8-substituted indanone quinoxaline aminothiourea compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an 8-substituted indanone quinoxaline aminothiourea compound as well as a preparation method and application thereof. The invention provides an 8-substituted indanone quinoxaline amino thiourea compound with a novel structure and a preparation method of the 8-substituted indanone quinoxaline amino thiourea compound. Experiments prove that the compound is combined with DNA (deoxyribonucleic acid) in an embedding manner, proliferation of various tumor cells is effectively inhibited, excellent anti-tumor activity is shown, the overall effect of the compound is more outstanding than that of a broad-spectrum anti-tumor drug adriamycin, and the compound has important medicinal value in the field of research and development of anti-tumor drugs; in addition, the preparation method of the compound is simple, mild in condition, low in cost and suitable for large-scale industrial production and application.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and more specifically, relates to an 8-substituted indanone-quinoxaline thiosemicarbazide compound and a preparation method and application thereof. Background Art

[0002] Cancer is a major disease that threatens human life. In 2022, there will be nearly 20 million new cancer cases worldwide, of which nearly half of new cancer cases (49.2%) and most cancer deaths (56.1%) occurred in Asia. Among various types of cancer, female breast cancer and colon cancer rank second and third in the incidence list, respectively, with their high incidence and high mortality rates, posing a huge threat to the life and health of humans around the world, especially women. Although cervical cancer ranks eighth in incidence, its harm to women's health cannot be ignored, especially in some areas where medical resources are relatively scarce, the incidence and mortality rates of cervical cancer remain high.

[0003] Currently, cancer, as a severe challenge facing the medical community, is driving continuous exploration and innovation in this field. Among the many means of cancer treatment, chemotherapy occupies an important position and has achieved remarkable clinical results. Doxorubicin (DOX), as one of the classic and broad-spectrum anticancer drugs, has shown significant therapeutic effects on many types of malignant tumors, becoming a highlight in the field of chemotherapy.

[0004] In clinical treatment, there is still a relative lack of therapeutic drugs for cancer patients. Therefore, in order to meet the challenges of cancer treatment, the medical community urgently needs to increase research and development efforts to develop more new drugs with high anti-cancer effects, in order to provide patients with more options and further improve treatment effects. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the current relative scarcity of therapeutic drugs for cancer patients, and to provide an 8-substituted indanone and quinoxaline thiosemicarbazide compound.

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

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

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

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

[0010]

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

[0012] The 8-substituted indanone and quinoxaline thiosemicarbazide compounds prepared by the present invention are closely combined with cancer cell DNA through intercalation. This binding process changes the double helix structure of DNA, and the stacking state of base pairs changes accordingly, resulting in a weakened interaction between electrons, thereby reducing the ability of bases to absorb ultraviolet light, and finally producing a color reduction effect. Importantly, the significant degree of the color reduction effect is proportional to the interaction intensity between the compounds and cancer cell DNA, that is, the more obvious the color reduction effect, the stronger the degree of change of the double helix structure of DNA by the compounds, thereby more effectively inhibiting the proliferation of cancer cells. In addition, this interaction may be further stabilized by a variety of non-covalent interactions, including hydrophobic interactions, hydrogen bonds and π-π stacking. The inventors have found that adjusting the group types (such as halogen atoms, -NO2, -CN, etc.) on the 8-substitution position of the compounds in the present invention can affect the strength of hydrogen bond formation. Specifically, when the electron-withdrawing effect of these groups is weakened, the hydrogen bonding effect will be significantly enhanced. This enhanced hydrogen bonding can strengthen the mutual binding between this type of compound and the DNA of cancer cells, making the color reduction effect more significant, thereby more effectively inhibiting the proliferation of cancer cells.

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

[0014] The present invention protects the preparation method of the above-mentioned 8-substituted indanone and quinoxaline thiosemicarbazide compounds, synthesizes

[0015] The route is as follows:

[0016]

[0017] The specific steps include:

[0018] S1. Compound 1 and compound 2 are mixed in a first solvent, an acidic catalyst is added, the mixture is fully reacted at 70 to 80° C., and post-treated to obtain an intermediate product 3;

[0019] S2. The intermediate product 3 obtained in step S1, compound 4, and an acidic catalyst are mixed in a second solvent, reacted at 70 to 80° C., and post-treated to obtain an 8-substituted indanone-quinoxaline thiosemicarbazide compound of formula (I);

[0020] The definition of R is consistent with the above.

[0021] The preparation method of this compound is simple, the conditions are mild, the raw materials are cheap and easily available, and the production cost is low. It provides a new reference approach 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 through the above-mentioned route, or can be purchased from the market.

[0023] Furthermore, 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 (1.1-2):1. This ratio setting makes compound 1 appropriately excessive relative to compound 2, which helps to ensure sufficient reaction.

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

[0026] Furthermore, 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, dissolving and mixing.

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

[0029] Furthermore, in step S1, the molar ratio of the compound 1 to the acidic catalyst is 1:(2-10). The acidic catalyst added within this ratio range has an acidic property that helps the reaction proceed, thereby accelerating the reaction rate.

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

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

[0032] Furthermore, the cooling is cooling the reaction mixture to room temperature.

[0033] Furthermore, the filtration is suction filtration, and the cooled mixture is suction filtered to collect the filter residue.

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

[0035] Furthermore, the drying is to dry the filter residue after washing.

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

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

[0038] Preferably, in step S2, the molar ratio of the intermediate product 3 to the compound 4 is 1:(1.1-2). This ratio setting makes the compound 4 appropriately excessive relative to the intermediate product 3, which helps to ensure sufficient reaction.

[0039] Furthermore, 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 the acidic catalyst can also significantly promote the reaction.

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

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

[0043] Furthermore, the filtration is to filter the reaction solution while it is hot and collect the filter residue.

[0044] Furthermore, the washing is to use hot methanol to wash the filter residue 1 to 3 times.

[0045] Furthermore, the drying is to dry the filter residue after washing.

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

[0047] The present invention protects the use of the above 8-substituted indanone and quinoxaline thiosemicarbazide compounds in the preparation of anti-tumor 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] The present invention provides an 8-substituted indanone and quinoxaline thiosemicarbazide compound with a novel structure. Experiments have shown that the compound binds to DNA in an intercalation manner, effectively inhibits the proliferation of various tumor cells, and exhibits excellent anti-tumor activity. The overall effect is more prominent than that of the broad-spectrum anti-tumor drug adriamycin, and has important medicinal value in the research and development of anti-tumor drugs; in addition, the preparation method of the compound is simple, the conditions are mild, and the cost is low, which is suitable for large-scale industrial production and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is the H NMR spectrum of the compound 8-chloro-11H-indeno[1,2-b]quinoxaline-11-one thiosemicarbazone provided in Example 1.

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

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

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

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

[0056] Figure 6 The C NMR spectrum of the compound 8-nitro-11H-indeno[1,2-b]quinoxaline-11-one thiosemicarbazone provided in Example 3;

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

[0058] Figure 8 The UV-visible spectrum of the compound 8-bromo-11H-indeno[1,2-b]quinoxaline-11-one thiosemicarbazone provided in Example 2 as the concentration of circulating tumor DNA changes;

[0059] Fig. 9 The UV-visible spectrum of the compound 8-nitro-11H-indeno[1,2-b]quinoxaline-11-one thiosemicarbazone provided in Example 3 as a function of circulating tumor DNA concentration;

[0060] Fig.10 The fluorescence spectrum of the compound 8-chloro-11H-indeno[1,2-b]quinoxaline-11-one thiosemicarbazone provided in Example 1;

[0061] Fig.11The fluorescence spectrum of the compound 8-bromo-11H-indeno[1,2-b]quinoxaline-11-one thiosemicarbazone provided in Example 2;

[0062] Fig.12 The fluorescence spectrum of the compound 8-nitro-11H-indeno[1,2-b]quinoxaline-11-one thiosemicarbazone provided in Example 3;

[0063] Fig.13 The circular dichroism spectrum of the compound 8-chloro-11H-indeno[1,2-b]quinoxaline-11-one thiosemicarbazone provided in Example 1;

[0064] Fig.14 The circular dichroism spectrum of the compound 8-bromo-11H-indeno[1,2-b]quinoxaline-11-one thiosemicarbazone provided in Example 2;

[0065] Fig.15 The circular dichroism spectrum of the compound 8-nitro-11H-indeno[1,2-b]quinoxaline-11-one thiosemicarbazone provided in Example 3;

[0066] Fig.16 A molecular docking 2D diagram of the compound 8-chloro-11H-indeno[1,2-b]quinoxaline-11-one thiosemicarbazone provided in Example 1;

[0067] Fig.17 A molecular docking 2D diagram of the compound 8-bromo-11H-indeno[1,2-b]quinoxaline-11-one thiosemicarbazone provided in Example 2;

[0068] Fig.18 The molecular docking 2D diagram of the compound 8-nitro-11H-indeno[1,2-b]quinoxaline-11-one thiosemicarbazone provided in Example 3. DETAILED DESCRIPTION

[0069] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0070] Unless otherwise specified, the 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 thiosemicarbazone The synthetic route of 8-chloro-11H-indeno[1,2-b]quinoxaline-11-one thiosemicarbazone is as follows:

[0072]

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

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

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

[0076] 0.069 g of the intermediate product 3-1 (0.26 mmol) prepared in step (1) was weighed into a 250 mL three-necked flask, 30 mL of dichloromethane was added to completely dissolve the solid, and 25 mL of methanol was added, heated and stirred, and then 0.0427 g of compound 4 (0.47 mmol) and 0.0155 g of p-toluenesulfonic acid (0.0901 mmol) were added in sequence, heated to 80 ° C and refluxed for 10 h. After the reaction was completed, it was filtered while hot, washed three times with hot methanol, and dried to obtain a yellow solid product, namely compound I-1 (8-chloro-11H-indeno[1,2-b]quinoxaline-11-one thiosemicarbazone), with a yield of 67.31%. Its NMR data are as follows:

[0077] H NMR spectrum data: 1 H-NMR (400MHz, DMSO-d6) δ12.50 (s, 1H), 9.10 (s, 1H), 8.85 (s, 1H), 8.24 (d, J = 2.3 Hz, 1H), 8.16 (d, J = 7.2 Hz, 1H), 8.12 (d, J = 8.9 Hz, 1H), 8.07 (d, J = 7.3 Hz, 1H), 7.90 (dd, J = 8.9, 2.4 Hz, 1H), 7.71 (td, J = 7.5, 1.1 Hz, 1H), 7.66 (td, J = 7.5, 1.2 Hz, 1H); its hydrogen nuclear magnetic resonance spectrum is as follows Figure 1 As shown;

[0078] NMR carbon spectrum data: 13 C-NMR (100MHz, DMSO-d6): δ179.21, 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 shown.

[0079] Mass spectral 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-indeno[1,2-b]quinoxaline-11-one thiosemicarbazone The synthetic route of 8-bromo-11H-indeno[1,2-b]quinoxaline-11-one thiosemicarbazone is as follows:

[0081]

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

[0083] Weigh 1.0636g of compound 2 (5.97mmol) and 1.1382g of compound 1-2 (6.12mmol) into a flask, add 25mL of ethanol, heat to 75°C, stir to dissolve, then add 1mL of glacial acetic acid (16.6mmol), heat to 80°C and reflux for 3h. After the reaction is completed, cool to room temperature, filter, wash three times with 30mL of ethanol, and dry to obtain a brown 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 the 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 completed, filter while hot, wash 3 times with hot methanol, and dry to obtain a dark green solid product, namely compound I-2 (8-bromo-11H-indeno[1,2-b]quinoxaline-11-one thiosemicarbazone), with a yield of 21.65%.

[0086] H NMR spectrum data: 1H-NMR (400MHz, DMSO-d6): δ12.48 (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 hydrogen nuclear magnetic resonance spectrum is as follows Figure 3 shown.

[0087] NMR carbon spectrum data: 13 C-NMR (100MHz, DMSO-d6): δ179.20, 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 shown.

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

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

[0090]

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

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

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

[0094] 0.0924g of the intermediate product 3-3 (0.33mmol) obtained in step (1) was weighed into a 250mL three-necked flask, and 40mL of dichloromethane was added in portions to dissolve the solid completely, and then 11mL of methanol was added, heated and stirred, and then 0.0369g of compound 4 (0.40mmol) and 0.013g of p-toluenesulfonic acid (0.075mmol) were added in sequence, and heated to 80°C and refluxed for 8h. After the reaction was completed, it was filtered while hot, washed three times with hot methanol, and dried to obtain an orange-red solid product, namely compound I-3 (8-nitro-11H-indeno[1,2-b]quinoxaline-11-one thiosemicarbazone), with a yield of 49.33%.

[0095] H NMR spectrum data: 1 H-NMR (400MHz, DMSO-d6): δ12.47 (s, 1H), 9.16 (s, 1H), 8.92 (s, 1H), 8.83 (d, J = 4 Hz, 1H), 8.62 (dd, J = 8, 4 Hz, 1H), 8.39 (d, J = 8.0 Hz, 1H), 8.21 (d, J = 8.0 Hz, 1H), 8.16 (d, J = 8.0 Hz, 1H), 7.77 (t, J = 8.0 Hz, 1H), 7.70 (t, J = 8.0 Hz, 1H); its hydrogen nuclear magnetic resonance spectrum is shown in Figure 5 shown.

[0096] NMR carbon spectrum data: 13 C-NMR (100MHz, DMSO-d6): δ179.28, 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 shown.

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

[0098] Experimental Example 1 UV-Vis Spectra of 8-Substituted Indanone-Quinoxaline Thiosemicarbazone 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 -5 mol L -1 While keeping the compound concentration constant, the concentration of circulating tumor DNA (ctDNA) was changed to perform UV absorption titration. The UV spectrum was recorded in the range of 200-600 nm, and the absorbance value was measured after incubation for 5 min.

[0101] According to the change of absorbance intensity at the maximum wavelength, the binding constant K of compounds I-1 to I-3 and DNA was calculated according to formula (1): b :

[0102]

[0103] Where [DNA] represents the DNA concentration, ε a represents the molar absorption coefficient at any compound concentration, ε f represents the molar absorption coefficient of the free compound, ε b represents the molar absorption coefficient of the fully bound compound. The association constant K b This can be done by plotting [DNA] versus (ε a -ε f ) / [DNA] ratio.

[0104] 2. Experimental results

[0105] Table 1 Association constants K of compounds I-1 to I-3 with DNA at the maximum wavelength b and the correlation coefficient R 2 Statistics

[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 to 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 mol / L increases to 1×10 -5 mol / L, the amount of compounds I-1 to I-3 inserted into DNA also increased, resulting in a decreasing trend in the absorption intensity of compounds I-1 to I-3. When these compounds bind to ctDNA, their color reduction rate (i.e., the percentage of ctDNA ultraviolet absorption intensity reduction at a certain wavelength (the wavelength range of this application is 259 to 300nm)) is between 43% and 70.8%, all exceeding the threshold of 30%. According to the data in Table 1, by comparing the inherent binding constants K of compounds I-1 to I-3 and DNA b, it was concluded that the binding ability of compound I-2 to ctDNA is better than that of I-1 and I-3. This significant color reduction phenomenon indicates that compounds I-1 to I-3 are effectively inserted into the base pairs of ctDNA by intercalation. Among them, the more significant the color reduction effect, the stronger the interaction between the compound and ctDNA, and the stronger the degree of change of the compound to the DNA double helix structure, so that the proliferation of cancer cells can be more effectively inhibited.

[0108] Experimental Example 2 Fluorescence Spectra of 8-Substituted Indonone-Quinoxaline Thiosemicarbazone Compounds

[0109] 1. Experimental Methods

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

[0111] 2. Experimental results

[0112] EB is a fluorescent probe that binds to DNA by intercalation. However, the fluorescence intensity of EB itself is relatively weak, but when its planar structure is successfully inserted between the base pairs of DNA, the fluorescence intensity of the entire system will increase significantly. Figures 10-12 As shown, when compounds I-1 to I-3 enter the system, they compete with EB for DNA binding sites. As the addition amount of compounds I-1 to I-3 gradually increases from 0 μL to 80 μL, their substitution rate for EB gradually increases, causing the fluorescence intensity of the system in the wavelength range of 590 to 630 nm to gradually decrease. This phenomenon shows that compounds I-1 to I-3 and EB use the same binding method, that is, they are embedded in the double helix structure of ctDNA by intercalation.

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

[0114] 1. Experimental Methods

[0115] Compounds I-1 to I-3 (1×10 -4 M) were added to ctDNA (4.37×10 -4 M). Circular dichroism spectra (CD spectra) were measured in the wavelength range of 200-300 nm, and the CD spectra of ctDNA without adding compounds (i.e., blank spectra) were subtracted from each measured curve. All samples were kept at 25°C for 3 min and then measured at a scanning rate of 100 nm / min.

[0116] 2. Experimental results

[0117] Depend on Figures 13-15 It can be seen that the CD spectrum of free DNA shows a positive peak at 275nm, which usually corresponds to the stacking effect of base pairs; and a negative peak at 245nm, which is usually related to the right-handed helical structure of DNA. As the addition amount of compounds I-1 to I-3 gradually increased from 0μL to 60μL, the CD spectrum changed significantly: the positive peak ellipticity at 275nm decreased significantly, while the negative peak ellipticity at 245nm increased. These changes clearly show that compounds I-1 to I-3 interact with DNA mainly through the intercalation mode and significantly disturb the folding structure of DNA. The decrease in the positive peak ellipticity may be due to the insertion of these compounds into the base pairs of ctDNA, thereby affecting the conformation of DNA, causing it to change from a relatively compact double helical structure to a more loose or deformed structure, which in turn leads to the unfolding of the helix. The increase in the negative peak ellipticity further indicates that the embedded compounds I-1 to I-3 can unwind the helical structure of DNA, thereby reducing the stability of DNA.

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

[0119] 1. Experimental Methods

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

[0121] 2. Experimental results

[0122] Depend on Figures 16 to 18It can be seen that the molecular docking results of compounds I-1 to I-3 show the receptor interface residues involved in the interaction. Molecular docking analysis shows that the distances between the O3 atom of Dc11 (cytosine 11) and the N4 atom on compounds I-1, I-2, and I-3 are These distances reflect the strength of the hydrogen bonding between the compound and DNA, among which the hydrogen bonding between compound I-2 and DNA is the most significant. The introduction of bromine atoms significantly reduces the hydrogen bonding distance, enhances the binding between the compound and DNA base pairs, and enhances the inhibitory effect of the compound on cancer cells. In addition, other residues around the compound are represented by crescent symbols (arcs and straight lines), which represent hydrophobic interactions.

[0123] Experimental Example 5 Biological Activity Test of 8-Substituted Indanone-Quinoxaline Thiosemicarbazone Compounds

[0124] The CCK-8 method was used to determine the half inhibitory concentration (IC50) of 8-substituted indanone and quinoxaline thiosemicarbazide compounds on human breast cancer MCF-7 cells, human cervical cancer Hela cells, and human colon cancer HCT-116 cells. 50 ), cells with a live cell ratio of more than 90% were used for the experiment, and the cell proliferation inhibition experiment was performed using EnoGeneCell TM Counting Kit-8 (CCK-8) cell viability detection kit, the specific steps 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, respectively, doxorubicin (DOX) was used as a positive control, DMSO was used as a solubilizing agent, and 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: Cells were digested, counted, and prepared into a concentration of 1×10 5 100 μL of cell suspension (1×10 cells / mL) was added to each well of a 96-well plate. 4cells); the 96-well plate was placed in a 37°C, 5% CO2 incubator for 24 hours; 100 μL of the corresponding drug-containing culture medium (compounds I-1 to I-3) was added to each well, and a negative control group, a solvent control group, and a positive control group were set up at the same time, with 5 replicates in each group; the 96-well plate was placed in a 37°C, 5% CO2 incubator for 4 hours; 20 μL of CCK-8 solution was added to each well, the culture plate was incubated in the incubator for 4 hours, and the optical density (OD) value at 450 nm was measured with an enzyme marker to calculate the IC values ​​of the above compounds for tumor cells (MCF-7, Hela, HCT-116) 50 Value. 50 : The drug concentration at which 50% of the cells survive. 】

[0129] 3. Experimental results

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

[0131]

[0132] From the data in Table 2, it can be seen that 8-substituted indanone and quinoxaline thiosemicarbazide compounds have significant proliferation inhibition effects on various cancer cell lines such as human breast cancer cells, human cervical cancer cell lines and human colon cancer cells. This inhibitory effect is mainly attributed to the group introduced by this type of compound at the 8-substitution position. As the electron-withdrawing effect of the group weakens, the hydrogen bonding effect is correspondingly enhanced, thereby enhancing the interaction between the compound molecule and the DNA molecule. This enhanced interaction leads to an enhanced hypochromic effect, which seriously interferes with the replication and transcription process of DNA, thereby achieving efficient inhibition of cancer cell proliferation. Specifically, the IC values ​​of the above three compounds on human breast cancer MCF-7 cells, human cervical cancer cell lines Hela and human colon cancer HCT-116 cells are as follows: 50 The values ​​were 14.21-16.13 μM, 7.578-27.63 μM, and 12.77-20.22 μM, respectively. Among them, the compound I-2 prepared in Example 2 showed the best anti-tumor activity, and its IC 50 The values ​​were 16.23μM, 7.578μM, and 12.77μM, respectively, which were better than the broad-spectrum anticancer drug doxorubicin (DOX). Therefore, 8-substituted indanone and quinoxaline thiosemicarbazone 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 implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An 8-substituted indanone-quinoxaline thiosemicarbazide compound, characterized in that: The structural formula of the 8-substituted indanone-quinoxaline thiosemicarbazide compound is shown in formula (I): Wherein, R is a halogen atom, NO2 or CN.

2. The 8-substituted indanone-quinoxaline thiosemicarbazide compound according to claim 1, characterized in that: The halogen atom is F, Cl or Br.

3. The method for preparing the 8-substituted indanone-quinoxaline thiosemicarbazide compound according to claim 1 or 2, characterized in that: The synthetic route is as follows: The specific steps include: S1. Compound 1 and compound 2 are mixed in a first solvent, an acidic catalyst is added, the mixture is fully reacted at 70 to 80° C., and post-treated to obtain an intermediate product 3; S2. The intermediate product 3 obtained in step S1, compound 4, and an acidic catalyst are mixed in a second solvent, reacted at 70 to 80° C., and post-treated to obtain an 8-substituted indanone-quinoxaline thiosemicarbazide compound of formula (I); The definition of R is consistent with that of claim 1.

4. The preparation method according to claim 3, 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.

5. The preparation method according to claim 3, characterized in that: In step S1, the first solvent includes one or more of ethanol, ethyl acetate, and chloroform.

6. The preparation method according to claim 3, characterized in that: In step S2, the second solvent includes one or more of dichloromethane, methanol, and tetrahydrofuran.

7. The preparation method according to claim 3, characterized in that: In step S1, the molar ratio of compound 1 to compound 2 is (1-2):

1.

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

9. Use of the 8-substituted indanone-quinoxaline thiosemicarbazide compound according to claim 1 or 2 in the preparation of antitumor drugs.

10. The use according to claim 9, characterized in that: The tumor is one or more of breast cancer, cervical cancer, and colon cancer.

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