An 8-substituted indanone-quinoxaline thiourea compound, its preparation method and application

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to an 8-substituted indanone-quinoxaline thiourea compound, its preparation method, and its applications. This invention provides an 8-substituted indanone-quinoxaline thiourea compound with a novel structure. Experiments have shown that this compound binds to DNA via an intercalation mechanism, effectively inhibiting the proliferation of various tumor cells and exhibiting excellent antitumor activity. Its overall effect is more prominent than that of the broad-spectrum antitumor drug doxorubicin, demonstrating significant pharmaceutical value in the field of antitumor drug development. Furthermore, the preparation method of this compound is simple, mild, and inexpensive, making it suitable for large-scale industrial production and application.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology. More specifically, it relates to an 8-substituted indanone-quinoxaline thiourea compound, its preparation method, and its application. Background Technology

[0002] Cancer is a major disease threatening human life. In 2022, nearly 20 million new cancer cases were diagnosed globally, with nearly half of these new cases (49.2%) and the majority of cancer deaths (56.1%) occurring in Asia. Among various cancers, breast cancer and colorectal cancer in women rank second and third in incidence, respectively, due to their high incidence and mortality rates, posing a significant threat to the health and lives of people worldwide, especially women. While cervical cancer ranks eighth in incidence, its harm to women's health is equally undeniable, especially in regions with relatively limited medical resources, where both incidence and mortality rates remain high.

[0003] Currently, cancer, as a serious challenge facing the medical community, is driving continuous exploration and innovation in this field. Among the many methods of cancer treatment, chemotherapy plays a crucial role and has achieved significant clinical results. Doxorubicin (DOX), as one of the classic and broad-spectrum anticancer drugs, has demonstrated significant therapeutic effects on many types of malignant tumors, becoming a highlight in the field of chemotherapy.

[0004] In clinical treatment, there remains a relative scarcity of drugs for cancer patients. Therefore, to address the challenges of cancer treatment, the medical community urgently needs to increase research and development efforts to develop more new drugs with highly effective anti-cancer effects, in order to provide patients with more options and further improve treatment outcomes. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiency and inadequacy of the current treatment drugs for cancer patients, and to provide an 8-substituted indanone-quinoxaline thiourea compound.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned 8-substituted indanone-quinoxaline thiourea compounds.

[0007] Another object of the present invention is to provide the use of the above-mentioned 8-substituted indanone-quinoxaline thiourea compounds in the preparation of antitumor drugs.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] This invention protects an 8-substituted indanone-quinoxaline thiourea compound, the structural formula of which is shown in formula (I):

[0010]

[0011] Where R is a halogen atom, NO2, or CN.

[0012] The 8-substituted indanone-quinoxaline thiourea compounds prepared in this invention bind tightly to cancer cell DNA through intercalation. This binding process alters the double helix structure of DNA, changing the base pair stacking state and weakening electron-electron interactions. Consequently, the bases' ability to absorb ultraviolet light decreases, ultimately producing a hypochromic effect. Importantly, the severity of the hypochromic effect is directly proportional to the strength of the interaction between the compound and cancer cell DNA; that is, the more pronounced the hypochromic effect, the stronger the alteration of the DNA double helix structure by the compound, thus more effectively inhibiting cancer cell proliferation. Furthermore, this interaction may be further stabilized through various non-covalent interactions, including hydrophobic interactions, hydrogen bonds, and π-π stacking. The inventors discovered that adjusting the type of groups at the 8-substitution position of these compounds (such as halogen atoms, -NO2, -CN, etc.) can affect the strength of hydrogen bond formation. Specifically, when the electron-withdrawing effect of these groups weakens, the hydrogen bond interaction is significantly enhanced. This enhanced hydrogen bonding strengthens the interaction between these compounds and cancer cell DNA, making the hypocoloration effect more pronounced and thus more effectively inhibiting cancer cell proliferation.

[0013] Furthermore, the halogen atom is F, Cl, or Br.

[0014] This invention protects the method for preparing the above-mentioned 8-substituted indanone-quinoxaline thiourea compounds, and synthesizing...

[0015] The route is as follows:

[0016]

[0017] Specifically, the following steps are included:

[0018] S1. Compound 1 and Compound 2 are mixed in a first solvent, an acidic catalyst is added, and the mixture is reacted at 70-80°C until fully reacted. After post-treatment, intermediate product 3 is obtained.

[0019] S2. Mix the intermediate product 3, compound 4, and acid catalyst obtained in step S1 in a second solvent, react them thoroughly at 70-80°C, and then treat them to obtain the 8-substituted indanone-quinoxaline thiourea compound shown in formula (I).

[0020] The definition of R is the same as described above.

[0021] The preparation method of this compound is simple, the conditions are mild, the raw materials are inexpensive and readily available, and the production cost is low. It provides a new reference route for the research and development of anti-tumor drugs and has potential medical value and broad application prospects.

[0022] Furthermore, the intermediate product 3 can be synthesized via the above-described route or obtained through market purchase.

[0023] Further, in step S1, the molar ratio of compound 1 to compound 2 is (1-2):1.

[0024] Preferably, in step S1, the molar ratio of compound 1 to compound 2 is (1.1 to 2):1. This ratio setting allows compound 1 to be in appropriate excess relative to compound 2, which helps to ensure a complete reaction.

[0025] Further, in step S1, the first solvent includes one or more of ethanol, ethyl acetate, and chloroform.

[0026] Further, in step S1 or step S2, the acidic catalyst includes one or more of p-toluenesulfonic acid, acetic acid, hydrochloric acid, phosphoric acid, and acetic acid.

[0027] Furthermore, in step S1, the mixing is performed by heating and dissolving the mixture.

[0028] Furthermore, the heating temperature is 70–80°C.

[0029] Further, in step S1, the molar ratio of compound 1 to the acidic catalyst is 1:(2-10). Adding an acidic catalyst within this ratio range, whose acidic properties facilitate the reaction, thereby accelerating the reaction rate.

[0030] Furthermore, in step S1, the time for the complete reaction is 2 to 3 hours.

[0031] Furthermore, in step S1, the post-processing includes cooling, filtering, washing, and drying.

[0032] Furthermore, the cooling refers to cooling the post-reaction mixture to room temperature.

[0033] Furthermore, the filtration is a vacuum filtration, in which the cooled mixture is filtered by vacuum and the filter residue is collected.

[0034] Furthermore, the washing process involves washing the filter residue with ethanol 1 to 3 times.

[0035] Furthermore, the drying process involves drying the washed filter residue.

[0036] Specifically, in step S1, the post-processing includes cooling the reaction mixture to room temperature, filtering the cooled mixture, collecting the filter residue, washing the filter residue with ethanol 1 to 3 times, and drying the washed filter residue.

[0037] Furthermore, in step S2, the molar ratio of intermediate product 3 to compound 4 is 1:(1-2).

[0038] Preferably, in step S2, the molar ratio of intermediate product 3 to compound 4 is 1:(1.1~2). This ratio setting allows compound 4 to be in appropriate excess relative to intermediate product 3, which helps to ensure a complete reaction.

[0039] Further, in step S2, the second solvent includes one or more of dichloromethane, methanol, and tetrahydrofuran.

[0040] Furthermore, in step S2, the molar ratio of the intermediate product 3 to the acidic catalyst is 1:(0.1~1). Within this ratio range, adding a relatively small amount of acidic catalyst can also significantly promote the reaction.

[0041] Furthermore, in step S2, the time for the complete reaction is 8 to 10 hours.

[0042] Furthermore, in step S2, the post-processing includes filtration, washing, and drying.

[0043] Furthermore, the filtration involves filtering the reaction solution while it is still hot and collecting the filter residue.

[0044] Furthermore, the washing process involves washing the filter residue 1 to 3 times with hot methanol.

[0045] Furthermore, the drying process involves drying the washed filter residue.

[0046] Specifically, in step S2, the post-processing includes filtering the reaction solution while it is hot, washing the filter residue with hot methanol 1 to 3 times, and then drying the washed filter residue.

[0047] This invention protects the use of the above-mentioned 8-substituted indanone-quinoxaline thiourea compounds in the preparation of antitumor drugs.

[0048] Furthermore, the tumor is one or more of breast cancer, cervical cancer, and colon cancer.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] This invention provides a novel 8-substituted indanone-quinoxaline thiourea compound. Experiments have demonstrated that this compound binds to DNA via an intercalation mechanism, effectively inhibiting the proliferation of various tumor cells and exhibiting excellent antitumor activity. Its overall effect is more pronounced than that of the broad-spectrum antitumor drug doxorubicin, making it of significant pharmaceutical value in the field of antitumor drug development. Furthermore, the preparation method of this compound is simple, mild, and inexpensive, making it suitable for large-scale industrial production and application. Attached Figure Description

[0051] Figure 1 The 1H NMR spectrum of compound 8-chloro-11H-indo[1,2-b]quinoxaline-11-one thiocarbazone provided in Example 1.

[0052] Figure 2 The carbon NMR spectrum of compound 8-chloro-11H-indo[1,2-b]quinoxaline-11-one thiocarbamate provided in Example 1;

[0053] Figure 3 The 1H NMR spectrum of compound 8-bromo-11H-indo[1,2-b]quinoxaline-11-one thiocarbamate provided in Example 2;

[0054] Figure 4 The carbon NMR spectrum of compound 8-bromo-11H-indo[1,2-b]quinoxaline-11-one thiocarbamate provided in Example 2;

[0055] Figure 5 The 1H NMR spectrum of compound 8-nitro-11H-indo[1,2-b]quinoxaline-11-one thiocarbazone provided in Example 3;

[0056] Figure 6 The carbon NMR spectrum of compound 8-nitro-11H-indo[1,2-b]quinoxaline-11-one thiocarbamate provided in Example 3;

[0057] Figure 7 The UV-Vis spectrum of compound 8-chloro-11H-indo[1,2-b]quinoxaline-11-one thiocarbamate provided in Example 1 as a function of circulating tumor DNA concentration;

[0058] Figure 8 The UV-Vis spectrum of compound 8-bromo-11H-indo[1,2-b]quinoxaline-11-one thiocarbamate provided in Example 2 as a function of circulating tumor DNA concentration;

[0059] Figure 9 The UV-Vis spectrum of compound 8-nitro-11H-indo[1,2-b]quinoxaline-11-one thiocarbazone provided in Example 3 as a function of circulating tumor DNA concentration;

[0060] Figure 10 The fluorescence spectrum of compound 8-chloro-11H-indo[1,2-b]quinoxaline-11-one thiocarbamate provided in Example 1;

[0061] Figure 11The fluorescence spectrum of compound 8-bromo-11H-indo[1,2-b]quinoxaline-11-one thiocarbamate provided in Example 2;

[0062] Figure 12 Fluorescence spectrum of compound 8-nitro-11H-indo[1,2-b]quinoxaline-11-one thiocarbamate provided in Example 3;

[0063] Figure 13 The circular dichroism spectrum of compound 8-chloro-11H-indo[1,2-b]quinoxaline-11-one thiocarbamate provided in Example 1;

[0064] Figure 14 The circular dichroism spectrum of compound 8-bromo-11H-indo[1,2-b]quinoxaline-11-one thiocarbamate provided in Example 2;

[0065] Figure 15 The circular dichroism spectrum of compound 8-nitro-11H-indo[1,2-b]quinoxaline-11-one thiocarbazone provided in Example 3;

[0066] Figure 16 2D diagram of molecular docking of compound 8-chloro-11H-indo[1,2-b]quinoxaline-11-one thiocarbamate provided in Example 1;

[0067] Figure 17 2D diagram of molecular docking of compound 8-bromo-11H-indo[1,2-b]quinoxaline-11-one thiocarbamate provided in Example 2;

[0068] Figure 18 2D molecular docking diagram of compound 8-nitro-11H-indo[1,2-b]quinoxaline-11-one thiocarbazone provided in Example 3. Detailed Implementation

[0069] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0070] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0071] Example 1 Preparation of 8-chloro-11H-indeno[1,2-b]quinoxaline-11-one thiocarbazone The synthetic route of 8-chloro-11H-indeno[1,2-b]quinoxaline-11-one thiocarbazone is as follows:

[0072]

[0073] (1) Preparation of intermediate product 3-1

[0074] Weigh 0.5080 g of compound 2 (2.85 mmol) and 0.4520 g of compound 1-1 (3.17 mmol) into a 250 mL flask, add 25 mL of ethanol, heat to 70 °C, stir to dissolve, then add 0.5 mL of glacial acetic acid (8.3 mmol), heat to 75 °C and reflux for 3 h. After the reaction is complete, cool to room temperature, filter, wash three times with ethanol, and dry to obtain a brownish-yellow solid product, which is intermediate 3-1, with a yield of 78.72%.

[0075] (2) Preparation of compound I-1

[0076] Weigh 0.069 g of intermediate product 3-1 (0.26 mmol) prepared in step (1) into a 250 mL three-necked flask, add 30 mL of dichloromethane to dissolve the solid completely, then add 25 mL of methanol, heat and stir, then add 0.0427 g of compound 4 (0.47 mmol) and 0.0155 g of p-toluenesulfonic acid (0.0901 mmol) sequentially, heat to 80 °C and reflux for 10 h. After the reaction is complete, filter while hot, wash three times with hot methanol, and dry to obtain a yellow solid product, which is compound I-1 (8-chloro-11H-indeno[1,2-b]quinoxaline-11-one thiocarbamate), with a yield of 67.31%. Its NMR data are as follows:

[0077] 1H NMR data: 1 ¹H-NMR (400MHz, DMSO-d⁶) δ 12.50 (s, 1H), 9.10 (s, 1H), 8.85 (s, 1H), 8.24 (d, J = 2.3Hz, 1H), 8.16 (d, J = 7.2Hz, 1H), 8.12 (d, J = 8.9Hz, 1H), 8.07 (d, J = 7.3Hz, 1H), 7.90 (dd, J = 8.9, 2.4Hz, 1H), 7.71 (td, J = 7.5, 1.1Hz, 1H), 7.66 (td, J = 7.5, 1.2Hz, 1H); its ¹H NMR spectrum is as follows: Figure 1 As shown;

[0078] Carbon NMR data: 13 C-NMR (100MHz, DMSO-d6): δ 179.21, 154.82, 148.04, 142.16, 138.83, 138.78, 136.35, 136.08, 135.38, 133.07, 131.67, 131.38, 131.10, 128.63, 122.93, 122.51; its carbon NMR spectrum is as follows: Figure 2 As shown.

[0079] Mass spectrometry data: HRMS (ESI, m / z): calcd.for C 16 H 11 ClN5S(M+H) + 340.0418, found340.0413.

[0080] Example 2 Preparation of 8-bromo-11H-indo[1,2-b]quinoxaline-11-one thiocarbazone The synthetic route of 8-bromo-11H-indo[1,2-b]quinoxaline-11-one thiocarbazone is shown below:

[0081]

[0082] (1) Preparation of intermediate product 3-2

[0083] Weigh 1.0636 g of compound 2 (5.97 mmol) and 1.1382 g of compound 1-2 (6.12 mmol) into a flask, add 25 mL of ethanol, heat to 75 °C, stir to dissolve, then add 1 mL of glacial acetic acid (16.6 mmol), heat to 80 °C and reflux for 3 h. After the reaction is complete, cool to room temperature, filter, wash three times with 30 mL of ethanol, and dry to obtain a brownish-yellow solid product, i.e., intermediate 3-2, with a yield of 77.31%.

[0084] (2) Preparation of compound I-2

[0085] Weigh 0.0808 g of intermediate product 3-2 (0.26 mmol) obtained in step (1) into a flask, add 15 mL of tetrahydrofuran to dissolve the solid completely, then add 9 mL of methanol, heat and stir, then add 0.0369 g of compound 4 (0.40 mmol) and 0.013 g of p-toluenesulfonic acid (0.075 mmol) in sequence, heat to 80 °C and reflux for 8 h. After the reaction is complete, filter while hot, wash 3 times with hot methanol, and dry to obtain a dark green solid product, which is compound I-2 (8-bromo-11H-indeno[1,2-b]quinoxaline-11-one thiocarbamate), with a yield of 21.65%.

[0086] 1H NMR data: 1¹H-NMR (400MHz, DMSO-d⁶): δ 12.48 (s, 1H), 9.11 (s, 1H), 8.85 (s, 1H), 8.37 (d, J = 1.8 Hz, 1H), 8.15 (d, J = 7.4 Hz, 1H), 8.05 (d, J = 7.4 Hz, 1H), 8.01 (s, 1H), 7.99 (dd, J = 8.9, 2.0 Hz, 1H), 7.70 (td, J = 7.5, 1.2 Hz, 1H), 7.65 (td, J = 7.5, 1.1 Hz, 1H); its ¹H NMR spectrum is as follows: Figure 3 As shown.

[0087] Carbon NMR data: 13 C-NMR (100MHz, DMSO-d6): δ 179.20, 154.73, 148.05, 142.38, 139.04, 138.80, 135.37, 133.69, 131.84, 131.41, 124.79, 122.90, 122.49; its carbon NMR spectrum is as follows: Figure 4 As shown.

[0088] Mass spectrometry data: HRMS (ESI, m / z): calcd.for C 16 H 11 BrN5S(M+H) + 383.9913, found383.9913.

[0089] Example 3 Preparation of 8-nitro-11H-indeno[1,2-b]quinoxaline-11-one thiocarbazone The synthetic route of 8-nitro-11H-indeno[1,2-b]quinoxaline-11-one thiocarbazone is shown below:

[0090]

[0091] (1) Preparation of intermediate product 3-3

[0092] Weigh 0.1025 g of compound 2 (0.58 mmol) and 0.1040 g of compound 1-3 (0.68 mmol) into a flask, add 25 mL of ethanol, heat to 80 °C, stir to dissolve, then add 0.25 mL of glacial acetic acid (4.15 mmol), heat to 80 °C and reflux for 3 h. After the reaction is complete, cool to room temperature, filter, wash three times with 30 mL of ethanol, and dry to obtain a dark yellow solid product, which is intermediate 3-3, with a yield of 74.04%.

[0093] (2) Preparation of compound I-3

[0094] Weigh 0.0924 g of intermediate product 3-3 (0.33 mmol) obtained in step (1) into a 250 mL three-necked flask. Add 40 mL of dichloromethane in portions to dissolve the solid completely. Then add 11 mL of methanol, heat and stir. Then add 0.0369 g of compound 4 (0.40 mmol) and 0.013 g of p-toluenesulfonic acid (0.075 mmol) in sequence, and heat to 80 °C and reflux for 8 h. After the reaction is complete, filter while hot, wash three times with hot methanol, and dry to obtain an orange-red solid product, which is compound I-3 (8-nitro-11H-indeno[1,2-b]quinoxaline-11-one thiocarbamate), with a yield of 49.33%.

[0095] 1H NMR data: 1 1H-NMR (400MHz, DMSO-d6): δ 12.47 (s, 1H), 9.16 (s, 1H), 8.92 (s, 1H), 8.83 (d, J = 4Hz, 1H), 8.62 (dd, J = 8, 4Hz, 1H), 8.39 (d, J = 8.0Hz, 1H), 8.21 (d, J = 8.0Hz, 1H), 8.16 (d, J = 8.0Hz, 1H), 7.77 (t, J = 8.0Hz, 1H), 7.70 (t, J = 8.0Hz, 1H); its 1H NMR spectrum is shown below. Figure 5 As shown.

[0096] Carbon NMR data: 13 C-NMR (100MHz, DMSO-d6): δ 179.28, 156.60, 149.77, 147.57, 144.59, 139.47, 139.08, 135.81, 135.06, 133.89, 131.82, 131.45, 125.28, 124.93, 123.50, 122.60; its carbon NMR spectrum is as follows. Figure 6 As shown.

[0097] Mass spectrometry data: HRMS(ESI, m / z): calcd.for C 16 H 11 N6O2S(M+H) + 351.0659, found351.0659.

[0098] Experimental Example 1: UV-Vis spectra of 8-substituted indanone-quinoxaline thiourea compounds

[0099] 1. Experimental Methods

[0100] Compounds I-1 to I-3 prepared in Examples 1 to 3 were dissolved in dimethyl sulfoxide (DMSO) and then diluted with Tris-HCl buffer to obtain a working solution of 3.0 × 10⁻⁶. -5 mol L -1 While keeping the compound concentration constant, ultraviolet absorption titration was performed by varying the concentration of circulating tumor DNA (ctDNA). The ultraviolet spectrum was recorded in the range of 200–600 nm, and the absorbance was measured after incubation for 5 minutes.

[0101] Based on the change in absorbance intensity at the maximum wavelength, the binding constant K between compounds I-1 to I-3 and DNA is calculated according to equation (1). b :

[0102]

[0103] In the formula, [DNA] represents the DNA concentration, and ε a ε represents the molar absorption coefficient at any compound concentration. f ε represents the molar absorption coefficient of the free compound. b This represents the molar absorption coefficient of a fully bound compound. Correlation constant K. b By plotting [DNA] and (ε) a -ε f The relationship between the ratio of DNA and DNA is determined by a graph.

[0104] 2. Experimental Results

[0105] Table 1. Correlation constants K between compounds I-1 to I-3 and DNA at the maximum wavelength. b and correlation coefficient R 2 Statistical table

[0106] compound <![CDATA[λ max / nm]]> <![CDATA[K b ×10 5 / M -1 ]]> <![CDATA[R 2 ]]> I-1 259 7.4659 0.9950 I-2 262 9.94521 0.9936 I-3 299 9.484578 0.9882

[0107] from Figures 7-9 It can be seen that within the maximum absorption wavelength range (259–300 nm), as the DNA concentration gradually increases from 2 × 10⁻⁶, the absorption rate decreases. -6 Increase mol / L to 1×10 -5 With increasing mol / L concentrations, the amount of DNA intercalated by compounds I-1 to I-3 also increases, leading to a decreasing trend in the absorption intensity of compounds I-1 to I-3. When these compounds bind to ctDNA, their decolorization rates (i.e., the percentage decrease in the UV absorption intensity of ctDNA at a specific wavelength (the wavelength range of this application is 259–300 nm)) range from 43% to 70.8%, all exceeding the 30% threshold. Based on the data in Table 1, by comparing the intrinsic binding constants K of compounds I-1 to I-3 with DNA... bThe results showed that compound I-2 had a superior binding affinity to ctDNA compared to I-1 and I-3. This significant hypochromic effect indicates that compounds I-1 to I-3 effectively intercalated into the base pairs of ctDNA via intercalation. The more pronounced the hypochromic effect, the stronger the interaction between the compound and ctDNA, and the greater the degree to which the compound alters the DNA double helix structure, thus more effectively inhibiting the proliferation of cancer cells.

[0108] Experimental Example 2: Fluorescence spectra of 8-substituted indanone-quinoxaline thiourea compounds

[0109] 1. Experimental Methods

[0110] A solution containing 1.2 μmol / L was prepared in Tris-HCl buffer at pH 7.4. -1 ctDNA and 2.8 μmol l -1 Ethidium bromide (EB)-DNA solution. Compounds I-1 to I-3 prepared in Examples 1-3 were dissolved in DMSO and then diluted with Tris-HCl buffer to obtain a working solution of 3.0 × 10⁻⁶. -5 mol L -1 In the fluorescence instrument, 2.5 mL of EB-ctDNA mixture was added to a cuvette. Initial fluorescence emission spectra were recorded within the excitation wavelength range of 492 nm and the emission wavelength range of 510–800 nm, with both excitation and emission slit widths set to 10 nm. Subsequently, 10 μL of the compound was added each time and incubated for 5 min, with fluorescence emission spectra recorded repeatedly within the aforementioned wavelength range to obtain fluorescence changes after each addition of the compound.

[0111] 2. Experimental Results

[0112] EB is a fluorescent probe that binds to DNA through intercalation. However, EB itself has relatively weak fluorescence intensity, but when its planar structure successfully inserts between base pairs in DNA, the overall fluorescence intensity of the system increases significantly. Figures 10-12 As shown, when compounds I-1 to I-3 are introduced into the system, they compete with EB for DNA binding sites. As the amount of compounds I-1 to I-3 gradually increases from 0 μL to 80 μL, their substitution rate for EB gradually increases, leading to a gradual decrease in fluorescence intensity in the 590–630 nm wavelength range. This phenomenon indicates that compounds I-1 to I-3 and EB use the same binding mechanism, namely, intercalation into the double helix structure of ctDNA.

[0113] Experimental Example 3: Circular dichroism spectroscopy of 8-substituted indanone-quinoxaline thiourea compounds

[0114] 1. Experimental Methods

[0115] Compounds I-1 to I-3 (1×10) -4 M) was added to ctDNA (4.37 × 10⁻⁶) respectively. -4 In M), circular dichroism (CD) spectra were measured in the wavelength range of 200–300 nm, and the CD spectrum of ctDNA without the addition of the compound (i.e., blank spectrum) was subtracted from each measured curve. All samples were incubated at 25 °C for 3 min before measurement at a scan rate of 100 nm / min.

[0116] 2. Experimental Results

[0117] Depend on Figures 13-15 It is known that the CD spectrum of free DNA exhibits a positive peak at 275 nm, which typically corresponds to the base pair stacking effect; while a negative peak is present at 245 nm, which is usually associated with the right-handed helical structure of DNA. As the amount of compounds I-1 to I-3 gradually increased from 0 μL to 60 μL, the CD spectrum changed significantly: the ellipticity of the positive peak at 275 nm decreased significantly, while the ellipticity of the negative peak at 245 nm increased. These changes clearly indicate that compounds I-1 to I-3 mainly interact with DNA through intercalation and significantly perturb the DNA folding structure. The decrease in the positive peak ellipticity may be due to these compounds intercalating into the base pairs of ctDNA, thereby affecting the DNA conformation, transforming it from a relatively compact double helix structure to a looser or deformed structure, leading to helix unwinding. The increase in the negative peak ellipticity further indicates that the intercalated compounds I-1 to I-3 can unwind the DNA helix structure, thereby reducing DNA stability.

[0118] Experimental Example 4: Molecular docking test of 8-substituted indanone-quinoxaline thiourea compounds

[0119] 1. Experimental Methods

[0120] Molecular docking studies were performed using AutoDock Vina version 1.1.2. During docking, a grid box containing the entire DNA was created along the x, y, and z axes. Grid point spacing is Other parameters are set to default. The docking results are visualized using PyMol.

[0121] 2. Experimental Results

[0122] Depend on Figures 16-18The molecular docking results of compounds I-1 to I-3 revealed the receptor interface residues involved in the interaction. Molecular docking analysis showed that the distances between the O3 atom of Dc11 (cytosine 11) and the N4 atoms of compounds I-1, I-2, and I-3 were respectively... These distances reflect the strength of hydrogen bonding between the compounds and DNA. Compound I-2 exhibits the most significant hydrogen bonding with DNA; the introduction of the bromine atom noticeably reduces the hydrogen bond distance, enhancing the binding of the compound to DNA base pairs and thus strengthening its inhibitory effect on cancer cells. Furthermore, other residues surrounding the compounds are represented by crescent-shaped symbols (arcs and straight lines), which represent hydrophobic interactions.

[0123] Experimental Example 5: Bioactivity Test of 8-substituted indanone-quinoxaline thiourea compounds

[0124] The half-maximal inhibitory concentration (IC50) of 8-substituted indanone-quinoxaline thiourea compounds against human breast cancer MCF-7 cells, human cervical cancer HeLa cells, and human colon cancer HCT-116 cells was determined using the CCK-8 assay. 50 Cells with a viable cell ratio of over 90% were used for experiments. The cell proliferation inhibition assay was performed using EnoGeneCell. TM The specific steps for using the Counting Kit-8 (CCK-8) cell viability assay kit are as follows:

[0125] 1. Experimental drugs

[0126] Compounds I-1 to I-3 prepared in Examples 1 to 3 were used as test drugs, and doxorubicin (DOX) was used as a positive control. DMSO was used as a dissolution aid. The test concentrations used in the experiment were 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, and 1.56 μM.

[0127] 2. Experimental Methods

[0128] CCK-8 staining method: Cell digestion, counting, and preparation of a 1×10⁻⁶ cell concentration. 5 Add 100 μL of cell suspension per well (1 × 10⁶ cells / mL) to each well of a 96-well plate. 4(1 cell); 96-well plates were incubated at 37°C in a 5% CO2 incubator for 24 h; 100 μL of the corresponding drug-containing medium (compounds I-1 to I-3) was added to each well, and negative control, solvent control, and positive control were set up, with 5 replicates per group; after incubating the 96-well plates at 37°C in a 5% CO2 incubator for 4 h; 20 μL of LCK-8 solution was added to each well, and the culture plates were incubated in the incubator for 4 h. The optical density (OD) value at 450 nm was measured using a microplate reader, and the IC50 of the above compounds against tumor cells (MCF-7, HeLa, HCT-116) was calculated. 50 Value. [IC] 50 Drug concentration at which 50% of cells survive.

[0129] 3. Experimental Results

[0130] Table 2. Antitumor activity data of compounds I-1 to I-3

[0131]

[0132] As shown in Table 2, 8-substituted indanone-quinoxaline thiourea compounds exhibit significant inhibitory effects on the proliferation of various cancer cell lines, including human breast cancer cells, human cervical cancer cells, and human colon cancer cells. This inhibitory effect is mainly attributed to the group introduced at the 8-substitution position. As the electron-withdrawing effect of this group weakens, the hydrogen bonding effect strengthens, thereby enhancing the interaction between the compound molecule and DNA molecules. This enhanced interaction leads to a stronger hypochromic effect, severely interfering with DNA replication and transcription, thus achieving highly efficient inhibition of cancer cell proliferation. Specifically, the three compounds mentioned above showed significant inhibitory effects on the IC50 of human breast cancer MCF-7 cells, human cervical cancer HeLa cells, and human colon cancer HCT-116 cells. 50 The values ​​were 14.21–16.13 μM, 7.578–27.63 μM, and 12.77–20.22 μM, respectively. Among them, compound I-2 prepared in Example 2 exhibited the best antitumor activity, with IC50 values ​​against the above three cell types. 50 The values ​​were 16.23 μM, 7.578 μM, and 12.77 μM, respectively, all superior to the broad-spectrum anticancer drug doxorubicin (DOX). Therefore, 8-substituted indanone-quinoxaline thiourea compounds have significant inhibitory activity against human breast cancer cells, human cervical cancer cells, and human colon cancer cells, and have potential clinical application value.

[0133] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of an 8-substituted indanone-quinoxaline thiourea compound in the preparation of anti-cervical cancer drugs; in, The structural formula of the 8-substituted indanone-quinoxaline thiourea compound is shown in formula (I): R is Br.

2. The application according to claim 1, characterized in that, The synthetic route for the 8-substituted indanone-quinoxaline thiourea compounds is as follows: Specifically, the following steps are included: S1. Compound 1 and Compound 2 are mixed in a first solvent, an acidic catalyst is added, and the mixture is reacted at 70-80°C until fully reacted. After post-treatment, intermediate product 3 is obtained. S2. Mix the intermediate product 3, compound 4, and acid catalyst obtained in step S1 in a second solvent, react them thoroughly at 70-80°C, and then treat them to obtain the 8-substituted indanone-quinoxaline thiourea compound shown in formula (I). The definition of R is consistent with that in claim 1.

3. The application according to claim 2, characterized in that, In step S1 or step S2, the acidic catalyst includes one or more of p-toluenesulfonic acid, acetic acid, hydrochloric acid, phosphoric acid, and acetic acid.

4. The application according to claim 2, characterized in that, In step S1, the first solvent includes one or more of ethanol, ethyl acetate, and chloroform.

5. The application according to claim 2, characterized in that, In step S2, the second solvent includes one or more of dichloromethane, methanol, and tetrahydrofuran.

6. The application according to claim 2, characterized in that, In step S1, the molar ratio of compound 1 to compound 2 is (1-2):

1.

7. The application according to claim 2, characterized in that, In step S2, the molar ratio of intermediate product 3 to compound 4 is 1:(1-2).

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