5,7,4'-trimethoxyflavone sulfamide derivatives, methods for their preparation and use

Abstract

The application discloses 5,7,4'-trimethoxyflavone sulfamide derivatives, a preparation method and application thereof, belongs to the technical field of medicines, and relates to two series of compounds of 3'-sulfamide-5,7,4'-trimethoxyflavone and 8,3'-disulfamide-5,7,4'-trimethoxyflavone and a preparation method thereof. 2-hydroxy-4,6-dimethoxyacetophenone and p-anisaldehyde or 4-methoxy-3-nitrobenzaldehyde are used as starting materials, a chalcone intermediate is synthesized through a Claisen-Schmidt reaction, and 5,7,4'-trimethoxyflavone sulfamide derivatives are synthesized through a chalcone oxidative ring-closing method (Algar-Flynn-Oyamada method), the preparation method is simple, the conditions are mild, and the yield is high. In-vivo and in-vitro activity test results show that the derivatives disclosed by the application have obvious in-vitro inhibitory activity on gastric cancer, esophageal cancer, colorectal cancer and lung cancer cell lines, are inhibitors of target proteins LRPPRC / STAT3 / CDK1, and can be applied to targeted treatment of gastric cancer, esophageal cancer, colorectal cancer, lung cancer and other cancers in clinical treatment.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically involving two series of 5,7,4'-trimethoxyflavonoid sulfonamide derivatives, their preparation methods, and applications. Background Technology

[0002] Esophageal cancer, gastric cancer, and colorectal cancer are common cancers of the digestive tract. Currently, there are few effective drugs for the clinical treatment of these cancers, and the treatment options are quite limited.

[0003] LRPPRC (Leucine-rich pentatricopeptide repeat motif containing protein) is a leucine-rich pentapeptide repeat protein, first discovered in human liver cancer cells. LRPPRC is highly expressed in lung adenocarcinoma, esophageal squamous cell carcinoma, gastric cancer, colon cancer, breast cancer, endometrial adenocarcinoma, and lymphoma, but is almost not expressed in surrounding non-tumor tissues. LRPPRC could serve as a novel target for cancer therapy.

[0004] Recently, our research found that 5,7,4'-trimethoxyflavonoids inhibit the progression of esophageal cancer by suppressing the binding of LRPPRC to STAT3 and the expression of LRPPRC, thus affecting the LRPPRC-JAK2-STAT3-c-MYC signaling pathway. It is an effective LRPPRC inhibitor without significant toxicity. 5,7,4'-trimethoxyflavonoids showed strong inhibitory effects in esophageal cancer cell lines (KYSE70, 410, 450, 510) and mouse models of xenograft tumors from esophageal cancer patients, providing new insights and evidence for clinical research on drugs for the treatment and prevention of esophageal cancer and other tumors (CN114224880A).

[0005] N,N-diethylsulfonamide disubstituted benzothiazole derivatives exhibit certain proliferative inhibitory activities against lung cancer, gastric cancer, colon cancer, and melanoma cell lines, and are KAT8 protein inhibitors. This class of compounds has good application prospects (CN115073392A).

[0006]

[0007] 5,7,4'-trimethoxyflavonoids are flavonoid compounds extracted from orange peel, but their content is only 0.046 mg / g (Ke et al. Food Anal. Methods 5(10): 1328–1338; 2017). Currently, we have efficiently obtained 5,7,4'-trimethoxyflavonoids through chemical synthesis. To improve their antitumor activity, we consider introducing sulfonamide at the 3' position of the C ring. In addition, we also consider modifying the structure by introducing sulfonamide at both the 6 position of the A ring and the 3' position of the C ring to enhance its antitumor activity.

[0008] The two series of 5,7,4'-trimethoxyflavonoid sulfonamide derivatives disclosed in this invention are inhibitors of target proteins LRPPRC, STAT3 and CDK1, and have potential application value in developing them into novel drugs for the treatment of esophageal cancer, gastric cancer, colorectal cancer, lung cancer and other cancers. Summary of the Invention

[0009] The purpose of this invention is to provide two series of 5,7,4'-trimethoxyflavonoid sulfonamide derivatives, their preparation methods, and applications.

[0010] To achieve the objective of this invention, the technical solution is as follows:

[0011] Two series of 5,7,4'-trimethoxyflavonoid sulfonamide derivatives have the following general structural formula:

[0012] R1, R2, and R3 are each independently selected from hydrogen, methyl, ethyl, cyclopropyl, 2,4-difluorobenzene ring, 4-fluorobenzene ring, 4-chlorobenzene ring, 2,3-dichlorobenzene ring, 3-bromobenzene ring, benzene ring, 4-methoxybenzene ring, 4-methylbenzene ring, 4-bromobenzene ring, 4-nitrobenzene ring, 4-cyanobenzene ring, 3-bromobenzene ring, 3-methoxybenzene ring, 3-chlorobenzene ring, and 2-fluorobenzene ring.

[0013] Preferably, the compound has the following structure:

[0014]

[0015]

[0016] This invention provides methods for preparing 5,7,4'-trimethoxyflavones and their two series of derivatives. The reaction route of general formula I is as follows:

[0017]

[0018] The synthesis process of general formula I is as follows:

[0019] (1) Dissolve 2-hydroxy-4,6-dimethoxyacetophenone (compound 1) and 4-methoxy-3-nitrobenzaldehyde (compound 2a) in the reaction solvent, add alkali, heat under reflux to undergo Claisen-Schmidt reaction. After the reaction is complete, cool to room temperature, pour the reaction solution into ice water, adjust the pH of the system to 5 ~ 6 with dilute hydrochloric acid, precipitate out, filter, wash, and obtain chalcone compound 3a, which can be directly used in the next step of the reaction;

[0020] (2) Dissolve compound 3a in the reaction solvent, add a catalytic amount of iodine, heat to 140±10℃ to carry out the Algar-Flynn-Oyamada reaction. After the reaction is complete, cool to room temperature, pour the reaction solution into ice water, precipitate out, filter, wash, recrystallize to obtain compound 4a.

[0021] (3) Compound 4a was dissolved in the reaction solvent, and reducing metal powder was added. Acid was added and refluxed to reduce the nitro group to an amino group. The compound was purified by silica gel column chromatography to obtain compound 5.

[0022] (4) Compound 5 was dissolved in the reaction solvent, and an acylation reaction catalyst and a base were added. The mixture was then reacted with commercially available sulfonyl chloride (compound 6) to synthesize a series of derivatives 7 of general formula I.

[0023] Preferably, in step (1) of synthesizing the derivative of general formula I, the molar ratio of Claisen-Schmidt reaction compound 1, compound 2a, and base is 1:(1.2~1.4):(2~5), and the solvent used is methanol, ethanol, tert-butanol, or acetonitrile; the base used is one or a mixture of two or more of potassium carbonate, sodium carbonate, sodium hydroxide, lithium hydroxide, and sodium hydride; the concentration of dilute hydrochloric acid is 5~10 mol / L; and the recrystallization solvent is methanol, ethanol, or tert-butanol.

[0024] In step (2), the catalytic equivalent of iodine is 5%~20%, and the solvent used is dimethyl sulfoxide, N,N-dimethylformamide or N,N-dimethylacetamide; the recrystallization solvent is methanol, ethanol, isopropanol or tert-butanol.

[0025] In step (3), the molar ratio of compound 4a, reducing metal powder, and acid is 1:(5~8):(5~10). The selected reaction solvent is methanol, ethanol, isopropanol, or tert-butanol. The reducing metal powder is iron powder, magnesium powder, or zinc powder. The acid is concentrated hydrochloric acid, concentrated sulfuric acid, or glacial acetic acid.

[0026] In step (4), the molar ratio of compound 5, commercially available sulfonyl chloride (compound 6), catalyst, and base is 1:(1.5~2):(1~2):(3~5). The selected reaction solvent is acetone, tetrahydrofuran, or 1,4-dioxane. The catalyst is DMAP or DCC. The base is triethylamine, pyridine, DIPEA, or DBU.

[0027] The reaction route of general formula II and 5,7,4'-trimethoxyflavonoid (compound 4b) is as follows:

[0028]

[0029] The synthesis process of Formula II is as follows:

[0030] (1) Dissolve 2-hydroxy-4,6-dimethoxyacetophenone (compound 1) and p-anisaldehyde (compound 2b) in a reaction solvent, add alkali, heat to 110±10℃ to carry out Claisen-Schmidt reaction. After the reaction is complete, cool to room temperature, pour the reaction solution into ice water, adjust the pH of the system to 5~6 with dilute hydrochloric acid, precipitate, filter, wash, recrystallize to obtain chalcone compound 3b;

[0031] (2) Compound 3b was added to chlorosulfonic acid, a sulfonating agent, at 0±5℃. After the addition was complete, the mixture was moved to room temperature to sulfonate chalcone. After the reaction was complete, the reaction was quenched by ice water, and a precipitate was formed. The precipitate was filtered, washed with pure water, and a solid was obtained. The prepared sulfonyl chloride (compound 8) can be used directly in the next step of the reaction.

[0032] (3) Dissolve compound 8 in the reaction solvent, add commercially available amine (compound 9), stir at room temperature until the reaction is complete, evaporate to dryness, dissolve the residue in ethyl acetate, wash with hydrochloric acid solution until neutral, wash with saturated brine, dry with anhydrous sodium sulfate, filter, concentrate, and purify by silica gel column chromatography to obtain compound 10, which can be directly used in the next step of the reaction.

[0033] (4) Dissolve compound 3b or 10 in the reaction solvent, add a catalytic amount of iodine, heat to 140±10℃ to carry out the Algar-Flynn-Oyamada reaction. After the reaction is complete, cool to room temperature, pour the reaction solution into ice water, precipitate out, filter, wash, recrystallize to obtain compound 5,7,4'-trimethoxyflavone (compound 4b), or purify by silica gel column chromatography to obtain a series of derivatives of general formula II, 11.

[0034] Preferably, in step (1) of synthesizing general formula II derivatives, the molar ratio of Claisen-Schmidt reaction compound 1, compound 2b, and base is 1:(1.2~1.4):(2~5), and the solvent used is methanol, ethanol, tert-butanol, or acetonitrile; the base used is one or a mixture of two or more of potassium carbonate, sodium carbonate, sodium hydroxide, lithium hydroxide, and sodium hydride; the concentration of dilute hydrochloric acid is 5~10 mol / L; and the recrystallization solvent is methanol, ethanol, or tert-butanol.

[0035] In step (2), the molar ratio of compound 1 to chlorosulfonic acid is 1:(17~18).

[0036] In step (3), the molar ratio of compound 8 to commercially available amine (compound 9) is 1:(1.5~2), and the solvent used is acetone, tetrahydrofuran or 1,4-dioxane;

[0037] In step (4), when preparing compound 4b or 11, the catalytic equivalent of iodine is 5%~20%, and the solvent used is dimethyl sulfoxide, N,N-dimethylformamide or N,N-dimethylacetamide; the recrystallization solvent is methanol, ethanol or tert-butanol.

[0038] The 5,7,4'-trimethoxyflavonoid sulfonamide derivative provided by this invention has a simple preparation method and potential application value. It is a targeted inhibitor of LRPPRC, STAT3 and CDK1. In particular, compounds 7e, 7k, 11e and 11g have good inhibitory activity against gastric cancer, esophageal cancer, colorectal cancer and lung cancer cells at concentrations of 1 to 8 μM. Clinically, it can be developed into a drug for targeted therapy of gastric cancer, esophageal cancer, colorectal cancer, lung cancer and other cancers, which can improve the survival rate of patients and improve their quality of life. Attached Figure Description

[0039] Figure 1 This paper presents the inhibitory effects of derivatives 7e, 7k, 11e, 11g, and 4b of the present invention on esophageal squamous cell carcinoma cells, gastric cancer cells, and colorectal cancer cells. Specifically, derivatives 7e, 7k, 11e, and 11g of the present invention, at concentrations ranging from 1 to 8 μM, inhibited the proliferation (A, B, and C) and colony formation (D, E, F, and G) of esophageal squamous cell carcinoma cells KYSE450, gastric cancer cells HGC27, and colorectal cancer cells HCT116. The figures show the tumor cell proliferation curves and statistical results and images of the number of colonies at different time points with different drug concentrations.

[0040] Figure 2 The therapeutic effects of derivatives 7e, 7k, 11e, 11g, and 4b, and capecitabine on tumor growth in a mouse model of human gastric cancer HGC27 cell xenografts are shown in the following figures: A shows the trend of tumor volume changes during drug administration; B shows the weight of the tumors after the mice were sacrificed at the end of the experiment; C shows tumor images of the control group and the drug administration group; D shows the trend of mouse body weight changes during drug administration; E, F, G, and H show the weight of the liver, spleen, and kidneys of the mice at the end of the experiment, as well as HE staining of the tumors, liver, and kidneys.

[0041] Figure 3The derivatives 7e, 7k, 11e, 11g, and 4b of this invention directly target LRPPRC, STAT3, and CDK1: where A, B, and C are the results of the hydrogen bromide-activated Sepharose 4B protein pull-down assay (target proteins LRPPRC, STAT3, and CDK1); D, E, and F are the results of the cell thermal displacement assay verifying the binding ability (target proteins LRPPRC, STAT3, and CDK1); and G, H, and I are the CETSA melting curves.

[0042] Note: Figures 1 to 3 In the study, compared with the control group, * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

[0043] Materials and Methods

[0044] 1. Materials

[0045] 1.1 Tumor cell lines

[0046] The esophageal cancer cell lines, gastric cancer cell lines, colorectal cancer cell lines, and lung cancer cell lines used in this invention are from the Sino-US (Henan) Hormel Cancer Institute.

[0047] 1.2 Reagents

[0048] Penicillin: North China Pharmaceutical Co., Ltd.;

[0049] Streptomycin: Shandong Lukang Pharmaceutical Co., Ltd.;

[0050] RPMI-1640 culture medium: Biological Industries, Israel;

[0051] 0.25% Pancreatic Enzyme: Shanghai Beyotime Biotechnology Co., Ltd.;

[0052] Serum-free cell cryopreservation solution: Suzhou Xinsaimei Biotechnology Co., Ltd.;

[0053] PBS powder: Beijing Solarbio Science & Technology Co., Ltd.

[0054] MTT powder: Beijing Solarbio Technology Co., Ltd.;

[0055] Crystal violet powder: Tianjin Kemeo Chemical Reagent Co., Ltd.;

[0056] DMSO: Tianjin Kemeio Chemical Reagent Co., Ltd.

[0057] Fetal bovine serum: BI, Inc., USA;

[0058] CNBr-activated Sepharose 4B beads: Cytiva Corporation, Japan;

[0059] LRPPRC antibody: Abcam, UK;

[0060] β-actin antibody: Proteintech, USA;

[0061] STAT3 antibody: Abcam, UK;

[0062] CDK1 antibody: Abcam, UK;

[0063] 0.4% Sodium Pentobarbital: Sinopharm Chemical Reagent Co., Ltd.;

[0064] 500ml bottle of physiological saline: Chenxin Pharmaceutical Co., Ltd.

[0065] 1.3 Instruments and Consumables:

[0066] 1.5ml centrifuge tubes: Axygen Corporation, USA;

[0067] 15ml centrifuge tubes: Corning Corporation, USA;

[0068] 96-well cell culture plate: Wuxi NiceBiotechnology Co., Ltd.;

[0069] 6-well cell culture plate: Wuxi NiceBiotechnology Co., Ltd.;

[0070] 10 cm cell culture dish: Wuxi NiceBiotechnology Co., Ltd.;

[0071] 15 cm cell culture dish: Thermo Fisher Scientific, USA;

[0072] Disposable pipettes: Guangzhou Jetech Biofiltration Co., Ltd.;

[0073] Ultra-sensitive multi-functional imager: Cytiva Corporation, Japan;

[0074] Pipettes: Eppendorf, Germany;

[0075] Dry CO2 incubator: Shanghai Yiheng Scientific Instruments Co., Ltd.;

[0076] High-speed low-temperature centrifuge: Eppendorf GmbH, Germany;

[0077] Vacuum suction pump: Haimen Qilin Bell Instrument Manufacturing Co., Ltd.;

[0078] Snowflake ice maker: SANYO Corporation, Japan;

[0079] Mili-Q water purifier: Millipore, USA;

[0080] Thermo clean bench; ophthalmic scissors, surgical forceps, scalpel, dissolving needles, syringes.

[0081] 1.4 Laboratory Animals

[0082] Five-week-old BALB / c-nu mice were purchased from Henan Xincheng Youkang Biotechnology Co., Ltd., and housed in a constant temperature and pressure environment with a 12-hour day-night cycle. Mice were only allowed to participate in experiments when their weight was approximately 18-20 g. Mouse feed was purchased from Beijing Huafu Biotechnology Co., Ltd. The experimental animals were housed in the animal facilities of the School of Life Sciences, Zhengzhou University, Henan Province, under conditions of constant temperature (25-27℃), constant humidity (45%-50%), fresh air, and a dust-free, sterile, and pathogen-free (SPF-grade) environment. The animals were initially placed in organic plastic boxes (Suzhou Fengshi Experimental Animal Equipment Co., Ltd.), and then placed in an IVC system. Aseptically treated feed was provided for free access. High-temperature sterilized bedding was changed every three days, and cages and drinking water were ultraviolet-sterilized every three days. Sterile distilled water was provided as drinking water. Strict aseptic techniques were followed when changing feeding supplies. The experimental animals were housed under a 12 / 12-hour light / dark cycle, with free access to food and water, and the room temperature was controlled at 25℃. Detailed Implementation

[0083] The present invention will be further described below with reference to the embodiments, in which the solvent used in column chromatography is a volume ratio.

[0084] Example 1: Preparation of compounds 4b, 7a-7m, and 11a-11m

[0085] (1) Preparation of compound 3a

[0086] Compound 1 (1.08 g, 5.25 mmol, 1.0 equiv) was dissolved in methanol (30 mL) and potassium hydroxide (703 mg, 10.5 mmol, 2.0 equiv) was added. Compound 2a (962 mg, 5.31 mmol, 1.01 equiv) was added to the reaction solution, and the mixture was refluxed for 4 hours until compound 1 was completely consumed. After cooling to room temperature, the reaction solution was poured into an ice-water mixture and the pH was adjusted to 5-6 with dilute hydrochloric acid, resulting in the formation of a large amount of yellow solid. The solid was filtered, washed, and dried to obtain 1.69 g of compound 3a, with a yield of 89.9%, which could be used directly in the next reaction.

[0087] (2) Preparation of compound 4a

[0088] Compound 3a (1.81 g, 5.04 mmol, 1.0 equiv) was dissolved in DMSO (40 mL), and a catalytic amount of iodine (255.7 mg, 1.01 mol, 0.2 equiv) was added. The reaction was carried out at 140°C for 45 minutes. After the reaction was completed, the mixture was cooled to room temperature and poured into an ice-water mixture. 1.5 g of sodium bisulfite was added, and a large amount of yellow solid precipitated. The solid was filtered, washed, and dried to give 1.62 g of compound 4a, with a yield of 89.1%, which can be used directly in the next reaction.

[0089] (3) Preparation of compound 5

[0090] Compound 4a (550.3 mg, 1.54 mmol, 1.0 equiv) was mixed thoroughly with ethanol (10 mL), and concentrated hydrochloric acid (2 mL) and reduced iron powder (860.9 mg, 15.4 mmol, 10.0 equiv) were added. The reaction mixture was refluxed for 4 hours. After the reaction was complete, diatomaceous earth was filtered while hot and washed with ethyl acetate. The filtrate was adjusted to pH 10-11 with sodium hydroxide solution (2 mol / L), extracted with ethyl acetate, and the organic phase was separated, washed with saturated brine, and dried over anhydrous sodium sulfate. After filtration and rotary evaporation, the brown residue was purified by silica gel column chromatography (DCM / MeOH = 50:1). 375.4 mg of yellow solid was obtained, yield: 74.5%. mp 119-123℃; 1 H NMR(400 MHz, DMSO-d6) δ 7.30 (m, 2H), 6.96 (d, J = 9.1 Hz, 1H), 6.74 (d, J = 2.3Hz, 1H), 6.50 (d, J = 2.2 Hz, 1H), 6.46 (s, 1H), 3.90 (s, 3H), 3.86 (s, 3H), 3.83 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 176.02, 164.06, 161.02, 160.75,159.60, 149.80, 137.63, 123.63, 116.06, 111.41, 111.06, 108.75, 107.03,96.66, 93.57, 56.54, 56.39, 56.04. HRMS (ESI) [M+H] + calcd for C 18 H 18 NO5:328.1185, found: 328.1187.

[0091] (4) Preparation of compounds (7a-7m) represented by general formula 7

[0092] Compound 5 (50.3 mg, 0.16 mmol, 1.0 equiv) was dissolved in tetrahydrofuran, and DMAP (39.1 mg, 0.32 mmol, 2 equiv) and pyridine (130 μL, 1.6 mmol, 10.0 equiv) were added. The reaction mixture was then added to a commercially available sulfonyl chloride compound 6 (0.32 mmol, 2.0 equiv), and the reaction was allowed to proceed overnight at room temperature. The reaction mixture was poured into dilute hydrochloric acid solution, extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness. The brown residue was purified by silica gel column chromatography (DCM / MeOH = 40:1) to give compounds 7a-7m.

[0093] Compound 7a: pale yellow solid, yield: 70.2%. mp 180-183℃; 1 H NMR (400 MHz, CDCl3)δ 8.08 (d, J = 1.5 Hz, 1H), 7.63 (d, J = 7.2 Hz, 1H), 7.01 (d, J = 8.5 Hz,1H), 6.85 (s, 1H), 6.62 (d, J = 4.0 Hz, 1H), 6.58 (d, J = 2.1 Hz, 1H), 6.39 (t, J = 3.3 Hz, 1H), 3.98 (d, J = 4.5 Hz, 3H), 3.96 (s, 3H), 3.93 (d, J = 6.9Hz, 3H), 3.13 (q, J = 7.3 Hz, 2H), 1.38 (t, J = 7.3 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 177.53, 164.07, 160.86, 159.93, 159.85, 151.08, 126.99, 125.00,123.27, 117.46, 112.56, 110.93, 109.21, 108.53, 96.32, 92.92, 56.48, 56.24,55.89, 46.06, 8.21. HRMS (ESI) [M+H] + calcd for C 20 H 22 NO7S: 420.1117, found:420.1116.

[0094] Compound 7b: pale yellow solid, yield: 86.7%. mp 193-196℃; 1 H NMR (400 MHz, CDCl3)δ 8.11 (d, J = 2.1 Hz, 1H), 7.64 (dd, J = 8.6, 2.1 Hz, 1H), 7.01 (d, J = 8.7Hz, 1H), 6.85 (s, 1H), 6.62 (s, 1H), 6.58 (d, J =2.2 Hz, 1H), 6.38 (d, J =2.2 Hz, 1H), 3.98 (s, 3H), 3.96 (s, 3H), 3.92 (s, 3H), 2.54 – 2.48 (m, 1H),1.21 (m, 2H), 1.00 – 0.92 (m, 2H). 13 C NMR (101 MHz, CDCl3) δ 177.52, 164.06,160.86, 159.98, 159.84, 151.82, 127.02, 124.81, 123.47, 118.59, 110.86,109.20, 108.40, 96.29, 92.91, 56.46, 56.22, 55.87, 30.03, 5.69. HRMS (ESI) [M+H] + calcd for C 21 H 22 NO7S: 432.1117, found: 432.1115.

[0095] Compound 7c: Off-white solid, yield: 86.1%. mp 174-178℃; 1 H NMR (400 MHz, CDCl3)δ 8.10 (td, J = 8.3, 6.1 Hz, 2H), 7.96 (s, 1H), 7.13 – 7.05 (m, 2H), 7.04 –6.94 (m, 2H), 6.64 (s, 1H), 6.57 (d, J = 2.2 Hz, 1H), 6.40 (d, J = 2.1 Hz,1H), 3.98 (s, 3H), 3.94 (s, 3H), 3.65 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ177.44, 166.77 (dd, J C,F= 258.8, 11.5 Hz), 164.14, 160.96, 160.23 (dd, J C,F =261.5, 12.9 Hz), 159.82, 159.77, 159.26, 134.42 (d, J C,F = 10.6 Hz), 131.60,130.13, 124.73, 124.19 (dd, J = 13.1, 4.0 Hz), 122.66, 112.01 (dd, J C,F =21.6, 3.4 Hz), 111.95, 109.21, 108.51, 105.44 (t, J C,F = 50.6, 25.3 Hz),96.30, 92.96, 56.52, 55.88, 55.79. HRMS (ESI) [M+H] + calcd for C 24 H 20 F2NO7S:504.0929, found: 504.0929.

[0096] Compound 7d: Off-white solid, yield: 65.6%. mp 198-202℃; 1 H NMR (400 MHz, CDCl3)δ 8.04 – 8.01 (m, 3H), 7.91 (dd, J = 8.8, 2.2 Hz, 1H), 7.59 (d, J = 2.2 Hz,1H), 7.26 (d, J = 8.5 Hz, 2H), 7.00 (d, J = 8.8 Hz, 1H), 6.57 (s, 1H), 6.46 (d, J = 2.2 Hz, 1H), 6.40 (d, J = 2.2 Hz, 1H), 3.97 (s, 3H), 3.94 (s, 3H), 3.57 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 177.31, 165.94 (d, J C,F = 255.6 Hz),164.22, 160.94, 159.73, 159.62, 158.89, 135.66 (d, J C,F= 3.2 Hz), 131.99,131.89, 130.62, 129.93, 124.55, 123.37, 116.20, 115.97, 112.60, 109.15,108.40, 96.47, 92.66, 56.49, 55.84, 55.72. HRMS (ESI) [M+H] + , calcd forC 24 H 21 FNO7S: 486.1023, found: 486.1021.

[0097] Compound 7e: white solid, yield: 82.1%. mp 158-162℃; 1 H NMR (400 MHz, CDCl3) δ7.94 – 7.92 (m, 3H), 7.63 (d, J = 2.2 Hz, 1H), 7.54 (t, J = 7.4 Hz, 3H), 6.99(d, J = 8.8 Hz, 1H), 6.62 (s, 1H), 6.49 (d, J = 2.2 Hz, 1H), 6.40 (d, J = 2.2Hz, 1H), 3.97 (s, 3H), 3.95 (s, 3H), 3.55 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ177.34, 164.21, 160.92, 159.72, 159.55, 158.82, 140.82, 138.04, 130.57,130.42, 129.98, 129.12, 124.61, 123.24, 112.60, 109.15, 108.43, 96.43, 92.75,56.50, 55.93, 55.68, 0.02. HRMS (ESI) [M+H] + calcd for C 24 H 21 ClNO7S: 502.0727, found: 502.0715.

[0098] Compound 7f: pale yellow solid, yield: 70.1%. mp 112-115℃; 1H NMR (400 MHz, CDCl3)δ 8.01 (dd, J = 7.9, 1.3 Hz, 1H), 7.98 (d, J = 2.1 Hz, 1H), 7.67 (s, 1H),7.63 (dd, J = 8.0, 1.4 Hz, 1H), 7.56 (dd, J = 8.7, 2.1 Hz, 1H), 7.30 (d, J =8.0 Hz, 1H), 6.86 (d, J = 8.7 Hz, 1H), 6.56 (d, J = 2.4 Hz, 2H), 6.38 (d, J =2.2 Hz, 1H), 3.96 (s, 3H), 3.94 (s, 3H), 3.82 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 177.48, 164.13, 160.91, 159.88, 159.79, 151.87, 138.47, 135.55,134.84, 130.43, 130.25, 127.26, 125.73, 124.54, 123.86, 118.60, 110.90,109.12, 108.26, 96.25, 92.88, 56.47, 56.00, 55.87. HRMS (ESI) [M+H] + , calcdfor C 24 H 20 Cl2NO7S: 536.0338, found: 536.0340.

[0099] Compound 7g: white solid, yield: 65.7%. mp 227-231℃; 1 H NMR (400 MHz, CDCl3) δ8.07 (d, J = 2.2 Hz, 1H), 7.97 (t, J = 1.8 Hz, 1H), 7.72 – 7.63 (m, 2H), 7.60 (dd, J = 8.7, 2.2 Hz, 1H), 7.30 (t, J = 8.0 Hz, 1H), 7.01 (s, 1H), 6.86 (d, J= 8.7 Hz, 1H), 6.65 (s, 1H), 6.60 (d, J = 2.3 Hz, 1H), 6.39 (d, J = 2.2 Hz,1H), 3.97 (s, 3H), 3.94 (s, 3H), 3.75 (s, 3H).13 C NMR (101 MHz, CDCl3) δ177.51, 164.12, 160.87, 159.83, 152.05, 140.74, 136.12, 130.35, 130.14,125.89, 125.86, 125.86, 124.75, 124.33, 122.85, 119.63, 110.93, 109.15,108.38, 96.34, 92.91, 56.49, 56.04, 55.93. HRMS (ESI) [M+H] + , calcd forC 24 H 20 B r NO7S: 546.0222, 548.0202, found: 546.0218, 548.0200.

[0100] Compound 7h: off-white solid, yield: 80.9%. mp 223-225℃; 1 H NMR (400 MHz, CDCl3)δ 7.98 (d, J = 1.2 Hz, 2H), 7.90 (dd, J = 8.8, 2.3 Hz, 1H), 7.69 (t, J = 7.4Hz, 2H), 7.57 (d, J = 8.0 Hz, 3H), 6.96 (d, J = 8.8 Hz, 1H), 6.50 (s, 1H), 6.49 (d, J = 2.3 Hz, 1H), 6.39 (d, J = 2.2 Hz, 1H), 3.97 (s, 3H), 3.94 (s,3H), 3.49 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 177.37, 164.08, 160.93, 159.77,159.74, 159.04, 139.77, 133.90, 130.89, 129.70, 128.95, 128.77, 124.35,123.54, 112.41, 109.20, 108.29, 96.28, 92.89, 56.47, 55.86, 55.59. HRMS (ESI)[M+H] + calcd for C 24 H 22 NO7S: 468.1117, found: 468.1116.

[0101] Compound 7i: Off-white solid, yield: 87.6%. mp 116-119℃; 1 H NMR (400 MHz, CDCl3)δ 8.04 (d, J = 2.2 Hz, 1H), 7.77 – 7.72 (m, 2H), 7.54 (dd, J = 8.6, 2.2 Hz,1H), 7.03 (s, 1H), 6.90 – 6.87 (m, 2H), 6.84 (d, J = 8.7 Hz, 1H), 6.58 (d, J= 1.3 Hz, 2H), 6.38 (d, J = 2.3 Hz, 1H), 3.96 (s, 3H), 3.94 (s, 3H), 3.81 (s,3H), 3.75 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 177.55, 164.06, 163.24, 160.88,160.02, 159.83, 151.68, 130.56, 129.51, 126.77, 124.54, 123.40, 118.48,114.09, 110.76, 109.20, 108.28, 96.26, 92.90, 56.46, 56.02, 55.85, 55.62.HRMS (ESI) [M+H] + calcd for C 25 H 24 NO8S: 498.1223, found: 498.1224.

[0102] Compound 7j: Off-white solid, yield: 74.2%. mp 118-123℃; 1 H NMR (400 MHz, CDCl3)δ 8.05 (d, J = 2.1 Hz, 1H), 7.69 (d, J = 8.2 Hz, 2H), 7.54 (dd, J = 8.6, 2.1Hz, 1H), 7.22 (d, J = 8.1 Hz, 2H), 7.05 (s, 1H), 6.83 (d, J = 8.7 Hz, 1H), 6.58 (s, 2H), 6.38 (d, J = 2.1 Hz, 1H), 3.96 (s, 3H), 3.94 (s, 3H), 3.73 (s,3H), 2.37 (s, 3H).13 C NMR (101 MHz, CDCl3) δ 177.55, 164.07, 160.88, 160.00,159.82, 151.65, 144.06, 136.05, 129.55, 127.33, 126.68, 124.55, 123.44,118.48, 110.77, 109.19, 108.28, 96.26, 92.90, 56.46, 55.99, 55.87, 21.55.HRMS (ESI) [M+H] + calcd for C 25 H 24 NO7S: 482.1273, found: 482.1277.

[0103] Compound 7K: off-white solid, yield: 75.7%; mp 248-251℃; 1 H NMR (400 MHz, CDCl3)δ 7.91 (dd, J = 8.8, 2.3 Hz, 1H), 7.86 – 7.83 (m, 3H), 7.73 – 7.70 (m, 2H), 7.63 (d, J = 2.3 Hz, 1H), 6.99 (d, J = 8.9 Hz, 1H), 6.57 (s, 1H), 6.49 (d, J= 2.3 Hz, 1H), 6.40 (d, J = 2.2 Hz, 1H), 3.97 (s, 3H), 3.96 (s, 3H), 3.54 (s,3H). 13 C NMR (101 MHz, CDCl3) δ 177.29, 164.20, 160.94, 159.72, 159.53,158.79, 138.60, 132.12, 130.57, 130.43, 129.97, 129.44, 124.65, 123.23,112.59, 109.19, 108.46, 96.42, 92.79, 56.49, 55.97, 55.68. HRMS (ESI) [M+H] + calcd for C 24 H 21 BrNO7S: 546.0222, 548.0202, found: 546.0226, 548.0199.

[0104] Compound 7l: Off-white solid, yield: 50.1%. mp 247-252℃; 1 H NMR (400 MHz, DMSO-d6) δ 8.53 (d, J = 8.8 Hz, 2H), 8.25 (dd, J = 8.8, 1.9 Hz, 1H), 8.16 (d, J =8.8 Hz, 3H), 7.85 (d, J = 1.9 Hz, 1H), 7.29 (d, J = 9.0 Hz, 1H), 6.83 (s,1H), 6.77 (d, J = 1.8 Hz, 1H), 6.53 (d, J = 1.9 Hz, 1H), 3.92 (s, 3H), 3.85(s, 3H), 3.47 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 176.04, 164.28, 160.72,159.56, 158.47, 151.34, 143.94, 131.43, 130.70, 130.53, 125.15, 124.40,122.17, 114.10, 108.74, 108.34, 96.92, 93.73, 56.57, 56.43, 56.40. HRMS (ESI)[M+H] + calcd for C 24 H 21 N2O9S: 513.0968, found: 513.0956.

[0105] Compound 7M: Off-white solid, yield: 58.5%. mp 230-234℃; 1 H NMR (400 MHz, CDCl3)δ 8.09 (d, J = 2.0 Hz, 1H), 7.89 (d, J = 8.3 Hz, 2H), 7.73 (d, J = 8.3 Hz, 2H), 7.63 (dd, J = 8.6, 2.0 Hz, 1H), 7.08 (s, 1H), 6.86 (d, J = 8.7 Hz, 1H), 6.61 (s, 1H), 6.58 (d, J = 2.1 Hz, 1H), 6.39 (d, J = 2.1 Hz, 1H), 3.96 (s,3H), 3.94 (s, 3H), 3.71 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 177.47, 164.16,160.92, 159.80, 159.57, 152.07, 143.17, 132.68, 127.87, 125.46, 124.91,124.61, 119.89, 117.11, 116.83, 111.03, 109.18, 108.49, 96.32, 92.89, 56.48,56.04, 55.89. HRMS (ESI) [M+H] + calcd for C 25 H 21 N2O7S: 493.1069, found:493.1066.

[0106] (5) Preparation of compound 3b

[0107] In a thick-walled, pressure-resistant bottle, compounds 1 (9.0 g, 0.0459 mol) and 2b (6.9 g, 0.504 mol) were dissolved in 225 mL of methanol. Potassium hydroxide (6.4 g, 0.115 mol) was added, and the mixture was reacted at 110 °C for 18 hours. After the reaction was complete, the mixture was cooled to room temperature, and the reaction solution was poured into ice water. The pH of the system was adjusted to 5-6 with 10 mol / L dilute hydrochloric acid, resulting in the precipitation of a yellow precipitate. The precipitate was filtered, washed with pure water, and recrystallized from 100 mL of ethanol to give 12.2 g of a pale yellow solid, with a yield of 84.7%. mp 112-114 °C; 1 H NMR (600 MHz, CDCl3) δ14.40 (s, 1H), 7.79 (d, J = 2.2 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H), 6.93 (d, J= 8.4 Hz, 2H), 6.11 (s, 1H), 5.96 (d, J = 1.3 Hz, 1H), 3.92 (s, 3H), 3.85 (s, 3H), 3.83 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ 192.61, 168.38, 166.03, 162.48,161.38, 142.46, 130.11, 128.36, 125.17, 114.38, 106.39, 93.85, 91.25, 55.84,55.57, 55.40. HRMS (ESI) [M+H] + calcd for C 18 H 19O5: 315.1238, found: 315.1232.

[0108] (6) Preparation of compounds (10a-10m) represented by general formula 10

[0109] At 0°C, compound 3b (130 mg, 0.41 mmol, 1.0 equiv) was slowly added to chlorosulfonic acid (1 mL), and the reaction was allowed to proceed to room temperature for 1 hour. The reaction solution was then poured into an ice-water mixture, and a large amount of yellow solid precipitated, which was sulfonyl chloride (compound 8). The solid was filtered, washed with pure water, and immediately proceeded to the next reaction. Compound 8 was dissolved in tetrahydrofuran, and pyridine (2.01 mmol, 5.0 equiv) and commercially available amine compound 9 (0.82 mmol, 2.0 equiv) were added. The mixture was reacted overnight at room temperature. After the reaction was complete, the reaction solution was poured into dilute hydrochloric acid, extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness. The brown residue was subjected to silica gel column chromatography (DCM / MeOH = 40: 1) to give compounds 10a-10m.

[0110] Compound 10a: pale yellow solid, yield: 60.5%. mp 85-89℃; 1 H NMR (400 MHz, CDCl3) δ11.01 (s, 1H), 8.10 (d, J = 2.1 Hz, 1H), 7.70 (dd, J = 8.6, 2.1 Hz, 1H), 7.37(d, J = 16.0 Hz, 1H), 7.01 (d, J = 3.5 Hz, 1H), 6.98 (d, J = 10.9 Hz, 1H),6.07 (s, 1H), 3.97 (d, J = 4.9 Hz, 6H), 3.86 (s, 3H), 3.41 – 3.28 (m, 8H),1.16 (t, J = 7.1 Hz, 6H), 1.11 (t, J = 7.1 Hz, 6H). 13 C NMR (101 MHz, CDCl3) δ192.80, 162.19, 160.93, 158.04, 157.38, 142.99, 133.82, 131.63, 129.74,127.79, 127.30, 112.50, 110.37, 106.32, 87.31, 56.34, 56.19, 56.03, 42.24,41.81, 14.28. HRMS (ESI) [M+H] + calcd for C26 H 37 N2O9S2: 585.1940, found:585.1942.

[0111] (7) Preparation of compounds (11a-11m) represented by general formula 11

[0112] Compound 10a-10m (1 equiv) was dissolved in DMSO (20.0 mL), followed by the addition of a catalytic amount of iodine (0.05 equiv). The reaction mixture was reacted at 140 °C for 45 minutes. After the reaction was complete, the mixture was cooled to room temperature, poured into an ice-water mixture, and sodium bisulfite was added. The mixture was extracted with ethyl acetate, and the organic phases were combined. The mixture was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness. The yellow residue was purified by silica gel column chromatography (DCM / MeOH = 30:1) to give compound 11a-11m.

[0113] Compound 11a: white solid, yield: 61.9%. mp. 208-213℃; 1 H NMR (400 MHz, DMSO-d6)δ 8.55 (d, J = 2.3 Hz, 1H), 8.46 (dd, J = 8.8, 2.4 Hz, 1H), 7.43 (d, J = 9.0Hz, 1H), 6.82 (s, 1H), 6.71 (s, 1H), 4.07 (s, 3H), 3.99 (s, 6H), 3.30 (dq, J= 14.5, 7.1 Hz, 8H), 1.10 (t, J = 7.1 Hz, 6H), 1.03 (t, J = 7.1 Hz, 6H). 13 CNMR (101 MHz, DMSO) δ 175.43, 163.61, 161.82, 159.16, 156.95, 133.23, 129.71,129.05, 122.97, 114.19, 110.42, 108.58, 108.09,93.59,57.59,57.09,57.07,42.85,41.95,40.60,40.39,40.18,39.97,39.77,39.56,39.35,15.20,14.70.HRMS (ESI) [M+H] + calcd for C 26 H 35 N2O9S2: 583.1784, found: 583.1783.

[0114] Compound 11b: white solid, yield: 66.5%. mp 175-178℃; 1 H NMR (400 MHz, DMSO-d6)δ 9.97 (s, 1H), 9.96 (s, 1H), 8.68 (d, J = 2.3 Hz, 1H), 8.38 (dd, J = 8.9,2.3 Hz, 1H), 7.36 (d, J = 9.0 Hz, 1H), 7.10 – 7.02 (m, 4H), 6.95 (dd, J =11.4, 8.5 Hz, 4H), 6.78 (s, 1H), 6.63 (s, 1H), 4.06 (s, 3H), 4.00 (s, 3H), 3.94 (s, 3H), 2.54 (s, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 175.27, 164.07,162.29, 159.41, 159.09, 157.29, 135.86, 135.48, 133.61, 133.29, 129.93,129.48, HRMS (ESI) [M+H] + , calcd forC 32 H 31 N2O9S2: 651.1471, found: 651.1466.

[0115] Compound 11c: white solid, yield: 63.1%. mp 171-176℃; 1 H NMR (400 MHz, DMSO-d6)δ 10.37 (s, 1H), 10.36 (s, 1H), 8.71 (d, J = 2.4 Hz, 1H), 8.41 (dd, J = 8.9,2.4 Hz, 1H), 7.38 (d, J = 9.0 Hz, 1H), 7.26 – 7.20 (m, 4H), 7.20 – 7.14 (m,4H), 6.84 (s, 1H), 6.63 (s, 1H), 4.02 (s, 3H), 3.98 (s, 3H), 3.95 (s, 3H).13 CNMR (101 MHz, DMSO-d6) δ 175.27, 164.28, 162.14, 159.38, 159.10, 157.43,137.62, 137.21, 133.93, 129.50, 129.43, 128.01, 127.96, 127.35, 122.90,121.20, 120.77, 114.17, 108.36, 108.27, 107.37, 93.47, 57.76, 57.29, 57.15.HRMS (ESI) [M+H] + calcd for C 30 H 25 Cl2N2O9S2: 691.0379, found: 691.0377.

[0116] Compound 11d: white solid, yield: 63.9%. mp 153-157℃; 1 H NMR (400 MHz, DMSO-d6)δ 10.37 (s, 1H), 10.36 (s, 1H), 8.71 (d, J = 2.2 Hz, 1H), 8.41 (dd, J = 8.9,2.3 Hz, 1H), 7.38 (d, J = 9.0 Hz, 1H), 7.23 (dd, J = 9.0, 3.3 Hz, 3H), 7.18 (dd, J = 8.9, 1.8 Hz, 3H), 6.84 (s, 1H), 6.63 (s, 1H), 4.02 (s, 3H), 3.99 (s,3H), 3.95 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 175.29, 164.28, 162.14,159.38, 159.09, 157.43, 137.61, 137.20, 133.93, 129.51, 129.43, HRMS (ESI) [M+H] + calcd for C 30 H 23Cl4N2O9S2:758.9599, 760.9570, found: 758.9595, 760.9568.

[0117] Compound 11e: Off-white solid, yield: 71.8%. mp 182-185℃; 1 H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 10.38 (s, 1H), 8.72 (d, J = 2.4 Hz, 1H), 8.41 (dd, J =8.9, 2.4 Hz, 1H), 7.40 – 7.31 (m, 5H), 7.16 – 7.09 (m, 4H), 6.84 (s, 1H), 6.63 (s, 1H), 4.02 (s, 3H), 3.98 (s, 3H), 3.95 (s, 3H). 13 C NMR (101 MHz, DMSO) δ 175.27, 164.29, 162.13, 159.38, 159.10, 157.44, 138.07, 137.67,133.95, 132.40, 132.33, 129.56, 127.31, 122.91, 121.46, 121.06, 115.98,114.19, 108.29, 107.35, 93.48, 57.76, 57.30, 57.16. HRMS (ESI) [M+H] + , calcdfor C 30 H 25 Br2N2O9S2: 778.9368, 780.9348, found: 778.9375, 780.9367.

[0118] Compound 11f: Off-white solid, yield: 68.5%. mp 124-128℃; 1H NMR (400 MHz, DMSO-d6) δ 10.49 (s, 1H), 10.44 (s, 1H), 8.76 (d, J = 2.4 Hz, 1H), 8.44 (dd, J =8.9, 2.4 Hz, 1H), 7.39 (d, J = 9.0 Hz, 1H), 7.36 (d, J = 1.2 Hz, 1H), 7.30 (d, J = 1.9 Hz, 1H), 7.20 (dt, J = 7.1, 2.0 Hz, 1H), 7.17 – 7.06 (m, 5H), 6.87 (s, 1H), 6.64 (s, 1H), 4.03 (s, 3H), 3.98 (s, 3H), 3.95 (s, 3H). 13 C NMR(101 MHz, DMSO-d6) δ 175.24, 164.40, 162.34, 159.40, 159.10, 157.48, 140.43,139.95, 134.06, 131.56, 131.46, 129.44, 127.41, 126.60, 126.38, 122.93,122.20, 122.14, 121.85, 121.07, 117.92, 117.53, 114.26, 108.28, 108.26,107.16, 93.50, 57.80, 57.29, 57.18. HRMS (ESI) [M+H] + , calcd forC 30 H 25 Br2N2O9S2: 778.9368, 780.9348, found: 778.9360, 780.9343.

[0119] Compound 11g: white solid, yield: 72.3%. mp 198-201℃; 1H NMR (400 MHz, DMSO-d6)δ 10.14 (s, 2H), 8.64 (d, J = 2.4 Hz, 1H), 8.40 (dd, J = 8.9, 2.4 Hz, 1H), 7.38 (d, J = 9.0 Hz, 1H), 7.21 – 7.13 (m, 4H), 7.01 (ddd, J = 15.1, 10.6, 5.6Hz, 4H), 6.79 (s, 1H), 6.64 (s, 1H), 4.04 (s, 3H), 4.00 (s, 3H), 3.94 (s,3H). 13 C NMR (101 MHz, DMSO-d6) δ 175.25, 164.19, 162.21, 159.20 (d, J C,F =238.9 Hz), 159.13 (d, J C,F = 239.0 Hz), 159.37, 159.06, 157.31, 134.59 (d, J C,F = 38.4 Hz), 134.56 (d, J C,F = 38.3 Hz), 133.78, 129.37, 127.48, 122.85, 122.33(d, J C,F = 8.1 Hz), 121.83 (d, J C,F = 8.1 Hz), 116.30, 116.07, 114.07, 108.30,108.13, 107.40, 93.42, 57.73, 57.24, 57.14. HRMS (ESI) [M+H] + , calcd forC 30 H 25 F2N2O9S2: 659.0970, found: 659.0973.

[0120] Compound 11h: white solid, yield: 66.5%. mp 173-177℃; 1H NMR (400 MHz, DMSO-d6)δ 10.16 (s, 1H), 10.15 (s, 1H), 8.73 (d, J = 2.3 Hz, 1H), 8.40 (dd, J = 8.9,2.4 Hz, 1H), 7.36 (d, J = 9.0 Hz, 1H), 7.16 (t, J = 5.2 Hz, 8H), 6.98 – 6.93(m, 2H), 6.80 (s, 1H), 6.63 (s, 1H), 4.04 (s, 3H), 3.99 (s, 3H), 3.93 (s,3H). 13 C NMR (101 MHz, DMSO) δ 175.27, 164.14, 162.27, 159.42, 159.12, 157.35,138.56, 138.20, 133.71, 129.50, 129.48, 127.69, 124.11, 124.05, 122.80,119.71, 119.50, 114.07, 108.25, 108.10, 107.60, 93.43, 57.72, 57.24, 57.11.HRMS (ESI) [M+H] + calcd for C 30 H 27 N2O9S2: 623.1158, found: 623.1150.

[0121] Compound 11i: white solid, yield: 59.8%. mp 271-275℃; 1H NMR (400 MHz, DMSO-d6)δ 10.40 (s, 1H), 10.31 (s, 1H), 8.84 (d, J = 2.3 Hz, 1H), 8.41 (dd, J = 8.9,2.3 Hz, 1H), 7.98 (d, J = 13.7 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.47 (d, J= 8.4 Hz, 1H), 7.36 (d, J = 9.0 Hz, 1H), 7.17 (s, 1H), 6.86 (s, 1H), 6.83(dd, J = 6.7, 2.2 Hz, 2H), 6.64 (s, 1H), 6.58 (m, 2H), 4.05 (s, 3H), 4.00 (s,3H), 3.95 (s, 3H), 3.69 (s, 3H), 3.42 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ175.25, 164.40, 163.40, 162.40, 159.33, 159.13, 158.38, 158.35, 157.52,140.47, 139.93, 139.53, 139.37, 133.89, 129.63, 127.23, 122.79, 114.22,113.45, 112.91, 108.27, 106.96, 103.06, 101.89, 93.59, 79.31, 78.97, 57.81,57.29, 57.15, 56.58, 56.00. HRMS (ESI) [M+H] + calcd for C 32 H 31 N2O 11 S2:683.1369, found: 683.1374.

[0122] Compound 11j: Off-white solid, yield: 57.7%. mp 281-287℃; 1H NMR (400 MHz, DMSO-d6) δ 9.78 (s, 2H), 8.58 (d, J = 2.4 Hz, 1H), 8.38 (dd, J = 8.9, 2.4 Hz, 1H), 7.37 (d, J = 9.0 Hz, 1H), 7.11 – 7.03 (m, 4H), 6.79 – 6.75 (m, 2H), 6.74 (s,1H), 6.72 – 6.67 (m, 2H), 6.65 (s, 1H), 4.08 (s, 3H), 4.03 (s, 3H), 3.94 (s,3H), 3.61 (s, 3H), 3.59 (s, 3H), 2.55 (s, 1H). 13 C NMR (101 MHz, CDCl3) δ175.01, 163.78, 162.04, 159.15, 158.79, 156.93, 156.41, 156.33, 133.31,130.72, 129.11, 127.51, HRMS (ESI) [M+H] + calcd for C 32 H 31 N2O 11 S2: 683.1369, found: 683.1370.

[0123] Compound 11k: off-white solid, yield: 85.8%. mp 193-197℃; 1H NMR (400 MHz, DMSO-d6) δ 9.91 (s, 1H), 9.90 (s, 1H), 8.42 (d, J = 2.2 Hz, 1H), 8.35 (dd, J =8.9, 2.3 Hz, 1H), 7.47 – 7.37 (m, 2H), 7.33 (td, J = 8.9, 6.3 Hz, 1H), 7.28 –7.20 (m, 1H), 7.20 – 7.11 (m, 1H), 7.00 (dd, J = 12.1, 4.9 Hz, 1H), 6.74 –6.65 (m, 3H), 4.05 (s, 3H), 3.98 (s, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 175.20,164.26, 162.89, 160.40 (dd, J C,F = 266.0, 19.1 Hz), 160.08 (dd, J C,F = 243.9,10.8 Hz), 159.28, 156.94 (dd, J C,F = 248.9, 12.4 Hz), 156.60, 156.30 (dd, J C,F = 248.7, 12.7 Hz), 155.76, 133.67, 129.59 (dd, J C,F = 10.0, 1.8 Hz), 128.73(dd, J C,F = 9.7, 1.5 Hz), 128.56, 128.38, 122.51, 121.59 (dd, J C,F = 13.3, 3.8Hz), 121.38 (dd, J C,F = 13.3, 3.8 Hz), 113.95, 112.05 (dd, J C,F = 22.4, 3.7Hz), 111.73 (dd, J C,F = 22.1, 3.6 Hz), 108.21, 108.06, 107.97, 105.04 (dd, J C,F = 24.5, 9.0 Hz), 104.77 (dd, J C,F= 24.6, 9.1 Hz), 93.50, 57.65, 57.16, 57.14.HRMS (ESI) [M+H] + calcd for C 30 H 23 F4N2O9S2: 695.0781, found: 695.0776.

[0124] Compound 11l: Off-white solid, yield: 68.1%. mp 148-152℃; 1 H NMR (400 MHz, DMSO-d6) δ 10.50 (s, 1H), 10.47 (s, 1H), 8.79 (d, J = 2.3 Hz, 1H), 8.43 (dd, J =8.9, 2.3 Hz, 1H), 7.39 (d, J = 9.0 Hz, 1H), 7.24 – 7.09 (m, 6H), 7.04 – 6.96 (m, 2H), 6.87 (s, 1H), 6.65 (s, 1H), 4.03 (s, 3H), 3.98 (s, 3H), 3.95 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 175.25, 164.38, 162.30, 159.38, 159.11,157.47, 140.29, 139.82, 133.94, 133.79, 133.75, 131.27, 131.18, 129.46,127.40, 123.69, 123.54, 122.90, 118.91, 118.30, 117.55, 117.21, 114.25,108.28, 108.26, 107.20, 93.57, 57.79, 57.29, 57.18. HRMS (ESI) [M+H] + , calcdfor C 30 H 25 Cl2N2O9S2: 691.0379, found: 691.0372.

[0125] Compound 11m: Off-white solid, yield: 88.1%. mp 147-152℃; 1H NMR (400 MHz, CDCl3)δ 8.46 (dd, J = 8.8, 2.2 Hz, 1H), 8.42 (d, J = 2.3 Hz, 1H), 7.73 – 7.61 (m,2H), 7.52 (d, J = 3.1 Hz, 1H), 7.31 (d, J = 3.0 Hz, 1H), 7.20 – 7.15 (m, 1H),7.06 (m, 1H), 7.03 – 6.89 (m, 5H), 6.60 (s, 1H), 6.37 (d, J = 3.4 Hz, 1H),4.11 (s, 3H), 4.09 (s, 3H), 4.02 (d, J = 4.9 Hz, 3H). 13 C NMR (101 MHz, CDCl3)δ 176.12, 164.67, 161.95, 159.68, 158.56, 157.41, 153.59 (d, J C,F = 242.1 Hz),153.41 (d, J C,F = 241.7 Hz), 133.90, 129.23, 127.04, 125.90 (d, J C,F = 7.8 Hz),125.88 (d, J C,F = 7.3 Hz), 125.04 (d, J C,F = 3.4 Hz), 125.01 (d, J C,F = 3.4 Hz),124.76 (d, J C,F = 29.2 Hz), 124.63 (d, J C,F = 29.4 Hz), 123.25, 122.98, 122.62,115.36 (d, J C,F = 19.4 Hz), 115.28 (d, J C,F = 19.4 Hz), 112.77, 109.06, 108.28,107.58, 91.56, 57.03, 56.75. HRMS (ESI) [M+H] + , calcd for C 30 H 25 F2N2O9S2:659.0970, found: 659.0971.

[0126] (8) Preparation of 5,7,4'-trimethoxyflavonoid (compound 4b)

[0127] In a single-necked reaction flask, compound 3 (12.2 g, 0.0388 mol) was dissolved in 180 mL of dimethyl sulfoxide, and iodine (1.3 g, 4.851 mmol) was added. The reaction was carried out at 140 °C for 40 minutes. After the reaction was completed, the mixture was cooled to room temperature and poured into ice water, resulting in the precipitation of a pale yellow precipitate. The precipitate was filtered, washed with pure water, and recrystallized from 80 mL of ethanol to give 10.1 g of an off-white solid, with a yield of 83.5%. mp 158-160 °C; 1 H NMR (600 MHz, CDCl3) δ 7.82 (d, J = 8.5 Hz, 2H), 7.00 (d, J = 8.6 Hz, 2H), 6.59 (s, 1H), 6.56 (s, 1H), 6.37 (s, 1H), 3.96 (s, 3H), 3.91 (s, 3H),3.88 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ 177.64, 163.92, 162.07, 160.94,160.69, 159.87, 127.61, 123.90, 114.37, 109.27, 107.73, 96.09, 92.85, 56.44,55.73, 55.48. HRMS (ESI) [M+H] + calcd for C 18 H 17 O5: 313.1076, found: 313.1081.

[0128] Example 2: Cell proliferation experiment

[0129] Gastric cancer cells HGC27, esophageal squamous cell carcinoma cells KYSE70 and KYSE450, colorectal cancer cells HCT116, and lung cancer cells A549 were seeded in 96-well plates, approximately 4000 cells per well, and incubated overnight at 37°C with 5% CO2. Then, different concentrations of 5,7,4'-trimethoxyflavonoids and their derivatives (0.1–40 μM) were added for drug screening. The efficacy of 5,7,4'-trimethoxyflavonoids and their derivatives (4b, 5, 7e, 7k, 11e, 11g) was compared by adding different concentrations (1–8 μM). After treatment for 0 h, 24 h, 48 h, and 72 h, the cells were incubated with MTT for 2 h, the supernatant was discarded, and 150 μL of DMSO was added. The absorbance was measured at 490 nm, and the IC50 of 5,7,4'-trimethoxyflavonoids and their derivatives was calculated. 50Values ​​and results are detailed in the [link / reference]. Figure 1 See Table 1.

[0130] Table 1. Inhibition of esophageal cancer, colon cancer, and gastric cancer cell lines by the derivatives of this invention. 50 (μM)

[0131]

[0132] Example 3: Plate Colony Formation Experiment

[0133] Esophageal squamous cell carcinoma cells KYSE450, gastric cancer cells HGC27, and colorectal cancer cells HCT116 (900 cells per well) were seeded in 6-well plates, and corresponding concentrations of compounds 4b, 5, 7e, 7k, 11e, and 11g were added. The plates were then incubated at 37 ℃ and 5% CO2 for about 10 days. The number of cell clones was photographed and counted, and the statistical results were analyzed.

[0134] Example 4: In vivo antitumor activity experiment in mice

[0135] After digesting and centrifuging HGC27 cells, the cell concentration was adjusted to 5 × 10⁻⁶ cells with PBS based on the cell count results. 7 / ml, place the cell suspension on ice, and after entering the animal room, inoculate each nude mouse with 100μL of cell suspension in a clean bench, which is approximately 5×10⁶ cells per nude mouse. 6 We used 10 cells to establish a human gastric cancer HGC27 cell xenograft mouse model (CDX).

[0136] One week after inoculation, nude mice were ear-tagged and weighed. Mice were then evenly distributed into groups of nine mice each, with eleven groups in total. Each group was administered physiological saline containing 10% DMSO, 200 mg / kg / day capecitabine, 50 mg / kg / day 5,7,4'-trimethoxyflavone (compound 4b), and 5, 10 mg / kg / day compounds 7e, 7k, 11e, and 11g, respectively, via gavage. Daily administration was initiated, and tumor appearance was monitored. Once tumors became visible to the naked eye, tumor size was measured. Tumor volume and body weight were measured at fixed intervals until the tumor volume reached 1000 mm². 3 At that point, the experiment was terminated, the tumor tissue was removed, the tumor weight was measured, and a photograph was taken.

[0137] Example 5: Hydrogen bromide-activated Sepharose 4B protein pull-down assay

[0138] A drug bead mixture was prepared by combining 2 mg of compounds 4b, 7e, 7k, 11e, 11g and 0.3 g of Sepharose 4B beads. The bead mixture was then mixed with cell lysates from esophageal cancer cells KYSE450, gastric cancer cells HGC27 and colorectal cancer cells HCT116 and incubated overnight at 4°C. After washing, the mixture was incubated in a 95°C metal bath for 5 min. Meanwhile, a 10% input sample was prepared. After the sample was prepared, it was detected by Western blot using LRPPRC, STAT3, and CDK1 antibodies.

[0139] Example 6: Cell thermal displacement experiment to verify binding ability

[0140] Esophageal cancer cells KYSE450, gastric cancer cells HGC27, and colorectal cancer cells HCT116 were collected, lysed, and centrifuged to collect the supernatant. 11 g (80 μM) of the compound and solvent were added, and the mixture was incubated at 37 °C for 2 hours. The mixture was then divided into 12 tubes and heated at gradient temperatures of 37, 41, 45, 49, 53, 57, 61, 65, and 69 °C for 3 minutes each. After centrifugation, the supernatant was collected and incubated in a 95 °C metal bath for 5 minutes. The target proteins LRPPRC, STAT3, and CDK1 were detected and quantified by Western blotting, and CETSA melting curves were plotted.

[0141] Experimental results

[0142] Figure 1 The growth inhibition of esophageal squamous cell carcinoma cells, gastric cancer cells, and colorectal cancer cells by the derivatives 7e, 7k, 11e, 11g, and 4b of the present invention is shown. The modified derivatives have better antitumor activity than 5,7,4'-trimethoxyflavone. In the figures, A, B, and C are bar graphs showing tumor cell proliferation at different time points with different drug concentrations. The results show that the derivatives 7e, 7k, 11e, and 11g of the present invention can inhibit the proliferation of esophageal squamous cell carcinoma KYSE450, gastric cancer cells HGC27, and colorectal cancer cells HCT116 at concentrations ranging from 1 to 8 μM, with the highest inhibition rate at a concentration of 8 μM. D, E, and F are the statistics of the number of clones formed by the derivatives 7e, 7k, 11e, and 11g of the present invention in inhibiting the formation of clones of esophageal squamous cell carcinoma KYSE450, gastric cancer cells HGC27, and colorectal cancer cells HCT116. G is a clone formation image. With the increase of drug concentration, the number of clones decreased significantly. The results indicate that the derivatives 7e, 7k, 11e, and 11g of the present invention can inhibit the formation of clones of esophageal squamous cell carcinoma KYSE450, gastric cancer cells HGC27, and colorectal cancer cells HCT116. The antitumor activity of compounds 7e, 7k, 11e, and 11g is superior to that of 5,7,4'-trimethoxyflavonoids. (Compared with the control group, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001)

[0143] Figure 2 This invention relates to the therapeutic effects of derivatives 7e, 7k, 11e, 11g, 4b, and capecitabine on tumor growth in a mouse model of human gastric cancer HGC27 cell xenografts. A shows the trend of tumor volume changes during drug administration; B shows the tumor weight after mouse sacrifice at the end of the experiment; and C shows tumor images of the control and drug-treated groups. The results indicate that compounds 7e, 7k, 11e, 11g, and 4b, at 50 mg / kg / day, significantly increase tumor growth. Compound 4b at 5 mg / kg / day significantly inhibited the volume and weight of tumors in a mouse model of human gastric cancer cell HGC27 xenografts. Compared with the positive control 200 mg / kg / day capecitabine, compounds 7e, 7k, 11e, and 11g showed more significant therapeutic effects, indicating that compounds 7e, 7k, 11e, and 11g can significantly inhibit the growth of tumors in a mouse model of human gastric cancer HGC27 xenografts and have significant therapeutic effects on tumor growth in a CDX mouse model of gastric cancer. D is a graph showing the trend of mouse body weight change during the administration period, E, F, and G are the weights of the liver, spleen, and kidneys of mice in each group after the experiment, and H is HE staining of the tumor, liver, and kidneys. The results show that compounds 7e, 7k, 11e, and 11g at 5 mg / kg / day and compound 4b at 50 mg / kg / day had no effect on the body weight, liver, spleen, and kidneys of mice, indicating that compounds 7e, 7k, 11e, and 11g have no obvious toxic side effects. (Compared with the control group, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001)

[0144] Figure 3 The derivatives 7e, 7k, 11e, 11g, and 4b of this invention can bind well to the target proteins LRPPRC, STAT3, and CDK1. A, B, and C are hydrogen bromide-activated Sepharose 4B protein pull-down assays, which show that the derivatives 7e, 7k, 11e, 11g, and 4b of this invention bind to the target proteins LRPPRC, STAT3, and CDK1. D, E, and F are cell thermal displacement assays, and G, H, and I are CETSA melting curves, which show that the derivative 11g of this invention binds to the target proteins LRPPRC, STAT3, and CDK1.

[0145] In summary, the 5,7,4'-trimethoxyflavonoid derivatives, when examined at 0, 24, 48, and 72 h after treatment, showed that they inhibited cell proliferation in esophageal cancer cell lines, gastric cancer cell lines, colorectal cancer cell lines, and lung cancer cell lines, with activity superior to 5,7,4'-trimethoxyflavonoids. Plate colony formation assays demonstrated that compounds 7e, 7k, 11e, and 11g inhibited the formation of esophageal cancer KYSE450, gastric cancer HGC27, and colorectal cancer HCT116 cell colonies. Hydrogen bromide-activated Sepharose 4B protein pull-down assays showed that compounds 7e, 7k, 11e, and 11g bound to target proteins LRPPRC, STAT3, and CDK1. Cell thermal shift assays further confirmed the binding of compound 11g to target proteins LRPPRC, STAT3, and CDK1. In a gastric cancer CDX model, compounds 7e, 7k, 11e, and 11g showed significant tumor therapeutic effects on mice with gastric cancer cell xenografts, providing assistance for the prevention and treatment of esophageal cancer, gastric cancer, and colorectal cancer in clinical practice.

Claims

1,5,7,4'-Trimethoxyflavonoid sulfonamide derivatives, characterized in that, Compounds having the following structure: 。 2. The method for preparing the 5,7,4'-trimethoxyflavonoid sulfonamide derivative according to claim 1, characterized in that, The synthesis route is as follows: The reaction routes of compounds 7e and 7k are as follows: The synthesis process is as follows: (1) Dissolve compound 1 and compound 2a in the reaction solvent, add alkali, heat under reflux to carry out Claisen-Schmidt reaction. After the reaction is complete, cool to room temperature, pour the reaction solution into ice water, adjust the pH of the system to 5 ~ 6 with dilute hydrochloric acid, precipitate out, filter, wash, and obtain chalcone compound 3a, which can be directly used in the next step of the reaction. (2) Dissolve compound 3a in the reaction solvent, add a catalytic amount of iodine, heat to 140±10℃ to carry out the Algar-Flynn-Oyamada reaction. After the reaction is complete, cool to room temperature, pour the reaction solution into ice water, precipitate out, filter, wash, and obtain compound 4a. (3) Compound 4a was dissolved in the reaction solvent, and reducing metal powder was added. Acid was added and refluxed to reduce the nitro group to an amino group. The compound was purified by silica gel column chromatography to obtain compound 5. (4) Compound 5 is dissolved in the reaction solvent, and when an acylation reaction catalyst and a base are added, it reacts with compound 6 to give compounds 7e and 7k, respectively. In step (1), the molar ratio of Claisen-Schmidt reaction compound 1, compound 2a, and base is 1:(1.2~1.4):(2~5), and the solvent used is methanol, ethanol, tert-butanol, or acetonitrile; the base used is one or a mixture of two or more of potassium carbonate, sodium carbonate, sodium hydroxide, lithium hydroxide, and sodium hydride; the concentration of dilute hydrochloric acid is 5~10. mol / L; In step (2), the catalytic equivalent of iodine is 5%~20%, and the solvent used is dimethyl sulfoxide, N,N-dimethylformamide or N,N-dimethylacetamide; In step (3), the molar ratio of compound 4a, reducing metal powder and acid is 1:(5~8):(5~10), the selected reaction solvent is methanol, ethanol, isopropanol or tert-butanol, the reducing metal powder is iron powder, magnesium powder or zinc powder, and the acid is concentrated hydrochloric acid, concentrated sulfuric acid or glacial acetic acid; In step (4), the molar ratio of compound 5, compound 6, catalyst and base is 1:(1.5~2):(1~2):(3~5), the selected reaction solvent is acetone, tetrahydrofuran or 1,4-dioxane, the catalyst is DMAP or DCC, and the base is triethylamine, pyridine or DBU; The reaction routes for compounds 11e and 11g are as follows: The synthesis process is as follows: (1) Dissolve compound 1 and compound 2b in the reaction solvent, add alkali, heat to 110 ± 10℃ to carry out Claisen-Schmidt reaction. After the reaction is completed, cool to room temperature, pour the reaction solution into ice water, adjust the pH of the system to 5 ~ 6 with dilute hydrochloric acid, precipitate out, filter, wash, and obtain chalcone compound 3b. (2) Add compound 3b to chlorosulfonic acid, a sulfonating agent, at 0±5 ℃. After the addition is complete, move to room temperature to sulfonate chalcone. After the reaction is complete, quench the reaction with ice water, precipitate, filter, wash with pure water, and obtain solid. The prepared compound 8 can be directly used in the next step of the reaction. (3) Dissolve compound 8 in the reaction solvent, add compound 9, stir at room temperature until the reaction is complete, evaporate to dryness, dissolve the residue in ethyl acetate, wash with hydrochloric acid solution until neutral, wash with saturated brine, dry with anhydrous sodium sulfate, filter, concentrate, and purify by silica gel column chromatography to obtain compound 10, which can be directly used in the next step of the reaction. (4) Dissolve compound 10 in the reaction solvent, add a catalytic amount of iodine, heat to 140±10℃ to carry out the Algar-Flynn-Oyamada reaction. After the reaction is complete, cool to room temperature, pour the reaction solution into ice water to precipitate the precipitate, filter, wash, and purify by silica gel column chromatography to obtain compounds 11e and 11g respectively. In step (1), the molar ratio of Claisen-Schmidt reaction compound 1, compound 2b, and base is 1:(1.2~1.4):(2~5), and the solvent used is methanol, ethanol, tert-butanol, or acetonitrile; the base used is one or a mixture of two or more of potassium carbonate, sodium carbonate, sodium hydroxide, lithium hydroxide, and sodium hydride; the concentration of dilute hydrochloric acid is 5~10 mol / L; in step (3), the molar ratio of compound 8 and compound 9 is 1:(1.5~2), and the solvent used is acetone, tetrahydrofuran, or 1,4-dioxane; in step (4), the catalytic equivalent of iodine is 5%~20%, and the solvent used is dimethyl sulfoxide, N,N-dimethylformamide, or N,N-dimethylacetamide.

3. The use of the 5,7,4'-trimethoxyflavonoid sulfonamide derivative according to claim 1 in the preparation of medicaments for treating gastric cancer, esophageal cancer, colorectal cancer or lung cancer.

4. The application according to claim 3, characterized in that, The derivative was able to significantly inhibit tumor growth in in vivo animal models.

5. The application according to claim 3, characterized in that, The derivative exerts its anti-tumor effect by inhibiting the proliferation of tumor cells.

6. The application according to claim 3, characterized in that, The derivative can bind to target proteins LRPPRC, STAT3, and CDK1.

7. The application according to claim 3, characterized in that, It can be used as an active ingredient or in combination with other drugs to prepare antitumor drugs targeting LRPPRC, STAT3 and CDK1 proteins, wherein the tumor is selected from gastric cancer, esophageal cancer, colorectal cancer and lung cancer.

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