Selenium-containing coumarin derivatives, preparation method thereof and application thereof in preparing antitumor drugs

Abstract

The present invention discloses selenium-containing coumarin derivatives, their preparation methods and their applications in the preparation of anti-tumor drugs, and belongs to the field of pharmaceutical chemistry. The selenium-containing coumarin compounds of the present invention have a simple preparation method, simple raw materials, convenient post-treatment, high compound yield and purity, with a purity of more than 98%, and their structures are verified by nuclear magnetic resonance and mass spectrometry. In vitro activity screening experiments have shown that compounds of this type of structure have anti-tumor activity, especially showing extremely strong killing activity against neuroblastoma SK-N-SH, among which compound 11f (half inhibitory concentration, IC 50 =2.92μM)、11i(IC 50 =2.51μM)、11q(IC 50 =2.79μM) has the strongest inhibitory activity and significantly inhibits the proliferation, migration and invasion of tumor cells. Therefore, this invention has substantial significance for the development of anti-tumor drugs for screening such compounds.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemistry, and specifically relates to selenium-containing coumarin derivatives and a preparation method thereof and application thereof in preparing anti-tumor drugs. Background Art

[0002] Coumarin is a compound containing a benzopyrone nucleus structure (as shown in Formula 1 below). It is a secondary metabolite of many higher plants and is mainly found in leaves, seeds and roots, especially in plants of the Rutaceae and Umbelliferae families. The first natural coumarin was isolated in 1820. These natural product derivatives containing special oxygen heterocycles have good fluorescence properties and excellent DNA embedding ability. So far, a large number of studies have shown that coumarin has a variety of pharmacological activities, such as anti-inflammatory, antibacterial, antiviral, antioxidant, analgesic, anti-tumor, anti-asthma, anti-depression, anti-HIV, anti-tuberculosis, anti-Alzheimer, anti-influenza, and anti-hyperlipidemic. Due to their different effects on diseases, less damage to normal cells, and unique pharmacological properties, they are very likely to be potential resources for the development of anticancer drugs.

[0003]

[0004] Due to the potential application of coumarins in cancer chemotherapy, the design and synthesis of coumarin derivatives have been widely studied in terms of anticancer activity. In order to improve the anticancer activity of coumarins against different tumor cells, different positions of their parent nuclei have been modified. Therefore, the modification of the coumarin nucleus has produced a large number of compounds with different mechanisms of action. However, from the perspective of drug development, obtaining a large amount of coumarins only from natural biological resources will lead to serious resource scarcity and environmental problems. Therefore, chemical synthesis is the preferred method to solve the problem of sample source. More importantly, the synthesis and structural optimization of active natural products can obtain more valuable drugs. In addition, nitrogen-containing heterocycles represent key structural groups in heterocyclic chemistry and occupy a prominent position in the construction of a variety of active compounds, providing ample opportunities for the discovery of new drugs.

[0005] The importance of selenium (Se) and its derivatives has been recognized since soon after Berzelius isolated the element and continues to this day. Several experimental models have long demonstrated that Se is involved in many physiological processes, mainly through selenoproteins, including at least 25 eukaryotic proteins. The most common are glutathione peroxidase (GPx), iodothyronine deiodinase, thioredoxin reductase (TrxR) and selenoproteins. In addition, Se supplementation directly or indirectly plays an antioxidant function, thereby increasing the cell's defense against oxidative stress. Several reports have demonstrated the important roles of Se in human health, including antioxidant, anti-inflammatory, antimutagenic, antitumor or chemopreventive, antiviral, antibacterial, antifungal, antiparasitic and neuroprotective effects.

[0006]

[0007] Although there are three main categories of selenium compounds with potential pharmacological properties (inorganic selenium compounds, organic selenium compounds, and selenoproteins), the most developed and studied are organic selenium derivatives. The main reason for this is that they have low toxicity and their biological activities are comparable to or even better than those of inorganic selenium compounds. There have been many reports on different organic selenium compounds, and selenium heterocyclic compounds have received widespread attention as drugs for the treatment of various diseases such as cancer, diabetes, stroke, Alzheimer's disease, and inflammatory processes. The most famous compound related to selenium-nitrogen heterocycles is 2-phenyl-1,2-benzisoselenazole-3-[2H]-one, named Ebselen (EBS). It was first synthesized in 1924, and in the 1980s, people found that EBS was a good antioxidant and therapeutic agent.

[0008] In recent years, the potential therapeutic effects of selenium in cancer therapy have attracted great interest. Several selenium compounds, including selenocyclodextrin, butyl phenylisoselenocyanate, sodium selenite, diarylseleno, 3,4-diaryl-1,2,5-selenadiazole analogs, and selenite aspirin analogs, have been shown to inhibit cancer cell growth in vitro or in various xenograft rodent models of different cancer types. Summary of the invention

[0009] Inspired by the synergistic effect of organic selenium compounds and chemotherapeutic drugs, and aimed at the above-mentioned problems existing in the prior art, the present invention provides three series of selenium-containing coumarin compounds. These compounds have different degrees of killing and inhibitory effects on different tumor cells, especially on neuroblastoma SK-N-SH.

[0010] The selenium-containing coumarin compound is characterized in that its general structural formula is as shown in any one of formulas (I), (II) and (III):

[0011]

[0012] In formula (I), R 1 is one of the following: hydrogen, fluorine, chlorine, bromine, iodine, methyl, halomethyl; R 2 is one of the following: hydrogen, fluorine, chlorine, bromine, iodine, C1-C4 alkyl, C1-C4 haloalkyl, methoxy; n is an integer of 1-5;

[0013]

[0014] In formula (II), R 1 is one of the following: hydrogen, fluorine, chlorine, bromine, iodine, methyl, selenyl; R 2is one of the following: hydrogen, fluorine, chlorine, bromine, iodine, C1-C4 alkyl, C1-C4 haloalkyl, methoxy;

[0015]

[0016] In formula (III), R is one of the following:

[0017] n=an integer of 1-5, preferably 2 or 3.

[0018] The selenium-containing coumarin compound is characterized in that in formula (I), R 1 is one of the following: fluorine, chlorine, bromine; R 2 is one of the following: fluorine, chlorine, bromine, trifluoromethyl, methyl, methoxy; n=1 or 2.

[0019] The selenium-containing coumarin compound is characterized in that in formula (II), R 1 is one of the following: fluorine, selenomethyl; R 2 It is trifluoromethyl.

[0020] The method for preparing the selenium-containing coumarin compound is characterized in that the selenium-containing coumarin compound represented by general formula (I) is selected from any one of compounds 10b, 10c, 10e, 10r, 11a-11r, and the synthesis route is as follows:

[0021] 1) Using DMF as solvent and the 3-carboxylic acid ethyl ester-7-hydroxy substituted coumarin derivative shown in formula 2 as raw material, reacting with 1-bromo-2-chloroethane or 1-bromo-3-chloropropane in K 2 CO 3 In the presence of , the reaction produces compound 3a or 3b;

[0022] 2) using ethanol-water in a volume ratio of 1:0.5-2 as a solvent, the compound 3a or 3b is hydrolyzed with lithium hydroxide to generate the compound 4a or 4b;

[0023] 3) using ethanol as solvent and 4-halogenated acetophenone as starting material, and reacting with 4-substituted-benzaldehyde in the presence of NaOH to obtain compound 6a-6r; the substituent of 4-substituted-benzaldehyde is one of the following: fluorine, chlorine, bromine, trifluoromethyl, methyl, methoxy; the halide of 4-halogenated acetophenone is fluorine, chlorine or bromine;

[0024] 4) Using methanol as solvent, compounds 6a-6r react with hydrazine hydrochloride to generate compounds 7a-7r;

[0025] 5) firstly subjecting compound 4a or 4b to acyl chloride reaction with thionyl chloride in the presence of a catalytic amount of DMF, and then reacting the acyl chloride reaction product with compound 7a-7r in the presence of triethylamine using dichloromethane as solvent to generate a key intermediate, wherein the key intermediate is any one of compounds 8b, 8c, 8e, 8r, and 9a-9r;

[0026] 6) Finally, using acetonitrile as solvent, the key intermediate obtained in step 5) is reacted with potassium selenocyanate in the presence of a catalytic amount of sodium iodide to generate the target compound; the synthesis route is as follows:

[0027]

[0028] The method for preparing the selenium-containing coumarin compound is characterized in that the selenium-containing coumarin compound represented by general formula (II) is selected from compound 12a or 12b, and the synthesis route is as follows:

[0029] 1) Using 4-methylseleno-acetophenone shown in formula 15 as a raw material, and then reacting it with trifluoromethylbenzaldehyde under alkaline conditions to obtain compound 16, and then reacting compound 16 with hydrochloric acid hydrazine hydrate to obtain compound 17; 2) Using compound 19 as a raw material, first using dichlorothionyl in the presence of a catalytic amount of DMF to perform an acyl chloride reaction on compound 19, and then using dichloromethane as a solvent, reacting the acyl chloride reaction product with compound 7r or 17 in the presence of triethylamine to generate compound 12a or 12b. The synthesis route is as follows:

[0030]

[0031] The method for preparing the selenium-containing coumarin compound is characterized in that the selenium-containing coumarin compound represented by general formula (III) is selected from compound 27a, 27b, 28a, 28b, 31a or 31b, and the synthesis route is as follows:

[0032] 1) reacting compound 23a or 23b with potassium selenocyanate to generate compound 24a or 24b;

[0033] 2) Compound 24a or 24b is then reacted with compound 26a, 26b or 30 under alkaline conditions to obtain the corresponding target compounds. The synthetic routes are as follows:

[0034]

[0035] The application of the selenium-containing coumarin compounds in the preparation of anti-tumor drugs.

[0036] The application of the selenium-containing coumarin compounds in the preparation of anti-tumor drugs, the present invention firstly uses CCK-8 tumor cell toxicity test to study the cytotoxicity of 30 selenium-containing coumarin molecules on 18 cell lines. The results show that the compounds of the present invention have different killing effects on different tumor cells, among which compounds 11f (IC50 = 2.92μM), 11i (IC50 = 2.51μM), and 11q (IC50 = 2.79μM) have the strongest inhibitory activity on SK-N-SH cells, and significantly inhibit the proliferation, migration and invasion of the tumor cells. Therefore, this invention has substantial significance for the research on anti-tumor drugs for screening such compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a graph showing compound 11f inhibiting the proliferation of SK-N-SH cells;

[0038] Figure 2 This is a graph showing compound 11i inhibiting the proliferation of SK-N-SH cells;

[0039] Figure 3 This is a graph showing that compound 11q inhibits the proliferation of SK-N-SH cells;

[0040] Figure 4 This is the graph showing that compound 11f inhibits cell SK-N-SH migration;

[0041] Figure 5 This is a graph showing compound 11f inhibiting cell invasion of SK-N-SH;

[0042] Figure 6 This is the graph showing the inhibition of cell SK-N-SH migration by compound 11i;

[0043] Figure 7 This is the graph showing the inhibition of cell invasion by compound 11i on SK-N-SH;

[0044] Figure 8 This is the graph of compound 11q inhibiting cell SK-N-SH migration;

[0045] Fig. 9 Compound 11q inhibits cell invasion of SK-N-SH; DETAILED DESCRIPTION

[0046] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0047] Example 1: Preparation of Selenium-Containing Coumarin Derivatives Series I.

[0048] (1) Synthesis of ethyl 7-hydroxy-2-oxo-2H-benzopyran-3-carboxylate (Compound 2)

[0049]

[0050] Under nitrogen protection, 2,4-dihydroxybenzaldehyde (compound 1, 43.4mmol) and diethyl malonate (52.1mmol) were added to a 100mL three-necked flask at room temperature, and then 75mL of anhydrous ethanol was added. Under stirring, the temperature was slowly raised to 78°C and the reaction was continued for 4 hours. TLC (petroleum ether: ethyl acetate = 1:1, v / v) monitored the complete reaction of the raw materials. The reaction solution was cooled to room temperature, and 10% hydrochloric acid aqueous solution was slowly added dropwise to acidify to pH about 1-2, producing a large amount of white solid. The obtained solid was filtered, the filter cake was washed with cold ethanol, and vacuum dried to obtain 13.1g of product 2 (white solid), with a yield of 64.4%.

[0051] Melting point: 170-172°C (literature, 170.5-171.8°C). 1 H NMR (500 MHz, DMSO-d 6 )δ=11.08(s,1H),8.87(s,1H),7.75(d,J=8.8Hz,1H),6.84(dd,J=8.8Hz,J=2. 4Hz, 1H), 6.72 (d, J = 2.4Hz, 1H), 4.26 (q, J = 7.2Hz, 2H), 1.30 (t, J = 7.2Hz, 3H).

[0052] (2) Synthesis of ethyl 7-(2-chloroethoxy)-2-oxo-2H-benzopyran-3-carboxylate (Compound 3a)

[0053]

[0054] At room temperature, compound 2 (21.4 mmol) was dissolved in 70 mL of DMF solution and K 2 CO 3 (42.7mmol). After mixing and stirring for 30 minutes, 1-bromo-2-chloroethane (21.4mmol) was added, and the reaction mixture was stirred and heated to 35°C, and stirred for 24 hours. After the reaction was completed, it was quenched with cold dilute hydrochloric acid (1M), and the obtained solid was filtered and washed with water to obtain a crude compound 3a. The obtained crude product was purified by silica gel column chromatography (dichloromethane / petroleum ether=3 / 1, v / v) to obtain a white solid compound 3a with a yield of 75.4%.

[0055] Melting point: 218-220℃; 1 H NMR (500 MHz, CDCl 3): δ = 8.52 (s, 1H), 7.53 (dd, J = 10.0Hz, 5.0Hz, 1H), 6.91 (dd, J = 10.0Hz, 5.0Hz, 1H), 6.85 (d, J = 5.0Hz, 1H), 4.42 (dd, J = 15. 0Hz,10.0Hz,2H),4.27-4.21(m,2H),3.77(t,J=5.0Hz,1H),3.77(t,J=5.0Hz,1H),2.41-2.29(m,2H),1.44(J=5.0Hz,3H).

[0056] (3) Synthesis of ethyl 7-(3-chloropropoxy)-2-oxo-2H-benzopyran-3-carboxylate (Compound 3b)

[0057]

[0058] The synthesis method of compound 3b is the same as that of 3a (the only difference is that 1-bromo-2-chloropropane replaces 1-bromo-3-chloroethane in the same molar amount). The obtained product is a white solid with a yield of 80.5%. Melting point: 209-211°C.

[0059] (4) Synthesis of 7-(2-chloroethoxy)-2-oxo-2H-benzopyran-3-carboxylic acid (Compound 4a)

[0060]

[0061] At room temperature, compound 3a or 3b (1.0 mmol) was added to a mixture of ethanol (2.0 mL) and water (2.0 mL). Under stirring, lithium hydroxide (1.2 mmol) was added. The reaction mixture was then stirred at room temperature for 8 hours. After the reaction was completed, the solvent was removed by rotary evaporator, and then dilute hydrochloric acid (1 M) was added to the concentrated crude product. The resulting solid was filtered and washed with water to obtain a light yellow solid product 4a or 4b, which was directly used in the next step without further purification.

[0062] Compound 4a is a light yellow solid with a yield of 88.4%. Melting point: 212-214°C; 1 HNMR (500MHz, CDCl 3 ): δ=8.72(s,1H),7.85(d,J=5.0Hz,1H),7.07-7.03(m,2H),4.41(t,J=5.0Hz,2H),3.99(t,J=5.0Hz,2H).

[0063] 7-(3-Chloropropoxy)-2-oxo-2H-benzopyran-3-carboxylic acid compound 4b, light yellow solid, yield 93.0%, melting point: 218-220°C.

[0064]

[0065] 1 H NMR (500MHz, DMSO-d6): δ = 8.71 (s, 1H), 7.84 (d, J = 10.0Hz, 1H), 7.07-7.03 (m, 2H), 4.25 (t, J = 5.0Hz, 2H), 3.81 (t, J = 5.0Hz, 2H), 2.24-2.19 (m, 2H).

[0066] (5) Synthesis of (E)-1,3-di(4-chlorophenyl)propyl-2-enyl-1-one (Compound 6a)

[0067]

[0068] Under nitrogen protection, compound 54-chloro-acetophenone (12.5mmol) and 4-chloro-benzaldehyde (12.5mmol) were added to a 100mL three-necked flask at room temperature, and then 20mL of anhydrous ethanol was added, stirred, and cooled to 0-5°C in an ice-water bath. 10% aqueous sodium hydroxide solution (0.57g of sodium hydroxide was dissolved in 5.7mL of water and prepared for standby use) was slowly added dropwise to the above reaction solution. After the addition was completed, the reaction was stirred at 0-5°C for 2 hours. The white solid produced by the reaction was filtered, and the filter cake was washed with cold methanol and dried to obtain 3.17g of off-white solid of compound 6a, with a yield of 88.4%. Melting point: 155-156°C (literature, 155-157°C).

[0069] 1 H NMR (500 MHz, CDCl 3 ); δ=7.98 (d, J=7.6Hz, 2H), 7.79 (d, J=16.6Hz, 1H), 7.59 (d, J=8.4Hz, 2H), 7.50-7.39 (m, 5H).

[0070] The synthesis methods of the following compounds 6b-6r refer to the synthesis process of 6a, the only difference is the replacement of the types of 4-halo-acetophenone and 4-substituted-benzaldehyde.

[0071] (E)-3-(4-bromo-phenyl)-1-(4-chloro-phenyl)propyl-2-enyl-1-one 6b was synthesized in the same manner as 6a.

[0072]

[0073] White solid, yield 89.2%. Melting point: 164-168°C. 1 H NMR (500 MHz, CDCl3 ); δ=7.88-7.74(3H,m),7.64-7.58(4H,m),7.41-6.99(3H,m).

[0074] (E)-3-(4-Fluoro-phenyl)-1-(4-chloro-phenyl)propyl-2-enyl-1-one 6c was synthesized in the same manner as 6a.

[0075]

[0076] Off-white solid, yield 90.4%. Melting point: 152-154°C. 1 H NMR (500 MHz, CDCl 3 ); δ=7.78-7.69(3H,m),7.60-7.48(4H,m),7.21-6.95(3H,m).

[0077] (E)-3-(4-chloro-phenyl)-1-(4-bromo-phenyl)propyl-2-enyl-1-one 6d was synthesized in the same manner as 6a.

[0078]

[0079] Off-white solid, yield 85.2%. Melting point: 172-173°C. 1 H NMR (500 MHz, CDCl 3 ); δ=7.72-7.66(3H,m),7.60-7.42(4H,m),7.31-6.99(3H,m).

[0080] The same method as that of synthesizing 6a was used to synthesize (E)-1,3-di(4-bromophenyl)propyl-2-enyl-1-one 6e. White solid, yield 87.5%. Melting point: 196-198°C.

[0081]

[0082] The same method as that of synthesizing 6a was used to synthesize (E)-3-(4-fluoro-phenyl)-1-(4-bromo-phenyl)propyl-2-enyl-1-one 6f. White solid, yield 88.4%. Melting point: 184-187°C.

[0083]

[0084] The same method as that of synthesizing 6a was used to synthesize (E)-3-(4-chloro-phenyl)-1-(4-fluoro-phenyl)propyl-2-enyl-1-one 6g. White solid, yield 91.4%. Melting point: 194-196°C.

[0085]

[0086] The same method as that of synthesizing 6a was used to synthesize (E)-3-(4-bromo-phenyl)-1-(4-fluoro-phenyl)propyl-2-enyl-1-one 6h. White solid, yield 89.2%. Melting point: 190-192°C.

[0087]

[0088] The same method as that for synthesizing 6a was used to synthesize (E)-1,3-di(4-fluoro-phenyl)propyl-2-enyl-1-one 6i, a white solid, with a yield of 92.5%. Melting point: 154-156°C.

[0089]

[0090] The same method as that of synthesizing 6a was used to synthesize (E)-3-(4-methyl-phenyl)-1-(4-chloro-phenyl)propyl-2-enyl-1-one 6j, a white solid, with a yield of 88.6%. Melting point: 158-160°C.

[0091]

[0092] The same method as that of synthesizing 6a was used to synthesize (E)-3-(4-methoxy-phenyl)-1-(4-chloro-phenyl)propyl-2-enyl-1-one 6k, a white solid, with a yield of 82.2%. Melting point: 166-167°C.

[0093]

[0094] The same method as that of synthesizing 6a was used to synthesize (E)-3-(4-trifluoromethyl-phenyl)-1-(4-chloro-phenyl)propyl-2-enyl-1-one 61, a white solid, with a yield of 87.5%. Melting point: 170-171°C.

[0095]

[0096] The same method as that of synthesizing 6a was used to synthesize (E)-3-(4-methyl-phenyl)-1-(4-bromo-phenyl)propyl-2-enyl-1-one 6m, a white solid, with a yield of 85.5%. Melting point: 174-178°C.

[0097]

[0098] The same method as that of synthesizing 6a was used to synthesize (E)-3-(4-methoxy-phenyl)-1-(4-bromo-phenyl)propyl-2-enyl-1-one 6n, a white solid, with a yield of 90.0%. Melting point: 148-150°C.

[0099]

[0100] The same method as that of synthesizing 6a was used to synthesize (E)-3-(4-trifluoromethyl-phenyl)-1-(4-bromo-phenyl)propyl-2-enyl-1-one 6o, a white solid, with a yield of 83.8%. Melting point: 179-181°C.

[0101]

[0102] The same method as that of synthesizing 6a was used to synthesize (E)-3-(4-methyl-phenyl)-1-(4-fluoro-phenyl)propyl-2-enyl-1-one 6p, a white solid, with a yield of 86.5%. Melting point: 178-179°C.

[0103]

[0104] The same method as that of synthesizing 6a was used to synthesize (E)-3-(4-methoxy-phenyl)-1-(4-fluoro-phenyl)propyl-2-enyl-1-one 6q, a white solid, with a yield of 78.5%. Melting point: 172-174°C.

[0105]

[0106] The same method as that of synthesizing 6a was used to synthesize (E)-3-(4-trifluoromethyl-phenyl)-1-(4-fluoro-phenyl)propyl-2-enyl-1-one 6r, a white solid, with a yield of 79.7%. Melting point: 155-157°C.

[0107]

[0108] (6) Synthesis of 3,5-bis(4-chlorophenyl)-4,5-dihydro-1H-pyrazole hydrochloride (Compound 7a)

[0109]

[0110] According to the literature, 4,4'-dichlorochalcone 6a (1.8 mmol) and hydrazine hydrochloride (1.89 mmol) were added to a 10 mL single-mouth bottle at room temperature, and 5 mL of anhydrous methanol was added. The temperature was raised to 55 ° C with stirring, and the reaction was stirred for 2.5 hours. TLC (petroleum ether: ethyl acetate = 10: 1, v / v) monitored the complete reaction of the raw materials. The reaction solution was cooled to room temperature and concentrated by rotary evaporator to remove methanol. The crude product was directly used in the next step without further purification. The synthesis method of 7b-7r is similar to that of 7a.

[0111] (7) Synthesis of 3-(5-(4-bromophenyl)-3-(4-chlorophenyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)-7-(2-chloro-ethoxy)-2H-chroman-2-one (Compound 8b)

[0112]

[0113] At room temperature, compound 4a or 4b (1.8 mmol, 1.0 equiv) was added to a reaction flask, and dichlorothionyl (10 mL) and a catalytic amount of DMF were added under stirring. 2 Under protection, the temperature was raised to reflux temperature and stirred at reflux for 2 h. After cooling to room temperature, the excess thionyl chloride was distilled off, anhydrous toluene (10 mL) was added to the concentrate, and the residual thionyl chloride was removed by distillation. The toluene removal of thionyl chloride was repeated twice.

[0114] Then, under nitrogen protection, dry dichloromethane (10 mL) was added to the reaction flask, and the desired compound 7a-r (1.8 mmol, 1.0 equiv) and Et 3 N (3.9 mmol, 2.2 equiv) was added to the reaction flask. After stirring at room temperature for 2 h, a red solid was obtained, which was filtered and washed with dichloromethane to obtain the target compounds 8b, 8c, 8e, 8r, and 9a-r, respectively, which were used directly in the next step without further purification.

[0115] (9a) 3-(3,5-Bis(4-chlorophenyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)-7-(3-chloro-ethoxy)-2-hydro-chroman-2-one.

[0116]

[0117] 1 H NMR (500 MHz, CDCl 3 ): δ7.95(s,1H),7.58(d,J=10.0Hz,2H),7.46(d,J=5.0Hz,1H),7.39-7.31(m,6H),6.92-6.89(m,2H),5.72(dd,J=15.0,5.0Hz,1 H), 4.24 (t, J = 5.0Hz, 2H), 3.83 (dd, J = 15.0, 10.0Hz, 1H), 3.79 (t, J = 5.0Hz, 2H), 3.15 (dd, J = 15.0, 5.0Hz, 1H), 2.34-2.30 (m, 2H).

[0118] (8) Synthesis of 3-(5-(4-bromo-phenyl)-3-(4-chloro-phenyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)-7-(2-cyanoselenethoxy)-dihydro-benzopyran-2-one (Compound 10b)

[0119]

[0120] Under nitrogen protection, compounds 8b, 8c, 8e, 8r, 9a-r (2.0 mmol) were added to a reaction bottle at room temperature, and 5 mL of acetonitrile was added to stir and dissolve, and KSeCN (3.0 mmol) and a catalytic amount of NaI were added to the reaction solution. The temperature was raised to reflux and the reaction was continued for 12 h. After the reaction was completed, the acetonitrile was removed by concentration, and the crude product was purified by column chromatography to obtain the target compounds 10b, 10c, 10e, 10r, 11a-r.

[0121] (10b) 3-(5-(4-bromo-phenyl)-3-(4-chloro-phenyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)-7-(2-cyanoselenethoxy)-dihydro-benzopyran-2-one

[0122]

[0123] Pale yellow solid, yield: 37%, melting point: 208-210°C, purity: 99.3% (HPLC).

[0124] 1 H NMR (500 MHz, CDCl 3 ): δ7.95(s,1H),7.58(d,J=10.0Hz,2H),7.52(d,J=10.0Hz,2H),7.49(s,1 H),7.33(dd,J=20.0Hz,10.0Hz,4H),6.95-6.91(m,2H),5.71(dd,J=15.0H z,5.0Hz,1H),4.51(t,J=5.0Hz,2H),3.84(dd,J=15.0,10.0Hz,1H),3.48(t,J=5.0Hz,2H),3.16(dd,J=20.0Hz,5.0Hz,1H).ESI-Mass:m / zcalculated forC 28 H 19 BrClN 3 O 4 Se:654.94; found:656.2[M+H] + .

[0125] (10c) 3-(3-(4-chloro-phenyl)-5-(4-fluoro-phenyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)-7-(2-cyanoselenethoxy)-dihydro-benzopyran-2-one

[0126]

[0127] Pale yellow solid, yield: 33.5%, melting point: 211-212°C, purity: 98.8% (HPLC).

[0128] 1 H NMR (500 MHz, CDCl 3 ): δ7.95(s,1H),7.59(d,J=5.0Hz,2H),7.50(d,J=10.0Hz,1H),7.41(t,J=5.0Hz ,2H),7.34(d,J=10.0Hz,2H),7.08(t,J=10.0Hz,2H),6.95-6.91(m,2H),5.75(d d,J=10.0Hz,5.0Hz,1H),4.51(t,J=5.0Hz,2H),3.84(dd,J=15.0,10.0Hz,1H),3.48(t,J=5.0Hz,2H),3.17(dd,J=20.0Hz,5.0Hz,1H).ESI-Mass:m / zcalculated for C 28 H 19 CIF 3 O 4 Se:595.02; found:596.1[M+H] + .

[0129] (10e) 3-(3,5-bis(4-bromophenyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)-7-(2-cyanoselenethoxy)-dihydro-benzopyran-2-one

[0130]

[0131] Pale yellow solid, yield: 34.5%, melting point: 218-220°C, purity: 98.5% (HPLC).

[0132] 1 H NMR (500 MHz, CDCl 3 ): δ7.95(s,1H),7.53-7.49(m,7H),7.32(d,J=5.0Hz,2H),6.95-6.91(m,2H),5.71(dd,J=15.0Hz,5.0Hz,1H),4.51(t ,J=5.0Hz,2H),3.84(dd,J=15.0Hz,10.0Hz,1H),3.48(t,J=5.0Hz,2H),3.15(dd,J=15.0Hz,5.0Hz,1H).ESI-Mass:m / z calculated for C 28 H 19 Br 2 N 3 O 4Se:698.89; found:700.1[M+H] + .

[0133] (10r) 3-(3-(4-fluoro-phenyl)-5-(4-trifluoromethyl-phenyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)-7-(2-cyanoselenethoxy)-dihydro-benzopyran-2-one

[0134]

[0135] Pale yellow solid, yield: 32.5%, melting point: 208-210°C, purity: 99.1% (HPLC).

[0136] 1 H NMR (500 MHz, CDCl 3 )δ3.17(dd,J=17.61,4.77Hz,1H),3.41-3.54(m,2H),3.81-3.94(m,1H),4.50(t,J=5.69Hz,2H),5.73-5.86(m,1H),6.91(s, 2H),7.06(s,2H),7.50(d,J=8.44Hz,1H),7.56(d,J=8.07Hz,2H),7.60-7.75(m,4H),7.96(s,1H).ESI-Mass:m / zcalculated for C 29 H 19 F 4 N 3 O 4 Se:629.05; found:629.8[M+H] + .

[0137] (11a) 3-(3,5-bis(4-chlorophenyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)-7-(3-cyanoselenylpropoxy)-dihydro-benzopyran-2-one

[0138]

[0139] Pale yellow solid, yield: 30.6%, melting point: 104-106°C, purity: 99.2% (HPLC).

[0140] 1 H NMR (500 MHz, CDCl 3): δ7.94(s,1H),7.57(d,J=10.0Hz,2H),7.47(d,J=5.0Hz,1H),7.38-7.32(m,6H),6.89-6.87(m,2H),5.72(dd,J=15.0,5.0Hz,1H),4.2 1(t,J=5.0Hz,2H),3.83(dd,J=15.0,10.0Hz,1H),3.30(t,J=5.0Hz,2H),3.14(dd,J=15.0,5.0Hz,1H),2.48-2.43(m,2H); ESI-Mass:m / z calculated for C 29 H 21 Cl 2 N 3 O 4 Se:625.01; found:626.2[M+H] + .

[0141] (11b) 3-(5-(4-bromophenyl)-3-(4-chlorophenyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)-7-(3-cyanoselenylpropoxy)-dihydro-benzopyran-2-one

[0142]

[0143] Pale yellow solid, yield: 31.2%, melting point: 115-117°C, purity: 98.9% (HPLC).

[0144] 1 H NMR (500 MHz, CDCl 3 ): δ7.95(s,1H),7.58(d,J=10.0Hz,2H),7.52(d,J=5.0Hz,2H),7.49-7.47( m,1H),7.33(dd,J=15.0,5.0Hz,4H),6.91-6.88(m,2H),5.71(dd,J=15.0,5 .0Hz,1H),4.23(t,J=5.0Hz,2H),3.84(dd,J=15.0,10.0Hz,1H),3.32(t,J= 5.0Hz, 2H), 3.15 (dd, J=15.0, 5.0Hz, 1H), 2.51-2.46 (m, 2H); ESI-Mass: m / z calculatedfor C 29 H 21 BrClN 3 O 4 Se:668.96; found:670.2[M+H] + .

[0145] (11c) 3-(3-(4-chlorophenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)-7-(3-cyanoselenylpropoxy)-dihydro-benzopyran-2-one

[0146]

[0147] Bright yellow solid, yield: 29.5%, melting point: 102-104°C, purity: 99.1% (HPLC).

[0148] 1 H NMR (500 MHz, CDCl 3 ): δ7.95(s,1H),7.59(d,J=10.0Hz,2H),7.48(d,J=5.0Hz,1H),7.42-7.40(m,2H), 7.35(d,J=10.0Hz,2H),7.08(t,J=10.0Hz,2H),6.90-6.89(m,2H),5.75(dd,J=10. 0,5.0Hz,1H),4.23(t,J=5.0Hz,2H),3.84(dd,J=15.0,10.0Hz,1H),3.32(t,J=5.0 Hz,2H),3.17(dd,J=15.0,5.0Hz,1H),2.51-2.46(m,2H); ESI-Mass:m / zcalculated for C 29 H 21 CIF 3 O 4 Se:609.04; found:610.2[M+H] + .

[0149] (11d) 3-(3-(4-bromophenyl)-5-(4-chlorophenyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)-7-(2-cyanoselenylpropoxy)-dihydro-benzopyran-2-one

[0150]

[0151] Pale yellow solid, yield: 32.7%, melting point: 108-110°C, purity: 99.2% (HPLC).

[0152] 1 H NMR (500 MHz, CDCl 3): δ7.95(s,1H),7.51(s,4H),7.49-7.47(m,1H),7.37(s,4H),6.91-6.88(m,2H),5.73(dd,J=10.0,5.0Hz,1H),4.23(t,J=5.0 Hz,2H),3.84(dd,J=15.0,10.0Hz,1H),3.32(t,J=5.0Hz,2H),3.15(dd,J=15.0,5.0Hz,1H),2.51-2.46(m,2H); ESI-Mass:m / z calculated for C 29 H 21 BrClN 3 O 4 Se:668.96; found:670.1[M+H] + .

[0153] (11e) 3-(3,5-bis(4-bromophenyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)-7-(3-cyanoselenylpropoxy)-dihydro-benzopyran-2-one

[0154]

[0155] Pale yellow solid, yield: 25.1%, melting point: 105-107°C, purity: 99.2% (HPLC).

[0156] 1 H NMR (500 MHz, CDCl 3 ): δ7.95(s,1H),7.53-7.47(m,7H),7.32(d,J=5.0Hz,2H),6.91-6.88(m,2H),5.71(dd,J=15.0,5.0Hz,1H),4.23(t,J=5.0H z,2H),3.84(dd,J=15.0,10.0Hz,1H),3.32(t,J=5.0Hz,2H),3.15(dd,J=15.0,5.0Hz,1H),2.51-2.46(m,2H); ESI-Mass:m / z calculated for C 29 H 21 Br 2 N 3 O 4 Se:712.91; found:714.2[M+H] + .

[0157] (11f) 3-(3-(4-bromophenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)-7-(3-cyanoselenylpropoxy)-dihydro-benzopyran-2-one

[0158]

[0159] Pale yellow solid, yield: 30.4%, melting point: 106-108°C, purity: 99.5% (HPLC).

[0160] 1 H NMR (500 MHz, CDCl 3 ): δ7.95(s,1H),7.53-7.47(m,5H),7.42-7.39(m,2H),7.08(t,J=10.0Hz,2H),6.91-6.88(m,2H),5.74(dd,J=15.0,5.0Hz,1H),4.23( t,J=5.0Hz,2H),3.84(dd,J=15.0,10.0Hz,1H),3.32(t,J=5.0Hz,2H),3.16(dd,J=15.0,5.0Hz,1H),2.51-2.46(m,2H).ESI-Mass:m / z calculated for C 29 H 21 BrFN 3 O 4 Se:652.99; found:654.1[M+H] + .

[0161] (11g) 3-(5-(4-chlorophenyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)-7-(3-cyanoselenylpropoxy)-dihydro-benzopyran-2-one

[0162]

[0163] Pale yellow solid, yield: 23.4%, melting point: 108-110°C, purity: 99.4% (HPLC).

[0164] 1 H NMR (500 MHz, CDCl 3): δ7.95(s,1H),7.65(dd,J=10.0Hz,5.0Hz,2H),7.48(d,J=10.0Hz,1H),7.3 9-7.35(m,4H),7.07(t,J=5.0Hz,2H),6.91-6.88(m,2H),5.73(dd,J=10.0Hz ,5.0Hz,1H),4.23(t,J=5.0Hz,2H),3.84(dd,J=20.0,10.0Hz,1H),3.32(t,J =5.0Hz,2H),3.16(dd,J=10.0,5.0Hz,1H),2.51-2.46(m,2H).ESI-Mass:m / z calculatedfor C 29 H 21 CIF 3 O 4 Se:609.04; found:610.2[M+H] + .

[0165] (11h) 3-(5-(4-bromophenyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)-7-(3-cyanoselenylpropoxy)-dihydro-benzopyran-2-one

[0166]

[0167] Pale yellow solid, yield: 27.3%, melting point: 100-102°C, purity: 99.1% (HPLC).

[0168] 1 H NMR (500 MHz, CDCl 3 ): δ7.95(s,1H),7.65(dd,J=10.0Hz,5.0Hz,2H),7.52(d,J=10.0Hz,2H),7.49-7.47 (m,1H),7.32(d,J=10.0Hz,2H),7.06(t,J=5.0Hz,2H),6.90-6.88(m,2H),5.71(dd, J=15.0Hz,5.0Hz,1H),4.23(t,J=5.0Hz,2H),3.84(dd,J=20.0Hz,15.0Hz,1H),3.32 (t,J=5.0Hz,2H),3.16(dd,J=20.0Hz,5.0Hz,1H),2.50-2.47(m,2H).ESI-Mass:m / z calculated for C 29 H 21 BrFN 3 O 4Se:652.99; found:654.1[M+H] + .

[0169] (11i) 3-(3,5-bis(4-fluorophenyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)-7-(3-cyanoselenylpropoxy)-dihydro-benzopyran-2-one

[0170]

[0171] Bright yellow solid, yield: 28.3%, melting point: 103-105°C, purity: 99.3% (HPLC).

[0172] 1 H NMR (500 MHz, CDCl 3 ): δ7.96(s,1H),7.57(dd,J=10.0Hz,5.0Hz,2H),7.39(d,J=10.0Hz,1H),7.33 (t,J=5.0Hz,2H),7.01-6.96(m,4H),6.82-6.80(m,2H),5.66(dd,J=10.0Hz,5 .0Hz,1H),4.15(t,J=5.0Hz,2H),3.76(dd,J=15.0Hz,10.0Hz,1H),3.23(t,J= 5.0Hz,2H),3.19(dd,J=20.0Hz,5.0Hz,1H),2.42-2.37(m,2H).ESI-Mass:m / z calculatedfor C 29 H 21 F 2 N 3 O 4 Se:593.07; found:594.2[M+H] + .

[0173] (11j) 3-(3-(4-chlorophenyl)-5-(4-methylphenyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)-7-(3-cyanoselenylpropoxy)-dihydro-benzopyran-2-one

[0174]

[0175] Bright yellow solid, yield: 26.8%, melting point: 107-109°C, purity: 99.2% (HPLC).

[0176] 1 H NMR (500 MHz, CDCl 3): δ7.85(s,1H),7.50(d,J=10.0Hz,2H),7.38(d,J=10.0Hz,1H),7.25(d,J=10.0Hz, 2H),7.22(d,J=5.0Hz,2H),7.11(d,J=10.0Hz,2H),6.81-6.79(m,2H),5.64(dd,J=15 .0,5.0Hz,1H),4.14(t,J=5.0Hz,2H),3.73(dd,J=20.0,15.0Hz,1H),3.23(t,J=10.0 Hz,2H),3.09(dd,J=15.0,5.0Hz,1H),2.42-2.37(m,2H),2.26(s,3H).ESI-Mass:m / z calculated for C 30 H 24 C1N 3 O 4 Se:605.06; found:606.3[M+H] + .

[0177] (11k) 3-(3-(4-chlorophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)-7-(3-cyanoselenylpropoxy)-dihydro-benzopyran-2-one

[0178]

[0179] Bright yellow solid, yield: 29.9%, melting point: 104-106°C, purity: 99.2% (HPLC).

[0180] 1 H NMR (500 MHz, CDCl 3 ): δ7.94(s,1H),7.59(d,J=10.0Hz,2H),7.47(d,J=10.0Hz,1H),7.34(d,J=10.0Hz,4H),6.93-6.87(m,4H),5.72(dd,J=10.0,5.0Hz, 1H),4.23(t,J=5.0Hz,2H),3.83-3.77(m,4H),3.32(t,J=5.0Hz,2H),3.18(dd,J=15.0,5.0Hz,1H),2.51-2.46(m,2H).ESI-Mass:m / z calculated for C 30 H 24 C1N 3 O 5 Se:621.06; found:621.9[M+H]+ .

[0181] (11l) 3-(3-(4-chlorophenyl)-5-(4-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)-7-(3-cyanoselenylpropoxy)-dihydro-benzopyran-2-one

[0182]

[0183] Pale yellow solid, yield: 21.3%, melting point: 107-109°C, purity: 99.2% (HPLC).

[0184] 1 H NMR (500 MHz, CDCl 3 ): δ7.97(s,1H),7.67(d,J=5.0Hz,2H),7.58(t,J=10.0Hz,4H),7.49(d,J= 10.0Hz,1H),7.35(d,J=10.0Hz,2H),6.91-6.90(m,2H),5.81(dd,J=10.0,5 .0Hz,1H),4.24(t,J=5.0Hz,2H),3.88(dd,J=15.0,10.0Hz,1H),3.32(t,J= 5.0Hz,2H),3.17(dd,J=20.0,5.0Hz,1H),2.51-2.46(m,2H).ESI-Mass:m / z calculatedfor C 30 H 21 CIF 3 N 3 O 4 Se:659.03; found:659.8[M+H] + .

[0185] (11m) 3-(3-(4-bromophenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)-7-(3-cyanoselenylpropoxy)-dihydro-benzopyran-2-one

[0186]

[0187] Pale yellow solid, yield: 22.5%, melting point: 106-108°C, purity: 99.2% (HPLC).

[0188] 1 H NMR (500 MHz, CDCl 3): δ7.93(s,1H),7.52-7.45(m,5H),7.30(d,J=5.0Hz,2H),7.19(d,J=5.0 Hz, 2H), 6.89-6.87 (m, 2H), 5.72 (dd, J = 15.0Hz, 5.0Hz, 1H), 4.22 (t, J = 5. 0Hz, 2H), 3.81 (dd, J = 20.0Hz, 15.0Hz, 1H), 3.31 (t, J = 10.0Hz, 2H), 3.17 (dd, J = 15.0Hz, 5.0Hz, 1H), 2.49-2.44 (m, 2H), 2.34 (s, 3H). ESI-Mass: m / z calculated forC 30 H 24 Bn 3 O 4 Se:649.01; found:649.8[M+H] + .

[0189] (11n) 3-(3-(4-bromophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)-7-(3-cyanoselenylpropoxy)-dihydro-benzopyran-2-one

[0190]

[0191] Pale yellow solid, yield: 24.7%, melting point: 102-104°C, purity: 99.2% (HPLC).

[0192] 1 H NMR (500 MHz, CDCl 3 ): δ7.94(s,1H),7.53-7.49(m,4H),7.46(d,J=5.0Hz,1H),7.34(d,J=10.0Hz,2H),6.92-6.89(m,4H),5.72(dd,J=10.0 ,5.0Hz,1H),4.23(t,J=5.0Hz,2H),3.83-3.77(m,6H),3.18(dd,J=20.0,5.0Hz,1H),2.34-2.29(m,2H).ESI-Mass:m / z calculated for C 30 H 24 Bn 3 O 5 Se:665.01; found:666.2[M+H] + .

[0193] (11o) 3-(3-(4-bromophenyl)-5-(4-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)-7-(3-cyanoselenylpropoxy)-dihydro-benzopyran-2-one

[0194]

[0195] Pale yellow solid, yield: 28.9%, melting point: 103-105°C, purity: 99.1% (HPLC).

[0196] 1 H NMR (500 MHz, CDCl 3 ): δ7.97(s,1H),7.66(d,J=5.0Hz,2H),7.57(d,J=5.0Hz,2H),7.51(s,4H ),7.49(d,J=10.0Hz,1H),6.91-6.89(m,2H),5.81(dd,J=10.0Hz,5.0Hz,1 H),4.24(t,J=5.0Hz,2H),3.88(dd,J=15.0Hz,10.0Hz,1H),3.32(t,J=5.0Hz,2H),3.17(dd,J=20.0Hz,5.0Hz,1H),2.51-2.46(m,2H).ESI-Mass:m / z calculated forC 30 H 21 F 3 N 3 O 4 Se:702.98; found:704.2[M+H] + .

[0197] (11p) 3-(3-(4-fluorophenyl)-5-(p-methylphenyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)-7-(3-cyanoselenylpropoxy)-dihydro-benzopyran-2-one

[0198]

[0199] Pale yellow solid, yield: 27.7%, melting point: 94-96°C, purity: 98.5% (HPLC).

[0200] 1 H NMR (500 MHz, CDCl 3): δ7.94(s,1H),7.65(dd,J=10.0Hz,5.0Hz,2H),7.47(d,J=10.0Hz,1H),7.31(d,J=5.0 Hz,2H),7.20(d,J=5.0Hz,2H),7.06(t,J=5.0Hz,2H),6.89-6.88(m,2H),6.73(dd,J=10 .0Hz,5.0Hz,1H),4.23(t,J=5.0Hz,2H),3.82(dd,J=20.0Hz,15.0Hz,1H),3.32(t,J=5. 0Hz,2H),3.19(dd,J=20.0Hz,5.0Hz,1H),2.50-2.46(m,2H),2.35(s,3H).ESI-Mass:m / z calculated for C30H24FN3O4Se:589.09; found:589.9[M+H] + .

[0201] (11q) 3-(3-(4-fluorophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)-7-(3-cyanoselenylpropoxy)-dihydro-benzopyran-2-one

[0202]

[0203] Pale yellow solid, yield: 29.5%, melting point: 96-98°C, purity: 98.8% (HPLC).

[0204] 1 H NMR (500 MHz, CDCl 3 ): δ7.94(s,1H),7.65(dd,J=10.0Hz,10.0Hz,2H),7.47(d,J=10.0Hz,1H),7 .35(d,J=10.0Hz,2H),7.06(t,J=10.0Hz,2H),6.93-6.88(m,4H),5.72(dd, J=10.0Hz,5.0Hz,1H),4.22(t,J=5.0Hz,2H),3.84-3.78(m,4H),3.32(t,J= 5.0Hz,2H),3.19(dd,J=20.0,5.0Hz,1H),2.50-2.45(m,2H).ESI-Mass:m / z calculated forC 30 H 24 FN 3 O 5 Se:605.09; found:605.9[M+H] + .

[0205] (11r) 3-(3-(4-fluorophenyl)-5-(4-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)-7-(3-cyanoselenylpropoxy)-dihydro-benzopyran-2-one

[0206]

[0207] Pale yellow solid, yield: 30.9%, melting point: 92-94°C, purity: 98.7% (HPLC).

[0208] 1 H NMR (500 MHz, CDCl 3 ): δ7.97(s,1H),7.67-7.64(m,4H),7.57(d,J=10.0Hz,2H),7.49(d,J=10.0 Hz,1H),7.07(t,J=10.0Hz,2H),6.91-6.89(m,2H),5.81(dd,J=10.0Hz,5.0H z,1H),4.24(t,J=5.0Hz,2H),3.88(dd,J=20.0Hz,10.0Hz,1H),3.32(t,J=5.0Hz,2H),3.18(dd,J=20.0Hz,5.0Hz,1H),2.51-2.46(m,2H).ESI-Mass:m / z calculated forC 30 H 21 F 4 N 3 O 4 Se:643.06; found:644.0[M+H] + .

[0209] Example 2: Preparation of Selenium-Containing Coumarin Derivatives Series II.

[0210] (1) Synthesis of 1-(4-cyanoselenylphenyl)ethyl-1-one (Compound 14)

[0211]

[0212] According to the literature, under nitrogen protection, at room temperature, compound 13 p-aminoacetophenone (7.4 mmol) was dissolved in 14 mL (2 M) of hydrochloric acid aqueous solution. The reaction solution was cooled to 0-5°C under stirring, and sodium nitrite aqueous solution (0.51 g of sodium nitrite was dissolved in 4.0 mL of water) was added dropwise, and the temperature of the reaction solution was controlled to be 0-5°C during the addition. After the addition was completed, the reaction solution was stirred at 0-5°C for 0.5 hours. 1.4 g of saturated sodium acetate aqueous solution was added to the above reaction solution, and acetic acid was added dropwise to adjust the pH value to 3-4.

[0213] Then potassium selenocyanate (8.9 mmol) was added to the reaction mixture, and the mixture was stirred at 0-5°C for 1 hour. After the reaction, sodium acetate was added to adjust the pH to 5-6. The mixture was extracted with dichloromethane, concentrated, and purified by silica gel chromatography (petroleum ether / ethyl acetate = 10:1, v / v) to obtain 0.91 g of yellow solid compound 14 with a yield of 54.9%.

[0214] (2) Synthesis of 1-(4-(methylseleno)phenyl)ethyl-1-one (Compound 15)

[0215]

[0216] According to the literature, compound 14 p-selenocyanoacetophenone (1.8 mmol) was dissolved in 10 mL of methanol at room temperature and cooled to 0-5°C in an ice-water bath. Under nitrogen protection, iodomethane (7.1 mmol) was added, followed by sodium borohydride (2.0 mmol) was slowly added, and the reaction was continued at 0-5°C with stirring for 2 hours.

[0217] The reaction of the raw material was monitored by TLC (petroleum ether: ethyl acetate = 10: 1, v / v). The reaction solution was concentrated to dryness under reduced pressure. Ethyl acetate and saturated aqueous ammonium chloride solution were added to the residue, extracted, separated, the ethyl acetate layer was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (mobile phase: petroleum ether / ethyl acetate = 20: 1, v / v) to obtain 0.3 g of the target compound 15 as a light yellow solid, with a yield of 54.9%.

[0218] (3) Synthesis of (E)-1-(4-(methylseleno)phenyl)-3-(4-(trifluoromethyl)phenyl)propyl-2-enyl-1-one (Compound 16)

[0219]

[0220] Under nitrogen protection, compound 15 (0.84 mmol) was added to a reaction bottle at room temperature, and ethanol (2.0 mL) was added, and the compound was dissolved under stirring. The reaction solution was cooled to 0°C in an ice-salt bath, and a 10% NaOH (0.5 mL) aqueous solution was added dropwise. After the addition was completed, the reaction mixture was stirred at 0°C for 20 minutes. Compound 4-(trifluoromethyl)benzaldehyde (0.93 mmol) was added to the above reaction solution and stirred at 0°C for 4 hours until the reaction was completed. After the reaction was completed, cold water was added, and the solid obtained under stirring was filtered, washed with water, and dried to obtain 0.31 g of a white solid compound, with a yield of 99.0%, a purity of 98.5% (HPLC), and a melting point of 161-163°C.

[0221] 1 H NMR (500 MHz, CDCl3 ): δ=7.93(d,J=5.0Hz,2H),7.82(d,J=15.0Hz,1H),7.75(d,J=10.0Hz,2H),7.69(d,J=5.0Hz ,2H),7.58(d,J=10.0Hz,1H),7.50(d,J=10.0Hz,2H),2.44(s,3H).ESI-Mass:m / zcalculated for C 17 H 13 F 3 OSe:370.01; found:371.3[M+H] + .

[0222] (4) Synthesis of 3-(4-(methylselenyl)phenyl)-5-(4-(trifluoromethyl)phenyl)-4,5-dihydro-pyrazole hydrochloride (Compound 17)

[0223]

[0224] At room temperature, hydrazine hydrochloride (0.7 mmol) was dissolved in methanol (10 mL) and stirred for 15 min until completely dissolved. Compound 16 (0.7 mmol) was then added. The reaction mixture was heated to 55 °C and stirred for 3 h. After the reaction was completed, the reaction mixture was concentrated to dryness to obtain a light yellow solid pyrazole hydrochloride 17, which was used directly in the next step without further purification.

[0225] (5) Synthesis of 7-methoxy-2-oxo-dihydro-benzopyran-3-carboxylic acid ethyl ester (Compound 18)

[0226]

[0227] According to the literature, 7-hydroxycoumarin-3-carboxylic acid ethyl ester 2 (8.5 mmol) was dissolved in 20 mL DMF at room temperature, and potassium carbonate (9.4 mmol) was added under stirring. After stirring for 10 min, iodomethane (9.4 mmol) was added, and the reaction was stirred at room temperature for 12 hours.

[0228] The reaction of the raw materials was monitored by TLC (petroleum ether: ethyl acetate = 3: 1, v / v). The reaction solution was poured into 200 mL of water and stirred. The obtained solid was filtered, the filter cake was washed with water, and dried to obtain 2.1 g of the target compound 18 as a white solid with a crude yield of 98.9%. It was used directly in the next step without further purification.

[0229] (6) Synthesis of 7-methoxy-2-oxo-dihydro-benzopyran-3-carboxylic acid (Compound 19)

[0230]

[0231] According to the literature, compound 187-methoxycoumarin-3-carboxylic acid ethyl ester (8.5 mmol) was dissolved in 100 mL DMF at room temperature, and lithium hydroxide monohydrate (42.7 mmol) was added under stirring. The reaction was stirred at room temperature for 4 hours. TLC (petroleum ether: ethyl acetate = 3:1, v / v) monitored the complete reaction of the raw materials. The reaction solution was adjusted to weak acidity with 2M hydrochloric acid, and the obtained solid was filtered, washed with water, and dried to obtain a pale yellow solid compound 19 crude product, which was directly used in the next step without further purification.

[0232] (7) Synthesis of 3-(3-(4-fluorophenyl)-5-(4-(trifluoromethyl)phenyl)-4,5-dihydro-pyrazole-1-carbonyl)-7-methoxy-dihydro-benzopyran-2-one (Compound 12a)

[0233]

[0234] Under nitrogen protection, crude compound 19 (2.3 mmol, 1.0 equiv) was added to a reaction flask at room temperature, followed by thionyl chloride (10 mL) and a catalytic amount of DMF. The temperature was raised to reflux under nitrogen protection and the reaction was continued for 2 h. After the reaction was completed, the mixture was cooled to room temperature, the excess thionyl chloride was distilled off, and anhydrous toluene (10 mL) was added to the crude acid chloride, and distillation was continued to remove the residual thionyl chloride. This process was repeated twice.

[0235] Under nitrogen protection, dry dichloromethane was added to the crude acyl chloride, and then the desired compound 7r (2.3 mmol, 1.0 equiv) and Et 3 N (5.0mmol, 2.2equiv) were added to the reaction solution. The reaction was stirred at room temperature for 3h. After the reaction was completed, the solvent dichloromethane was removed under vacuum to obtain a crude product, which was purified by column chromatography (dichloromethane / methanol=300 / 1) to obtain a bright yellow solid target compound 12a. Yield: 30.9%, melting point: 262-264°C, purity: 99.2% (HPLC);

[0236] 1 H NMR (500 MHz, CDCl 3): δ7.98(s,1H),7.68-7.65(m,4H),7.58(d,J=10.0Hz,2H),7.48(d,J=5.0Hz,1H),7.07(t,J=5.0Hz,2H),6.92(d,J=5.0Hz, 2H),5.82(dd,J=10.0,5.0Hz,1H),3.94(s,3H),3.89(dd,J=15.0,10.0Hz,1H),3.18(dd,J=15.0,5.0Hz,1H).ESI-Mass:m / z calculated for C 27 H 18 F 4 N 2 O 4 :510.12; found:511.1[M+H] + .

[0237] (8) Synthesis of 3-(3-(4-methylseleno)-5-(4-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazole-1-carbonyl)-7-methoxy-dihydro-benzopyran-2-one (Compound 12b)

[0238]

[0239] The synthesis method is the same as that of compound 12a (the only difference is that the same molar amount of compound 17 replaces compound 7r). Pale yellow solid, yield: 19.1%, melting point: 217-219°C, purity: 98.7% (HPLC);

[0240] 1 H NMR (500 MHz, CDCl 3 ): δ7.97(s,1H),7.67(d,J=5.0Hz,2H),7.58(d,J=5.0Hz,2H),7.54(d,J=5.0Hz,2H),7.48(d,J=5.0Hz,1H),7.39(d,J=5.0Hz,2H),6.92-6.90(m ,2H),5.81(dd,J=10.0Hz,5.0Hz,1H),3.94(s,3H),3.88(dd,J=15.0Hz,10.0Hz,1H),3.17(dd,J=15.0Hz,5.0Hz,1H),2.38(s,3H).ESI-Mass:m / z calculated for C 28 H 21 F 3 N 2 O 4Se:586.06; found:586.8[M+H] + .

[0241] Example 3: Preparation of selenium-containing coumarin derivatives series III.

[0242] (1) Synthesis of 2,4-dichloro-6-methoxy-1,3,5-triazine (Compound 21)

[0243]

[0244] According to the literature, 45 mL of methanol and compound 20 cyanuric chloride (27.1 mmol) were added to a 100 mL three-necked flask at room temperature, and the mixture was cooled to 0°C in an ice-salt bath. Sodium bicarbonate (27.1 mmol) was slowly added to the reaction solution in batches. After the addition, the reaction solution was slowly warmed to room temperature and stirred for 2 hours.

[0245] Sample TLC (petroleum ether: ethyl acetate = 3: 1, v / v) was taken to monitor the complete reaction of the raw materials. 50 mL of water was added and extracted with dichloromethane three times (70 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography (mobile phase: petroleum ether: ethyl acetate = 3: 1, v / v) to obtain 4.1 g of compound 21 as a white solid with a yield of 84%. Melting point: 115-117 ° C, 1 H NMR (500 MHz, CDCl 3 ):δ=4.17(s,3H).

[0246] (2) N-(2-bromoethyl)-4-chloro-6-methoxy-1,3,5-triazine-2-amine (Compound 23a) and N-(2-bromopropyl)-4-chloro-6-methoxy-1,3,5-triazine-2-amine (Compound 23b)

[0247]

[0248] At room temperature, 10 mL of acetone, 2,4-dichloro-6-methoxy-1,3,5-triazine compound 21 (11.1 mmol), 2-bromopropylamine hydrobromide (12.2 mmol) or 3-bromopropylamine hydrobromide (12.2 mmol) were added to a 50 mL three-necked flask. Under nitrogen protection, stirring was turned on and triethylamine (24.5 mmol) was slowly added. After the addition, the reaction was stirred at room temperature for 4 hours. TLC (petroleum ether: ethyl acetate = 3:1) monitored the complete reaction of the raw materials. The reaction solution was concentrated to dryness, and the crude product was purified by column chromatography (petroleum ether: ethyl acetate = 6:1) to obtain 1.5 g of white solid 23a, with a yield of 51.8%, or 2.2 g of white solid 23b, with a yield of 69.7%.

[0249] (3) 4-Chloro-6-methoxy-N-(2-selenocyanoethyl)-1,3,5-triazine-2-amine (Compound 24a) and 4-Chloro-6-methoxy-N-(3-selenocyanopropyl)-1,3,5-triazine-2-amine (Compound 24b)

[0250]

[0251] At room temperature, add 10 mL of acetone, 23a (7.5 mmol) or 23b (7.1 mmol) to a 50 mL three-necked flask. Under nitrogen protection, stir and add potassium selenocyanate (1.2 g, 8.2 mmol or 1.1 g, 7.8 mmol). After the addition, stir at room temperature for 12 hours. TLC (petroleum ether: ethyl acetate = 1:1) monitors the complete reaction of the raw materials. The reaction solution is concentrated to dryness, and the crude product is purified by column chromatography (petroleum ether: ethyl acetate = 3:1) to obtain 1.8 g of white solid 24a, with a yield of 80.0% or 1.7 g of white solid 24b, with a yield of 77.6%.

[0252] (4) 8-Hydroxy-4-methyl-dihydro-naphthopyran-2-one (Compound 26a) and 9-Hydroxy-1-methyl-dihydro-naphthopyran-3-one (Compound 26b)

[0253]

[0254] Compound 251, 4-naphthalene diol (40.0 mmol) and ethyl acetoacetate (100 mmol) were added to a three-necked flask and cooled to 0°C in an ice bath under nitrogen protection. 40 mL of a pre-prepared 80% aqueous sulfuric acid solution was slowly added dropwise to the above reaction solution, and the temperature of the addition was controlled at 0-5°C. After the addition was completed, the reaction solution was heated to room temperature and stirred for 24 hours.

[0255] TLC (petroleum ether: ethyl acetate = 1:1) showed that the raw material was completely reacted. The reaction solution was poured into 350 mL of ice water and stirred overnight. The obtained solid was filtered and washed with water. The solid was then dissolved in 150 mL of 10% sodium hydroxide aqueous solution, filtered again, and the filtrate was adjusted to acidity. The obtained solid was filtered, washed with water, and recrystallized with acetic acid to obtain 3.66 g of compound 26a. 1 H NMR (500 MHz, CDCl 3 )δ10.23(s,1H),8.28(s,1H),7.76(d,J=10.0Hz,1H),7.62(s,1H),7.17(d,J=5 .0Hz, 1H), 7.13 (dd, J = 10.0Hz, 5.0Hz, 1H), 6.34 (d, J = 5.0Hz, 1H), 2.52 (s, 3H).

[0256] The filtrate was allowed to stand overnight, and the obtained solid was filtered, washed with water, and recrystallized from acetic acid to obtain 2.7 g of compound 26b. 1 H NMR (500 MHz, CDCl 3 )δ10.15(s,1H),8.05(d,J=10.0Hz,1H),8.01(s,1H),7.91(d,J=10.0Hz,1H),7 .29(d,J=5.0Hz,1H),7.14(dd,J=10.0Hz,5.0Hz,1H),6.43(s,1H),2.86(s,3H).

[0257] (5) 8-((4-methoxy-6-((2-((2-propionyl)seleno)ethyl)amine)-1,3,5-triazine-2-yl-oxo)-4-methyl-dihydro-naphthopyran-2-one (Compound 27a)

[0258]

[0259] At room temperature, add 20 mL of acetone, compound 26a (1.1 mmol), compound 24a (1.1 mmol) and potassium carbonate (2.2 mmol) to a 50 mL single-necked bottle. After the addition, turn on the stirring and react at 45 ° C for 2 days under nitrogen protection. TLC (dichloromethane: methanol = 20: 1) monitors the complete reaction of the raw materials. The reaction solution is concentrated to dryness, and the obtained crude product is purified twice by column chromatography (mobile phase: dichloromethane: methanol = 300: 1-200: 1-150: 1) to obtain 0.16 g of white solid compound 27a, with a yield of 29.0%.

[0260] 1 H NMR (500 MHz, CDCl 3 )δ2.26-2.38(m,3H),2.59(dd,J=3.30Hz,1.10Hz,3H),2.68-2.84(m,2H),2.70-2.86(m,1H),3.1 1-3.29(m,2H),3.60(d,J=6.24Hz,1H),3.74(q,J=6.60Hz,1H),3.89-4.01(m,3H),5.75-5.91(m, 1H),6.37(s,1H),6.35-6.42(m,1H),7.31-7.41(m,1H),7.33-7.40(m,1H),7.65-7.69(m,1H),7. 69(s,1H),7.96(d,J=8.80Hz,1H),7.94-7.99(m,1H),8.06-8.15(m,1H),8.11(d,J=2.93Hz,1H). ESI-Mass:m / z calculated for C23 H 22 N 4 O 5 Se:514.08; found:514.8[M+H] + .

[0261] (6) 8-((4-methoxy-6-((2-((2-propionyl)seleno)propyl)amine)-1,3,5-triazine-2-yl-oxo)-4-methyl-dihydro-naphthopyran-2-one (Compound 27b)

[0262]

[0263] The synthesis method of compound 27b is similar to that of 27a (24b is used instead of 24a), and 0.09 g of white solid compound 27b is obtained with a purity of 99.4% (HPLC) and a yield of 15.7%.

[0264] 1 H NMR (500 MHz, CDCl 3 )δ1.83-2.00(m,2H),2.27-2.37(m,3H),2.51-2.60(m,4H),2.63(t,J=7.34Hz,1 H),3.13-3.25(m,2H),3.41(d,J=6.60Hz,1H),3.54(d,J=6.24Hz,1H),3.86-4.0 1(m,4H),6.36(s,1H),7.35(ddd,J=13.20,8.99,2.02Hz,1H),7.66(dd,J=7.89, 1.65Hz, 1H), 7.70 (d, J = 3.30Hz, 1H), 7.91-8.01 (m, 1H), 8.11 (d, J = 8.07Hz, 1H). ESI-Mass: m / z calculated for C 24 H 24 N 4 O 5 Se:528.09; found:529.2[M+H] + .

[0265] (7) 9-((4-methoxy-6-((2-((2-propionyl)seleno)ethyl)amine)-1,3,5-triazine-2-yl-oxo)-1-methyl-dihydro-naphthopyran-2-one (Compound 28a)

[0266]

[0267] The synthesis method of compound 28a is similar to that of 27a, and 0.19 g of white solid compound 28a is obtained with a purity of 98.8% (HPLC) and a yield of 34.6%.

[0268] 1 H NMR (600 MHz, CDCl 3 )δ2.25-2.37(m,3H),2.73(s,1H),2.81(s,1H),2.90(d,J=2.57Hz,3H),3.09-3.29(m,2H),3.56-3.65(m,1H) ,3.72(s,1H),3.85-4.03(m,3H),6.39(s,1H),7.48(dd,J=8.80,1.83Hz,2H),7.88-8.05(m,2H),8.41(s,1H). ESI-Mass:m / zcalculated for C 23 H 22 N 4 O 5 Se:514.08; found:515.2[M+H] + .

[0269] (8) 9-((4-methoxy-6-((2-((2-propionyl)seleno)propyl)amine)-1,3,5-triazine-2-yl-oxo)-1-methyl-dihydro-naphthopyran-2-one (Compound 28b)

[0270]

[0271] The synthesis method of compound 28b is similar to that of 27a, and 0.08 g of white solid compound 28b is obtained with a purity of 99.0% (HPLC) and a yield of 13.9%.

[0272] 1 H NMR (600 MHz, CDCl 3 )δ1.85-2.00(m,1H),1.87-1.99(m,1H),2.32(d,J=11.37Hz,3H),2.50-2.69(m,2H),2.90(d,J=1.47Hz,3H),3.10-3.28(m, 2H), 3.37-3.59 (m, 2H), 3.83-4.04 (m, 3H), 6.39 (s, 1H), 7.48 (dd, J = 8.80, 2.93Hz, 2H), 7.95 (d, J = 8.80Hz, 2H), 8.41 (s, 1H). ESI-Mass:m / z calculated for C 24 H 24 N 4O 5 Se:528.09; found:529.1[M+H] + .

[0273] (9) 7-Hydroxy-4-methyl-dihydro-naphthopyran-2-one (Compound 30)

[0274]

[0275] Compound 291, 5-naphthalene diol (31.2 mmol) and ethyl acetoacetate (78.0 mmol) were added to a three-necked flask at room temperature, and cooled to 0°C in an ice bath under nitrogen protection. 60 mL of 80% sulfuric acid aqueous solution prepared in advance was slowly added dropwise to the above reaction solution, and the temperature of the addition was controlled at 0-5°C. After the addition was completed, the reaction solution was heated to room temperature and stirred for 24 hours. TLC (petroleum ether: ethyl acetate = 1:1) showed that the raw materials were completely reacted.

[0276] The reaction solution was poured into 500 mL of ice water and stirred overnight. The obtained solid was filtered and washed with water. The solid was then dissolved in 200 mL of 10% sodium hydroxide aqueous solution, filtered again, and the filtrate was adjusted to acidic. The obtained solid was filtered, washed with water, and recrystallized from acetic acid to obtain 3.3 g of compound 30 as pale yellow crystals. The yield was 46.7%. mp: 270-272°C. (Literature: 271-272°C).

[0277] (10) 7-((4-methoxy-6-((2-((2-propionyl)seleno)ethyl)amine)-1,3,5-triazine-2-yl-oxo)-4-methyl-dihydro-naphthopyran-2-one (Compound 31a)

[0278]

[0279] The synthesis method of compound 31a is similar to that of 27a, and 0.07 g of white solid compound 31a is obtained with a purity of 99.2% (HPLC) and a yield of 12.3%.

[0280] 1 H NMR (600 MHz, CDCl 3)δ2.27-2.39(m,3H),2.55(s,3H),2.61(t,J=6.79Hz,1H),2.80(t,J=6.79Hz,1H),3.07-3.27(m,2H),3.48(d,J=6.60Hz,1H),3.71(d,J=6 .60Hz,1H),3.86-3.98(m,3H),6.43(s,1H),7.47(dd,J=12.65,7.52Hz,1H),7.60-7.72(m,2H),7.77-7.87(m,1H),8.52(d,J=8.44Hz,1H). ESI-Mass:m / z calculated for C 23 H 22 N 4 O 5 Se:514.08; found:515.1[M+H] + .

[0281] (11) 7-((4-methoxy-6-((2-((2-propionyl)seleno)ethyl)amine)-1,3,5-triazine-2-yl-oxo)-4-methyl-dihydro-naphthopyran-2-one (Compound 31b)

[0282]

[0283] The synthesis method of compound 31b is similar to that of 27a, and 0.06 g of white solid compound 31b is obtained with a purity of 99.1% (HPLC) and a yield of 10.6%.

[0284] 1 H NMR (600 MHz, CDCl 3 )δ1.69-1.81(m,1H),1.93(t,J=7.15Hz,1H),2.32(d,J=6.60Hz,3H),2.42(t,J=7.15 Hz,1H),2.54(d,J=1.83Hz,3H),2.61(t,J=7.15Hz,1H),3.09-3.25(m,2H),3.29(d,J =6.60Hz,1H),3.50(d,J=6.60Hz,1H),3.82-4.00(m,3H),6.42(s,1H),7.46(dd,J=11 .19,7.52Hz,1H),7.58-7.71(m,2H),7.81(d,J=9.17Hz,1H),8.51(d,J=8.44Hz,1H). ESI-Mass:m / z calculated for C 24 H 24 N4 O 5 Se:528.09; found:529.1[M+H] + .

[0285] Example 4: Screening experiment of 30 selenium-containing coumarin derivatives for CCK-8 tumor cell toxicity.

[0286] (1) Experimental materials:

[0287] 18 cell lines H1299, A549, HCC-827, MCF-7, K562, AGS, HepG2, 786-O, A375, MRC-5, MDA-MB-231, PC-3, HT-29, M17, SK-N-AS, SH-SY5Y, IMR-32, SK-N-SH, 30 selenium-containing coumarins 27a, 2 7b, 28a, 28b, 31a, 31b, 12a, 12b, 10r, 10b, 10c, 10e, 11a, 11b, 11c, 11d, 11e, 11f, 11g, 11h, 11i, 11j, 11k, 11l, 11m, 11n, 11o, 11p, 11q, and 11r were all dissolved in DMSO solution to prepare a 10mM stock solution for later use.

[0288] (2) Experimental methods:

[0289] a) Cell culture

[0290] b) Cell plating

[0291] Collect cells in the logarithmic growth phase, spread them in a 96-well plate with the optimal cell amount, and add 95uL of cell suspension to each well. Set up blank control wells, add the same volume of culture medium, and subtract background interference. And add 100uL PBS to the wells with cells to prevent the culture medium in the 96-well plate from volatilizing and affecting the experimental results. Place the well plate in a 37℃ constant temperature incubator and let it adhere to the wall.

[0292] c) Dosing

[0293] Dilute the compound stock solution with DMSO to the required concentration, prepare a system with a volume ratio of DMSO: culture medium = 1:4 in an Eppendorf tube, mix well, add 5 μL per well to the corresponding well of a 96-well plate, and mark the lid. The control group is 1% DMSO. Place in an incubator and culture for 60 hours.

[0294] d) Add CCK-8 reagent and detect

[0295] Insert the tip of the gun below the liquid surface, add 10uL of CCK-8 detection reagent to each well, place in a 37℃ constant temperature incubator and incubate for a suitable time. When the liquid in the well turns yellow, use an enzyme marker to detect the absorbance. Calculate the cell survival rate.

[0296] The present invention tests the cytotoxicity of 30 selenium-containing coumarin compounds on 18 cell lines (H1299, A549, HCC-827, MCF-7, K562, AGS, HepG2, 786-O, A375, MRC-5, MDA-MB-231, PC-3, HT-29, M17, SK-N-AS, SH-SY5Y, IMR-32, SK-N-SH) by CCK-8 method, with 1% DMSO cell culture fluid as reference; paclitaxel is used as positive control. Afterwards, the selected compounds with better effects are further finely screened by concentration gradient, and the corresponding IC 50 The experimental results are shown in Table 1. Table 1 is the IC values ​​of 30 selenium-containing coumarin compounds for cytotoxicity screening determined by CCK-8 method. 50 (μM) value summary table. The results showed that compound 11f (IC 50 =2.92μM)、11i(IC 50 =2.51μM)、11q(IC 50 =2.79 μM) had the strongest growth inhibitory ability on neuroblastoma SK-N-SH, and selenium-containing coumarin compounds had little toxicity to normal cells MRC-5.

[0297] Table 1 IC values ​​of 30 selenium-containing coumarins against various tumor cell lines 50 Value (μM), paclitaxel was used as positive control

[0298]

[0299] ND: After preliminary anti-tumor cell activity screening, the compound had poor tumor inhibition effect, so no further screening was performed, and therefore, no IC50 value was obtained.

[0300] Example 5: Experiment to investigate the effects of compounds 11f, 11i, and 11q on SK-N-SH cell proliferation

[0301] Experimental materials: cells SK-N-SH; compounds 11f, 11i, 11q.

[0302] Experimental methods:

[0303] a) Cell cloning

[0304] SK-N-SH cells in the logarithmic growth phase were digested and plated into a 12-well plate at a density of 2000 cells / well / 1mL. After the cells adhered to the wall, the corresponding concentration of compounds were added and cultured for 24 hours. The culture medium in the wells was discarded, and the wells were slowly rinsed with PBS 2-3 times. The culture medium without the compound was replaced and cultured for another week. The culture was terminated when a single clone colony containing more than 50 cells was observed under a microscope.

[0305] b) Dyeing

[0306] Discard the original culture medium in the wells, rinse slowly with PBS 2-3 times to remove cell debris, and fix with 4% neutral paraformaldehyde for 10 minutes. Discard the paraformaldehyde and rinse with neutral PBS buffer 2-3 times. Evenly cover the bottom of the well plate with crystal violet staining solution and stain for 20 minutes. Rinse slowly with neutral PBS buffer several times to make the background crystal violet color of the well plate lighter.

[0307] c) Photo analysis

[0308] Remove the liquid from the well plate, take a photo with a white background, and use Image J software for counting and analysis.

[0309] Unlimited proliferation of tumor cells is the basis of tumor development, and the inhibition of tumor cell proliferation has a crucial impact on the treatment of cancer. The present invention has proved that single cells can proliferate indefinitely through cell plate cloning experiments. Under the interference of compounds 11f, 11i, and 11q, the number of cell clones in the drug-treated group was significantly reduced compared with the control group, and it was concentration-dependent. This shows that the selenium-containing coumarin compounds of the present invention can effectively inhibit the proliferation and cell viability of neuroblastoma (the results are shown in Figure 1 , 2 , 3).

[0310] Example 6: Experiment to investigate the effects of compounds 11f, 11i, and 11q on the migration and invasion of SK-N-SH cells.

[0311] Experimental materials: cells SK-N-SH; compounds 11f, 11i, 11q.

[0312] Experimental methods:

[0313] a) Preparation of Matrigel

[0314] Take out the matrix gel from the -20℃ refrigerator and thaw it at 4℃ for 30 minutes. Pre-cool the required pipette tip at 4℃, mix it with serum-free medium at a volume of 1:8 on the ice box, and then drop it into the Trans-well chamber with a membrane pore size of 8μm and place it in a 24-well plate. Let it stand in a 37℃ constant temperature incubator for 4-5 hours to solidify the matrix gel. (Trans-well experiments to verify cell migration do not require the addition of matrix gel, and the cell suspension can be directly inoculated into the chamber. The subsequent operations are the same as below)

[0315] b) Cell plating

[0316] Remove the chamber and discard the excess matrix gel with a pipette. Digest the cells in the logarithmic growth phase and wash the cells with serum-free medium to remove the serum. Resuspend the cells to a count dilution concentration of 50,000 / 100 μL, add the corresponding concentration of the compound, mix well, and drop into the upper chamber. Add 60 μL of culture medium containing 30% FBS to each well in the lower chamber, and ensure that no bubbles are generated during the process. Place in a constant temperature incubator for 48 hours.

[0317] c) Cell processing

[0318] After the end, the cell suspension in the chamber was aspirated and discarded, and washed with neutral PBS buffer. Fix with 4% neutral paraformaldehyde at room temperature for 10 minutes. After the end, rinse off the paraformaldehyde with neutral PBS buffer. Stain with crystal violet solution in the dark for 15 minutes to ensure that the stain covers the cell surface and stains fully. After the end, remove the residual liquid and wash again with neutral PBS buffer. Invert on filter paper for 5-10 minutes to air dry, and place under a microscope to take pictures.

[0319] The present invention uses Trans-well experiments to find that after 48 hours of compound action, the number of SK-N-SH cells passing through the membrane pores is less than that of the control group, further verifying that compared with the control group, the compound group has a significant inhibitory effect on tumor migration and invasion ability (see the results). Figure 4 , 5 , 6, 7, 8, 9).

Claims

1. A selenium-containing coumarin compound, characterized in that Its general structural formula is shown in any one of formula (I) and (III): In formula (I), n=an integer of 1-2, when n=1, R1=4-chloro and R2=4-bromo; when n=2, R1=4-bromo and R2=4-fluoro, or R1=4-fluoro and R2=4-fluoro, or R1=4-chloro and R2=4-trifluoromethyl, or R1=4-bromo and R2=4-methyl, or R1=4-bromo and R2=4-trifluoromethyl, or R1=4-fluoro and R2=4-methoxy, or R1=4-fluoro and R2=4-trifluoromethyl; In formula (III), R is: n＝2。 2. The method for preparing the selenium-containing coumarin compound according to claim 1, characterized in that The selenium-containing coumarin compound represented by the general formula (I) is selected from any one of compounds 10b, 11l, 11m, 11o, 11r, 11f, 11i, and 11q, and its synthesis route is as follows: 1) using DMF as solvent and the 3-carboxylic acid ethyl ester-7-hydroxy substituted coumarin derivative shown in formula 2 as raw material, reacting with 1-bromo-2-chloroethane or 1-bromo-3-chloropropane in the presence of K2CO3 to generate compound 3a or 3b; 2) using ethanol-water in a volume ratio of 1:0.5-2 as a solvent, the compound 3a or 3b is hydrolyzed with lithium hydroxide to generate the compound 4a or 4b; 3) using ethanol as solvent and 4-halogenated acetophenone as starting material, and carrying out aldol condensation reaction with 4-substituted-benzaldehyde in the presence of NaOH to obtain compound 6; 4) Using methanol as solvent, compound 6 reacts with hydrazine hydrochloride to generate compound 7; 5) First, compound 4a or 4b is subjected to chlorination reaction with thionyl chloride in the presence of a catalytic amount of DMF, and then the chlorination reaction product is reacted with compound 7 in the presence of triethylamine using dichloromethane as solvent to generate a key intermediate, wherein the key intermediate is compound 8; the substituents R1 and R2 in compound 6, compound 7 and compound 8 are the same as those in formula (I): 6) Finally, using acetonitrile as solvent, the key intermediate obtained in step 5) is reacted with potassium selenocyanate in the presence of a catalytic amount of sodium iodide to generate the target compound; the synthesis route is as follows:

3. The method for preparing the selenium-containing coumarin compound according to claim 1, characterized in that The selenium-containing coumarin compound represented by general formula (III) is selected from compound 31a, and its synthesis route is as follows: 1) reacting compound 23a with potassium selenocyanate to generate compound 24a; 2) Compound 24a is then reacted with compound 30 under alkaline conditions to obtain the corresponding target compounds. The synthetic routes are as follows:

4. Use of the selenium-containing coumarin compound according to claim 1 in the preparation of an anti-tumor drug, wherein the anti-tumor drug is used to inhibit human breast cancer cells MDA-MB-231 or SK-N-SH cells.

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