related biological research, providing a new approach and means to solve pigment-related problems.

[0029] (5) When used as antioxidants, the indole derivatives provided by the present invention can scavenge free radicals within cells and reduce oxidative stress-induced cell damage. This has a positive effect on the prevention and treatment of various oxidative stress-related diseases, such as cardiovascular disease and neurodegenerative diseases. They also have broad application prospects in areas such as food preservation and cosmetic antioxidants, helping to extend the shelf life of products and protect the skin from oxidative damage.

[0030] (6) The indole derivatives provided by the present invention have potential application value in the treatment of lung cancer. They may inhibit the growth and proliferation of lung cancer cells or induce apoptosis through a specific mechanism of action, providing new drug candidates or therapeutic strategies for the treatment of lung cancer. This is of great significance for improving the treatment effect and prognosis of lung cancer patients and is expected to bring new breakthroughs and hope to the treatment of lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings are included to provide a further understanding of the embodiments and are incorporated into and constitute a part of this specification. The accompanying drawings illustrate the embodiments and, together with the description, serve to explain the principles of the present invention. Other embodiments and many of the expected advantages of the embodiments will be readily apparent as they become better understood by reference to the following detailed description. The elements of the drawings are not necessarily to scale with respect to each other. Like reference numerals designate corresponding similar parts.

[0032] Figure 1 is a flow chart of a method for preparing an indole derivative according to one embodiment of the present invention;

[0033] Figure 2 This is a graph showing the experimental results of YM818 on monophenolase according to one embodiment of the present invention;

[0034] Figure 3 is a line graph showing the inhibitory effect of YM818 on diphenolase according to one embodiment of the present invention;

[0035] Figure 4 1 is a graph showing the experimental results of the inhibition mechanism and type of mTYR by YM818 according to one embodiment of the present invention;

[0036] Figure 5 is a graph showing the experimental results of the effect of YM818 on mTYR fluorescence emission according to one embodiment of the present invention;

[0037] Figure 6 is a molecular docking simulation diagram of YM818 and mTYR according to one embodiment of the present invention;

[0038] Figure 7 This is a micrograph of a Masson-Fontana-stained B16F10 cell experiment showing the inhibitory effect of YM818 on melanin production according to one embodiment of the present invention;

[0039] Figure 8 1 is a graph and a bar graph showing the inhibitory effect of YM818 on the melanin content of B16F10 cells according to one embodiment of the present invention;

[0040] Figure 9The effect of YM818 on key proteins in intracellular melanin synthesis according to one embodiment of the present invention;

[0041] Figure 10 The effect of YM818 on cellular melanin production under AZD5363 treatment according to one embodiment of the present invention;

[0042] Figure 11 This is a stereoscopic image of melanin content on the surface of zebrafish observed by YM818 according to one embodiment of the present invention;

[0043] Figure 12 The effect of YM818 on a key protein in zebrafish melanin production according to one embodiment of the present invention;

[0044] Figure 13 This is an experimental image taken with a Zeiss LSM 780 confocal microscope of the inhibition of melanin transfer of HaCAT cells by YM818 according to one embodiment of the present invention;

[0045] Figure 14 YM818 inhibits melanin transfer in HaCAT cells by flow cytometry according to an embodiment of the present invention;

[0046] Figure 15 This is the effect of YM818 on melanin transfer protein in a co-culture system according to one embodiment of the present invention;

[0047] Figure 16 1 is a graph showing the experimental results of the ABTS and DPPH free radical scavenging ability of YM818 according to one embodiment of the present invention;

[0048] Figure 17 The effect of YM818 on reactive oxygen species in B16F10 cells according to one embodiment of the present invention;

[0049] Figure 18 1 is a graph showing the experimental results of YM818 inhibiting A549 cell viability and colony formation ability according to one embodiment of the present invention;

[0050] Figure 19 These are experimental images captured using a TE2000-U phase contrast microscope showing the effect of YM818 on A549 cell morphology according to one embodiment of the present invention;

[0051] Figure 20 This is the experimental result of the effect of YM818 on the migration and invasion ability of A549 cells according to one embodiment of the present invention;

[0052] Figure 21This is the effect of YM818 on the A549 cell cycle according to one embodiment of the present invention;

[0053] Figure 22 This is a graph showing the experimental results of YM818 inducing apoptosis in A549 cells according to one embodiment of the present invention;

[0054] Figure 23 This is a diagram showing the experimental results of YM818-induced mitochondrial dysfunction in A549 cells according to one embodiment of the present invention. DETAILED DESCRIPTION

[0055] The following will be combined with the accompanying drawings of the present invention to clearly and completely describe the technical solutions in the embodiments. Similar component numbers in the drawings represent similar components. Obviously, the embodiments described below are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0056] It will be understood that when used in this specification and the appended claims, the terms “comprises” and “comprising” indicate the presence of described features, integers, steps, operations, elements and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof.

[0057] It should also be understood that the terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the present invention. As used in the specification and appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly indicates otherwise.

[0058] In the first aspect, the present invention provides a flow chart of a method for preparing an indole derivative, as shown in FIG. Figure 1 As shown, the method includes:

[0059] S101, preparing a reaction medium, dissolving copper acetate and triethylamine in isopropanol, and stirring until completely dissolved to obtain a reaction medium.

[0060] In some specific embodiments, 0.4 g, 2 mmol, of copper acetate and 0.2 g, 2 mmol, of triethylamine were weighed and placed in a reaction flask. 10 ml of isopropanol was added to the reaction flask and stirred for 10 min to completely dissolve the mixture to obtain a reaction medium.

[0061] S102, preparing an indole derivative solution, adding 5-bromoisatin and indole to the reaction medium, and stirring thoroughly at room temperature until the reactants are completely converted.

[0062] In some specific embodiments, 4.52 g (20 mmol) of 5-bromoisatin and 2.8 g (24 mmol) of indole were added to a reaction flask, and the mixture was stirred at room temperature to allow the post-coupling reaction to occur. The reaction progress was monitored by thin layer chromatography.

[0063] S103, extracting the indole derivative powder, distilling off the reaction solvent to obtain a crude product, purifying and recrystallizing to obtain a high-purity solid powder of the indole derivative.

[0064] In some specific embodiments, the isopropanol solvent in the reaction system is removed by reduced pressure distillation using a rotary evaporator, and the residue is collected to obtain a crude product of the reactant. The crude product is added to a mixed solvent of petroleum ether and dichloromethane, stirred and dissolved, and then recrystallized to obtain a solid powder of the indole derivative.

[0065] In a second aspect, an embodiment of the present invention provides an indole derivative, which is a light brown solid powder prepared by the preparation method described in the first aspect. The full Chinese name is 3-hydroxy-5-bromo-(3-indolyl)-2-carbonyl indole, and the chemical structure is:

[0066]

[0067] Example 1

[0068] Copper acetate (Cu(OAC)2·H2O, 0.4 g, 2 mmol, 10 mol%) and triethylamine (0.2 g, 2 mmol, 10 mol%) were accurately weighed and placed in a glass bottle. 10 mL of isopropanol was added and stirred for 10 min to dissolve the drug components. 5-Bromoisatin (4.52 g, 20 mmol) and indole (2.8 g, 24 mmol) were accurately weighed and added to the glass bottle. The mixture was stirred thoroughly at room temperature and the reaction was monitored by thin layer chromatography (TLC) until the reaction of the raw materials was complete. The solvent was removed by vacuum distillation and the residue was purified to obtain a light brown solid powder, namely the crude product YM818. A mixed solvent of petroleum ether and dichloromethane was then added for recrystallization, ultimately yielding 0.318 g of a light brown solid powder with a purity of over 93%, namely the indole derivative YM818. The molecular weight of YM818 was determined by LC-MS (m / z) mass spectrometry to be 364.99 [M + Na] + The actual calculated molecular weight is 343.90[M] + The results of H-NMR spectrum data are: 1H NMR (500MHz, DMSO-d6) δ11.04 (s, 1H), 10.50 (s, 1H), 7.44 (d, J = 8.1 Hz, 1H), 7.41-7.28 (m, 3H), 7.12 (s, 1H), 7.05 (t, J = 7.5 Hz, 1H), 6.90 (dd, J = 11.2, 8.1 Hz, 2H), 6.52 (s, 1H). The results of the carbon nuclear magnetic spectrum are as follows: 13 C NMR (126MHz, DMSO-d6) δ177.90,140.93,136.80,135.85,131.72,127.33,124. 67,123.58,121.18,120.01,118.66,114.67,113.33,111.76,111.62,74.93cm -1 The chemical structure of YM818 is shown below.

[0069]

[0070] The indole derivative YM818 prepared in Example 1 was used as the research object, and mushroom tyrosinase (mTYR), mouse melanoma cells B16F10, zebrafish, human immortalized epidermal keratinocytes HaCAT, and B16F10-HaCAT cell co-cultures were used as models to systematically study the inhibitory mechanism of YM818 on tyrosinase activity, melanin production, and melanin transfer. Through in vitro enzymatic experiments, experiments on YM818's effects on cellular melanin production, zebrafish experiments, experiments on YM818's effects on cellular melanin transfer, YM818 antioxidant activity assays, and YM818-induced mitochondrial-mediated apoptosis experiments in the A549 cell line, a comprehensive and systematic analysis of its mechanism as a tyrosinase inhibitor and antioxidant, as well as its application in inhibiting intracellular melanin production and / or metastasis and treating lung cancer was carried out.

[0071] Application Example 1

[0072] In vitro enzymatic experiments include four experiments: the effect of YM818 on the monophenolase activity of mTYR, the effect of YM818 on the diphenolase activity of mTYR, the inhibition mechanism and inhibition type of YM818 on mTYR, and the interaction between YM818 and mTYR.

[0073] In an experiment investigating the inhibitory effect of YM818 on mTYR monophenolase activity, tyrosine was used as a substrate to determine its effect. YM818 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 30 mmol / L stock solution. This solution was subsequently aliquoted and diluted according to experimental requirements. To a 3 mL reaction system, 1.60 mL of double-distilled water, 750 μL of 0.05 mol / L phosphate buffer, 500 μL of a 1 mg / mL tyrosine solution, and 50 μL of various YM818 concentrations were added in sequence. Finally, 100 μL of a 0.5 mg / mL enzyme solution was added. The mixture was rapidly mixed and placed in a microplate reader. The absorbance at 475 nm was measured at a constant temperature of 37°C. After completion of the reaction, a curve was generated using GRAPHWIN software. The slope of the line represents the steady-state enzyme activity, and the intercept represents the reaction lag time. The residual enzyme activity was plotted against the YM818 concentration, and the results were analyzed using the Statistical Package for Social Sciences (SPSS software) to obtain the IC value of YM818 for monophenolase. 50 value.

[0074] refer to Figure 2 , Figure 2 The experimental results of the inhibition of monophenolase by YM818 according to the present invention are shown in FIG. Figure 2 A represents the kinetic cycle diagram of the oxidation reaction of mTYR monophenolase catalyzed by YM818. Figure 2 1-6 in A correspond to YM818 concentrations of 0mmol / L, 0.1mmol / L, 0.2mmol / L, 0.3mmol / L, 0.4mmol / L, and 0.5mmol / L, respectively. Figure 2 B represents the steady-state tyrosinase activity diagram, Figure 2 C represents the hysteresis time of the reaction catalyzed by YM818 on monophenolase. Tyrosinase has monophenolase activity and catalyzes L-tyrosine (L-Tyr) to produce L-3,4-dihydroxyphenylalanine (L-DOPA) in melanin synthesis. Using L-Tyr as a substrate, the inhibitory effect of YM818 on mTYR monophenolase activity was determined. The results are shown in Figure 2. Figure 2 shown. Figure 2 A represents the kinetic curve of the oxidation reaction of mTYR monophenolase catalyzed by YM818. The initial product accumulation rate is slow, and the product accumulation increases rapidly after a certain period of reaction. As the concentration of YM818 increases, the activity of the steady-state enzyme gradually decreases (e.g. Figure 2 B). Figure 2C represents the hysteresis of the catalytic activity reaction time of monophenolase in the presence of YM818. As the concentration of YM818 increases, the hysteresis reaction time of the monophenolase catalytic reaction is prolonged. When the YM818 concentration is 0.5mmol / L, the enzyme activity drops to 22.59% and the hysteresis time is 15.5min. The results show that YM818 has a significant inhibitory effect on monophenolase, and its mechanism of inhibiting the catalytic action of the enzyme is related to prolonging the hysteresis time of the enzyme catalytic reaction and reducing the activity of the monophenolase. The IC value of YM818 on monophenolase was calculated by SPSS software. 50 The value is 0.261mmol / L.

[0075] In the experiment to study the inhibitory effect of YM818 on the activity of mTYR diphenolase, the effect of YM818 on the activity of mTYR diphenolase was determined using L-DOPA substrate. YM818 was dissolved in DMSO solution to prepare a 30 mmol / L mother solution for standby use, which was subsequently diluted to solutions of different concentrations according to experimental needs. In a reaction system with a total volume of 3 mL, 1.80 mL of double distilled water, 750 μL of 0.05 mmol / L phosphate buffer, 300 μL of 1 mg / ml L-DOPA solution, 50 μl of YM818 solution of different concentrations were added in sequence, and finally 100 μL of 0.5 mg / ml enzyme solution was added, mixed quickly, placed in a microplate reader and the absorbance at 475 nm was measured at a constant temperature of 37°C. The slope of the straight line measured at the end of the reaction was the steady-state enzyme activity, and the relative residual enzyme activity was plotted against different YM818 concentrations. SPSS software was used to analyze and obtain the IC value of YM818 on diphenolase. 50 value.

[0076] Experimental results reference Figure 3 , Figure 3 The line graph shows the inhibitory effect of YM818 on diphenolase according to the present invention. Tyrosinase has diphenolase activity. Under the action of the enzyme, L-DOPA is oxidized to generate dopaquinone, which is then converted into melanin through a series of reactions. Figure 3 It can be seen that with the increase of YM818 concentration, the enzyme activity gradually decreased and showed a certain concentration-dependent effect. When the YM818 concentration was 0.5 mmol / L, the enzyme activity dropped to 35.50%. The IC50 of YM818 for inhibiting diphenolase activity was calculated by SPSS software. 50 It is 0.376mmol / L.

[0077] Further references Figure 4 , Figure 4The following graph shows the results of an experiment on the mTYR inhibition mechanism and type of inhibition according to the present invention. The mechanism by which YM818 inhibits mTYR diphenolase activity was further investigated in a 3 mL activity measurement system. The concentration of the substrate L-DOPA was kept constant, while the concentrations of the enzyme and YM818 were varied to measure the mTYR diphenolase activity. The relative residual activity of the enzyme was plotted ( Figure 4 A) yields five straight lines passing through the origin. The slopes of these lines decrease as the YM818 concentration increases, indicating that YM818's inhibitory effect on mTYR activity is reversible. While YM818 reduces the rate at which mTYR diphenolase catalyzes substrate conversion, it does not permanently denature or inactivate the enzyme. In a 3 mL activity assay, the mTYR concentration was kept constant while varying the L-DOPA substrate concentration and the YM818 concentration to further investigate the type of YM818 inhibition of mTYR diphenolase activity. The results are shown in Figure 1. Figure 4 As shown in B, five straight lines intersecting in the second quadrant were obtained by plotting the double reciprocal 1 / v against 1 / [S]. The results showed that YM818 can affect the maximum reaction rate (V m ), can also affect the Michaelis constant (K m ). Under the action of YM818, V m Reduce K m The value increases, indicating a mixed inhibition type. The slope and vertical intercept are plotted against the concentration of YM818, and two straight lines are obtained ( Figure 4 C. Figure 4 D), calculate the inhibition constant (K I )=0.155mmol / L, inhibition constant (K IS )=0.976mmol / L. K IS The value is much larger than K I The value of indicates that the affinity of YM818 to the tyrosinase-substrate complex is greater than that to the free tyrosinase.

[0078] In experiments investigating the interaction between YM818 and mTYR, intrinsic fluorescence quenching was utilized to measure the intrinsic fluorescence intensity of mTYR in response to varying YM818 concentrations using a Varian CaryEclipse fluorescence spectrophotometer. Parameters were set to an excitation wavelength of 280 nm, an emission slit width of 10 nm, and an excitation slit width of 5 nm. Changes in tyrosinase fluorescence intensity were measured within the 320-400 nm range. Before the experiment, the instrument was zeroed with 2 mL of double-distilled water. A 0.2 mg / mL tyrosinase solution was prepared with double-distilled water. 2 mL of the enzyme solution was placed in a light-transmitting cuvette. A 50 mmol / L YM818 solution was then added to the cuvette, 2 μL at a time. The solution in the cuvette was mixed and allowed to react for one minute. The fluorescence intensity of the solution was then scanned and measured. The quenching rate of both the compound and the fluorescent substance follows the Stern-Volomer curve equation: F0 / F = 1 + K. SV [Q], where F0 is the intrinsic fluorescence intensity before quenching, F is the intrinsic fluorescence intensity after quenching, [Q] is the concentration of quenching, and KSV is the quenching reaction constant. Based on the quenching reaction results, the Stern-Volmer curve of YM818 on the fluorescence intensity of mTYR can be drawn to determine the type of fluorescence quenching mechanism. If the quenching constant K SV More than 100M -1 The equilibrium constant (K) of the reaction can be obtained by the Scatchard equation. A ) and binding constant (n): lg[(F0-F) / F]=lgK A +nlg[Q].

[0079] Molecular Operating Environment software (MOE (2020) software) can be used to simulate the interaction mechanism between YM818 and mTYR and further study the interaction between YM818 and tyrosinase. The mTYR protein structure (PDB: 2Y9X) was downloaded from the Protein DataBank database (PDB database), and MOE software was used to remove protein crystal water, original ligands, etc. The chemical structure of YM818 was drawn using ChemDraw software and energy minimization was performed. The region close to the copper ion active center was selected for simulated docking, highlighting the binding mode of YM818 and mTYR and the interacting amino acid residues. The results were analyzed using the simulated docking diagram.

[0080] The experimental results are as follows Figure 5 and Figure 6 As shown, Figure 5 and Figure 6Figures 2 and 3 show the experimental results and molecular docking simulations of the effect of YM818 on mTYR fluorescence emission, respectively. Tyrosinase contains a tryptophan aromatic group, which can be excited and emit intrinsic fluorescence. Binding of the compound to the enzyme results in quenching of the intrinsic fluorescence, which can be used to explore the interaction between the compound and the enzyme. Figure 5 A represents the fluorescence spectrum curve, Figure 5 Curves 1-8 in A represent YM818 concentrations of 0 mmol / L, 0.05 mmol / L, 0.1 mmol / L, 0.15 mmol / L, 0.2 mmol / L, 0.25 mmol / L, 0.3 mmol / L, and 0.35 mmol / L, and curve 9 represents the absorption peak of YM818; Figure 5 B represents the mTYR fluorescence quenching effect; Figure 5 C represents the Stern-Volmer curve, Figure 5 A and Figure 5 As shown in Figure 2, with the increase of YM818 concentration, the fluorescence intensity of mTYR showed a regular peak weakening and a slight red shift, indicating that YM818 and mTYR had a coordinated reaction and new substances were generated in the reaction. The Stern-Volmer curve (F0 / F versus YM818 concentration [I]) was plotted to show a linear relationship. Figure 5 C), Stern-Volmer equation is: y = 12.33x + 1, R 2 =0.9683, K SV The value is 1.233×10 4 M -1 Because K SV More than 100M -1 , so the quenching type of YM818 on mTYR is static quenching. Plot lg[(F0-F) / F] against lg[I], and the results are shown in Figure 5 D, fitting Scatchard equation is y = 1.247x-1.512, R 2 =0.9641. The equilibrium constant (K A ) is 3.27×105M -1 , the binding constant (n) was 1.247, indicating that YM818 could form a complex with an mTYR.

[0081] Figure 6 The results of the simulated docking of YM818 and mTYR are shown. Figure 6 A represents the three-dimensional diagram of the interaction between YM818 and mTYR. Figure 6 B represents the interaction plane, Figure 6 C represents the interaction surface form diagram, Figure 6D represents a local 3D diagram. The figure shows that YM818 has a good binding effect with TYR, and the binding energy of the interaction is -5.344kcal / mol. Although YM818 cannot directly interact with the copper ion in the active center of mTYR, it can affect the activity of tyrosinase by forming hydrogen bonds and hydrophobic forces. The carbonyl group and amino group in YM818 can form hydrogen bonds with the amino acid residues Gln307 and Asp312 of the side chain donor, respectively, and the hydrogen bond lengths are YM818 also interacts hydrophobically with the side chains of the active site, including Lys376, Tyr311, Glu356, Asp357, Thr308, and Lys379. This suggests that YM818 forms hydrogen bonds with mTYR and generates hydrophobic forces, which in turn affect the structure of mTYR and inhibit enzyme activity.

[0082] Application Example 2

[0083] The experiments on the effect of YM818 on cellular melanin production include the experiment on the effect of YM818 on the melanin content and synthesis of B16F10 cells, the experiment on the expression level of key proteins in melanin production detected by Western Blot, and the experiment on the change of melanin synthesis by YM818 under the treatment of AKT inhibitor AZD5363.

[0084] In studying the effect of YM818 on melanin synthesis in B16F10 cells, a Masson-Fontana melanin staining kit (Solarbio) was used for cytochemical staining observation. B16F10 cells were cultured in a six-well plate with a 24mm diameter sterile cell slide. The cells were treated with 30μmol / L and 60μmol / L YM818 for 48 hours, respectively. The positive control was 60μmol / L arbutin. After the drug treatment, the old culture medium was removed and the cells were washed three times with PBS (Phosphate Buffered Saline). 4% polymethanol solution was added to fix the cells for 20 minutes. After washing with double distilled water, the Fontana ammonia silver solution in the kit was added and placed in a 56°C incubator to react for 30 minutes in the dark. Immerse in double-distilled water and wash several times, add Hypo solution for 5 minutes, rinse with double-distilled water for 3 minutes, add neutral red stain for 5 minutes, wash with double-distilled water, and finally add 95% ethanol and anhydrous ethanol for dehydration. Use neutral gum to seal the cell slides and store them in a light-proof environment. Use a Leica upright fluorescence microscope to observe and take pictures.

[0085] Masson-Fontana melanin dye can reduce silver ammonia solution to metallic silver through chemical reaction, and the melanin particles appear obvious black after being stained. B16F10 cells were treated with 0μmol / L, 30μmol / L and 60μmol / L YM818 respectively, and stained after 48 hours. The melanin coloration in B16F10 cells was observed and images were taken using an upright fluorescence microscope to obtain Figure 7 The B16F10 cell experiment shown in Figure 1 is a micrograph of the B16F10 cell experiment. Figure 7 It can be seen that the number of melanin granules in the control group was larger and the staining was darker. After drug treatment, the coloration of the melanin granules in the cells became significantly lighter, and the inhibitory effect of the 60μmol / L YM818 treatment group was better, which was better than the inhibitory effect of arbutin at the same concentration (60μmol / L).

[0086] The cell density of cultured B16F10 cells was adjusted to 10 5 CFU / mL, culture medium containing 0μmol / L, 30μmol / L, 40μmol / L, 50μmol / L, and 60μmol / L YM818 was added and incubated in an incubator for 48 hours. After washing with PBS, the cells were digested and transferred to a 1.5mL centrifuge tube and resuspended in PBS for washing. Centrifuge at 12000 rpm and 4°C for 3 minutes, then the supernatant was aspirated and the cell pellet was lysed by adding 150μL of 1mol / L NaOH solution to the centrifuge tube. After mixing, the cell pellet was heated at 95°C in a wet-dryer for 0.5 hours. After cooling to room temperature, the solution was transferred to a 96-well plate and the absorbance at 405nm was measured using a microplate reader. Three replicates were set for each drug concentration. A standard melanin concentration curve was determined using a standard melanin sample to calculate the cellular melanin content. The relative melanin content was plotted against the YM818 concentration.

[0087] refer to Figure 8 , Figure 8 The graph and bar chart show the inhibitory effect of YM818 according to the present invention on melanin production in B16F10 cells. Figure 8 A represents the appearance of B16F10 cell melanin lysate and melanin precipitation. Figure 8 B represents the bar graph of melanin content. B16F10 cells were treated with YM818 at concentrations of 0μmol / L, 30μmol / L, 40μmol / L, 50μmol / L and 60μmol / L for 48 hours and then the changes in intracellular melanin content were measured. The results are shown in Figure 2. Figure 8 As shown in the melanin cleavage diagram ( Figure 8A) It can be observed that the melanin content of B16F10 cells is significantly reduced after the treatment of YM818, and the color of the melanin becomes lighter. Figure 8 As shown in Figure B, YM818 at concentrations of 30 μmol / L, 40 μmol / L, 50 μmol / L, and 60 μmol / L significantly reduced intracellular melanin content compared to the control group, exhibiting a concentration-dependent effect. In the 60 μmol / L YM818-treated group, melanin content decreased to 35.8% compared to the control group, demonstrating that YM818 significantly inhibits melanin production in B16F10 cells.

[0088] Continue to refer Figure 9 , Figure 9 The effect of YM818 on key proteins in intracellular melanin synthesis is shown. Figure 9 A represents the target protein band. Figure 9 B. Figure 9 C and Figure 9 D represents the expression levels of TYR, TRP2, and MITF proteins, respectively. Western blot analysis was performed to determine the effects of YM818 at different concentrations of 30 μmol / L, 40 μmol / L, 50 μmol / L, and 60 μmol / L for 48 h on the expression of TYR, TRP2 (tyrosinase-related protein 2), and MITF (microphthalmia-related transcription factor) proteins in melanin production in B16F10 cells. Figure 9 As shown, with increasing YM818 concentration, TYR, TRP2, and MITF all showed a decreasing trend, with TYR and MITF showing a concentration-dependent effect. At concentrations of 50 μmol / L and 60 μmol / L, the inhibitory effects on TYR and TRP2 protein expression were significantly higher than those in the control group (P < 0.01). TRP2 expression showed no significant difference at 30 μmol / L YM818, while MITF protein expression showed a significant change compared to the control group. These results indicate that YM818 inhibits the expression of TYR, MITF, and TRP2, key proteins in melanogenesis in B16F10 cells.

[0089] refer to Figure 10 , Figure 10 The effect of YM818 on cellular melanin production under AZD5363 treatment according to the present invention is shown. AZD5363 is an inhibitor of protein kinase B (AKT), inhibiting AKT phosphorylation, which promotes cell apoptosis. Western blot analysis was used to examine changes in the expression of phosphorylated AKT protein (p-AKT protein) in B16F10 cells. Figure 10 A represents the expression of p-AKT protein in B16F10 cells. Figure 10 B represents the melanin content and the apparent figure of the lysate. After B16F10 cells were pretreated with 10 μmol / L AKT inhibitor AZD5363 for 1 h, 60 μmol / L YM818 was added to the medium and incubated for 48 h. Figure 10 A is the change in the expression level of p-AKT protein after drug treatment. The expression level of p-AKT protein was significantly reduced in the 0.5 μmol / L AZD5363 treatment group compared with the control group. The expression level of p-AKT was significantly increased in the experimental group treated with 0.5 μmol / L AZD5363 and 60 μmol / L YM818 compared with the control group. The up-regulation level of the treatment group using 60 μmol / L YM818 alone was higher than that of the treatment group using AZD5363 and YM818 simultaneously. Figure 10 B is the apparent figure and the amount of melanin generated by the cells. It can be seen that the melanin content of the inhibitor treatment group is significantly increased. The melanin generation of the Y818 treatment group is significantly reduced compared with the control group, the inhibitor group and the inhibitor and YM818 co-treatment group. It can be seen that YM818 can significantly inhibit the melanin generation of B16F10 cells. By regulating the activated AKT signaling pathway to down-regulate the expression level of MITF protein, the generation of B16F10 melanin is inhibited.

[0090] Application Example 3

[0091] Reference for stereoscopic microscope observation of zebrafish melanin apparent content experiment Figure 11 , Figure 11 The figure shows the stereoscopic microscope observation image of the zebrafish body surface melanin content according to YM818. After zebrafish embryos were treated with 0 μmol / L, 200 μmol / L, 300 μmol / L, 400 μmol / L and 500 μmol / L YM818 solution for 48 h, the melanin deposition on the fish body surface was observed under the stereoscopic microscope and photographed. Figure 11 It can be seen that the yolk sac, eyes and lateral spine of the control group zebrafish have a large amount of melanin generation, and the melanin particle area is large and the color is dark. Compared with the control group, the melanin particles in the yolk sac and the spine of the YM818 treatment group are significantly reduced, and the melanin color is light, indicating that YM818 has an inhibitory effect on the melanin deposition on the zebrafish body surface.

[0092] The expression level of TYR, TRP1, TRP2 and a-MSH in zebrafish was detected by Western blot method after 0 μmol / L, 200 μmol / L, 300 μmol / L, 400 μmol / L and 500 μmol / L YM818 solution was used for 48 h, and β-actin protein was used as an internal control. The influence of YM818 on the expression level of TYR, TRP1, TRP2 and a-MSH in zebrafish was studied. As shown in Figure 12 As shown, Figure 12 A is a representative image of the target protein band; Figure 12 B. Figure 12 C. Figure 12 D. Figure 12 E are relative protein expression levels. Under the action of YM818, the expression levels of TYR, TRP1 and α-MSH proteins were significantly downregulated compared with the control group, and showed a concentration effect, while the expression level of TRP2 was not affected.

[0093] Application Example 4

[0094] The experiments on the effect of YM818 on cellular melanin transfer included fluorescent microsphere phagocytosis assay to detect the effect of YM818 on HaCAT cell melanin transfer, flow cytometry assay to detect the effect of YM818 on melanin transfer in co-cultured cell systems, and Western Blot assay to detect the effect of YM818 on key proteins of melanin transfer.

[0095] In the experiment of observing the effect of YM818 on HaCAT cell phagocytosis by confocal microscopy, the density of HaCAT cells was adjusted to 10 5 CFU / mL, cultured HaCAT cells were transferred to a 12-well plate containing a sterile slide and cultured in a cell culture incubator for 12 hours. Once the cells were adherent and in good growth condition with an appropriate growth density, the original culture medium was removed and cultured for another 6 hours in serum-free DMEM medium containing 10 μmol / L Ser-Leu-Ile-Gly-Arg-Leu-NH2 (abbreviated as Sligrl) containing YM818 (40, 60 μmol / L) or arbutin (100 μmol / L). The original culture medium was removed and culture medium containing FluoSpheres™ carboxyl-modified microspheres (Thermo Fisher Scientific) (1 μm in diameter) was added at a concentration of 100 cells / cell and cultured for 16 hours. The cells were washed three times with PBS to remove excess microspheres and fixed with 4% paraformaldehyde in a dark environment for 15 minutes. DAPI stain was added to the cell area, and the slide was inverted and placed flat on a glass slide to stain the cells. Care was taken to avoid bubbles that would affect image observation. The slide was fixed with neutral gum and sealed. A Zeiss LSM 780 confocal microscope was used to observe and capture images, and ZEN2.3.0 software was used to process and analyze the images.

[0096] Figure 13 The experimental images taken with a Zeiss LSM 780 confocal microscope show the inhibition of melanin transfer of HaCAT cells by YM818 according to the present invention. Figure 13 A indicates that the confocal magnification is 200X. Figure 13B indicates that the confocal magnification is 400X. Protease-activated receptor-2 (PAR-2), also known as protease-activated peptide, Sligrl-NH2 (Ser-Leu-Ile-Gly-Arg-Leu-NH2), referred to as Sligrl, has the effect of activating PAR-2 ​​and promoting melanin transfer. FluoSpheresTM carboxyl-modified microspheres are fluorescent microspheres with a diameter of 1 μm. In the phagocytosis experiment, the microspheres and HaCAT cells were co-cultured at a ratio of 100:1 to simulate the process of cell transfer of melanin particles. The distribution and density of the fluorescent microspheres in HaCAT cells were observed using a confocal microscope. Figure 13 As shown, the addition of 10 μmol / L sligrl stimulated HaCAT to transfer melanin, and the number of fluorescent microspheres increased significantly compared with the control group. In the 40 μmol / L and 60 μmol / L YM818 treatment groups, the number of fluorescent microspheres was less, showing a concentration effect. The 60 μmol / L YM818 treatment group was better than the positive control 100 μmol / L arbutin treatment group. Therefore, the migration effect of HaCAT cells was significantly inhibited under the action of YM818.

[0097] In the flow cytometry experiment to detect the effect of YM818 on melanin transfer in the co-cultured cell system:

[0098] (1) Construction of control group system: The control group is divided into single culture cells and co-culture cells. The single culture cell system is B16F10 cells stained with CFDA fluorescent labeling and HaCAT cells stained with incubated antibodies; the co-culture cells are single-stained HaCAT cells, single-stained B16F10 cells and cells without staining. B16F10 were cultured in two 6cm culture dishes. When the cells were in good growth state and the growth density was appropriate, PBS buffer containing 2mmol / L CFDA SE was added to one of the dishes and stained at 37℃ in the dark for 40 minutes. The cells in the other dish were not labeled and then transferred to a six-well plate for culture. HaCAT cells were also transferred to a six-well plate to construct the single culture or co-culture system of the control group. After the cells were cultured for 48 hours, the original DMEM medium was removed and washed three times with PBS solution. Trypsin was added to digest the cells for 2 minutes. The B16F10 cells in the single culture and co-culture cell systems were collected and transferred into a centrifuge tube. HaCAT cells in single culture and co-culture systems need to be digested for another 8 minutes, and the cells are collected and transferred to a centrifuge tube and centrifuged at 300g for 3 minutes. The supernatant is aspirated, and 500μL of 4% polymethanol fixative is added to the pellet and fixed at 4°C for 20 minutes. The cells are incubated with 200μL of primary antibody (Pan-Keratin) at 4°C for 30 minutes, followed by incubation with 200μL of secondary antibody CoraLite594-conjugated GoatAnti-Mouse IgG (H+L) at 4°C for 30 minutes. The B16F10 cells collected from the co-culture group are added to the treated HaCAT cells. Flow cytometer analysis is performed using a Fortessa (BD, USA).

[0099] (2) Experimental group reaction steps: B16F10 cells were stained with PBS containing 2 mmol / L CFDA SE at 37°C in the dark for 40 minutes, and co-cultured for 12 hours at a ratio of B16F10 cells: HaCAT cells of 1:3. The cells were observed to be attached to the wall and growing well. They were treated with YM818 or arbutin for 48 hours, rinsed three times with PBS, digested with trypsin for 2 minutes, and rinsed three times with PBS to remove B16F10 cells from the system. The remaining HaCAT cells were incubated with antibodies and then analyzed by flow cytometry. The number of double-positive signals detected in HaCAT cells in the co-culture system is the number of B16F10 cell metastasis. If the number of CFDA SE-positive cells in HaCAT cells decreases, it indicates that the drug has an inhibitory effect on the metastasis of B16F10 cells.

[0100] refer to Figure 14 , Figure 14The flow cytometric method for detecting the changes of melanin transfer of HaCAT cells by YM818 according to the present invention is shown. Figure 14 A is the negative control group, Figure 14 B is the experimental group, Figure 14 C is the level of melanin transferred by HaCAT cells. CFDA SE cell tracking dye can fluorescently label living cells. Once it enters the cells, it can be catalyzed by esterase to produce CFSE that emits strong green fluorescence. The fluorescence in the labeled cells is stable and uniform, and the adjacent cells are not stained. In the experiment, B16F10 was stained with CFDA SE, and keratin in HaCAT cells was labeled with Pan-Keratin antibody. Then, the cells were incubated with secondary antibody CoraLite594-conjugated Goat Anti-Mouse IgG (H+L). After the incubation, the cells were stained with the coupled primary antibody to emit fluorescence. Figure 14 Figures ae and c are the staining results for the control groups, representing staining of B16F10 cells cultured alone, HaCAT cells cultured alone, co-cultured cells stained with either B16F10 or HaCAT alone, and co-cultured cells unstained. The results show that single-cell staining is superior, and the staining of co-cultured cells does not interfere with each other. Figures fj are the results for the experimental groups, representing the control group, 10 μmol / L sligrl, and groups treated with YM818 (40 and 60 μmol / L) and arbutin (100 μmol / L), both containing 10 μmol / L sligrl. In the experimental groups, the proportion of HaCAT cells with double-positive signals in the control group was 13.5%, which increased to 18.6% after sligrl stimulation. The proportions decreased to 10.8% and 8.5% in the 40 and 60 μmol / L YM818-treated groups, respectively, and to 14.1% in the 100 μmol / L arbutin-treated group. The above results indicate that YM818 can significantly inhibit cell melanin metastasis, and its effect is better than arbutin.

[0101] Continue to refer Figure 15 , Figure 15 The effect of YM818 of the present invention on melanin transfer protein in the co-culture system is shown. Figure 15 A is a representative image of the target protein band; Figure 15 B is the relative protein expression level. The expression of PAR-2 ​​protein in B16F10-HaCAT co-culture system cells after 48h treatment with different concentrations of YM818 (30μmol / L, 40μmol / L, 50μmol / L and 60μmol / L) is shown in Figure 3. Figure 14As shown in the figure, compared with the control group, the expression of PAR-2 ​​protein in the four concentration treatment groups was significantly downregulated, showing a concentration effect. The results indicate that YM818 can inhibit the role of promoting melanin metastasis by reducing the expression of PAR-2 ​​protein in cells.

[0102] Application Example 5

[0103] The indole derivative YM818 prepared in Example 1 was used as an antioxidant. The antioxidant effect of YM818 included an in vitro antioxidant test of YM818 and a flow cytometry test to detect the effect of YM818 on intracellular ROS levels.

[0104] In the in vitro antioxidant activity test, this study used the 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid free radical scavenging test (ABTS method) and the 1,1-diphenyl-2-trinitrophenylhydrazine free radical scavenging test (DPPH method) to detect the free radical scavenging ability of YM818. YM818 was prepared with DMSO solvent into solutions of different concentrations of 0mmol / L, 0.5mmol / L, 1.0mmol / L, 1.5mmol / L, 2.0mmol / L, and 2.5mmol / L for use. Vitamin C was used as a positive control sample. The method steps are as follows:

[0105] ABTS assay: Weigh 0.0768g of ABTS and dissolve it in 20mL of double-distilled water. Weigh 0.0134g of potassium persulfate and dissolve it in 20mL of double-distilled water. Mix the two solutions in equal proportions and incubate at room temperature in the dark for 16 hours. This solution is used as the ABTS stock solution. Before the experiment, dilute it with PBS to an ABTS working solution. The absorbance at 734nm is 0.70±0.02, and the solution is stored in a dark environment. In a 96-well plate, add 180μL of ABTS solution and 20μL of YM818 solution of varying concentrations to each well. Incubate at room temperature in the dark for 6 minutes. Place the plate in a microplate reader and measure the absorbance of the reaction solution at 734nm. Double-distilled water was used as the control group, and three replicates were performed for each sample.

[0106] DPPH Assay: Accurately weigh 14 mg of DPPH solid powder using a balance, dissolve it in 95% methanol, and adjust the volume to 200 mL. Store the solution in a refrigerator at 4°C, protected from light, as this DPPH stock solution. Prepare the solution immediately before use. First, adjust the concentration of the DPPH working stock solution using methanol to an absorbance between 0.7 and 0.8 at 517 nm. After adjustment, add 1.5 mL of DPPH solution and 50 μL of various YM818 concentrations to a centrifuge tube. Mix thoroughly, incubate in the dark for 30 minutes, and measure the absorbance of the reaction solution at 517 nm using a microplate reader. A methanol solution was used as a control. Three replicates were performed for each sample.

[0107] The free radical scavenging rate of YM818 can be calculated based on the absorbance of the reaction solution. A scavenging rate curve corresponding to YM818 concentration is plotted. The results are expressed in mmol / L at different YM818 concentrations. The antioxidant capacity of YM818 is calculated as follows: Antioxidant capacity (%) = (1 – A1 / A0) × 100%, where A0 is the absorbance of the control solution and A1 is the absorbance of the drug-treated solution.

[0108] Experimental results reference Figure 16 , Figure 16 The figure shows the experimental results of the ABTS and DPPH free radical scavenging ability of YM818 of the present invention. Figure 16 A represents the scavenging rate of YM818 on ABTS free radicals, Figure 16 B represents the scavenging rate of YM818 against DPPH free radicals. The catalytic reaction of tyrosinase involves multiple oxidation reactions. In the enzyme catalysis, most TYR inhibitors also have antioxidant effects. The chemical structure of YM818 contains a carbonyl functional group, a nitrogen atom heterocyclic ring, and a hydroxyl group, which can act as an oxygen hydrogen donor, thus having the effect of scavenging free radicals. The antioxidant capacity of YM818 was analyzed by ABTS and DPPH experiments. The results are as follows: Figure 16 As shown in the figure, YM818 has a significant antioxidant effect and can effectively remove ABTS ( Figure 16 A) and DPPH( Figure 16 B) Free radicals, IC 50 The values ​​were 0.67 and 1.69 mmol / L, respectively, which are comparable to those of vitamin C (0.59 and 1.93 mmol / L, respectively). The antioxidant capacity of YM818 and vitamin C is summarized in Table 1. In summary, YM818 has a strong antioxidant effect.

[0109] Table 1 Antioxidant effects of YM818 and vitamin C

[0110]

[0111] In the flow cytometry experiment to detect the effect of YM818 on the level of ROS in B16F10 cells, B16F10 cells were cultured and the density was adjusted to 10 5CFU / mL, the cells were transferred to six-well plates, and cultured for 12 h. After the cells adhered, the original culture medium was removed, and culture medium containing YM818 at concentrations of 0 μmol / L, 15 μmol / L, 30 μmol / L, 45 μmol / L, and 60 μmol / L was added, respectively. The cells were placed in a cell incubator and cultured for 48 h. The B16F10 cells were digested and transferred to a centrifuge tube, centrifuged at 2000 rpm for 3 min, and the supernatant was discarded. The cells were resuspended in serum-free DMEM medium and washed three times. An active oxygen detection kit (Beyotime Company) was used to load the fluorescent probe DCFH-DA, and the specific operation was as follows: DCFH-DA was diluted to 10 μmol / L at a ratio of 1:1000 with serum-free cell culture solution. The cells were collected and transferred to the diluted medium, and reacted at 37°C for 20 min. The centrifuge tube was inverted every 5 min to mix the probe and cells thoroughly. After the reaction, the cells were centrifuged and the original culture medium was removed. The cells were resuspended in serum-free medium and washed three times to completely remove the DCFH-DA that did not enter the cells. Then, the culture solution was filtered with a 300-mesh filter and transferred to a flow tube (BD Company). The flow cytometer FC500 was used for on-machine detection, and the WinMDI2.9 software was used for analysis of the detection results.

[0112] Reference is made to the experimental results Figure 17 , Figure 17 Figure 6 shows the effect of YM818 on the intracellular reactive oxygen species of B16F10 cells, wherein Figure 17 a- Figure 17e represents the YM818 concentration of 0μmol / L, 15μmol / L, 30μmol / L, 45μmol / L and 60μmol / L respectively. Under the action of reactive oxygen species in the cells, the fluorescent probe DCFH-DA penetrates the cell membrane and is hydrolyzed to produce DCFH, which can be oxidized to produce a fluorescent substance DCF. The ROS content in the cells can be quantitatively measured by fluorescence detection using a flow cytometer. From the above analysis of the antioxidant capacity of YM818, it can be seen that YM818 has a high efficiency in scavenging free radicals. The experiment was carried out by loading the fluorescent probe and using a flow cytometer to detect the changes in the fluorescence signal in B16F10 cells. The fluorescence intensity is proportional to the level of ROS in the cells. After treating B16F10 cells with YM818 at different concentrations of 0μmol / L, 15μmol / L, 30μmol / L, 45μmol / L, and 60μmol / L for 48 hours, intracellular ROS levels were measured. Compared with the control group, the ROS levels in the YM818-treated groups were all reduced, and a decreasing trend was observed with increasing YM818 concentration. In the 60μmol / L YM818-treated group, the ROS level was 26.4, a 60% decrease compared to the control (66.0). These results indicate that YM818 has strong antioxidant properties in cells and can significantly reduce ROS levels in B16F10 cells.

[0113] The results of enzymatic experiments showed that YM818 inhibited monophenolase IC 50 The value is 0.261mmol / L, and the IC value of diphenolase inhibition is 0.261mmol / L. 50 The value was 0.372 mmol / L, indicating reversible mixed inhibition of diphenolase. Fluorescence quenching and MOE molecular docking results indicated that YM818 inhibited enzyme activity through both hydrogen bonding and hydrophobic interactions. Regarding melanin production, YM818 significantly reduced melanin content and TYR activity in B16F10 cells. By activating the AKT pathway, it downregulated MITF expression, further downregulating the expression of TYR family members, ultimately inhibiting melanin production. Zebrafish experiments demonstrated that YM818 inhibited both melanin content and TYR activity, suppressing melanin production by downregulating the expression of TYR, TRP1, and α-MSH proteins. Regarding melanin transfer, YM818 significantly inhibited melanin transfer and downregulated the expression of PAR-2, a key protein in melanin transfer. Antioxidant assays demonstrated that YM818 possessed antioxidant capacity and significantly reduced intracellular ROS levels.

[0114] Application Example 6

[0115] The indole derivative YM818 prepared in Example 1 was used in the treatment of lung cancer. The experiments on YM818 inducing mitochondrial-mediated apoptosis in the A549 cell line included experiments on YM818's inhibition of A549 cell viability and colony formation ability, the effects of YM818 on A549 cell morphology, cell migration and invasion ability, cell cycle, cell migration, and mitochondrial dysfunction.

[0116] In the experiment of studying the inhibition of A549 cell viability and colony formation ability by YM818, in order to test the effect of YM818 on the viability of human non-small cell lung cancer cell line A549, A549 cells were treated with different concentrations of YM818 for 24h and 48h, and then the cell viability was detected using CCK-8 assay. Figure 18 ,like Figure 18 As shown in A, YM818 inhibited the growth of A549 cells in a dose-dependent and concentration-dependent manner. After 24h and 48h of YM818 treatment, IC 50 The values ​​were 128.1μM and 101.0μM respectively. After 48h of YM818 treatment, the number and diameter of A549 cell colonies decreased significantly (e.g. Figure 18 B).

[0117] In the experiment to study the effect of YM818 on the morphology of A549 cells, the cells were plated at 5×10 5 The cells were plated at 100 μl / well and treated with DMSO and YM818 for 24 hours respectively the next day. A 10 μL pipette tip was used to artificially scratch a cell wound in each well of the six-well plate, and then washed with PBS and cultured for another 24 hours. Finally, the adherent cells were stained with crystal violet and the artificially scratched cell wound was photographed using a phase contrast microscope (TE2000-U, Nikon, Tokyo, Japan, equipped with NIS-Element software). The photographic results are referenced to Figure 19 , Figure 19 The experimental images taken with a TE2000-U phase contrast microscope show the effect of YM818 on A549 cell morphology. Figure 19 A shows the effect of YM818 treatment on A549 cell morphology under optical microscope. Figure 19 B shows the effect of YM818 treatment on the morphology of A549 cells under Giemsa staining. After 48 hours of treatment with different concentrations of YM818, the morphology of A549 cells showed obvious changes. Figure 19 As shown, the cells in the control group were spindle-shaped, plump and adherent. After drug treatment, the cell number decreased significantly, the cells became curved and wrinkled, and a large number of suspended cells appeared in the high-concentration group.

[0118] In the experiment to study the effect of YM818 on the migration and invasion ability of A549 cells, a cell migration assay was used to detect the invasion ability of the A549 cell line. After 48 hours, the cells on the top surface of the bottom of the upper chamber were wiped off with a cotton swab, and the cells that had migrated or invaded to the other side of the upper chamber were then fixed with paraformaldehyde for 30 minutes, and then stained with 0.5% crystal violet for 15 minutes. The upper chamber was then washed three times with PBS and dried. Finally, a phase contrast microscope was used to randomly select 5 areas to take pictures and count the cells that successfully migrated or invaded in the area. Experimental results reference Figure 20 , Figure 20 A represents the effect of YM818 on the migration ability of A549 cells. Figure 20 B shows the effect of YM818 on the invasion ability of A549 cells. Figure 20 C shows the effect of YM818 on the protein levels of β-catenin and MMP-2 in A549 cells. After cell scratching, compared with the cells in the control group, which gradually migrated to the middle area, almost no cells in the high-concentration drug treatment group migrated to the middle area, indicating that YM818 inhibited the migration ability of A549 cells (refer to Figure 20 A). In the cell migration assay, as the concentration of YM818 increased, fewer and fewer cells were able to penetrate the bottom membrane (polycarbonate membrane) and reach the lower chamber, indicating that YM818 significantly inhibited the invasive ability of A549 cells (refer to Figure 20 B). Western blot results clearly showed that the downregulation of β-catenin and MMP-2 proteins was dose-dependent (refer to Figure 20 C).

[0119] In the experiment to study the effect of YM818 on the A549 cell cycle, after 24 hours of YM818 treatment of A549 cells, the cells were collected and washed. The cells were fixed with 75% ethanol, then resuspended and treated with 5uL of 10mg / mL RNase at 37°C for 30min; after that, DNA was treated with 5uL of 10mg / mL PI at 4°C in the dark for 30min. Finally, a flow cytometer (FC500, Beckman Coulter) was used to detect the DNA content in the cells, and the data were output by ModFit LT 5.0 software. Experimental results refer to Figure 21 , Figure 21 A represents the results of treating A549 cells with different concentrations of YM818 for 24 hours and measuring them by flow cytometry (FCM). Figure 21 A (top); the bar graph shows the quantitative results. Figure 21B shows the effect of YM818 on the expression of A549 cell cycle-related proteins. In order to further study the inhibitory effect of YM818 on the proliferation of A549 cells, the changes in the cell cycle after YM818 treatment were quantitatively detected by PI staining. The distribution of cell cycle is shown in Figure 2. Figure 21 As shown in A, YM818 can induce cell cycle arrest in A549 cells at the G0 / G1 phase. Western blot results showed that the expression level of CDK2 protein was significantly reduced in a drug concentration-dependent manner ( Figure 21 B).

[0120] For the Annexin V-FITC / PI double staining assay, apoptosis was detected using the Annexin V-FITC / PI Apoptosis Detection Kit, used according to the manufacturer's instructions, and assayed by flow cytometry. Cells were plated in six-well plates and treated with YM818 for 48 hours, then trypsinized and resuspended in buffer. Subsequently, 5 μL of Annexin V-FITC and 5 μL of propidium iodide (PI) were added, incubated for 15 minutes, and apoptotic cells were analyzed using an FC500 flow cytometer.

[0121] The experimental results are as follows Figure 22 As shown, Figure 22 FIG2 shows the experimental results of YM818 inducing apoptosis of A549 cells according to one embodiment of the present invention. Figure 22 A indicates the effect of YM818 on A549 cells after Hoechst 33258 staining. Figure 22 B represents the cell apoptosis analysis after Annexin V-FITC / PI staining by flow cytometry, and the bar graph shows the cell apoptosis rate; Figure 22 C shows the effect of YM818 on the expression of apoptosis-related proteins in A549 cells. Figure 22 As shown in Figure A, the A549 cells in the control group exhibited weak blue fluorescence, while the A549 cells in the YM818-treated group for 48 hours emitted intensive strong blue light, indicating that YM818 may induce apoptosis by inhibiting the proliferation of A549 cells. This conclusion was also confirmed by flow cytometry analysis. Figure 22 As shown in Figure B, as the concentration of YM818 increases, the apoptosis rate of A549 cells also increases. To reveal its molecular mechanism, we performed a Western blot experiment, and the results are shown in Figure 2. Figure 22As shown in Figure C, the Bax / Bcl-2 ratio increased. In addition, the precursors of caspase-9, caspase-3, and poly(ADP-ribose) polymerase-1 (PARP-1) were cleaved, and the expression of their cleaved forms increased. These results indicate that YM818 induces apoptosis in A549 cells through the mitochondrial pathway.

[0122] In experiments investigating YM818-induced mitochondrial dysfunction in A549 cells, cells were plated in six-well plates for 12 hours and then treated with YM818 at various concentrations, including 50 μmol / L, 100 μmol / L, and 150 μmol / L, for 48 hours. For ROS analysis, treated cells were harvested and resuspended in DCFH-DA. The cells were incubated in the dark for 30 minutes before being harvested and analyzed using an FC500 flow cytometer. Separately, the harvested cells were treated with JC-1 staining solution for one hour. Finally, ΔΨm values ​​were quantified using an FC500 flow cytometer. Typically, the ratio of red fluorescence to green fluorescence of 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) reflects changes in ΔΨm.

[0123] Experimental results reference Figure 23 , Figure 23 The figure shows the experimental results of YM818-induced mitochondrial dysfunction in A549 cells. Figure 23 A represents the fluorescence image of the changes in mitochondrial transmembrane potential in A549 cells after YM818 treatment. Figure 23 Figure B shows changes in mitochondrial transmembrane potential in A549 cells after YM818 treatment. The fluorescent probe JC-1 can be used to monitor mitochondrial membrane polarity and the involvement of mitochondrial pathways. As shown in the figure, in the mitochondria of A549 cells, multimeric JC-1 exhibited bright red fluorescence in the control group. However, in the YM818-treated group, the red fluorescence gradually dimmed with increasing drug concentration, and green fluorescence slowly emerged. This shift from red to green fluorescence indicates that YM818 treatment causes a gradual decrease in the ΔΨm of A549 cells (a sign of apoptosis).

[0124] It is clear that a person skilled in the art can make various modifications and alterations to the embodiments of the application without departing from the spirit and scope of the application. In this manner, the application is also intended to cover these modifications and alterations if they come within the scope of the claims of the application and their equivalents. The word "comprising" does not exclude the presence of elements or steps not listed in a claim. The mere fact that different claims enumerate mutually different measures does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

Claims

1. An indole derivative, characterized in that The chemical structural formula of the indole derivative is shown below:

2. The method for preparing the indole derivative according to claim 1, characterized in that: The preparation method comprises: S1, preparing a reaction medium, dissolving copper acetate and triethylamine in isopropyl alcohol, and stirring until completely dissolved to obtain the reaction medium; S2, preparing an indole derivative solution, adding 5-bromoisatin and indole to the reaction medium, and stirring thoroughly at room temperature until the reactants are completely converted; S3, extracting the indole derivative powder, distilling off the reaction solvent to obtain a crude product, purifying and recrystallizing to obtain a high-purity solid powder of the indole derivative.

3. The method for preparing an indole derivative according to claim 2, wherein: The S1 step includes the following: S11, weigh 0.4 g (2 mmol) of copper acetate and 0.2 g (2 mmol) of triethylamine and place them in a reaction flask; S12, adding 10 ml of isopropanol to the reaction flask and stirring for 10 minutes to completely dissolve the isopropanol to obtain the reaction medium.

4. The method for preparing an indole derivative according to claim 3, wherein: The S2 step includes adding 4.52 g (20 mmol) of 5-bromoisatin and 2.8 g (24 mmol) of indole into the reaction flask, stirring the mixture sufficiently at room temperature to allow a post-coupling reaction to occur, and monitoring the reaction progress by thin layer chromatography.

5. The method for preparing an indole derivative according to claim 2, wherein: The S3 step includes: S31, performing reduced pressure distillation using a rotary evaporator to remove the isopropanol solvent in the reaction system, collecting the residue, and obtaining the crude product of the reactant; S32, adding the crude product to a mixed solvent of petroleum ether and dichloromethane, stirring thoroughly to dissolve, and then recrystallizing to obtain a solid powder of the indole derivative.

6. Use of the indole derivative according to claim 1 or the indole derivative prepared by the method for preparing the indole derivative according to any one of claims 2 to 5 in the preparation of a medicament for inhibiting the production and / or transfer of melanin in cells.

7. Use of the indole derivative according to claim 1 or the indole derivative prepared by the method for preparing the indole derivative according to any one of claims 2 to 5 in the preparation of an antioxidant.

8. Use of the indole derivative according to claim 1 or the indole derivative prepared by the method for preparing the indole derivative according to any one of claims 2 to 5 in the preparation of a medicament for treating lung cancer.

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