Synthesis method of seven-membered spiro oxoindole compound

Abstract

The invention discloses a synthesis method of a seven-membered spiro oxoindole compound, and belongs to the technical field of organic synthesis. According to the invention, a key continuous quaternary carbon and tertiary carbon three-dimensional center in an oxindole molecule is constructed in one step based on coordination induction of chiral sulfinamide as a prothetic group and metal copper and catalysis of a Michael addition cyclization reaction of amide alpha-position arylation tandem ketene ester by cuprous iodide; the preparation method comprises the following steps: preparing a seven-membered spiro-oxoindole compound, removing tert-butylsulfinyl through a subsequent acidic condition, carrying out a series condensation cyclization reaction to prepare an amino-protected seven-membered spiro-oxoindole compound, and further removing 4-methoxy benzyl protection of nitrogen atoms on an oxoindole ring to obtain the key seven-membered spiro-oxoindole compound. The compound has obvious biological activity on human lung cancer cells A549 and H1299, and the IC50 (half maximal inhibitory concentration) of the compound is 18.73 mu M / L and 15.81 mu M / L respectively. The preparation method provided by the invention is mild in reaction condition and low in energy consumption, and adopts cheap metal cuprous iodide as a catalyst; the method also has the characteristics of better atom economy and high product yield; the method has the advantages of short synthetic route and simple reaction operation process, and can be widely popularized and applied.

Description

Technical Field

[0001] The invention belongs to the technical field of organic synthesis, and particularly relates to a method for synthesizing a seven-membered spirocyclic oxidized indole compound. Background Art

[0002] Spiroindoles are a class of unique spirocyclic skeleton unit compounds with a fused spiro ring at the C3 position of the spirocyclic core. These special and interesting spiroindoles heterocyclic structural units are widely present in natural products and drug molecules, and have a wide range of biological activities, such as anti-tumor, antibacterial, anti-HIV, anti-malarial, antiviral, antipyretic and sodium channel blockers. For example: Spiroindoles alkaloid natural products Spirotryprostatin A and Spirotryprostatin B are two biologically active spiroindoles alkaloids isolated from Aspergillus fumigatus fungi in 1996. The natural product Spirotryprostatin B can inhibit mouse breast cancer cells (tsFT2l0) in the G2 / M phase, and it has been confirmed that it has cytotoxic activity against human chronic myeloid leukemia (K562) cells and human promyelocytic leukemia (HL-60) cells. From the perspective of the structure of spiro-oxidized indole compounds, spiro-oxidized indole is considered to be a rigid spirocyclic system with good affinity for three-dimensional proteins. Compared with monocyclic structures, spiro-oxidized indole compounds can improve certain physical and chemical properties of compounds, such as lipophilicity, water solubility and metabolic stability. In recent years, with the development of synthetic chemistry science, the application of spiro-oxidized indole structures in drug discovery has received great attention. Due to their good biological activity, physiological activity and high drug-making rate, they have an important position in the fields of biomedicine and pharmacy, and continue to receive extensive attention from synthetic chemists and pharmacists. Their synthesis has always been a research hotspot and difficulty in the field of organic chemical synthesis. Due to the special chemical structure and important physiological activity of spiro-oxidized indole compounds, it is particularly important to develop new reactions for the efficient synthesis of spiro-oxidized indole compounds, which is of great significance for drug research and development. In 2005, the famous medicinal chemist Wang Shaomeng developed a new anti-tumor drug MDM2 inhibitor based on the key spiroindole core structure in the spiroindole alkaloids Spirotryprostatin A and Alstonisine natural products. Then in 2010, the patent of his MDM2 inhibitor was transferred to Sanofi-Aventis, the world's third largest pharmaceutical company, for US$398 million. This is a successful case of a new drug developed based on the key spiroindole structure in the spiroindole alkaloids Spirotryprostatin A and Alstonisine natural products as the core skeleton unit. Summary of the invention

[0003] The purpose of the present invention is to provide a method for synthesizing a seven-membered spiro ring oxidized indole compound. The present invention is based on the induction of chiral sulfenamide as a cofactor and coordination with metallic copper, and the catalysis of cuprous iodide to arylate the α-position of the amide and the Michael addition cyclization reaction of the ketone ester in series, so as to construct the key continuous quaternary carbon and tertiary carbon stereocenters in the oxidized indole molecule in one step, and then remove the tert-butyl sulfenyl group in series condensation cyclization reaction under subsequent acidic conditions to obtain an amino-protected seven-membered spiro ring oxidized indole compound, and further remove the 4-methoxybenzyl protection of the nitrogen atom on the oxidized indole ring to obtain the key seven-membered spiro ring oxidized indole compound. The compound has obvious biological activity against human lung cancer cells A549 and H1299, and its IC50 is 18.73 μM / L and 15.81 μM / L, respectively. At the same time, sufficient seven-membered spiro ring oxidized indole compound samples can be provided for clinical trial research, which has important theoretical and practical significance.

[0004] To achieve the above object, the present invention provides the following technical solutions:

[0005] One of the technical solutions of the present invention is to provide a method for synthesizing a seven-membered spirocyclic oxidized indole compound, comprising the following steps:

[0006] Reducing a chiral sulfenyl imine compound containing a 4-methoxybenzyl protected amino group to prepare an amino protected chiral sulfenamide compound; reacting the amino protected chiral sulfenamide compound with an inorganic base and a nitrogen alkylating agent in a solvent to prepare an N-alkylated amino protected chiral sulfenamide compound; heating the N-alkylated amino protected chiral sulfenamide compound with cuprous iodide and lithium bistrimethylsilylamide in a solvent under a protective gas, and then adding (E)-3-oxohex-4-enoic acid ethyl ester to continue the reaction to prepare an oxidized indole compound with continuous quaternary carbon and tertiary carbon stereo centers; converting the oxidized indole compound with continuous quaternary carbon and tertiary carbon stereo centers into an amino protected seven-membered spiro oxidized indole compound under acidic conditions; removing the 4-methoxybenzyl group on the nitrogen atom of the oxidized indole ring in the amino protected seven-membered spiro oxidized indole compound to obtain a seven-membered spiro oxidized indole compound;

[0007] The structural formula of the chiral sulfenyl imine compound containing a methoxybenzyl protected amino group is shown in Formula 1:

[0008]

[0009] Preferably, when reducing the chiral sulfenyl imine compound containing a 4-methoxybenzyl protected amino group, the reducing agent is sodium borohydride and the reduction temperature is an ice water bath.

[0010] More preferably, the molar ratio of the chiral sulfenyl imide compound containing a 4-methoxybenzyl protected amino group to the sodium borohydride is 1:3.

[0011] Preferably, the inorganic base is sodium hydride; the nitrogen alkylating agent is benzyl bromide; and the reaction temperature of the amino-protected chiral sulfenamide compound with the inorganic base and the nitrogen alkylating agent is an ice-water bath.

[0012] More preferably, the molar ratio of the amino-protected chiral sulfenamide compound, the sodium hydride and the benzyl bromide is 1:3:1.5.

[0013] Preferably, the molar ratio of the N-alkylated amino-protected chiral sulfenamide compound, the cuprous iodide, the lithium bis(trimethylsilyl)amide and the (E)-3-oxohex-4-enoate is 10:1:20:15.

[0014] Preferably, the conversion under acidic conditions is specifically to dissolve the oxidized indole compound with continuous quaternary carbon and tertiary carbon stereocenters in an anhydrous solvent, and add hydrochloric acid solution to convert the oxidized indole compound with continuous quaternary carbon and tertiary carbon stereocenters into an amino-protected seven-membered spirocyclic oxidized indole compound.

[0015] More preferably, the concentration of the hydrochloric acid solution is 3 mmol / mL, and the dosage ratio of the oxidized indole compound having consecutive quaternary carbon and tertiary carbon stereocenters to the hydrochloric acid solution is 1 mmol:1 mL.

[0016] Preferably, when removing the 4-methoxybenzyl group, the amino-protected seven-membered spirocyclic indole oxide compound is dissolved in an anhydrous solvent, trifluoromethanesulfonic acid is added in an ice-water bath, the reaction is carried out for 2 hours, and then the temperature is returned to room temperature, and the reaction is continued to complete the removal of the methoxybenzyl group.

[0017] More preferably, the molar ratio of the amino-protected seven-membered spirocyclic oxidized indole compound to the trifluoromethanesulfonic acid is 1:4.

[0018] The second technical solution of the present invention is to provide a seven-membered spirocyclic oxidized indole compound synthesized according to the above-mentioned synthesis method.

[0019] The structure of the synthesized seven-membered spirocyclic indole compound is shown in Figure 7 .

[0020] The third technical solution of the present invention is to provide an application of the above-mentioned seven-membered spirocyclic indole compound in the preparation of drugs for treating lung cancer.

[0021] The beneficial technical effects of the present invention are as follows:

[0022] The invention provides a novel synthesis method of a seven-membered spiro ring oxidized indole compound. The method comprises the following steps: using a chiral sulfenamide as an auxiliary group and inducing coordination with metallic copper, catalyzing a cuprous iodide-catalyzed amide position arylation reaction and a Michael addition cyclization reaction of a ketone ester in series, constructing key continuous quaternary carbon and tertiary carbon stereocenters in an oxidized indole molecule in one step, removing the tert-butyl sulfenyl group under subsequent acidic conditions and carrying out a series of condensation cyclization reactions, and obtaining an amino-protected seven-membered spiro ring oxidized indole compound. The key seven-membered spiro ring oxidized indole compound is further obtained by removing the 4-methoxybenzyl protection of the nitrogen atom on the oxidized indole ring.

[0023] The preparation method provided by the invention has mild reaction conditions, low energy consumption, and adopts cheap metal cuprous iodide as a catalyst; the method also has good atom economy and high product yield; the method has a short synthesis route and a relatively simple reaction operation process, and can be widely promoted and applied. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a flow chart for the preparation of the seven-membered spirocyclic oxidized indole compound in Example 1.

[0025] Figure 2 This is the reaction equation diagram of compound 2.

[0026] Figure 3 This is the reaction equation diagram of compound 3.

[0027] Figure 4 This is the reaction equation diagram of compound 5.

[0028] Figure 5 This is the reaction equation diagram of compound 6.

[0029] Figure 6 This is the reaction equation diagram of compound 7.

[0030] Figure 7 This is the X-ray single crystal diffraction structure of compound 7.

[0031] Figure 8 This is the relationship between the inhibition rate of A549 cells and the drug concentration.

[0032] Fig. 9 This is the relationship between the inhibition rate of H1299 cells and the drug concentration. DETAILED DESCRIPTION

[0033] Now, various exemplary embodiments of the present invention are described in detail, and this detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention.

[0034] In addition, for the numerical range in the present invention, it is understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0035] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] Example 1

[0038] Preparation steps of seven-membered spirocyclic indole oxide compounds (preparation process as shown in Figure 1 shown):

[0039] (1) Synthesis of compound 2, reaction formula is shown in Figure 2 :

[0040] In a 100mL double-necked round-bottom flask, compound 1 (5.26g, 10.0mmol) was weighed and dissolved in 100mL of anhydrous methanol solvent, and then sodium borohydride solid (1.14g, 30.0mmol, 3.0equiv) was slowly added in an ice-water bath at 0°C, and the reaction was continued to stir for 5h. Post-treatment: After evaporating most of the organic solvent, 60mL of water was added, and then extracted with ethyl acetate (3×100mL), dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure, and silica gel column chromatography (petroleum ether 60-90°C: ethyl acetate = 1:1, v / v) was performed to obtain a light yellow liquid compound 2 (4.1g, 93%).

[0041] The characterization data of compound 2 are as follows:

[0042] 1 H NMR (400 MHz, CDCl 3)δ(ppm):7.92(dt,J=7.9,1.4Hz,1H),7.22(td,J=7.6,1.2Hz,1H),7.09(d,J=8.6H z,2H),7.04(t,J=7.7Hz,1H),6.77(d,J=8.8Hz,2H),6.72-6.67(m,1H),5.57(d,J= 14.0Hz,1H),3.87(dd,J=14.0,2.4Hz,1H),3.76(s,3H),3.66-3.56(m,1H),3.21-3 .15(m,1H),3.08-3.00(m,1H),2.07-1.91(m,2H),1.89-1.84(m,2H),1.15(s,9H). 13 C NMR (100 MHz, CDCl 3 )δ(ppm):171.51,171.45,158.74,143.47,139.97,139.91,130.71,130.66,130.49,130.48,129.69,129.10,129.06,12 8.79,128.75,113.47,100.34,100.31,55.31,54.95,50.71,44.72,44.70,31.62,25.84,22.47.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 22 H 30 IN 2 O 3 S:529.1017, found 529.1015.

[0043] (2) Synthesis of compound 3, reaction formula is shown in Figure 3 :

[0044] 5.28 g of compound 2 (10 mmol) was weighed into a 200 mL single-mouth round-bottom flask, and then 100 mL of anhydrous tetrahydrofuran solvent was added to dissolve. Under an ice-water bath, 1.2 g of 60% sodium hydride (30 mmol, 3 equiv) and 1.78 ml of benzyl bromide (15 mmol, 1.5 equiv) were slowly added, and then the temperature was restored to room temperature and the reaction was continued for 10 h. Post-treatment: 50 mL of saturated saline solution was added, stirred for 1 hour, and then extracted with ethyl acetate (3×100 mL), the solvent was evaporated under reduced pressure, and silica gel column chromatography (petroleum ether 60-90° C.: ethyl acetate = 2:1, v / v) was performed to obtain 5.69 g of light yellow compound 3 (92%).

[0045] The characterization data of compound 3 are as follows:

[0046] 1 H NMR (400 MHz, CDCl 3 )δ7.92(d,J=8.0Hz,1H),7.32-7.15(m,6H),7.07(d,J=8.4Hz,2H),7.07(t,J=8.4Hz,1H),7 .05(t,J=3.6Hz,1H),6.77(d,J=8.4Hz,2H),6.62(td,J=8.4,1.6Hz,1H),5.56(dd,J=14.4,5 .6Hz,1H),4.27(dd,J=15.6,1.6Hz,1H),4.09(dd,J=15.6,2.8Hz,1H),3.83(dd,J=14.0,3.2 Hz,1H),3.78(s,3H),2.98-2.86(m,1H),2.78-2.69(m,1H),1.95-1.65(m,4H),1.16(s,9H). 13 C NMR (100 MHz, CDCl 3 )δ171.40,158.99,143.67,140.22,140.19,137.14,130.96,130.90,130.73,129.87,129.29,129.25,129.09,128.52,128.41,127.33, 113.70,100.52,100.48,58.21,58.19,55.24,50.94,50.91,32.16,32.09,24.22,23.91,23.79,23.36,23.34.HRMS(ESI-TOF)m / z[M+H] + calcdfor C 29 H 36 IN 2 O 3 S:619.1486, found 619.1486.

[0047] (3) Synthesis of compound 5, reaction formula is shown in Figure 4 :

[0048] In a 50mL double-necked round-bottom flask, 38mg of cuprous iodide catalyst (0.2mmol, 0.1equiv) and 1.23g of compound 3 (2.0mmol) were weighed, and 30mL of anhydrous toluene solvent was added to dissolve, and then degassed continuously for 3 times under the protection of inert gas high-purity nitrogen. At room temperature, 4.0mL of lithium bis(trimethylsilyl)amide solution (1MinTHF, 2.0equiv) was injected with a syringe, and then degassed continuously for 2 times. Then, the mixture was heated in an oil bath at 85°C for 6h, and then 468mg of (E)-3-oxohex-4-enoic acid ethyl ester reagent (3.0mmol, 1.5equiv) was added, and the mixture was continued to react for 5h under the condition of oil bath heating at 85°C. Note that the reaction process needs to be carried out under a high-purity nitrogen environment. Post-treatment: add 15 mL of a saturated solution of ammonium chloride, then extract with ethyl acetate (3×40 mL), and then dry it over anhydrous sodium sulfate. The filtrate is placed on a rotary evaporator and concentrated under reduced pressure, followed by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1, v / v), and finally 1.06 g of a light yellow viscous liquid compound 5 (82%) is obtained.

[0049] The characterization data of compound 5 are as follows:

[0050] 1 H NMR (400 MHz, CDCl 3 )δ7.25-7.12(m,5H),7.10-7.00(m,3H),6.91-6.80(m,2H),6.73-6.62(m,3H),4.74- 4.54(m,2H),4.15-4.00(m,3H),3.94(d,J=15.3Hz,1H),3.66(s,2H),3.66(s,1H),3.2 4(s,1H),3.15(s,1H),2.51-2.35(m,3H),2.30-2.00(m,3H),1.95-1.84(m,1H),1.20- 1.09(m,3H),1.05(s,4.5H),1.04(s,4.5H),0.84-0.75(m,1H),0.64(d,J=6.6Hz,2H). 13 C NMR (100 MHz, CDCl 3)δ201.76,201.72,201.25,178.07,177.86,166.88,166.72,158.95,142.69,136.88,136.85,129.82,128.92,128.87,128.81,128.58 ,128.55,128.49,128.45,128.40,128.19,128.10,128.02,127.88,127.85,127.45,127.37,123.15,123.09,123.00,122.67,122.57,1 14.02,109.04,108.99,61.27,61.23,58.18,57.99,57.96,55.12,53.22,53.16,52.85,52.80,49.45,49.35,49.32,44.66,44.50,43. 07,35.71,35.68,35.41,33.64,33.48,32.99,32.87,24.15,23.27,23.26,23.16,14.82,14.27,14.02,13.98.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 37 H 47 N 2 O 6 S:647.3150,found647.3151.

[0051] (4) Synthesis of compound 6, reaction formula is shown in Figure 5 :

[0052] In a 50mL single-mouth round-bottom flask, weigh 646mg of compound 5 (1.0mmol), add 10mL of anhydrous dichloromethane / methanol (1:1, v / v) solvent to dissolve, then place the reaction bottle under room temperature, add 3mmol / mL of dilute hydrochloric acid (1.0mL, 3.0equiv), and stir to react for 5h. Post-treatment: add saturated sodium bicarbonate solution until the pH of the solution is >7, then extract with ethyl acetate (3×40mL), then dry it with anhydrous sodium sulfate, put the filtrate on a rotary evaporator, continue to concentrate under reduced pressure, and then use silica gel column chromatography (petroleum ether: ethyl acetate = 2:1, v / v) to finally obtain 456mg of light yellow viscous liquid compound 6 (87%).

[0053] The characterization data of compound 6 are as follows:

[0054] 1 H NMR (400 MHz, CDCl 3)δ7.31-7.26(m,2H),7.23-7.12(m,5H),7.07(t,J=7.5Hz,1H),7.01(d,J=7.1Hz,1H),6.95(d,J=7.4Hz,2H),6.76(d,J=8.4 Hz,2H),6.65(d,J=7.6Hz,1H),4.88-4.79(m,2H),4.68(d,J=17.3Hz,2H),4.56(d,J=16.5Hz,1H),4.37(d,J=16.5Hz,1H),4 .14(d,J=14.6Hz,1H),3.95(d,J=14.1,7.0Hz,2H),3.70(s,3H),3.50(dd,J=14.5,10.0Hz,1H),2.91(dd,J=15.6,5.6Hz,1H ),2.15-2.07(m,1H),1.86(dd,J=14.8,11.2Hz,1H),1.65(dd,J=15.2,6.0Hz,1H),1.27-1.12(m,4H),0.74(d,J=6.8Hz,3H). 13 C NMR (100 MHz, CDCl 3 )δ179.03,169.47,166.63,159.00,141.99,136.68,134.56,128.80,128.67,128.05,127.75,127.37,126.76,122.76,122.02, 114.13,108.87,83.95,58.49,55.70,55.28,51.26,46.11,42.87,39.55,37.54,29.10,18.28,14.70.HRMS(ESI-TOF)m / z[M+H] + Calculate for C 33 H 37 N 2 O 4 :525.2748,found 525.2750.

[0055] (5) Synthesis of compound 7, reaction formula is shown in Figure 6 :

[0056] In a 50mL single-mouth round-bottom flask, 524mg of compound 6 (1.0mmol) was weighed, 15mL of anhydrous dichloromethane solvent was added to dissolve, and then the reaction bottle was placed in an ice-water bath, 0.32mL of trifluoromethanesulfonic acid (4.0mmol, 4.0equiv) was slowly added, and the reaction was continued in an ice-water bath for 2h, and then the reaction was placed at room temperature and stirred for 12h. Post-treatment: saturated sodium bicarbonate solution was added until the pH of the solution was >7, and then extracted with dichloromethane (3×20mL), and then dried with anhydrous sodium sulfate. The extract was placed on a rotary evaporator, and continued to be concentrated under reduced pressure, and then silica gel column chromatography (petroleum ether: ethyl acetate = 1:2, v / v) was used to finally obtain 327mg of colorless viscous liquid compound 7 (81%).

[0057] The characterization data of compound 7 are as follows:

[0058] 1 H NMR (400 MHz, CDCl 3 )δ7.73(d,J=9.2Hz,1H),7.31-7.26(m,2H),7.24-7.09(m,5H),7.02-6.94(m,2H),6.78(d,J=7.6Hz,1H),4.76(dd,J= 15.6,10.8Hz,1H),4.65(s,1H),4.56(d,J=16.4Hz,1H),4.36(d,J=16.4Hz,1H),4.14(d,J=14.8Hz,1H),4.01(d,J=8. 8Hz,1H),3.97(d,J=7.2Hz,1H),3.41(dd,J=14.4,10Hz,1H),2.91(dd,J=15.6,5.6Hz,1H),2.13-2.03(m,1H),1.85(d d,J=14.8,11.2Hz,1H),1.72(dd,J=15.2,6.0Hz,1H),1.27-1.16(m,1H),1.15(t,J=7.2Hz,3H),0.79(d,J=6.8Hz,3H). 13 C NMR (100 MHz, CDCl 3 )δ181.04,169.57,166.65,139.84,136.74,135.20,128.90,127.94,127.47,126.82,122.88,122.46,1 09.35,84.06,58.60,55.79,51.64,46.14,39.59,37.35,29.03,18.21,14.79.HRMS(ESI-TOF)m / z[M+H]+ Calculate for C 25 H 29 N 2 O 3 :405.2173,found 405.2171.

[0059] The X-ray single crystal diffraction structure of compound 7 is shown in Figure 7 .

[0060] X-single crystal data for compound 7: Crystal data for ptf-21:C 25 H 28 N 2 O 3 ,M=404.49, α=90°, β=96.877(4)°, γ=90°, T=157(2)K, space group P21 / C, Z=2, μ(Cu Kα)=0.670mm -1 ,4008reflections measured,3687independent reflections(R int =0.0703).The final R 1 values ​​were 0.0751(I>2σ(I)).The final wR(F 2 )values ​​were 0.0786(I>2σ(I)).Thegoodness offit onF 2 was 1.194.

[0061] It can be seen from Example 1 that the preparation method provided by the present invention has a high product yield, a short synthesis route, a simple reaction operation process, and good atom economy.

[0062] Compound 7 is a seven-membered spirocyclic oxidized indole compound, which has obvious biological activity against human lung cancer cells A549 and H1299, with IC50 values ​​of 18.73 μM / L and 15.81 μM / L, respectively.

[0063] IC50 was calculated by MTS method.

[0064] The steps of the MTS method are as follows:

[0065] The concentration gradients of the seven-membered spirocyclic oxidized indole compound 7 were set to 2.5, 5, 10, 20, 40, 50, 60 and 80 (μM), and three replicate wells were set for each concentration gradient.

[0066] 1. Cell culture: Digest and centrifuge A549 / H1299 cells in the logarithmic growth phase, wash three times with PBS and make a PBS suspension, count with a cell counter, and take the average value of three counts. After counting, add 100 μL of complete medium to each well of the 96-well plate, inoculate 1000 cells per well of the control well and experimental well into the 96-well plate, shake thoroughly to make the cells evenly distributed in the well plate, and do not inoculate cells in the blank well. After standing for a few minutes, place in an incubator and culture overnight to allow the cells to adhere to the wall.

[0067] 2. Adding drugs: After the cells in the experimental wells adhere to the wall, mix the drugs with the culture medium thoroughly, add drugs of different concentration gradients from low concentration to high concentration, shake gently, let it stand for a few minutes, and then culture in an incubator.

[0068] 3. MTS treatment: 72 hours after the cells were treated with drugs, 20 μL of MTS was added to each of the blank wells, control wells and experimental wells, and the cells were incubated in the incubator for another 4 hours to ensure that the MTS reagent was reduced to a colored product.

[0069] 4. ELISA detection: After incubation, place the 96-well plate in an ELISA instrument, detect the absorbance of each well at a wavelength of 490 nm, and save the test results.

[0070] 5. Data processing: Cell survival rate = (absorbance of experimental well - absorbance of blank well) / (absorbance of control well - absorbance of blank well)*100%.

[0071] 6. Calculate the IC50 based on the cell survival rate using GraphpadPrism and make a graph.

[0072] The relationship between the inhibition rate of A549 cells and drug concentration is shown in Figure 8 .

[0073] The relationship between the inhibition rate of H1299 cells and drug concentration is shown in Fig. 9 .

[0074] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for synthesizing a seven-membered spirocyclic indole compound, characterized in that: The following steps are involved: Reducing a chiral sulfenyl imine compound containing a 4-methoxybenzyl protected amino group to prepare an amino protected chiral sulfenamide compound; reacting the amino protected chiral sulfenamide compound with an inorganic base and a nitrogen alkylating agent in a solvent to prepare an N-alkylated amino protected chiral sulfenamide compound; heating the N-alkylated amino protected chiral sulfenamide compound with cuprous iodide and lithium bistrimethylsilylamide in a solvent under a protective gas, and then adding (E)-3-oxohex-4-enoic acid ethyl ester to continue the reaction to prepare an oxidized indole compound with continuous quaternary carbon and tertiary carbon stereo centers; converting the oxidized indole compound with continuous quaternary carbon and tertiary carbon stereo centers into an amino protected seven-membered spiro oxidized indole compound under acidic conditions; removing the 4-methoxybenzyl group on the nitrogen atom of the oxidized indole ring in the amino protected seven-membered spiro oxidized indole compound to obtain a seven-membered spiro oxidized indole compound; The structural formula of the chiral sulfenyl imine compound containing a methoxybenzyl protected amino group is shown in Formula 1:

2. The method for synthesizing the seven-membered spirocyclic indole compound according to claim 1, characterized in that: When reducing the chiral sulfinyl imine compound containing a methoxybenzyl protected amino group, the reducing agent is sodium borohydride and the reduction temperature is an ice water bath.

3. The method for synthesizing the seven-membered spirocyclic indole compound according to claim 1, characterized in that: The inorganic base is sodium hydride; the nitrogen alkylating agent is benzyl bromide; and the reaction temperature of the amino-protected chiral sulfenamide compound, the inorganic base and the nitrogen alkylating agent is an ice-water bath.

4. The method for synthesizing the seven-membered spirocyclic indole compound according to claim 1, characterized in that: The conversion under acidic conditions is specifically to dissolve the oxidized indole compound with continuous quaternary carbon and tertiary carbon stereo centers in an anhydrous solvent, and add hydrochloric acid solution to convert the oxidized indole compound with continuous quaternary carbon and tertiary carbon stereo centers into an amino-protected seven-membered spirocyclic oxidized indole compound.

5. The method for synthesizing the seven-membered spirocyclic indole compound according to claim 1, characterized in that: When removing the 4-methoxybenzyl group, specifically, the amino-protected seven-membered spirocyclic indole oxide compound is dissolved in an anhydrous solvent, trifluoromethanesulfonic acid is added under ice-water bath conditions, the reaction is restored to room temperature after 2 hours, and the reaction is continued to complete the removal of the methoxybenzyl group.

6. A seven-membered spirocyclic indole oxide compound synthesized according to the synthesis method according to any one of claims 1 to 5.

7. Use of the seven-membered spirocyclic oxidized indole compound according to claim 6 in the preparation of drugs for treating lung cancer.

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