A method for preparing a fluorinated polycyclic coumarin derivative and its application

Abstract

This invention discloses a method for preparing a fluorinated fused-ring coumarin derivative and its application. Specifically, a 4-(N-methyl-N-acrylamido)coumarin compound of formula (I), a thiodifluoroarylacetic acid compound of formula (II), an oxidant, and a catalyst are added to a reaction solvent. Under nitrogen protection, the reaction is stirred at 60-85°C for 6-12 hours. The resulting reaction solution is purified to obtain the target compound, a fluorinated fused-ring coumarin derivative as shown in formula (III). The reaction formula is as follows: In formula (I), the H on the benzene ring is substituted by a R group. 1 Substitution or non-substitution; when substituted, the substituent R 1 Selected from alkyl, halogen, or methoxy groups, the H on the benzene ring in formula (II) is substituted by a R group. 2 Substitution or non-substitution; when substituted, the substituent R 2 The compounds are selected from alkyl, methoxy, or halogen compounds. This invention enables the synthesis of difluoromethylated / cyclized fused-ring coumarin compounds with antitumor activity under mild reaction conditions and with high yields.

Description

Technical Field

[0001] This invention belongs to the field of organic synthesis technology, specifically relating to a method for preparing fluorinated fused-ring coumarin derivatives and their applications. Background Technology

[0002] Coumarins, also known as 1,2-benzopyranones or o-oxonaphthones, are a class of oxygen-containing heterocyclic compounds widely found in nature. They play important roles in anticancer, antibacterial, anti-inflammatory, anti-HIV, and anticoagulant activities. The development of direct and effective synthetic methods for compounds with coumarin lead structures possessing potential pharmacological activity has attracted widespread attention. Chinese patent CN102584841B discloses a one-pot synthesis of quinoline coumarin compounds and its antitumor activity testing. Studies show that these new compounds have good inhibitory effects on MCF-7 and A-549, with some compounds exhibiting high IC50 values. 50 The value reached 0.52 μM.

[0003] In addition, in the field of medicinal chemistry, fluoroalkyl groups can serve as bioisosteres with poor stability. Scientists often use CF bonds to replace CH bonds in drug molecules, thereby improving drug selectivity, stability, lipophilicity, and membrane permeability. The presence of sulfur atoms also increases the lipophilicity of the parent molecule. In 2010, Wang Sheng synthesized coumarin compounds or their corresponding fluorinated analogs and investigated the antitumor activity of some products using human liver cancer cells (HepG-2) as the tumor cell line and 5-fluorouracil (5-Fu) as the positive control. He found that the antitumor activity of the fluorinated compounds was stronger than that of the corresponding non-fluorinated coumarins (Master's thesis, Synthesis and Antibacterial and Antitumor Activity Study of Coumarin Compounds and Their Fluorinated Derivatives, Jiangsu University, 2010). Summary of the Invention

[0004] To address the above problems, the present invention aims to provide a method for preparing and using a fluorinated fused-ring coumarin derivative.

[0005] The specific technical solution is as follows:

[0006] A method for preparing a fluorinated fused-ring coumarin derivative includes the following steps: adding a 4-(N-methyl-N-acrylamido)coumarin compound of formula (I), a thiodifluoroarylacetic acid compound of formula (II), an oxidant, and a catalyst to a reaction solvent; stirring the reaction mixture at 60-85°C for 6-12 hours under nitrogen protection; and purifying the resulting reaction solution to obtain the target compound, a fluorinated fused-ring coumarin derivative of formula (III), as shown in the following reaction formula:

[0007]

[0008] In formula (Ⅰ), the H on the benzene ring is replaced by the R substituent. 1 Substitution or non-substitution; when substituted, the substituent R 1 Selected from alkyl, halogen, or methoxy groups, the H on the benzene ring in formula (II) is substituted by a R group. 2 Substitution or non-substitution; when substituted, the substituent R 2 It is selected from alkyl, methoxy or halogen.

[0009] Further, the reaction solvent is tetrahydrofuran:water = 1:0.2-2, acetonitrile:water = 1:0.2-2 or acetone:water = 1:0.2-2, preferably acetonitrile:water = 1:1.5, and the total volume of the reaction solvent is 5-15 mL / mmol, preferably 6-10 mL / mmol, based on the amount of 4-(N-methyl-N-acrylamido)coumarin compound shown in formula (I).

[0010] Further, the oxidant is potassium persulfate, ammonium persulfate or tert-butyl hydroperoxide, preferably potassium persulfate, and the molar ratio of the oxidant to the 4-(N-methyl-N-acrylamido)coumarin compound shown in formula (I) is 1.2-3.5:1, preferably 2.0-2.5:1.

[0011] Further, the catalyst is cuprous iodide, cuprous chloride, or silver carbonate, preferably cuprous chloride, and the amount of catalyst used is 5-30 mol% of the 4-(N-methyl-N-acrylamido)coumarin compound shown in formula (I), preferably 15-20 mol%.

[0012] Furthermore, the reaction temperature is 70-80℃, and the reaction time is 8-10h.

[0013] Furthermore, the molar ratio of the 4-(N-methyl-N-acrylamido)coumarin compound shown in formula (I) to the thiodifluoroarylacetic acid compound shown in formula (II) is 1:1.5-3.0, preferably 1:2-2.5.

[0014] Further, the necessary purification steps are as follows: after the reaction is completed, the organic solvent is recovered under reduced pressure, extracted with ethyl acetate, the organic layers are combined, dried with anhydrous Na2SO4, and the crude product is recovered under reduced pressure. The crude product is purified by silica gel column chromatography with a petroleum ether / ethyl acetate mixed solvent in a volume ratio of 6 to 10:1 to obtain the fluorinated fused-ring coumarin derivative as shown in formula (III).

[0015] An application of a fluorinated fused-ring coumarin derivative prepared by the above preparation method, wherein the application is the use of the fluorinated fused-ring coumarin derivative in the preparation of inhibitors for melanoma B16-F10 and lung cancer A549 cells.

[0016] Furthermore, the fluorinated fused-ring coumarin derivatives are compounds represented by formulas (IIIa), (IIIb), (IIIc), (IIId), (IIIe), (IIIf), (IIIg), (IIIh), (IIIi), or (IIIj).

[0017]

[0018] The beneficial effects of this invention are as follows:

[0019] 1) This invention uses 4-(N-methyl-N-acrylamido)coumarin compounds and thiodifluoroarylacetic acid compounds as raw materials, and can achieve the synthesis of difluoromethylated / cyclized fused-ring coumarin compounds with antitumor activity by inexpensive metal catalysis. The reaction conditions are mild and the reaction yield is high.

[0020] 2) This invention uses a one-step synthesis method to prepare the target compound. Compared with conventional techniques, this method is simple to operate, does not require prefunctionalization of the reaction substrate, has high atom economy, low cost, and advanced process route.

[0021] 3) The compounds synthesized in this invention have novel structures. Preliminary pharmacological activity screening shows that most compounds have significant in vitro inhibitory effects on melanoma B16-F10 cell lines and lung cancer A-549 cell lines. Some compounds have significant inhibitory effects on proliferation, which are superior to the positive control drug cisplatin, and have the potential to be developed into new anti-tumor drugs. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0023] Example 1 6-(2,2-difluoro-2-(phenylthio)ethyl)-4,6-dimethylpyrano[2,3,4-ij]isoquinoline-2,5(4H,6H)-dione

[0024]

[0025] Compound 4-(N-methyl-N-acrylamido)coumarin (121.5 mg, 0.5 mmol, 1.0 equivalent), compound thiodifluoroarylacetic acid (204 mg, 1.0 mmol), CuCl (20 mol% of compound 4-(N-methyl-N-acrylamido)coumarin), and potassium persulfate (270 mg, 1.0 mmol) were added to the mixture. Acetonitrile:water = 1:1 (5.0 mL) was added. After purging with nitrogen three times, the reaction system was stirred at 70 °C for 10 hours. Acetonitrile was recovered under reduced pressure. The aqueous layer was extracted three times with ethyl acetate. The organic layers were combined, dried over anhydrous Na2SO4, and ethyl acetate was recovered under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent was a mixture of petroleum ether and ethyl acetate in a volume ratio of 8:1) to obtain 280.7 mg of the target product, with a yield of 70%.

[0026] 1 H NMR (400MHz, CDCl3) δ7.57 (t, J=8.1Hz, 1H), 7.37 (dd, J=14.3, 7.3Hz, 3H), 7.32 -7.19(m,4H),5.83(s,1H),3.46-3.35(m,4H),2.97-2.80(m,1H),1.67(s,3H).

[0027] 13 C NMR (101MHz, CDCl3) δ171.8,161.6,152.1,148.5,137.3,136.1,132.3,130.0,128.9,127.9(d,J C-F =280.8Hz), 125.7, 121.52(d,J) C-F =1.7Hz),115.4,108.7,92.8,49.9(t,J C-F =21.8Hz), 43.9(d,J C-F =2.1Hz), 31.7, 29.5.

[0028] 19 F NMR(376MHz, CDCl3)δ-65.36--73.32(m).

[0029] Examples 2-9: Preparation of fluorinated polycyclic coumarin (III-a). The preparation methods of each example are as shown in Example 1. The difference lies in changing certain reaction conditions (such as the type of solvent, the amount of raw materials (I) and (II) added, the type of catalyst, the type of oxidant, the temperature, etc.) to determine the optimal reaction conditions. The specific changes in the conditions and the corresponding reaction effects of each example are shown in Tables 1-2 below.

[0030] Table 1 Summary of reaction conditions in Examples 2-5

[0031]

[0032] Table 2 Summary of reaction conditions in Examples 6-9

[0033]

[0034]

[0035] Referring to Tables 1-2, in Examples 2 and 3, the changes were in the type of reaction solvent and the amount of catalyst. The reaction solvents were replaced with THF:H2O = 1:1.5 and CH3CN:H2O = 1:1.5, respectively, and the catalyst amount was replaced with 15 mol%. In Examples 4 and 5, the changes were in the type and amount of oxidant, replacing (NH4)2S2O8 (2.0 equivalents) and (NH4)2S2O8 (1.5 equivalents), respectively. In Examples 6 and 7, the changes were in the molar ratio of reactant compound (I) and compound (II) and the type of catalyst, replacing them with 1:2.5, 1:3.0, and CuI and Ag2CO3, respectively. In Examples 8 and 9, the changes were in the temperature, replacing them with 75℃ and 80℃, respectively. The results show that the type and amount of solvent, catalyst, oxidant, and temperature all have a certain impact on the reaction yield.

[0036] Example 10 6-(2,2-difluoro-2-(phenylthio)ethyl)-4,6,7-trimethylpyrano[2,3,4-ij]isoquinoline-2,5(4H,6H)-dione

[0037]

[0038] Compound 4-(N-methyl-N-acrylamido)-6-methylcoumarin (128.5 mg, 0.5 mmol), compound thiodifluoroarylacetic acid (204.0 mg, 1.0 mmol), CuCl (20 mol% of 4-(N-methyl-N-acrylamido)-6-methylcoumarin), and potassium persulfate (270 mg, 1.0 mmol) were added to the mixture. Acetonitrile:water = 1:1 (5.0 mL) was added. After purging with nitrogen three times, the reaction system was stirred at 70 °C for 10 hours. Acetonitrile was recovered under reduced pressure. The aqueous layer was extracted three times with ethyl acetate. The organic layers were combined, dried over anhydrous Na2SO4, and ethyl acetate was recovered under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent was a mixture of petroleum ether and ethyl acetate in a volume ratio of 8:1) to obtain 290.5 mg of the target product, with a yield of 77%.

[0039] 1 H NMR (400MHz, CDCl3) δ7.45-7.32(m,4H),7.34-7.25(m,2H),7.19(d,J=8.5Hz,1H),5.79(s,1H),3.57-3.26(m,5H),2.54(s,3H),1.76(s,3H).

[0040] 13 C NMR (101MHz, CDCl3) δ173.1,161.5,151.2,148.8,137.1,136.1,133.9,131.6(d,J C-F =1.5Hz),130.0,129.0,128.23(t,J C-F =280.8Hz),125.8,116.0,109.1,92.6,46.0(t,J C-F =21.5Hz), 45.3(d,J C-F =2.1Hz), 30.0, 27.8, 22.1.

[0041] 19 F NMR (376MHz, CDCl3) δ-71.5 (d, J = 205.8Hz), -72.7 (d, J = 205.6Hz).

[0042] Example 11 7-Bromo-6-(2,2-difluoro-2-(phenylthio)ethyl)-4,6-dimethylpyrano[2,3,4-ij]isoquinoline-2,5(4H,6H)-dione

[0043]

[0044] Compound 4-(N-methyl-N-acrylamido)-6-bromocoumarin (160.5 mg, 0.5 mmol), compound thiodifluoroarylacetic acid (204.0 mg, 1.0 mmol), CuCl (20 mol% of 4-(N-methyl-N-acrylamido)-6-bromocoumarin), and potassium persulfate (270 mg, 1.0 mmol) were added to the mixture. Acetonitrile:water = 1:1 (5.0 mL) was added. After purging with nitrogen three times, the reaction system was stirred at 70 °C for 10 hours. Acetonitrile was recovered under reduced pressure. The aqueous layer was extracted three times with ethyl acetate. The organic layers were combined, dried over anhydrous Na2SO4, and ethyl acetate was recovered under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent was a mixture of petroleum ether and ethyl acetate in a volume ratio of 8:1) to obtain 335.3 mg of the target product, with a yield of 75%.

[0045] 1 H NMR (400MHz, CDCl3) δ7.77(d,J=8.9Hz,1H),7.46-7.25(m,5H),7.16(d,J=8.9Hz,1H),5.82(s,1H), 4.15(ddd,J=20.5,15.5,9.9Hz,1H),3.43(s,3H),3.30(ddd,J=23.3,15.5,7.7Hz,1H),1.92(s,3H).

[0046] 13 C NMR (101MHz, CDCl3) δ172.1,160.6,152.0,147.8,139.2,136.2,134.5,130.0,129.0,128.49(t,J C-F =280.8Hz), 125.8(t,J) C-F =2.2Hz), 117.7, 116.3 (d, J) C-F =1.8Hz), 111.0, 45.6(d,J) C-F =1.9Hz), 44.5(t,J C-F =21.2Hz), 30.1, 26.3.

[0047] 19 F NMR (376MHz, CDCl3) δ-71.4 (d, J = 205.2Hz), -72.3 (d, J = 205.3Hz).

[0048] Example: 12,7-chloro-6-(2,2-difluoro-2-(phenylthio)ethyl)-4,6-dimethylpyrano[2,3,4-ij]isoquinoline-2,5(4H,6H)-dione

[0049]

[0050] Compound 4-(N-methyl-N-acrylamido)-6-chlorocoumarin (138.5 mg, 0.5 mmol), compound thiodifluoroarylacetic acid (204.0 mg, 1.0 mmol), CuCl (20 mol% of 4-(N-methyl-N-acrylamido)-6-chlorocoumarin), and potassium persulfate (270 mg, 1.0 mmol) were added to the mixture. Acetonitrile:water = 1:1 (5.0 mL) was added. After purging with nitrogen three times, the reaction system was stirred at 70 °C for 10 hours. Acetonitrile was recovered under reduced pressure. The aqueous layer was extracted three times with ethyl acetate. The organic layers were combined, dried over anhydrous Na2SO4, and ethyl acetate was recovered under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent was a mixture of petroleum ether and ethyl acetate in a volume ratio of 8:1) to obtain 313.2 mg of the target product, with a yield of 72%.

[0051] 1 H NMR (400MHz, CDCl3) δ7.56 (d, J = 8.9 Hz, 1H), 7.45-7.25 (m, 6H), 5.83 (s, 1H), 3.94 (ddd, J =20.0,15.5,10.3Hz,1H),3.43(s,3H),3.34(ddd,J=23.5,15.5,8.4Hz,1H),1.88(s,3H).

[0052] 13 C NMR (101MHz, CDCl3) δ172.1,160.7,151.4,147.8,136.1,135.4,133.0,130.3,129.9,128.34(t,J C-F =281.8Hz),128.0,125.7,117.4,110.5,93.1,44.9(d,J C-F =2.0Hz), 44.6(d,J C-F =21.3Hz), 30.0, 26.1.

[0053] 19 F NMR (376MHz, CDCl3) δ-71.66 (d, J = 205.0Hz), -72.42 (d, J = 205.0Hz).

[0054] Example 136-(2,2-difluoro-2-(phenylthio)ethyl)-7-methoxy-4,6-dimethylpyrano[2,3,4-ij]isoquinoline-2,5(4H,6H)-dione

[0055]

[0056] Compound 4-(N-methyl-N-acrylamido)-6-methoxycoumarin (136.5 mg, 0.5 mmol), compound thiodifluoroarylacetic acid (204.0 mg, 1.0 mmol), CuCl (20 mol% of 4-(N-methyl-N-acrylamido)-6-methoxycoumarin), and potassium persulfate (270 mg, 1.0 mmol) were added to the mixture. Acetonitrile:water = 1:1 (5.0 mL) was added. After purging with nitrogen three times, the reaction system was stirred at 70 °C for 10 hours. Acetonitrile was recovered under reduced pressure. The aqueous layer was extracted three times with ethyl acetate. The organic layers were combined, dried over anhydrous Na2SO4, and ethyl acetate was recovered under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent was a mixture of petroleum ether and ethyl acetate in a volume ratio of 8:1) to obtain 318.9 mg of the target product, with a yield of 74%.

[0057] 1 H NMR (400MHz, CDCl3) δ7.43-7.33(m,3H),7.31-7.24(m,3H),7.15(d,J=9.1Hz,1H),5.79(s,1H), 3.89(s,3H),3.74-3.58(m,1H),3.41(s,3H),3.27(ddd,J=21.1,15.0,10.1Hz,1H),1.73(s,3H).

[0058] 13 C NMR (101MHz, CDCl3) δ173.1,161.9,153.2,148.7,146.5,136.1,129.9,129.0,128.6,(d,J C-F =280.8Hz),126.2,123.0,116.52,115.8,109.6,93.1,56.0,45.5(t,J C-F =21.4Hz), 43.7(d,J C-F =2.2Hz), 29.8, 26.9.

[0059] 19 F NMR (376MHz, CDCl3) δ-71.3 (d, J = 204.4Hz), -72.0 (d, J = 204.3Hz).

[0060] Example 14 6-(2,2-difluoro-2-(phenylthio)ethyl)-4,6,8-trimethylpyrano[2,3,4-ij]isoquinoline-2,5(4H,6H)-dione

[0061]

[0062] Compound 4-(N-methyl-N-acrylamido)-7-methylcoumarin (128.5 mg, 0.5 mmol), compound thiodifluoroarylacetic acid (204.0 mg, 1.0 mmol), CuCl (20 mol% of 4-(N-methyl-N-acrylamido)-7-methylcoumarin), and potassium persulfate (270 mg, 1.0 mmol) were added to the mixture. Acetonitrile:water = 1:1 (5.0 mL) was added. After purging with nitrogen three times, the reaction system was stirred at 70 °C for 10 hours. Acetonitrile was recovered under reduced pressure. The aqueous layer was extracted three times with ethyl acetate. The organic layers were combined, dried over anhydrous Na2SO4, and ethyl acetate was recovered under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent was a mixture of petroleum ether and ethyl acetate in a volume ratio of 8:1) to obtain 311.2 mg of the target product, with a yield of 75%.

[0063] H NMR (400MHz, CDCl3) δ7.44-7.22(m,5H),7.08-6.98(m,2H),5.75(s,1H),3.46 -3.30(m,4H),2.86(ddd,J=18.7,15.1,9.9Hz,1H),2.45(s,3H),1.65(s,3H).

[0064] 13 C NMR (101MHz, CDCl3) δ172.0,161.9,152.2,148.6,143.5,137.0,136.1,130.0,128.9,127.9(t,J C-F =281.8Hz), 125.8, 122.4 (d, J) C-F =1.6Hz),115.6,106.3,91.9,49.9(t,J C-F =21.8Hz), 43.8(d,J C-F =2.1Hz), 31.7, 29.5, 21.9.

[0065] 19 F NMR (376MHz, CDCl3) δ-68.8 (d, J = 205.0Hz), -70.2 (d, J = 205.0Hz).

[0066] Example 15 6-(2,2-difluoro-2-(p-toluenethio)ethyl)-4,6-dimethylpyrano[2,3,4-ij]isoquinoline-2,5(4H,6H)-dione

[0067]

[0068] Compound 4-(N-methyl-N-acrylamido)coumarin (121.5 mg, 0.5 mmol), compound p-methylthiodifluoroarylacetic acid (218.1 mg, 1.0 mmol), CuCl (20 mol% of 4-(N-methyl-N-acrylamido)coumarin), and potassium persulfate (270 mg, 1.0 mmol) were added to the mixture. Acetonitrile:water = 1:1 (5.0 mL) was added. After purging with nitrogen three times, the reaction system was stirred at 70 °C for 10 hours. Acetonitrile was recovered under reduced pressure. The aqueous layer was extracted three times with ethyl acetate. The organic layers were combined, dried over anhydrous Na2SO4, and ethyl acetate was recovered under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent was a mixture of petroleum ether and ethyl acetate in a volume ratio of 8:1) to obtain 311.2 mg of the target product, with a yield of 76%.

[0069] 1 H NMR (400MHz, CDCl3) δ7.57 (t, J = 8.1Hz, 1H), 7.34-7.16 (m, 4H), 7.09 (d, J = 7.9Hz, 2H), 5. 80(s,1H),3.42(s,3H),3.40-3.33(m,1H),2.99-2.75(m,1H),2.31(s,3H),1.67(s,3H).

[0070] 13 C NMR (101MHz, CDCl3) δ171.9,161.6,152.1,148.5,140.4,137.3,136.1,132.2,129.7,127.8(t,J C-F =280.8Hz), 122.1, 121.5(d,J) C-F =1.7Hz),115.3,108.7,92.7,49.9(t,J C-F =21.8Hz), 43.82(d,J C-F =2.1Hz), 31.6, 29.5, 21.2.

[0071] 19 F NMR (376MHz, CDCl3) δ-69.1 (d, J = 204.7Hz), -70.9 (d, J = 204.9Hz).

[0072] Example 16 6-(2,2-difluoro-2-((4-methoxyphenyl)thio)ethyl)-4,6-dimethylpyrano[2,3,4-ij]isoquinoline-2,5(4H,6H)-dione

[0073]

[0074] Compound 4-(N-methyl-N-acrylamido)coumarin (121.5 mg, 0.5 mmol), compound p-methoxythiodifluoroarylacetic acid (234.0 mg, 1.0 mmol), CuCl (20 mol% of 4-(N-methyl-N-acrylamido)coumarin), and potassium persulfate (270 mg, 1.0 mmol) were added to the mixture. Acetonitrile:water = 1:1 (5.0 mL) was added. After purging with nitrogen three times, the reaction system was stirred at 70 °C for 10 hours. Acetonitrile was recovered under reduced pressure. The aqueous layer was extracted three times with ethyl acetate. The organic layers were combined, dried over anhydrous Na2SO4, and ethyl acetate was recovered under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent was a mixture of petroleum ether and ethyl acetate in a volume ratio of 8:1) to obtain 310 mg of the target product, with a yield of 72%.

[0075] 1 H NMR (400MHz, CDCl3) δ7.57(t,J=8.1Hz,1H),7.34-7.26(m,2H),7.23(ddd,J=15.4,8.1,0.9Hz,2H),6.85-6.77(m, 2H),5.80(s,1H),3.77(s,3H),3.42(s,3H),3.40-3.29(m,1H),2.84(ddd,J=19.3,15.1,9.2Hz,1H),1.67(s,3H).

[0076] 13 C NMR (101MHz, CDCl3) δ171.9,161.7,161.2,152.2,148.6,138.0,137.4,132.3,127.8(t,J C-F =281.8Hz), 121.6(d,J C-F =1.8Hz), 116.2(t,J C-F =2.2Hz),115.4,114.5,108.8,92.8,55.3,49.8(t,J C-F =21.8Hz), 43.9(d,J C-F =2.0Hz), 31.7, 29.6.

[0077] 19 F NMR (376MHz, CDCl3) δ-69.64 (d, J = 204.9Hz), -71.52 (d, J = 204.9Hz).

[0078] Example 17 6-(2,2-difluoro-2-((4-fluorophenyl)thio)ethyl)-4,6-dimethylpyrano[2,3,4-ij]isoquinoline-2,5(4H,6H)-dione

[0079]

[0080] Compound 4-(N-methyl-N-acrylamido)coumarin (121.5 mg, 0.5 mmol), compound p-fluorothiodifluoroarylacetic acid (222.0 mg, 1.0 mmol), CuCl (20 mol% of 4-(N-methyl-N-acrylamido)coumarin), and potassium persulfate (270 mg, 1.0 mmol) were added to the mixture. Acetonitrile:water = 1:1 (5.0 mL) was added. After purging with nitrogen three times, the reaction system was stirred at 70 °C for 10 hours. Acetonitrile was recovered under reduced pressure. The aqueous layer was extracted three times with ethyl acetate. The organic layers were combined, dried over anhydrous Na2SO4, and ethyl acetate was recovered under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent was a mixture of petroleum ether and ethyl acetate in a volume ratio of 8:1) to obtain 318.4 mg of the target product, with a yield of 76%.

[0081] 1 H NMR (400MHz, CDCl3) δ7.59(t,J=8.1Hz,1H),7.42-7.31(m,2H),7.24(t,J=8.8Hz,2H),6.98(t ,J=8.6Hz,2H),5.82(s,1H),3.43(s,3H),3.41-3.34(m,1H),2.99-2.76(m,1H),1.68(s,3H).

[0082] 13 C NMR (101MHz, CDCl3) δ171.8,165.2,162.7,161.5,152.1,148.4,138.4(d,J=8.8Hz),137.2,132.3,127.7(t,J=281.2Hz),12 1.5 (d, J = 1.7Hz), 120.9 (d, J = 2.3Hz), 116.3, 116.1, 115.4, 108.7, 92.8, 49.7 (t, J = 21.7Hz), 43.8 (d, J = 2.2Hz), 31.7, 29.5.

[0083] 19 F NMR (376MHz, CDCl3) δ-69.1 (d, J = 204.3Hz), -70.9 (d, J = 204.3Hz), -110.0.

[0084] Example 18 6-(2-((4-chlorophenyl)thio)-2,2-difluoroethyl)-4,6-dimethylpyrano[2,3,4-ij]isoquinoline-2,5(4H,6H)-dione

[0085]

[0086] Compound 4-(N-methyl-N-acrylamido)coumarin (121.5 mg, 0.5 mmol), compound p-chlorothiodifluoroarylacetic acid (238.0 mg, 1.0 mmol), CuCl (20 mol% of 4-(N-methyl-N-acrylamido)coumarin), and potassium persulfate (270 mg, 1.0 mmol) were added to the mixture. Acetonitrile:water (1:1, 5.0 mL) was added. After purging with nitrogen three times, the reaction system was stirred at 70 °C for 10 hours. Acetonitrile was recovered under reduced pressure. The aqueous layer was extracted three times with ethyl acetate. The organic layers were combined, dried over anhydrous Na2SO4, and ethyl acetate was recovered under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent was a mixture of petroleum ether and ethyl acetate in a volume ratio of 8:1) to obtain 339.3 mg of the target product, with a yield of 78%.

[0087] 1 H NMR (400MHz, CDCl3) δ7.59(t,J=8.1Hz,1H),7.39-7.13(m,6H),5.82(s,1H),3.44(s,3H),3.42-3.31(m,1H),2.97-2.80(m,1H),1.68(s,3H).

[0088] 13 C NMR (101MHz, CDCl3) δ171.8,161.5,152.2,148.4,137.3,137.1,136.7,132.3,129.2,124.1(d,J C-F =2.6Hz), 121.5(d,J C-F =1.8Hz),115.5,108.7,92.9,49.8(t,J C-F =21.7Hz), 43.8(d,J C-F =2.3Hz), 31.7, 29.6.

[0089] 19 F NMR (376MHz, CDCl3) δ -68.62 (d, J = 204.3Hz), -70.39 (d, J = 204.2Hz).

[0090] Example 19 Antitumor Activity Test:

[0091] The activity of the compound against the proliferation of melanoma B16-F10 cell line and lung cancer A549 cell line were as follows:

[0092] In vitro antitumor activity was tested using the SRB method, with cisplatin as a control. First, B16-F10 and A549 cells were seeded at 5000 cells / well in sterile 96-well plates. An equal volume of culture medium or PBS buffer was added to the wells surrounding the cells. After overnight cell adhesion, the test compound (0, 1, 2, 4, 8, 16, 32, 64, 128 μM) was added to each well according to the designed concentration gradient. B16-F10 cells were cultured for 24 h, while A549 cells were cultured for 48 h after drug administration. At the drug administration time, the old culture medium was discarded, and 100 μL of pre-chilled 10% trichloroacetic acid (TCA) solution was added to each well to fix the cells. The plates were then placed at 4°C for more than 1 h. The TCA solution in the wells was removed, and the plates were washed at least five times with deionized water and the residual water was removed. The 96-well plates were then dried in a 60°C oven for approximately 1 h. Remove the dried 96-well plate and add 80 μL of SRB solution prepared with 1% acetic acid to each well. Incubate at room temperature for 20 min to stain. Discard the SRB staining solution from each well. Wash gently five times with 1% acetic acid to remove excess unbound SRB dye, and shake off any remaining liquid. Place the 96-well plate in a 60℃ oven to dry for approximately 1 hour. Remove the dried 96-well plate and add 100 μL of 10 mM Tris-base solution (pH 10.5) to each well to dissolve the SRB within the cells. Shake on a plate shaker for 15 min to fully dissolve the SRB within the cells. Measure the absorbance at 540 nm using a microplate reader. Calculate the viability based on the OD values ​​measured by the microplate reader using the following formula:

[0093] Survival rate (%) = (OD administered / OD control) × 100%, and IC was calculated using Graphpad Prism 8.0 software. 50 value.

[0094] Experimental Results: Compared with the blank control group, the half-maximal inhibitory concentration (IC50) of the test compound on tumor cell growth was determined after 24 h and 48 h of administration. 50 The specific test results are shown in Table 3.

[0095] Table 3. Antitumor activity of compounds III-a to III-j

[0096]

[0097]

[0098] Note: Cisplatin is the control drug. a IC50 : The concentration of the test compound that inhibits cell growth by 50%.

[0099] Table 3 shows that the antitumor activity test results indicate that most of the compounds have good inhibitory activity against MCF-7 and A-549 tumor cell lines.

[0100] 1) Among all compounds, compounds III-a, III-b, III-c, III-d, III-g, and III-j showed good inhibitory activity against the melanoma B16-F10 cell line, with an IC50 value of [missing information]. 50 The values ​​were 21.78 μM, 8.57 μM, 20.41 μM, 18.35 μM, 14.92 μM, and 16.02 μM, respectively. Its in vitro inhibitory activity against tumor cells was greater than that of the positive control drug cisplatin (26.52 μM), among which compound III-b showed the best inhibitory activity.

[0101] 2) Among all compounds, compounds III-b, III-c, III-g, III-h, and III-j exhibited excellent inhibitory activity against human lung cancer cells A-549, with IC50 values ​​of [missing information]. 50 The values ​​were 63.72 μM, 55.08 μM, 47.69 μM, 58.44 μM, and 16.04 μM, respectively. Among them, compounds III-g and III-j showed greater in vitro tumor cell inhibition activity than the positive control drug cisplatin (48.28 μM). Compound III-j exhibited the best inhibitory activity.

[0102] 3) It can be seen that the difluoromethylated / fused-ring coumarin derivatives provided by the present invention have good in vitro anti-tumor cell activity and have the potential to be developed into anti-tumor drugs.

[0103] The contents described in this specification are merely an enumeration of the implementation forms of the inventive concept, and the scope of protection of this invention should not be regarded as limited to the specific forms described in the embodiments.

Claims

1. A method for preparing a fluorinated fused-ring coumarin derivative, characterized in that, The process includes the following steps: adding 4-(N-methyl-N-acrylamido)coumarin compounds as shown in formula (I), thiodifluoroarylacetic acid compounds as shown in formula (II), an oxidant, and a catalyst to the reaction solvent; stirring the mixture at 60-85°C for 6-12 hours under nitrogen protection; and purifying the resulting reaction solution to obtain the target compound, a fluorinated fused-ring coumarin derivative as shown in formula (III), with the following reaction formula: In formula (Ⅰ), the H on the benzene ring is replaced by the R substituent. 1 Substitution or non-substitution; when substituted, the substituent R 1 Selected from halogens or methoxy groups, the H on the benzene ring in formula (II) is replaced by a substituent R. 2 Substitution or non-substitution; when substituted, the substituent R 2 Selected from methoxy or halogen; The fluorinated fused-ring coumarin derivatives shown in formula (III) are also selected from the following structures: The reaction solvent is tetrahydrofuran:water = 1:0.2-2, acetonitrile:water = 1:0.2-2, or acetone:water = 1:0.2-2; The oxidizing agent is potassium persulfate or ammonium persulfate; The catalyst is cuprous iodide, cuprous chloride, or silver carbonate.

2. The preparation method according to claim 1, characterized in that, The reaction solvent is acetonitrile:water = 1:1.

5.

3. The preparation method according to claim 1, characterized in that, The total volume of the reaction solvent, expressed as the amount of 4-(N-methyl-N-acrylamido)coumarin compound represented by formula (I), is 5–15 mL / s. mmol.

4. The preparation method according to claim 1, characterized in that, The molar ratio of the oxidant to the 4-(N-methyl-N-acrylamido)coumarin compound shown in formula (Ⅰ) is 1.2-3.5:

1.

5. The preparation method according to claim 4, characterized in that, The molar ratio of the oxidant to the 4-(N-methyl-N-acrylamido)coumarin compound shown in formula (Ⅰ) is 2.0-2.5:

1.

6. The preparation method according to claim 1, characterized in that, The amount of catalyst used is 5-30 mol% of the 4-(N-methyl-N-acrylamido)coumarin compound shown in formula (Ⅰ).

7. The preparation method according to claim 1, characterized in that, The reaction temperature is 70-80℃, and the reaction time is 8-10 hours.

8. The preparation method according to claim 1, characterized in that, The molar ratio of the 4-(N-methyl-N-acrylamido)coumarin compound shown in formula (Ⅰ) to the thiodifluoroarylacetic acid compound shown in formula (Ⅱ) is 1:1.5-3.

0.

9. The preparation method according to claim 1, characterized in that, The purification steps are as follows: After the reaction is completed, the organic solvent is recovered under reduced pressure, extracted with ethyl acetate, the organic layers are combined, dried with anhydrous Na2SO4, and the crude product is obtained by recovering ethyl acetate under reduced pressure. The crude product is purified by silica gel column chromatography with petroleum ether / ethyl acetate mixed solvent in a volume ratio of 6 to 10:1 to obtain the fluorinated fused-ring coumarin derivative as shown in formula (III).

10. An application of a fluorinated fused-ring coumarin derivative prepared by the preparation method described in claims 1-9, characterized in that, The application is the use of fluorinated fused-ring coumarin derivatives in the preparation of inhibitors for melanoma B16-F10 and lung cancer A549 cells.

11. The application as described in claim 10, characterized in that, Fluorinated fused-ring coumarin derivatives are compounds represented by formulas (IIIa), (IIIb), (IIIc), (IIId), (IIIe), (IIIf), (IIIg), (IIIh), (IIIi), or (IIIj).

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