KR20230036769A - 2,4-diphenyl indenopyridin-5-one derivatives containing halogen and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a halogen-containing 2,4-diphenyl indenopyridin-5-one derivative that enhances the anticancer response of a clinically used topoisomerase inhibitor and a method for producing the same. The antiproliferative activity of 2,4-diphenyl indenopyridin-5-one derivatives with high dual Topo I and IIα inhibitory activity showed a strong correlation with the Topo I and IIα expression levels of the tested cancer cells, and in particular, it was confirmed that the bromine-substituted derivative acts as a DNA indirect catalytic inhibitor for Topo I and IIα while lowering the probability of normal cytotoxicity. At the same time, the burden of general toxicological problems associated with Topo poisoning, such as genotoxic stress and myelosuppression, was greatly alleviated.

Description

2,4-diphenyl indenopyridin-5-one derivatives containing halogen and manufacturing method thereof}

The present invention is a halogen-containing 2,4-diphenyl indenopyridin-5-one derivative that enhances the anticancer response of topoisomerase inhibitors used clinically and its preparation provides a way

Cancer has long been regarded as one of the major diseases to overcome due to the increase in human life expectancy. Numerous studies have proposed various molecules as effective therapeutic targets and attempted to develop new drugs with excellent anticancer activity, and DNA topoisomerase (Topo) is one of the targets that have been intensively studied for decades. As ubiquitous nuclear factors, their essential functions in regulating various topological barriers during processes related to cell proliferation such as DNA replication, transcription, recombination, and chromosome segregation have made molecular enzymes universal therapeutic targets for various cancer types, According to structure, catalytic function, sequence, etc., topoisomerases are mainly classified into Topo I and Topo II. Various inhibitors of both Topo I and Topo II have been clinically demonstrated to have outstanding anticancer activity in a variety of cancer treatment settings. Topo I has a number of camptothecin-derived agents, and Topo II inhibition has several Anthracycline, anthracenedione and epipodophyllotoxins such as doxorubicin or etoposide are widely applied. These substances of most clinical importance are generally classified as topo poisons, which block DNA recombination by binding to and stabilizing cleaved DNA-topo complexes. This induces high levels of DNA strand cleavage, eventually leading to apoptosis. The superior anticancer activity of the anticancer agents in this group is based on the fact that cancer cells are relatively more proliferative than normal cells and are susceptible to DNA damage. However, genotoxic stress caused by drugs inevitably causes serious damage to normal tissues, and despite its efficacy, fatal genotoxic stress problems such as myelosuppression continue to occur. Therefore, in order to solve these problems, research is still being actively conducted to find new topoisomerase catalyst inhibitors and to explore new uses of these agents.

1. Republic of Korea Patent Publication No. 10-2016-0041547.

An object of the present invention is to provide a 2,4-diphenyl indenopyridin-5-one derivative represented by Formula 1 below or a pharmaceutically acceptable salt thereof. there is.

[Formula 1]

Another object of the present invention is a topoisomer comprising the 2,4-diphenyl indenopyridin-5-one derivative or a pharmaceutically acceptable salt thereof as an active ingredient. It is to provide a composition for inhibiting Topoisomerase I and IIα.

Another object of the present invention is cancer disease comprising the 2,4-diphenyl indenopyridin-5-one derivative or a pharmaceutically acceptable salt thereof as an active ingredient It is to provide a pharmaceutical composition for prevention or treatment.

Another object of the present invention is 1) the 2,4-diphenyl indenopyridin-5-one derivative of any one of claims 1 or 2 or a pharmaceutically acceptable product thereof possible salts and 2) etoposide, camptothecin, teniposide, doxorubicin, epirubicin, daunorubicin, idarubicin ), topotecan, irinotecan, and belotecan (belotecan) to provide a pharmaceutical composition for preventing or treating cancer disease containing at least one anticancer agent selected from the group consisting of as an active ingredient.

Another object of the present invention is cancer disease comprising the 2,4-diphenyl indenopyridin-5-one derivative or a pharmaceutically acceptable salt thereof as an active ingredient It is to provide a health functional food composition for prevention or improvement.

Another object of the present invention is i) mixing benzaldehyde, indenene-1,3-dione and bromophenyl methyl ketone; ii) irradiating the mixture of step i) with microwaves and then cooling; iii) washing the cooled mixture of step ii) with water and then extracting with an organic solvent to form an organic layer; and iv) drying and filtering the organic layer of step iii), followed by purification using an organic solvent; 2,4-diphenyl indenopyridin-5-one (2, It is to provide a method for producing 4-diphenyl indenopyridin-5-one) derivatives.

In order to achieve the above object, the present invention is a 2,4-diphenyl indenopyridin-5-one derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof provides

[Formula 1]

In Formula 1, R is selected from bromine (Br).

In addition, the present invention is a topoisomerase comprising the 2,4-diphenyl indenopyridin-5-one derivative or a pharmaceutically acceptable salt thereof as an active ingredient A composition for inhibiting (Topoisomerase) I and IIα is provided.

In addition, the present invention is a cancer disease prevention or treatment comprising the 2,4-diphenyl indenopyridin-5-one derivative or a pharmaceutically acceptable salt thereof as an active ingredient. It provides a pharmaceutical composition for treatment.

In addition, the present invention 1) the 2,4-diphenyl indenopyridin-5-one derivative of any one of claims 1 or 2 or a pharmaceutically acceptable salt thereof and 2) etoposide, camptothecin, teniposide, doxorubicin, epirubicin, daunorubicin, idarubicin, Provided is a pharmaceutical composition for preventing or treating cancer, comprising at least one anticancer agent selected from the group consisting of topotecan, irinotecan, and belotecan as an active ingredient.

In addition, the present invention is a cancer disease prevention or treatment comprising the 2,4-diphenyl indenopyridin-5-one derivative or a pharmaceutically acceptable salt thereof as an active ingredient. It provides a health functional food composition for improvement.

In addition, the present invention includes i) mixing benzaldehyde, indenene-1,3-dione and bromophenyl methyl ketone; ii) irradiating the mixture of step i) with microwaves and then cooling; iii) washing the cooled mixture of step ii) with water and then extracting with an organic solvent to form an organic layer; and iv) drying and filtering the organic layer of step iii), followed by purification using an organic solvent; 2,4-diphenyl indenopyridin-5-one (2, A method for preparing a 4-diphenyl indenopyridin-5-one) derivative is provided.

The present invention is a 2,4-diphenyl indenopyridin-5-one derivative containing a halogen that enhances the anticancer response of topoisomerase inhibitors used clinically, and It relates to a preparation method thereof, which has a strong dual topoisomerase I and IIα inhibitory effect and an antiproliferative effect that is dependent on the expression level of topoisomerase in various cancer cells, thereby enhancing anticancer effects and reducing toxicity. It has a great advantage in mitigating the risk of related problems, so it can be used for cancer treatment and health food.

1 shows a schematic diagram of a 2,4-diphenyl indenopyridin-5-one derivative design strategy.
Figure 2 shows the synthesis routes of 2,4-diphenyl indenopyridin-5-one derivatives.
Figure 3 shows 2,4-diphenyl indenopyridin-5-one derivative structures.
Figure 4 shows the relationship between Topoisomerase (hereinafter referred to as Topo) I and IIα inhibitory activities of 2,4-diphenyl indenopyridin-5-one derivatives.
5 shows a comparison of Topo I and IIα inhibitory abilities according to substituents of 2,4-diphenyl indenopyridin-5-one derivatives.
6 shows the degree of inhibition of proliferation of various cancer cells by 2,4-diphenyl indenopyridin-5-one derivatives.
7 is 2-(4-bromophenyl)-4-(phenyl)-5H-indeno[1,2-b]pyridin-5-one (2-(4-Bromophenyl)-4-(phenyl)- 5H-indeno[1,2-b]pyridin-5-one; hereinafter referred to as compound 9) is a DNA non-interfering Topo I and IIα catalyst inhibitor, and it is shown that it was confirmed that the cytotoxic effect on normal cells is minimized.
Figure 8 shows an effective strategy of combining Topo I and IIα catalyst inhibitors with Topo poisons (topoisomerase poison) to maximize anti-cancer activity.
9 shows a new strategy of combining Topo I and IIα catalyst inhibitors with Topo toxicants to alleviate general genotoxic stress and myelotoxicity.

Hereinafter, the present invention will be described in more detail.

The genotoxic stress caused by existing Topo I and Topo II inhibitory drugs inevitably causes serious damage to normal tissues, and fatal toxicity problems such as myelosuppression continue to occur despite anticancer effects, making it safer and safer. In order to identify an effective anti-cancer drug, ortho- to the 2-phenyl ring of 2,4-diphenyl indenopyridin-5-one , By substituting bromine (Br) at the meta- or para- position, the present invention was completed.

The present invention provides a 2,4-diphenyl indenopyridin-5-one derivative represented by Formula 1 below or a pharmaceutically acceptable salt thereof.

[Formula 1]

In Formula 1, R may be selected from bromine (Br).

The 2,4-diphenyl indenopyridin-5-one derivative represented by Formula 1 may be the following compounds 7 to 9.

(Compound 7) 2-(2-Bromophenyl)-4-(phenyl)-5H-indeno[1,2-b]pyridin-5-one (2-(2-Bromophenyl)-4-(phenyl) -5H-indeno[1,2-b]pyridin-5-one);

(Compound 8) 2-(3-Bromophenyl)-4-(phenyl)-5H-indeno[1,2-b]pyridin-5-one (2-(3-Bromophenyl)-4-(phenyl) -5H-indeno[1,2-b]pyridin-5-one);

(Compound 9) 2-(4-Bromophenyl)-4-(phenyl)-5H-indeno[1,2-b]pyridin-5-one (2-(4-Bromophenyl)-4-(phenyl) -5H-indeno[1,2-b]pyridin-5-one).

The present invention relates to a topoisomerase containing the 2,4-diphenyl indenopyridin-5-one derivative or a pharmaceutically acceptable salt thereof as an active ingredient. ) provides a composition for inhibiting I and IIα.

The present invention is a pharmaceutical for preventing or treating cancer disease comprising a 2,4-diphenyl indenopyridin-5-one derivative or a pharmaceutically acceptable salt thereof as an active ingredient. composition is provided.

The pharmaceutical composition can kill cancer cells by selectively inhibiting Topoisomerase I and IIα.

The cancer diseases include colorectal cancer, prostate cancer, stomach cancer, lung cancer, liver cancer, small intestine cancer, pancreatic cancer, brain cancer, bone cancer, melanoma, breast cancer, scleroderma, uterine cancer, cervical cancer, head and neck cancer, esophageal cancer, thyroid cancer, parathyroid cancer, kidney cancer , sarcoma, urethral cancer, bladder cancer, and hematological cancer.

The present invention relates to 1) a 2,4-diphenyl indenopyridin-5-one derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 or 2, and 2 ) etoposide, camptothecin, teniposide, doxorubicin, epirubicin, daunorubicin, idarubicin, topotecan (topotecan), irinotecan (irinotecan) and belotecan (benotecan) It provides a pharmaceutical composition for preventing or treating cancer disease containing at least one anticancer agent selected from the group consisting of as an active ingredient.

The present invention relates to a 2,4-diphenyl indenopyridin-5-one (2,4-diphenyl indenopyridin-5-one) derivative or a pharmaceutically acceptable salt thereof as an active ingredient for prevention or improvement of cancer disease health A nutraceutical composition is provided.

The present invention includes i) mixing benzaldehyde, indenene-1,3-dione and bromophenyl methyl ketone; ii) irradiating the mixture of step i) with microwaves and then cooling; iii) washing the cooled mixture of step ii) with water and then extracting with an organic solvent to form an organic layer; and iv) drying and filtering the organic layer of step iii), followed by purification using an organic solvent; 2,4-diphenyl indenopyridin-5-one (2, A method for preparing a 4-diphenyl indenopyridin-5-one) derivative is provided.

The bromophenyl methyl ketone is 1- (2-bromophenyl) ethanone (1- (2-bromophenyl) ethanone), 1- (3-bromophenyl) ethanone (1- (3 It may be one selected from the group consisting of -bromophenyl)ethanone) and 1-(4-bromophenyl)ethanone.

Step ii) may be irradiated with microwaves of 200 to 220 W at 100 to 140 °C for 8 to 20 minutes.

In one embodiment of the present invention, cancer containing the 2,4-diphenyl indenopyridin-5-one derivative or a pharmaceutically acceptable salt thereof as an active ingredient A pharmaceutical composition for preventing or treating a disease can be used in any one formulation selected from the group consisting of injections, granules, powders, tablets, pills, capsules, suppositories, gels, suspensions, emulsions, drops or liquids according to conventional methods. can

In another embodiment of the present invention, cancer disease containing a 2,4-diphenyl indenopyridin-5-one derivative or a pharmaceutically acceptable salt thereof as an active ingredient Pharmaceutical compositions for prevention or treatment are suitable carriers, excipients, disintegrants, sweeteners, coating agents, swelling agents, lubricants, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersants, and surfactants commonly used in the manufacture of pharmaceutical compositions. , It may further include one or more additives selected from the group consisting of binders and lubricants.

Specifically, carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil may be used, and solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules. These solid preparations may be prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions for oral use, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base material of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

According to one embodiment of the present invention, the pharmaceutical composition is administered in a conventional manner through intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes. can be administered to the subject as

A preferred dosage of the 2,4-diphenyl indenopyridin-5-one derivative or a pharmaceutically acceptable salt thereof is the condition and weight of the subject, the type and degree of disease , Depending on the drug form, administration route and period, it can be appropriately selected by those skilled in the art. According to one embodiment of the present invention, but not limited thereto, the daily dosage may be 0.01 to 200 mg/kg, specifically 0.1 to 200 mg/kg, and more specifically 0.1 to 100 mg/kg. Administration may be administered once a day or divided into several administrations, and the scope of the present invention is not limited thereby.

In the present invention, the 'subject' may be a mammal including a human, but is not limited to these examples.

In addition, the present invention is a cancer disease prevention or treatment containing the 2,4-diphenyl indenopyridin-5-one derivative or a pharmaceutically acceptable salt thereof as an active ingredient. A health functional food composition for improvement can be provided.

The health food is used with other foods or food additives in addition to the 2,4-diphenyl indenopyridin-5-one derivative, and can be appropriately used according to a conventional method. there is. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use thereof, for example, prevention, health or therapeutic treatment.

The effective dose of the compound contained in the health food may be used according to the effective dose of the therapeutic agent, but may be less than the above range in the case of long-term intake for the purpose of health and hygiene or health control. Since there is no problem in terms of safety, it is certain that the components can be used in an amount greater than the above range.

There is no particular limitation on the type of health food, and examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes; and the like.

Hereinafter, examples and the like will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. Examples of the present invention and the like are provided to more completely explain the present invention to those skilled in the art.

[Preparation Example 1] Reagents and Instruments

Starting materials and reagents used were Sigma-Aldrich, TCI Chemicals, Alfa-Aesar and Junsei Chemical Co. Ltd and used without further purification. HPLC grade methanol and acetonitrile were purchased from Burdick and Jackson. Flash column chromatography was performed using silica gel (Kieselgel 60, 230-400 mesh, Merck), and thin-layer chromatography (TLC) was performed using Kieselgel 60 F254 (Merck). All synthetic compounds containing aromatic rings were detected on a TLC plate using UV light (254 nm; short wavelength and 356 nm; long wavelength). Flash column chromatography for compound purification was performed using a Biotage Isolera One system and an internal tunable dual wavelength (200 nm to 400 nm) diode array detector. Dry sample loading was used by packaging the sample cartridge with silica in the SNAP cartridge. Microwave-assisted synthesis was performed using a Biotage Initiator microwave synthesizer. Proton and carbon NMR spectra for structure determination were obtained using a Bruker AMX 250 MHz spectrometer. Chemical shifts values (δ) were reported in ppm and corrected by TMS (Tetra-MethylSilane), residual peaks of the solvent used, and coupling constants (J values) in Hertz (Hz). Melting points were determined in open capillaries using a digital melting point apparatus (Electrothermal 1A 9100). The purity of synthetic compounds was assessed by gradient-controlled high-performance liquid chromatography (SCL-10A VP) equipped with two Shimadzu LC-10AT pumps, a photodiode array detector (SPD-M10A VP) and a Shimadzu System Controller (SCL-10A VP) utilizing the Shimadzu Lab Solution Program. recorded using a high performance liquid chromatography (HPLC) system. ESI LC/MS analysis was performed and mass (m/z) was calculated using an Advion type compact mass spectrometer and the Advion Data Express program. As the LC/MS ionization conditions, a capillary temperature of 250 °C and a capillary voltage of 180 V were used.

[ synthesis example 1] 2,4-diphenyl indenopyridine -5-on (2,4- diphenyl indenopyridin -5-one) derivative synthesis

1. Common synthesis process of compounds 1 to 15 using microwave

According to Figures 1 and 2, aryl methyl ketone (aryl methyl ketone; 2.00 mmol, 1.0 equiv), benzaldehyde (benzaldehyde; 2.00 mmol, 1.0 equiv), indenene-1,3-dione (indene) in a 5 mL microwave reaction vial -1,3-dione; 2.00 mmol, 1.0 equiv) and ammonium acetate (NH ₄ OAc; 3.00 mmol, 1.5 equiv) were dissolved in dimethylformamide (DMF; 3ml), mixed and sealed. The mixture was irradiated at 120° C. for 8 to 20 minutes with a microwave of 200 to 220 W, cooled to room temperature, poured into cold water, and extracted with ethyl acetate (70 mL). The resulting organic layer was washed with water (30mL x 3) and brine solution (30mL x 3), dried over magnesium sulfate, filtered and evaporated to dryness under reduced pressure. Crude products were purified using a mixed solution of ethyl acetate/hexanes by flash column chromatography (Isolera). As a result, solid compounds 1 to 15 were synthesized as shown in FIG. 3 in a yield of 6 to 16% by rotary evaporation of the eluate.

2. 2-(2- fluorophenyls )-4-(phenyl)-5H- Indeno[1,2-b]pyridine Synthesis of -5-one (2-(2-Fluorophenyl)-4-(phenyl)-5H-indeno[1,2-b]pyridin-5-one; hereinafter referred to as Compound 1)

Compound 1 is indenene-1,3-dione (0.29 g, 2.00 mmol), 1- (2-fluorophenyl) ethanone (1- (2-fluorophenyl) ethanone; 0.24 mL , 2.00 mmol), benzaldehyde (benzaldehyde; 0.2 mL, 2.00 mmol) and ammonium acetate (NH ₄ OAc; 0.23 g, 3.00 mmol) were dissolved in dimethylformamide (DMF; 3ml), and in the synthesis process 200 W microwave was irradiated for 10 minutes according to the method, to obtain Compound 1 as a yellow solid in 128 mg yield (0.36 mmol, 18.2 %).

TLC (ethyl acetate/hexane = 1:15) R _f = 0.30; mp: 190.6-191.6 °C; HPLC: Retention time: 7.97 min, purity: 77.3%; ESI LC/MS: m/z Calcd for C ₂₄ H ₁₅ FNO [MH] ⁺ 352.10; found 352.2. ¹ H NMR (250 MHz, CDCl ₃ ) δ 8.23 (td, J = 7.76, 2.03 Hz, 1H), 7.96 (d, J = 7.47 Hz, 1H), 7.76 - 7.62 (m, 4H), 7.57 (d, J = 7.10 Hz, 2H), 7.54 - 7.35 (m, 6H), 7.31 (t, J = 7.35 Hz, 1H), 7.18 (dd, J = 11.64, 8.14 Hz, 1H). ¹³ C NMR (63 MHz, CDCl ₃ ) δ 190.99, 166.14, 160.77 (d, J = 251.5 Hz), 156.86 (d, J = 2.81 Hz), 149.25, 142.98, 135.26 (d, J = 6.68 Hz), 134.86 , 131.36 (2C) (dd, J = 5.59, 3.11 Hz), 129.58, 129.17 (3C), 128.19 (3C), 125.27 (d, J = 10.8 Hz), 124.60 (d, J = 3.6 Hz), 123.72, 122.67, 120.90, 116.38 (d, J = 23.2 Hz).

3. 2-(3- fluorophenyls )-4-(phenyl)-5H- Indeno[1,2-b]pyridine Synthesis of -5-one (2-(3-Fluorophenyl)-4-(phenyl)-5H-indeno[1,2-b]pyridin-5-one; hereinafter referred to as Compound 2)

Compound 2 is indenene-1,3-dione (0.29 g, 2.00 mmol), 1- (3-fluorophenyl) ethanone (1- (3-fluorophenyl) ethanone; 0.25 mL , 2.00 mmol), benzaldehyde (benzaldehyde; 0.2 mL, 2.00 mmol) and ammonium acetate (NH ₄ OAc; 0.23 g, 3.00 mmol) were dissolved in dimethylformamide (DMF; 3ml), and in the synthesis process 200 W of the microwave was irradiated for 10 minutes according to the method, to obtain Compound 2 as a yellow solid in 78 mg yield (0.22 mmol, 11.1 %).

TLC (ethyl acetate/hexane = 1:20) R _f = 0.28; mp: 187.0-187.5 °C; HPLC: Retention time: 7.48 min, purity: 99.8%; ESI LC/MS: m/z Calcd for C ₂₄ H ₁₅ FNO [MH] ⁺ 352.10; found 352.3. ¹ HNMR (250 MHz, CDCl ₃ )δ 8.01-7.83 (m, 3H), 7.70-7.61 (m, 3H), 7.58 (dd, J = 7.45, 1.16 Hz, 1H), 7.55 - 7.37 (m, 6H) , 7.15 (td, J = 8.48, 2.60 Hz, 1H). ¹³ C NMR (63 MHz, CDCl ₃ )δ 190.84, 166.45, 163.30 (d, J = 245.91 Hz), 159.51 (d, J = 2.8 Hz), 149.66, 142.86, 140.60 (d, J = 7.62 Hz), 135.32 ( d, J = 15.18 Hz), 134.91, 131.10, 130.31 (d, J = 8.17 Hz), 129.67, 129.04 (3C), 128.26 (3C), 123.74, 123.04, 122.84 (d, J = 2.8 Hz), 121.11 ( d, J = 16.0 Hz), 116.90 (d, J = 21.4 Hz), and 114.41 (d, J = 23.0 Hz).

4. 2-(4- fluorophenyls )-4-(phenyl)-5H- Indeno[1,2-b]pyridine Synthesis of -5-one (2-(4-Fluorophenyl)-4-(phenyl)-5H-indeno[1,2-b]pyridin-5-one; hereinafter referred to as compound 3)

Compound 3 is indenene-1,3-dione (0.29 g, 2.00 mmol), 1- (4-fluorophenyl) ethanone (1- (4-fluorophenyl) ethanone; 0.24 mL , 2.00 mmol), benzaldehyde (benzaldehyde; 0.2 mL, 2.00 mmol) and ammonium acetate (NH ₄ OAc; 0.23 g, 3.00 mmol) were dissolved in dimethylformamide (DMF; 3ml), and in the synthesis process 200 W microwave was irradiated for 20 minutes according to the method, to obtain compound 3 as a yellow solid in 98 mg yield (0.28 mmol, 13.9 %).

TLC (ethyl acetate/hexane = 1:7) R _f = 0.25; mp: 190.4-190.8 °C; HPLC: Retention time: 7.50 min, purity: 98.8%; ESI LC/MS: m/z Calcd for C ₂₄ H ₁₅ FNO [MH] ⁺ 352.10; found 352.2. ^1H NMR (250 MHz, CDCl ₃ )δ8.20-8.10(m,2H),7.95(d, J = 7.30 Hz, 1H), 7.70 - 7.62 (m, 3H), 7.58 (td, J = 7.53, 1.23 Hz, 1H), 7.54 - 7.45 (m, 4H), 7.41 (td, J = 7.46, 1.03 Hz, 1H), 7.23 - 7.11 (m, 2H). ¹³ CNMR (63MHz, CDCl ₃ )δ190.91,166.44,164.13 (d, J = 250.57 Hz), 159.87, 149.56, 142.85, 135.35 (d, J = 9.42 Hz), 134.80, 134.42 (d, J = 3.14 Hz), 131.01, 129.60, 129.41, 129.27, 129.01 (3C), 128.22 (3C), 123.66, 122.46, 120.86, 120.70, 115.81 (d, J = 21.7 Hz).

5. 2-(2- chlorophenyl )-4-(phenyl)-5H- Indeno[1,2-b]pyridine Synthesis of -5-one (2-(2-Chlorophenyl)-4-(phenyl)-5H-indeno[1,2-b]pyridin-5-one; hereinafter referred to as Compound 4)

Compound 4 is indenene-1,3-dione (0.29 g, 2.00 mmol), 1- (2-chlorophenyl) ethanone (1- (2-chlorophenyl) ethanone; 0.26 mL, 2.00 mmol), benzaldehyde (benzaldehyde; 0.2 mL, 2.00 mmol) and ammonium acetate (NH ₄ OAc; 0.23 g, 3.00 mmol) were dissolved in dimethylformamide (DMF; 3ml) and, according to the above synthetic procedure, Irradiated with a microwave of 220 W for 20 minutes, yielding 45 mg of compound 4 as a yellow solid (0.12 mmol, 6.1%) was obtained.

TLC (ethyl acetate/hexane = 1:15) R _f = 0.22; mp: 193.5-194.5 °C; HPLC: Retention time: 7.30 min, purity: 99.7%; ESI LC/MS: m/z Calcd for C ₂₄ H ₁₅ ClNO [MH] ⁺ 368.07; found 368.1. ^1H NMR (250 MHz, CDCl ₃ )δ 7.94 (d, J = 7.38 Hz, 1H), 7.76 (dd, J = 6.95, 2.55 Hz, 1H), 7.72 - 7.63 (m, 3H), 7.58 (t , J = 7.54 Hz, 1H), 7.55 - 7.31 (m, 8H). ¹³ CNMR(63MHz,CDCl ₃ )δ191.03,166.24,160.60,148.70,142.98,138.45,135.26,135.05,134.95,132.37,131.62,131.03,130.40,130.25,129.64,129.21(2C),128.22(2C),127.11, 125.89,123.79,122.73,121.04.

6. 2-(3- chlorophenyl )-4-(phenyl)-5H- Indeno[1,2-b]pyridine Synthesis of -5-one (2-(3-Chlorophenyl)-4-(phenyl)-5H-indeno[1,2-b]pyridin-5-one; hereinafter referred to as compound 5)

Compound 5 is indenene-1,3-dione (0.29 g, 2.00 mmol), 1- (3-chlorophenyl) ethanone (1- (3-chlorophenyl) ethanone; 0.26 mL, 2.00 mmol), benzaldehyde (benzaldehyde; 0.2 mL, 2.00 mmol) and ammonium acetate (NH ₄ OAc; 0.23 g, 3.00 mmol) were dissolved in dimethylformamide (DMF; 3ml) and, according to the above synthetic procedure, Irradiated with a microwave of 200 W for 10 minutes, yielding 96 mg of compound 5 as a yellow solid (0.26 mmol, 13.1%) was obtained.

TLC (ethyl acetate/hexane = 1:20) R _f = 0.28; mp: 177.0-178.0 °C; HPLC: Retention time: 8.22 min, purity: 90.7%; ESI LC/MS: m/z Calcd for C ₂₄ H ₁₅ ClNO [MH] ⁺ 368.07; found 368.1. ¹ H NMR (250 MHz, CDCl ₃ )δ8.18(s,1H),8.06-7.92(m,2H),7.72-7.56(m,4H),7.56-7.47(m,4H),7.47-7.37( m,3H). ¹³ C NMR (63 MHz, CDCl ₃ )δ ¹³ CNMR (63MHz, CDCl ₃ )δ 190.86, 166.43, 159.32, 149.61, 142.75, 139.97, 135.34, 135.08, 134.96, 134.93, 131.11, 130.04, 129.97 (129.97) ),128.25(2C),127.52,125.34,123.73,122.97,121.23,120.99.

7. 2-(4- chlorophenyl )-4-(phenyl)-5H- Indeno[1,2-b]pyridine Synthesis of -5-one (2-(4-Chlorophenyl)-4-(phenyl)-5H-indeno[1,2-b]pyridin-5-one; hereinafter referred to as compound 6)

Compound 6 is indenene-1,3-dione (0.29 g, 2.00 mmol), 1- (4-chlorophenyl) ethanone (1- (4-chlorophenyl) ethanone; 0.26 mL, 2.00 mmol), benzaldehyde (benzaldehyde; 0.2 mL, 2.00 mmol) and ammonium acetate (NH ₄ OAc; 0.23 g, 3.00 mmol) were dissolved in dimethylformamide (DMF; 3ml) and, according to the above synthetic procedure, Irradiated with a microwave of 200 W for 15 minutes, yielding 91 mg of compound 6 as a yellow solid (0.25 mmol, 12.4 %) was obtained.

TLC (ethyl acetate/hexane = 1:20) R _f = 0.25; mp: 196.0-197.0 °C; HPLC: Retention time: 9.19 min, purity: 92.8%; ESI LC/MS: m/z Calcd for C ₂₄ H ₁₅ ClNO [MH] ⁺ 368.07; found 368.2. ¹ H NMR (250 MHz, CDCl ₃ )δ 8.10 (d, J = 8.59 Hz, 2H), 7.95 (d, J = 7.32 Hz, 1H), 7.72 - 7.54 (m, 4H), 7.54 - 7.35 (m , 7H). ¹³ CNMR(63MHz,CDCl ₃ )δ190.80,166.46,159.67,149.61,142.86,136.70,136.30,135.47,135.26,134.83,131.06,129.63,129.03(4C),128.64(2C,128.7.2C),128.64(2C),128.64(2C) ,120.91,120.83.

8. 2-(2- bromophenyl )-4-(phenyl)-5H- Indeno[1,2-b]pyridine Synthesis of -5-one (2-(2-Bromophenyl)-4-(phenyl)-5H-indeno[1,2-b]pyridin-5-one; hereinafter referred to as Compound 7)

Compound 7 is indene-1,3-dione (0.29 g, 2.00 mmol), 1- (2-bromophenyl) ethanone (1- (2-Bromophenyl) ethanone; 0.27 mL , 2.00 mmol), benzaldehyde (benzaldehyde; 0.2 mL, 2.00 mmol) and ammonium acetate (NH ₄ OAc; 0.23 g, 3.00 mmol) were dissolved in dimethylformamide (DMF; 3ml), and in the synthesis process 200 W microwave was irradiated for 15 minutes according to the method, to obtain 218 mg yield (0.27 mmol, 13.2%) of compound 7 as a yellow solid.

TLC (ethyl acetate/hexane = 1:20) R _f = 0.20; mp: 199.5-200.4 °C; HPLC: Retention time: 7.36 min, purity: 99.8%; ESI LC/MS: m/z Calcd for C ₂₄ H ₁₅ BrNO [MH] ⁺ 412.02; found 412.2. ^1H NMR (250 MHz, CDCl ₃ )δ 7.94 (d, J = 7.39 Hz, 1H), 7.75 - 7.63 (m, 5H), 7.58 (td, J = 7.49, 1.08 Hz, 1H), 7.52 - 7.37 (m, 6H), 7.29 (td, J = 7.77, 1.75 Hz, 1H). ¹³ CNMR(63MHz,CDCl ₃ )δ191.01,166.13,161.94,148.58,142.88,140.35,135.19,134.96,133.58,131.45,131.02,130.35,129.65,129.19(2C), 128.19 (2C), 127.65, 125.83, 123.77, 122.68, 121.77, 121.04.

9. 2-(3- bromophenyl )-4-(phenyl)-5H- Indeno[1,2-b]pyridine Synthesis of -5-one (2-(3-Bromophenyl)-4-(phenyl)-5H-indeno[1,2-b]pyridin-5-one; hereinafter referred to as Compound 8)

Compound 8 is indene-1,3-dione (0.29 g, 2.00 mmol), 1- (3-bromophenyl) ethanone (1- (3-Bromophenyl) ethanone; 0.27 mL , 2.00 mmol), benzaldehyde (benzaldehyde; 0.2 mL, 2.00 mmol) and ammonium acetate (NH ₄ OAc; 0.23 g, 3.00 mmol) were dissolved in dimethylformamide (DMF; 3ml), and in the synthesis process The mixture was irradiated with microwave at 200 W for 15 minutes, yielding 94 mg of Compound 8 as a yellow solid (0.23 mmol, 11.4 %).

TLC (ethyl acetate/hexane = 1:20) R _f = 0.27; mp: 159.5-160.5 °C; HPLC: Retention time: 9.66 min, purity: 80.3%; ESI LC/MS: m/z Calcd for C ₂₄ H ₁₅ BrNO [MH] ⁺ 412.02; found 412.1 ¹ HNMR (250MHz, CDCl ₃ )δ8.33(t, J = 1.73 Hz, 1H), 8.05 (dt, J = 7.83, 1.26 Hz, 1H), 7.98 (d, J = 7.35 Hz, 1H), 7.72 - 7.53 (m, 5H), 7.57 - 7.43 (m, 5H), 7.50 - 7.28 (m, 4H). ¹³ C NMR (63 MHz, CDCl ₃ )δ 190.70, 166.43, 159.22, 149.64, 142.83, 140.30, 135.45, 135.18, 134.86, 132.87, 131.07, 130.46, 130.25, 129.65, 129.1, 24.05(2C) 123.70,123.16,123.05,121.17,121.01.

10. 2-(4- bromophenyl )-4-(phenyl)-5H- Indeno[1,2-b]pyridine Synthesis of -5-one (2-(4-Bromophenyl)-4-(phenyl)-5H-indeno[1,2-b]pyridin-5-one; hereinafter referred to as Compound 9)

Compound 9 is indenene-1,3-dione (0.29 g, 2.00 mmol), 1- (4-bromophenyl) ethanone (1- (4-Bromophenyl) ethanone; 0.40 g , 2.00 mmol), benzaldehyde (benzaldehyde; 0.2 mL, 2.00 mmol) and ammonium acetate (NH ₄ OAc; 0.23 g, 3.00 mmol) were dissolved in dimethylformamide (DMF; 3ml), and in the synthesis process According to the microwave irradiation at 220 W for 8 minutes, compound 9 in a yellow solid state was obtained in a yield of 91 mg (0.22 mmol, 11.0 %).

TLC (ethyl acetate/hexane = 1:20) R _f = 0.29; mp: 226.9-227.9 °C; HPLC: Retention time: 9.72 min, purity: 95.1%; ESI LC/MS: m/z Calcd for C ₂₄ H ₁₅ BrNO [MH] ⁺ 412.02; found 412.3. ¹ H NMR (250 MHz, CDCl ₃ )δ 8.03 (d, J = 8.52 Hz, 2H), 7.96 (d, J = 7.36 Hz, 1H), 7.72 - 7.55 (m, 6H), 7.55 - 7.46 (m , 4H), 7.42 (t, J = 7.39 Hz, 1H). ¹³ C NMR (63 MHz, CDCl ₃ )δ 190.83, 166.50, 159.77, 149.66, 142.88, 137.18, 135.49, 135.26, 134.86, 132.02 (2C), 131.09, 129.66, 129.04 (2C), 128.90 (2C), 91 ), 124.73, 123.73, 122.85, 120.95, 120.85.

11. 2-(2- trifluorophenyl )-4-(phenyl)-5H- Indeno[1,2-b]pyridine Synthesis of -5-one (2-(2-Trifluoromethylphenyl)-4-(phenyl)-5H-indeno[1,2-b]pyridin-5-one; hereinafter referred to as Compound 10)

Compound 10 is indene-1,3-dione (0.29 g, 2.00 mmol), 1- [2- (trifluoromethyl) phenyl] ethanone (1- [2- (trifluoromethyl )phenyl]ethanone; 0.3 ml, 2.00 mmol), benzaldehyde (0.2 mL, 2.00 mmol) and ammonium acetate (NH ₄ OAc; 0.23 g, 3.00 mmol) in dimethylformamide (DMF; 3ml). Dissolved, and irradiated with a microwave at 200 W for 10 minutes according to the synthesis process to obtain Compound 10 as a yellow solid in 56 mg yield (0.14 mmol, 7.0%).

TLC (ethyl acetate/hexane = 1:25) R _f = 0.30; mp: 182.6-183.6 °C; HPLC: Retention time: 8.43 min, purity: 97.2%; ESI LC/MS: m/z Calcd for C ₂₅ H ₁₅ F ₃ NO [MH] ⁺ 402.10; found 402.2. ^1H NMR (250 MHz, CDCl ₃ )δ 8.71 (dd, J = 6.64, 3.15 Hz, 2H), 8.31 (dd, J = 6.57, 3.15 Hz, 2H), 8.08 (d, J = 7.31 Hz, 1H) ), 7.79 (d, J = 7.24 Hz, 1H), 7.69 (td, J = 7.46, 1.24 Hz, 1H), and 7.62 - 7.48 (m, 7H). ¹³ C NMR (63 MHz, CDCl ₃ )δ 190.07, 174.73, 167.30, 162.95, 141.09, 137.03, 135.48, 135.28, 134.97, 132.95, 131.92, 131.54, 130.23 (2C), 129.30 (2C), 129.30 (2C), 5 (2C), 124.09, 121.88, 118.86.

12. 2-(3- trifluorophenyl )-4-(phenyl)-5H- Indeno[1,2-b]pyridine Synthesis of -5-one (2-(3-Trifluoromethylphenyl)-4-(phenyl)-5H-indeno[1,2-b]pyridin-5-one; hereinafter referred to as Compound 11)

Compound 11 is indenene-1,3-dione (0.29 g, 2.00 mmol), 1- [3- (trifluoromethyl) phenyl] ethanone (1- [3- (trifluoromethyl )phenyl]ethanone; 0.3 ml, 2.00 mmol), benzaldehyde (0.2 mL, 2.00 mmol) and ammonium acetate (NH ₄ OAc; 0.23 g, 3.00 mmol) in dimethylformamide (DMF; 3ml). Dissolved, and irradiated with a microwave at 200 W for 10 minutes according to the synthesis process to obtain Compound 11 as a yellow solid in 49 mg yield (0.12 mmol, 6.1%).

TLC (ethyl acetate/hexane = 1:20) R _f = 0.28; mp: 189.0-190.0 °C; HPLC: Retention time: 7.99 min, purity: 91.7%; ESI LC/MS: m/z Calcd for C ₂₅ H ₁₅ F ₃ NO [MH] ⁺ 402.10; found 402.2. ¹ HNMR (250 MHz, CDCl ₃ )δ 8.44(s, 1H), 8.32 (d, J = 7.77 Hz, 1H), 7.99 (d, J = 7.43 Hz, 1H), 7.79 - 7.34 (m, 11H). ¹³ CNMR(63MHz,CDCl ₃ )δ190.75,166.52,159.22,149.77,142.74,139.03,135.39,135.07,134.97,131.19,130.46,129.74,129.31,129.04(2C), 128.29 (2C), 126.50, 124.27, 123.78, 123.14, 121.27, 121.06.

13. 2-(4- trifluorophenyl )-4-(phenyl)-5H- Indeno[1,2-b]pyridine Synthesis of -5-one (2-(4-Trifluoromethylphenyl)-4-(phenyl)-5H-indeno[1,2-b]pyridin-5-one; hereinafter referred to as Compound 12)

Compound 12 is indenene-1,3-dione (0.29 g, 2.00 mmol), 1- [4- (trifluoromethyl) phenyl] ethanone (1- [4- (trifluoromethyl )phenyl]ethanone; 0.376 g, 2.00 mmol), benzaldehyde (benzaldehyde; 0.2 mL, 2.00 mmol) and ammonium acetate (NH ₄ OAc; 0.23 g, 3.00 mmol) in dimethylformamide (DMF; 3ml). Dissolved, and irradiated with microwave at 200 W for 10 minutes according to the synthesis process to obtain Compound 12 as a yellow solid in 70 mg yield (0.18 mmol, 8.7%).

TLC (ethyl acetate/hexane = 1:20) R _f = 0.22; mp: 199.0-200.0 °C; HPLC: Retention time: 8.09 min, purity: 99.9%; ESI LC/MS: m/z Calcd for C ₂₅ H ₁₅ F ₃ NO [MH] ⁺ 402.10; found 402.4. ¹ HNMR (250 MHz, CDCl ₃ )δ 8.25 (d, J = 8.19 Hz, 2H), 7.97 (d, J = 7.36 Hz, 1H), 7.75 (d, J = 8.20 Hz, 2H), 7.71 - 7.55 ( m, 5H), 7.55 - 7.47 (m, 3H), 7.43 (t, J = 7.44 Hz, 1H). ¹³ CNMR(63MHz,CDCl ₃ )δ190.80,166.49,159.26,149.68,142.71,141.51,135.32,134.99,131.20,129.77,129.02(3C), 128.28 (3C), 127.65 ( dd = 3C), 125.65 (3C) , 10.0 Hz), 123.80, 123.17, 121.61, 120.98.

14. 2-(2- trifluoromethoxyphenyl )-4-(phenyl)-5H- Indeno[1,2-b]pyridine Synthesis of -5-one (2-(2-Trifluoromethoxyphenyl)-4-(phenyl)-5H-indeno[1,2-b]pyridin-5-one; hereinafter referred to as Compound 13)

Compound 13 is indenene-1,3-dione (0.29 g, 2.00 mmol), 1-[2-(trifluoromethoxy)phenyl]ethanone (1-[2-( trifluoromethoxy)phenyl]ethanone; 0.32 mL, 2.00 mmol), benzaldehyde (benzaldehyde; 0.2 mL, 2.00 mmol) and ammonium acetate (NH ₄ OAc; 0.23 g, 3.00 mmol) were mixed with dimethylformamide (DMF; 3ml). was dissolved in, and irradiated with a microwave at 200 W for 10 minutes according to the above synthesis process, yielding 132 mg of compound 13 as a yellow solid (0.32 mmol, 15.8%).

TLC (ethyl acetate/hexane = 1:20) R _f = 0.21; mp: 132.3-133.3 °C; HPLC: Retention time: 7.13 min, purity: 99.7%; ESI LC/MS: m/z Calcd for C ₂₅ H ₁₅ F ₃ NO ₂ [MH] ⁺ 418.09; found 418.2. ^1H NMR (250 MHz, CDCl ₃ )δ 8.07-7.99(m, 1H), 7.95(d, J = 7.39 Hz, 1H), 7.75 - 7.63 (m, 3H), 7.63 - 7.54 (m, 2H) , 7.54 - 7.34 (m, 7H). ¹³ C NMR (63 MHz, CDCl ₃ )δ 191.03, 166.28, 158.05, 149.04, 146.59, 142.90, 135.22, 135.01, 134.99, 132.86, 131.87, 131.07, 130.75, 129.65, 28.2, 3, 12(2C) 125.54, 123.79, 122.71, 121.51, 120.98.

15. 2-(3- trifluoromethoxyphenyl )-4-(phenyl)-5H- Indeno[1,2-b]pyridine Synthesis of -5-one (2-(3-Trifluoromethoxyphenyl)-4-(phenyl)-5H-indeno[1,2-b]pyridin-5-one; hereinafter referred to as Compound 14)

Compound 14 is indenene-1,3-dione (0.29 g, 2.00 mmol), 1-[3-(trifluoromethoxy)phenyl]ethanone (1-[3-( trifluoromethoxy)phenyl]ethanone; 0.33 mL, 2.00 mmol), benzaldehyde (0.2 mL, 2.00 mmol) and ammonium acetate (NH ₄ OAc; 0.23 g, 3.00 mmol) in dimethylformamide (DMF; 3ml) was dissolved in, and irradiated with a microwave at 200 W for 10 minutes according to the above synthesis process, yielding 120 mg of compound 14 as a yellow solid (0.29 mmol, 14.4%).

TLC (ethyl acetate/hexane = 1:20) R _f = 0.27; mp: 139.3-139.8 °C; HPLC: Retention time: 8.16 min, purity: 99.7%; ESI LC/MS: m/z Calcd for C ₂₅ H ₁₅ F ₃ NO ₂ [MH] ⁺ 418.09; found 418.2. ^1H NMR (250 MHz, CDCl ₃ )δ8.09-8.01(m,2H),7.98(d, J = 7.36 Hz, 1H), 7.71 - 7.56 (m, 4H), 7.56 - 7.47 (m, 5H) , 7.43 (t, J = 7.44 Hz, 1H), 7.38 - 7.27 (m, 1H). ¹³ C NMR (63 MHz, CDCL ₃ ) Δ190.78,166.48,149.84,149.71,142.77,140.40,135.11,134.95,131.16,1316,129.716.716.716.04 (2C), 128.28 (2C), 128 (2C), 125 123.12, 122.20, 121.28, 121.03, 120.09.

16. 2-(4- trifluoromethoxyphenyl )-4-(phenyl)-5H- Indeno[1,2-b]pyridine Synthesis of -5-one (2-(4-Trifluoromethoxyphenyl)-4-(phenyl)-5H-indeno[1,2-b]pyridin-5-one; hereinafter referred to as Compound 15)

Compound 15 is indenene-1,3-dione (0.29 g, 2.00 mmol), 1-[4-(trifluoromethoxy)phenyl]ethanone (1-[4-( trifluoromethoxy)phenyl]ethanone; 0.409 g, 2.00 mmol), benzaldehyde (0.2 mL, 2.00 mmol) and ammonium acetate (NH ₄ OAc; 0.23 g, 3.00 mmol) were mixed with dimethylformamide (DMF; 3ml). was dissolved in, and irradiated with a microwave at 200 W for 10 minutes according to the above synthesis process, yielding 135 mg of compound 15 as a yellow solid (0.32 mmol, 16.2%).

TLC (ethyl acetate/hexane = 1:20) R _f = 0.28; mp: 141.5-142.3 °C; HPLC: Retention time: 8.20 min, purity: 99.4%; ESI LC/MS: m/z Calcd for C ₂₅ H ₁₅ F ₃ NO ₂ [MH] ⁺ 418.09; found 418.2. ¹ HNMR (250 MHz, CDCl ₃ )δ8.19 (d, J = 8.79 Hz, 2H), 7.96 (d, J = 7.35 Hz, 1H), 7.72 - 7.56 (m, 4H), 7.56 - 7.47 (m, 4H) ), 7.43 (t, J = 7.44 Hz, 1H), 7.34 (d, J = 8.26 Hz, 2H). ¹³ C NMR (63 MHz, CDCl ₃ )δ 190.85, 166.50, 159.53, 150.58, 149.67, 142.82, 136.86, 135.42, 135.18, 134.90, 131.12, 129.69, 129.03(2C), 128.97(2C, 26.26(2C), 128.97(2C) ,122.80,121.08,121.04,120.93.

[ Experimental example One] In vitro DNA from topoisomerase ( Topo ) I and Iα Mitigation analysis

Supercoiled pBR322 plasmid DNA (Thermo Scientific, USA) and recombinant human DNA Topo I or IIα enzyme (Inspiralis, UK) were tested in a test buffer with or without the synthesized compounds 1 to 15. Incubated and the reaction was stopped by adding stop solution. The reaction material was electrophoresed on 0.8% agarose gels at 50 V for 1 hour using 1x TAE as a running buffer. The gel was stained with EtBr solution and visualized using UV. The amount of supercoiled DNA was quantified using an Alpha Tech Imager ^™ (Alpha Innotech Corp., USA).

[Experimental Example 2] Cleavage complex assay

Supercoil DNA (Thermo, USA; pBR322 125 ng) was incubated at 37 °C with Topo I or IIα enzyme (Inspiralis, UK) for 30 minutes, and the reaction was stopped with Topo I or II stop buffer. Digestion was completed with 2 μL of proteinase K (#P4850, Sigma, USA) at 45° C. for 30 minutes. After addition of the gel-loading dye, the specimens were electrophoresed on a 1.5% agarose gel containing 0.5 µg/mL EtBr in 1x TAE buffer, and cleaved or linear DNA bands were visualized according to the same DNA Topo relaxation assay method.

[Experimental Example 3] EtBr displacement assay

Competitive EtBr migration assay was performed, ctDNA (calf thymus DNA; Sigma, 30 μM) was pre-mixed with EtBr (20 μM) in tris buffer (10 mM, pH 7.2) and shaken at room temperature for 30 minutes cultured. Then, mAMSA (TopoGEN Inc., USA) and synthesized compounds 1 to 15 were added to a 96-well white plate (NuncTM, Denmark) containing the pre-incubated mixture of EtBr, ctDNA and Tris buffer, respectively. After all compounds were added, the plates were immediately incubated in the dark for 30 minutes. The fluorescence intensity of EtBr-ctDNA was measured using a Microplate Reader M200 Pro (Tecan Group Ltd., Switzerland) with an excitation of 471 nm and an emission spectrum of 520 to 700 nm.

[Experimental Example 4] Alkaline comet assay

To evaluate DNA damage, Comet assay was performed using single cell gel electrophoresis and Comet Assay Kit (Trevigen, USA) according to the manufacturer's instructions. The cells were dispensed in a 6-well plate at 1 × 10 ⁵ cells/well, and two days later, the cells were treated with the above compounds 1 to 15 for 24 hours, collected by trypsinization, and the cells were added to 1 mL of 1x PBS. redeposited.

Cells were mixed with LM agarose at a ratio of 1:10 and immediately coagulated on comet slides. The slide was further dissolved, electrophoresed under alkaline conditions, and finally stained with SYBR Gold (Vitrogen, Invitrogen, USA). DNA comet tail images were confirmed using an Axio Observer 7 fluorescence microscope (Carl Zeiss, Germany).

[Experimental Example 5] Cell culture and cell viability analysis

The cytotoxicity of compounds 1 to 15 was evaluated using four different cancer cell lines: HCT15 (human colon cancer cell line), T47D (human breast cancer cell line), DU145 (human prostate cancer cell line) and Hela (human cervical cancer cell line), The four cancer cell lines and M2-10b4 (murine bone marrow cells) cells were cultured in RPMI1640 or DMEM supplemented with 10% Fetal Bovine Serum (FBS; Corning, USA) and 1% penicillin-streptomycin (Thermo, USA). Cultured in high glucose medium (Welgene, Korea).

For cell viability analysis, cells were dispensed into 96-well culture plates at 1×10 ⁴ cells/well and incubated for 24 hours, then incubated in serum-free medium for 4 hours, and then containing the above compounds 1 to 15. It was incubated for 72 hours in serum-free medium. Ez-cytoX (5 μL; Dogen, Korea) was then added to each well for 1 to 4 hours, and absorbance was read at 450 nm using a Tecan Infinite M200 PRO Multi-Detection Microplate Reader (Tecan Group Ltd., Switzerland). Cell viability was evaluated by measuring.

[ Experimental example 6] of various cancer cell lines Topo Database analysis of gene expression levels

Topo gene expression levels in various cancer cell lines were evaluated using the gene expression omnibus (GEO) and Cancer Cell Line Encyclopedia (CCLE) data sets. GSE57083 used was obtained from the National Center for Biotechnology Information (NCBI) GEO, and the CCLE dataset was downloaded from the CCLE website (RCLE RNAseq Gene Expression Data 1019 Cell Lines (RPKM)). Topo I and IIα expression was evaluated using the respective probe IDs, ENSG00000198900.5 and ENSG00000131747.10. Heatmap was visualized by facilitating the GENE-E software hosted at the Broad Institute ( http://www.broadinstitute.org/cancer/software/GENE-E/ ).

[Experimental Example 7] Western blot analysis

Western blotting was performed according to previously known experimental methods. Before the experiment, HCT15 and DU145 were dispensed into a 6-well plate, cultured until reaching 60 to 70% confluency, and the compounds 1 to 15 were treated for 24 hours, respectively. The culture medium was changed to a serum-free state immediately before treatment with the compounds.

[Experimental Example 8] In vivo Xenograft mouse model in

HCT15 (5 × 106 cells/100 μL) single cell suspension was inoculated subcutaneously into the right flank of 6-week-old BALB/c female nude mice (Orient bio Inc., Seongnam, South Korea). Mice were randomly distributed when tumors reached an average size of 200 to 300 mm ³ . Compound 9 was prepared at the concentrations indicated in FIG. 8 in a DMAC/Tween80/saline (5:10:85) mixed solution and was administered daily for 6 days via intraperitoneal (ip) injection. The length (Length; L) and width (Width; W) of the tumor were measured every 2 days, and the dose was calculated using the formula (L × W ² )/2. Mice were treated 20 days after the first drug administration. After sacrifice, tumors were immediately isolated from each mouse. Ewha Womans University Animal Care and Use Committee (IACUC number: 20-008) was approved.

[Experimental Example 9] Immunohistochemistry (IHC) analysis

The tumor tissue slides derived from the xenograft mouse model of Experimental Example 8 were deparaffinized by changing xylene three times (5 minutes each), and gradually decreasing concentrations of ethanol (100, 95, 70 for 5 minutes each). , 50 and 30%), and for Antigen retrieval, slides were sonicated for 20 min in 10 mM sodium citrate buffer (pH 6.0). The slides were then incubated with 0.3% hydrogen peroxide for 15 minutes to block endogenous peroxidase activity and subsequently treated with blocking solution (1.5% horse serum in PBS) for 30 minutes. Slides were finally developed using 3,3'-diaminobenzidine tetrachloride (DAB) solution (Dako, USA) according to the manufacturer's protocol. After counterstaining with hematoxylin (Scytek, USA), the slides were washed in running tap water for 10 minutes, followed by increasing concentrations of ethanol (50, 70, 95, 100% for 3 minutes each) and xylene. were dehydrated in three changes (3 min each). Images were obtained at 200x magnification using the Axiophot 2 apparatus (Carl Zeiss, Germany), and finally, staining intensity was quantified using Image J software.

[ Example 1] of compounds 1 to 15 Topo I/ Iα Inhibitory activity and rescue activity correlation

Referring to FIG. 4 , Topo I and II relaxation assays were performed at a concentration of 100 μM to evaluate the Topo I and IIα inhibitory activities of the compounds 1 to 15, respectively. In FIG. 4 (A), Lane D is treated with only pBR322 DNA, Lane T is treated with pBR322 DNA + Topo I, Lane C is treated with pBR322 DNA + Topo I + camptothecin, and Lanes 1 to 15 are treated with pBR322 DNA + Topo I + means treatment with compounds 1 to 15, respectively. In FIG. 4 (B), Lane D is treated with only pBR322 DNA, Lane T is treated with pBR322 DNA + Topo IIα, and Lane C is treated with pBR322 DNA + Topo IIα + Treatment with etoposide and Lanes 1 to 15 indicate treatment with pBR322 DNA + Topo IIα + compounds 1 to 15, respectively.

As a result, according to Table 1, almost all 2,4-diphenyl indenopyridin-5-ones containing chlorine (Cl), bromine (Br) or trifluoromethyl compounds (2,4 -diphenyl indenopyridin-5-one) derivatives showed strong dual Topo I and IIα inhibitory activity, whereas fluoro (F) and trifluoromethoxy compound derivatives inhibited all Topo I except Compound 14. and IIα inhibitory activity were found to be weak. Compounds 5 to 9, 11, 12 and 14 inhibited Topo I (80.4 to 100%) more strongly than camptothecin at 100 μM. In the case of Topo IIα, compounds 4 to 11 showed stronger inhibitory activity (83.1 to 98.1%) than etoposide at 100 μM.

In addition, according to the upper panel of FIG. 5 , regardless of the substituted position, all compounds containing bromine (Br) show strong Topo I inhibition, especially when bromine is 2,4-diphenyl indeno. It can be explained that pyridin-5-one (2,4-diphenyl indenopyridin-5-one) plays an important role in the Topo I inhibitory activity, and according to the lower panel of FIG. 5, bromine (Br), chlorine (Cl) or the presence of a trifluoromethyl substituent compared to hydroxy (OH), fluoro (F) or trifluoromethoxy substituents in 4-diphenyl indenopyridin-5-one (2,4 -diphenyl indenopyridin-5-one) was also confirmed to have an effect on the Topo IIα inhibitory activity.

[Example 2] Confirmation of inhibition of cancer cell proliferation by compounds 1 to 15

According to Table 2, DU145, HCT15, Hela and T47D ( human breast cancer) were evaluated in 4 different human cancer cell lines. The four cancer cell lines expressed levels of Topo I and IIα (DU145 and HCT15; Topo I and IIα high expression cell lines; hereinafter referred to as Topo ^high , HeLa and T47D; Topo I and IIα low expression cell lines; hereinafter referred to as Topo ^low ) ) was finally selected based on To test the expression levels of Topo I and IIα, the databases of Experimental Example 6 (GSE57083 and CCLE databases) demonstrating the basic gene expression levels of various cancer cell lines were utilized. Figure 6 (A) shows the gene expression levels of Topo I and IIα in four different cancer cell lines DU145, HCT15, HeLa and T47D selected from GSE57083 and CCLE databases, and Figure 6 (B) shows the IC for the cancer cell lines tested. A value of ₅₀ indicates the number of compounds in Synthesis Example 1 above 50.

As a result, according to Table 3, 2,4-diphenyl indenopyridin-5-one, which shows Topo I and IIα inhibitory activity, was found to be effective in Topo ^high cells and Topo ^low cells. It was observed that the cytotoxic effect tended to be higher than that in cells. It was confirmed that this tendency appeared more clearly in Compounds 4 to 12, which showed the most distinct difference in Topo inhibitory activity between the oxidized and reduced forms. In addition, the IC ₅₀ value showed a difference of more than 2-fold between the upper cell line and the lower cell line. These results confirm that the anticancer effects of 2,4-diphenyl indenopyridin-5-one derivatives generally depend on Topo inhibition.

[ Example 3] DNA that minimizes cytotoxic effects on normal cells non-interfering Topo I and IIα Catalytic Inhibitors

Since compound 9 showed the strongest antiproliferative activity in the Topo ^high cell line, compound 9 was finally investigated further, mainly to find the exact mode of action (hereinafter referred to as MOA). conducted the test.

To first evaluate whether compound 9 acts as a Topo toxicant or catalyst inhibitor, Topo I and IIα were used as positive controls, respectively, with etoposide and camptothecin, which have well-known Topo toxic properties. A cleavage complex assay was performed on all. Excess Topo I and IIα were separately incubated with supercoil plasmid DNA and pBR322 in the presence of different concentrations of compound 9. The cleavage complex in each reaction sample was finally digested together with proteinase K, and the plasmid DNA phase change was analyzed by agarose gel electrophoresis. Since Topo I introduces single-strand breaks and Topo II induces double-strand breaks into DNA, different results should have been obtained when the Topo-DNA cleavage complex was stabilized by Topo toxicity inhibitors. 7 (A) and (B), camptothecin (100 and 250 μM) significantly increased the level of nick DNA, and etoposide (250 μM) effectively produced linearized DNA. However, at all concentrations, compound 9 was equivalent to etoposide and camptothecin in both Topo I- or Topo IIα-mediated assay systems. didn't make a difference Overall, these results confirm that compound 9 acts as a catalyst inhibitor for both Topo I and IIα, not a 　Topo toxic substance.

To elucidate the detailed MOA of compound 9, an EtBr displacement assay using Amsacrine (hereinafter referred to as mAMSA) was additionally performed as a DNA intercalative agent positive control. As a result, according to FIG. 7 (C), it was observed that the fluorescence intensity induced by the EtBr-ctDNA (calf thymus DNA) complex rapidly cooled with the gradual increase in the dose of mAMSA, but Compound 9 did not show any effect. This confirmed that compound 9 acts as a non-intercalative agent, unlike mAMSA, which competitively replaces EtBr in ctDNA and loses fluorescence.

As described above, genotoxic stress caused by toxicity inevitably causes serious toxicity problems related to various types of normal cells, which is a general basis for developing catalyst inhibitors. Compound 9 is finally non-interfering. As it was found to be a Topo I and IIα catalytic inhibitor, the cytotoxicity of compound 9 was further tested against normal mammalian fibroblast line MRC5 and bone marrow stratified cell line M2-10b4. Since the determined IC ₅₀ values of compound 9 against the Topo ^high cell line were less than 10 μM, we treated both MRC5 and M2-10b4 cell lines with compound 9 up to 10 μM for 72 hours. As a result, as shown in FIGS. 7 (D) and (E), Compound 9 showed an IC ₅₀ value of 10 μM or more and did not show a noticeable cytotoxic effect in the two cell lines. In particular, in the case of M2-10b4, it was confirmed that at all tested concentrations (1, 2, 5, 10 μM), the growth inhibition rates of etoposide and Compound 9 showed statistical significance and a difference of at least 5 times.

[ Example 4] As an effective strategy to maximize anticancer activity Topo Combination therapy of toxic substances and catalyst inhibitors

To investigate whether the use of compound 9 together with Topo toxicants as a dual Topo I and IIα catalyst inhibitor can be used as an effective modulatory strategy to improve overall therapeutic efficacy and overcome general toxicity problems associated with toxicity. The following tests were performed.

First, to evaluate the synergy between Compound 9 and the known Topo I and IIα toxicants, HCT15 cells were treated together with various combinations of Compound 9 and etoposide or camptothecin at various concentrations. To reduce the burden of undesirable toxicity problems caused by toxic inhibitors, both etoposide and camptothecin were applied below IC ₅₀ values. While compound 9 was treated up to 20 μM for IC ₅₀ values, concentrations that could exhibit synergistic anticancer effects with toxic substances were extensively investigated. After 72 hours of co-culture (co-incubation), the growth inhibition rate was measured through WST-1 analysis, and potential synergy was evaluated to calculate the combination index (CI) value through CompuSyn software. Here, synergism, additivity, and antagonism, defined as <1, =1, or >1, respectively, were evaluated. 8 (A) and (B), compound 9 at 10 μM or less exhibited synergistic growth inhibitory activity with all tested concentrations of etoposide or camptothecin. The optimal synergistic concentration combinations of compound 9 and camptothecin were found to be 5 μM and 1 nM, respectively, and calculated to be 5 μM and 0.2 μM for compound 9 and etoposide, respectively. However, at a concentration of 20 μM, which is more than twice the IC ₅₀ value, compound 9 had an additive or even antagonistic antiproliferative effect with etoposide in most cases. This confirmed that the synergistic anticancer activity of Compound 9 and etoposide or camptothecin was shown in low-dose combinations lower than individual IC ₅₀ values.

To further evaluate the effect of the co-treatment strategy on apoptosis, compound 9 (2 or 5 μM) was treated with either camptothecin 1 nM or etoposide ( After co-treatment with 0.2 μM of etoposide, changes in several apoptosis markers (cleaved PARP; c-PARP and cleaved caspase 7; c-casp7) were investigated. According to (C) and (D) of FIG. 8, it was confirmed that cell death was significantly promoted compared to the treatment of each compound individually.

It was confirmed that the same tendency was observed in the in vivo tumor xenograft model inoculated with the HCT15 cell line, and according to FIG. 8 (E), (F) and (G), the tumor growth retardation effect was compound 9 (4 mg / kg) and 2 mg/kg of etoposide (hereinafter referred to as Etopo ^Low ) was most clearly observed in the co-administered group, confirming once again the synergistic anticancer efficacy between Compound 9 and the toxic inhibitor. . Also, according to (H) of FIG. 8 , it was confirmed that Ki-67, a proliferation marker, was also most remarkably reduced in the tumor part of co-treated mice.

Based on the above results, it was confirmed that concomitant administration of Compound 9 and a toxic inhibitor in a low concentration combination can be provided as an alternative strategy to the use of high-concentration Topo toxic substances and has excellent therapeutic efficacy.

[ Example 5] genotoxic stress ( genotoxic stress) and bone marrow toxicity (myelotoxicity) to alleviate as a new treatment strategy for Topo Binding of Toxins and Compound 9

In addition to the synergistic activity for overall anticancer activity, the effect of compound 9 administration on general genotoxic stress and myelotoxicity related to toxicity was further evaluated.

As a result of treating HCT15 with camptothecin (100 nM) and etoposide (2 μM), according to (A) and (B) of FIG. 9, Ub ₁ -γ along with γ-H2AX -H2AX generation was markedly induced, confirming that high concentrations of toxic substances induce genotoxic stress in addition to apoptosis for cells. The above results were confirmed once again through Comet analysis. According to (C) of FIG. 9, high concentrations of camptothecin (100 nM) and etoposide (2 μM) were significant It was confirmed that it generated a fragmented DNA tail of . In contrast, treatment with low concentrations, such as 1 nM camptothecin, 0.2 μM etoposide, and Compound 9 (2 and 5 μM), did not cause the same change as treatment with high concentrations. It was confirmed that this did not induce genotoxicity due to DNA damage. In addition, co-treatment with 1 nM camptothecin or 0.2 μM etoposide and 2 or 5 μM of Compound 9, respectively, also did not induce formation of Ub ₁ -γ-H2AX and γ It was confirmed that only the level of -H2AX was increased, and a minimal level of tail DNA was also observed in Comet analysis under the same experimental conditions. Considering that a significant synergistic effect on anticancer activity was clearly demonstrated in this combination, upregulated γ-H2AX can be interpreted as a resultant event of markedly accelerated apoptosis, and these Topo toxicants and Compound 9 confirmed that the joint treatment strategy can act to reduce the burden of genotoxicity.

In addition to the evaluation at the cellular level, a complete blood cell count (CBC) test was also performed to evaluate the effect of Compound 9 on the toxicity-induced myelosuppressive action in the body. Although there was no statistical significance, according to (D) and (E) of FIG. 9, white blood cell (WBC) count and absolute neutrophil count (ANC) in the etoposide-treated group ) While a decreasing trend was observed, it was confirmed that the compound 9 treated case showed a marginal change compared to the control group (hereinafter referred to as control). In particular, in the case of the group simultaneously treated with Etopo ^Low and Compound 9, the effect of Etopo ^Low treatment compared to the control, average WBC (control vs. Etopo ^Low ; 1.8 vs. 1.0) and ANC value (control vs. Etopo ^Low ; 1.2 vs. 0.56) Considering that they induced 1.8-fold and 2-fold reductions, respectively, it was confirmed that treatment with compound 9 was interpreted as a beneficial treatment strategy in terms of mitigating the risk of neutropenia. In the case of red blood cells, according to (F) and (G) of FIG. 9, treatment with 20 mg/kg of etoposide (hereinafter, referred to as Etopo ^High ) compared to the control group, red blood cells (hereinafter, referred to as RBC) ) and hemoglobin (Hb) levels were significantly decreased, showing signs of anemia overall. In particular, hemoglobin levels in the Etopo ^High- treated group ranged from 11.0 to 15.1, which was lower than the normal range. In addition, according to (H) and (I) of FIG. 9, the RBC index calculated as mean corpuscular hemoglobin (MCH) and red cell distribution width (RDW) was higher than that of the control. , showed a significant increase, which was confirmed to reflect that Etopo ^High treatment induces macrocytosis and anisocytosis. In contrast, Etopo ^Low treatment group and Etopo ^Low All red blood cell parameters measured in the group treated simultaneously with compound 9 did not show significant differences from those of the control group, and the values were generally within the normal range and did not show any noticeable abnormality. This means that compound 9 does not have normal cytotoxicity problems in vivo , and co-treatment with Etopo ^Low does not cause any additional effects on etoposide-mediated myelotoxicity . Considered comprehensively, co-treatment of compound 9 with low-concentration toxicants serves as a novel treatment strategy that can alleviate genotoxic stress and myelotoxicity induced by the use of high-concentration toxicants, while at the same time better anticancer efficacy can be expected. I checked.

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims (11)

A 2,4-diphenyl indenopyridin-5-one derivative represented by Formula 1 below or a pharmaceutically acceptable salt thereof:
[Formula 1]

In Formula 1, R is characterized in that bromine (Br). The method according to claim 1, wherein the 2,4-diphenyl indenopyridin-5-one derivative represented by Formula 1 is 2,4-diphenyl indenopyridin-5-one, characterized in that the following compounds 7 to 9 ( 2,4-diphenyl indenopyridin-5-one) derivatives or pharmaceutically acceptable salts thereof:
(Compound 7) 2-(2-Bromophenyl)-4-(phenyl)-5H-indeno[1,2-b]pyridin-5-one (2-(2-Bromophenyl)-4-(phenyl) -5H-indeno[1,2-b]pyridin-5-one);
(Compound 8) 2-(3-Bromophenyl)-4-(phenyl)-5H-indeno[1,2-b]pyridin-5-one (2-(3-Bromophenyl)-4-(phenyl) -5H-indeno[1,2-b]pyridin-5-one);
(Compound 9) 2-(4-Bromophenyl)-4-(phenyl)-5H-indeno[1,2-b]pyridin-5-one (2-(4-Bromophenyl)-4-(phenyl) -5H-indeno[1,2-b]pyridin-5-one). A topo comprising the 2,4-diphenyl indenopyridin-5-one derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 or 2 as an active ingredient A composition for inhibiting Topoisomerase I and IIα. Cancer comprising the 2,4-diphenyl indenopyridin-5-one derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 or 2 as an active ingredient A pharmaceutical composition for preventing or treating disease. The pharmaceutical composition for preventing or treating cancer disease according to claim 4, wherein the pharmaceutical composition kills cancer cells by selectively inhibiting Topoisomerase I and IIα. The method according to claim 4, wherein the cancer disease is colorectal cancer, prostate cancer, stomach cancer, lung cancer, liver cancer, small intestine cancer, pancreatic cancer, brain cancer, bone cancer, melanoma, breast cancer, scleroderma, uterine cancer, cervical cancer, head and neck cancer, esophageal cancer, thyroid cancer, A pharmaceutical composition for preventing or treating cancer, characterized in that it is selected from the group consisting of parathyroid cancer, kidney cancer, sarcoma, urethral cancer, bladder cancer and blood cancer. 1) the 2,4-diphenyl indenopyridin-5-one derivative of any one of claims 1 or 2 or a pharmaceutically acceptable salt thereof; and
2) Etoposide, camptothecin, teniposide, doxorubicin, epirubicin, daunorubicin, idarubicin, topo Tekan (topotecan), irinotecan (irinotecan) and Belotecan (benotecan) at least one anti-cancer agent selected from the group consisting of; a pharmaceutical composition for preventing or treating cancer disease containing as an active ingredient. Cancer comprising the 2,4-diphenyl indenopyridin-5-one derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 or 2 as an active ingredient Health functional food composition for preventing or improving disease. i) mixing benzaldehyde, indene-1,3-dione and bromophenyl methyl ketone;
ii) irradiating the mixture of step i) with microwaves and then cooling;
iii) washing the cooled mixture of step ii) with water and then extracting with an organic solvent to form an organic layer; and
iv) drying and filtering the organic layer of step iii), followed by purification using an organic solvent; 2,4-diphenyl indenopyridin-5-one represented by the following formula 1 (2,4 -diphenyl indenopyridin-5-one) derivative preparation method:
[Formula 1]

In Formula 1, R is characterized in that bromine (Br). The method according to claim 9, wherein the bromophenyl methyl ketone (bromophenyl methyl ketone) is 1- (2-bromophenyl) ethanone (1- (2-bromophenyl) ethanone), 1- (3- bromophenyl) ethanone It is 2,4-diphenyl, characterized in that it is selected from the group consisting of (1- (3-bromophenyl) ethanone) and 1- (4-bromophenyl) ethanone (1- (4-bromophenyl) ethanone). Method for preparing a 2,4-diphenyl indenopyridin-5-one derivative. The method according to claim 9, wherein step ii) is 2,4-diphenyl indenopyridin-5-one (2,4-diphenyl Indenopyridin-5-one) derivative manufacturing method.

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