A method for synthesizing a seven-membered spirocyclic indole compound

Abstract

This invention discloses a method for synthesizing a seven-membered spirocyclic indole oxide compound, belonging to the field of organic synthesis technology. Based on a chiral sulfinamide as a cofactor and copper coordination-induced, cuprous iodide-catalyzed Michael addition cyclization reaction of α-arylization of the amide followed by ketene esterification, the key continuous quaternary and tertiary carbon stereocenters in the indole oxide molecule are constructed in one step. Then, a subsequent acidic tandem condensation cyclization reaction to remove the tert-butyl sulfinyl group yields an amino-protected seven-membered spirocyclic indole oxide compound. Further removal of the 4-methoxybenzyl group protecting the nitrogen atom on the indole oxide ring yields the crucial seven-membered spirocyclic indole oxide compound. This compound exhibits significant biological activity against human lung cancer cells A549 and H1299, with IC50 values ​​of 18.73 μM / L and 15.81 μM / L, respectively. The preparation method provided by this invention has mild reaction conditions and low energy consumption, and uses inexpensive metal cuprous iodide as a catalyst; the method also has good atom economy and high product yield; the synthetic route is short and the reaction operation is relatively simple, and it can be widely promoted and applied.

Description

Technical Field

[0001] This invention belongs to the field of organic synthesis technology, specifically relating to a method for synthesizing a seven-membered spirocyclic indole compound. Background Technology

[0002] Spirocyclic indole is a unique class of spirocyclic skeletal unit compounds, with a fused spirocyclic ring at the C3 position of the spirocyclic core. These particularly interesting spirocyclic indole heterocyclic structural units are widely found in natural products and drug molecules, exhibiting a wide range of biological activities, such as antitumor, antibacterial, anti-HIV, antimalarial, antiviral, antipyretic, and sodium channel blocker effects. For example, the spirocyclic indole alkaloids Spirotryprostatin A and Spirotryprostatin B are two bioactive spirocyclic indole alkaloids isolated from Aspergillus fumigatus in 1996. Spirotryprostatin B can inhibit mouse breast cancer cells (tsFT2l0) in the G2 / M phase and has been confirmed to have cytotoxic activity against human chronic myeloid leukemia (K562) cells and human promyelocytic leukemia (HL-60) cells. Structurally, spirocyclic indole compounds are considered rigid spirocyclic systems with excellent affinity for three-dimensional proteins. Compared to monocyclic structures, spirocyclic indole compounds can improve certain physicochemical properties, such as lipophilicity, water solubility, and metabolic stability. In recent years, with the development of synthetic chemistry, the application of spirocyclic indole structures in drug discovery has received considerable attention. Due to their excellent biological and physiological activities and high drug yield, they hold an important position in the biomedical and pharmaceutical fields and continue to attract widespread attention from synthetic chemists and pharmacologists. Their synthesis has always been a research hotspot and challenge in the field of organic chemistry synthesis. Given the unique chemical structure and important physiological activities of spirocyclic indole compounds, developing new reactions for the efficient synthesis of spirocyclic indole compounds is particularly important and of great significance for drug development. In 2005, renowned medicinal chemist Wang Shaomeng developed a novel anti-tumor drug, the MDM2 inhibitor, based on the key spirocyclic indole core structure of the spirocyclic indole alkaloids Spirotryprostatin A and Alstonisine. Subsequently, in 2010, the patent for his MDM2 inhibitor was transferred to Sanofi-Aventis, the world's third-largest pharmaceutical company, for $398 million. This is a successful case of developing a novel drug based on the key spirocyclic indole structure of the spirocyclic indole alkaloids Spirotryprostatin A and Alstonisine as the core scaffold unit. Summary of the Invention

[0003] The purpose of this invention is to provide a method for synthesizing a seven-membered spirocyclic indole oxide compound. This invention is based on a Michael addition cyclization reaction of α-arylation of an amide with a chiral sulfinamide as a cofactor and copper coordination induction, catalyzed by cuprous iodide, to construct the key continuous quaternary and tertiary carbon stereocenters in the indole oxide molecule in one step. Then, through a subsequent acidic condition tandem condensation cyclization reaction to remove the tert-butyl sulfinyl group, an amino-protected seven-membered spirocyclic indole oxide compound is obtained. Further, by removing the 4-methoxybenzyl group protection from the nitrogen atom on the indole oxide ring, the key seven-membered spirocyclic indole oxide compound is obtained. This compound exhibits significant biological activity against human lung cancer cells A549 and H1299, with IC50 values ​​of 18.73 μM / L and 15.81 μM / L, respectively. Simultaneously, it can provide sufficient samples of the seven-membered spirocyclic indole oxide compound for clinical trials, which has important theoretical and practical significance.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

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

[0006] A chiral sulfinamide compound with an amino-protected 4-methoxybenzyl-protected amino group was prepared by reducing the chiral sulfinamide compound. The amino-protected chiral sulfinamide compound was then reacted with an inorganic base and a nitrogen-alkylating agent in a solvent to prepare an N-alkylated amino-protected chiral sulfinamide compound. Under a protective atmosphere, the N-alkylated amino-protected chiral sulfinamide compound was further reacted with cuprous iodide and lithium bis(trimethylsilylamino)amine in a solvent under heating. Then, ethyl (E)-3-oxohexyl-4-enoate was added to continue the reaction and prepare an indole oxide compound with continuous quaternary and tertiary carbon stereocenters. The indole oxide compound with continuous quaternary and tertiary carbon stereocenters was converted into an amino-protected seven-membered spirocyclic indole oxide compound under acidic conditions. The 4-methoxybenzyl group on the nitrogen atom of the indole ring in the amino-protected seven-membered spirocyclic indole compound was removed to obtain the seven-membered spirocyclic indole oxide compound.

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

[0008]

[0009] Preferably, when reducing chiral sulfinimide compounds containing 4-methoxybenzyl protected amino groups, the reducing agent is sodium borohydride, and the reduction temperature is an ice-water bath.

[0010] More preferably, the molar ratio of the chiral sulfinimide compound containing 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 sulfinamide 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 sulfinamide 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 sulfinamide compound, the cuprous iodide, the bis(trimethylsilylamino)lithium, and the (E)-3-oxohex-4-enoic acid ethyl ester is 10:1:20:15.

[0014] Preferably, the transformation under acidic conditions specifically involves dissolving the indole oxide compound with continuous quaternary and tertiary carbon stereocenters in an anhydrous solvent and adding hydrochloric acid solution, thereby transforming the indole oxide compound with continuous quaternary and tertiary carbon stereocenters into an amino-protected seven-membered spirocyclic indole oxide compound.

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

[0016] Preferably, the removal of 4-methoxybenzyl is specifically carried out by dissolving the amino-protected seven-membered spirocycloindole compound in an anhydrous solvent, adding trifluoromethanesulfonic acid under ice-water bath conditions, reacting for 2 hours, restoring to room temperature, and continuing the reaction to complete the removal of methoxybenzyl.

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

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

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

[0020] The third technical solution of the present invention provides 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] This invention provides a novel synthetic method for a seven-membered spirocyclic indole oxide compound. Based on a chiral sulfinamide as a cofactor and copper coordination-induced, cuprous iodide-catalyzed amide-position arylation tandem enone ester Michael addition cyclization reaction, the key continuous quaternary and tertiary carbon stereocenters in the indole oxide molecule are constructed in one step. Then, through a subsequent acidic condition tandem condensation cyclization reaction to remove the tert-butyl sulfinyl group, an amino-protected seven-membered spirocyclic indole oxide compound is obtained. Further, by removing the 4-methoxybenzyl protection of the nitrogen atom on the indole oxide ring, the key seven-membered spirocyclic indole oxide compound is obtained.

[0023] The preparation method provided by this invention has mild reaction conditions and low energy consumption, and uses inexpensive metal cuprous iodide as a catalyst; the method also has good atom economy and high product yield; the synthetic route is short and the reaction operation is relatively simple, and it can be widely promoted and applied. Attached Figure Description

[0024] Figure 1 This is a flowchart illustrating the preparation process of the seven-membered spirocyclic indole compound in Example 1.

[0025] Figure 2 The reaction equation diagram for compound 2 is shown.

[0026] Figure 3 The reaction equation diagram for compound 3 is shown.

[0027] Figure 4 The reaction equation diagram for compound 5 is shown.

[0028] Figure 5 The reaction equation diagram for compound 6 is shown.

[0029] Figure 6 The reaction equation diagram for compound 7 is shown.

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

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

[0032] Figure 9 The relationship between the inhibition rate of H1299 cells and drug concentration. Detailed Implementation

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0034] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

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

[0037] Example 1

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

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

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

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

[0042] 1H NMR (400MHz, CDCl3) δ (ppm): 7.92 (dt, J=7.9, 1.4Hz, 1H), 7.22 (td, J=7.6, 1.2Hz, 1H), 7 .09(d,J=8.6Hz,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 (100MHz, CDCl3) 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, 1 29.06,128.79,128.75,113.47,100.34,100.31,55.31,54.95,50.71,44.72,44.70,31.62,25.84,22.47.HRMS(ESI-TOF)m / z[M+H] + calcd for C 22 H 30 IN2O3S:529.1017, found 529.1015.

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

[0044] 5.28 g (10 mmol) of compound 2 was weighed into a 200 mL single-necked round-bottom flask and dissolved in 100 mL of anhydrous tetrahydrofuran. 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 the mixture was allowed to return to room temperature for 10 h. Post-treatment: 50 mL of saturated saline solution was added, and the mixture was stirred for 1 hour. Extraction was then performed 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) yielded 5.69 g (92%) of pale yellow compound 3.

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

[0046] 1 H NMR (400MHz, CDCl3) 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 ,3.2Hz,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 (100MHz, CDCl3) δ171.40,158.99,143.67,140.22,140.19,137.14,1 30.96,130.90,130.73,129.87,129.29,129.25,129.09,128.52,128.41 ,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 IN2O3S:619.1486, found 619.1486.

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

[0048] In a 50 mL double-necked round-bottom flask, 38 mg of cuprous iodide catalyst (0.2 mmol, 0.1 equiv) and 1.23 g of compound 3 (2.0 mmol) were weighed and dissolved in 30 mL of anhydrous toluene. The mixture was then degassed three times under the protection of high-purity nitrogen. At room temperature, 4.0 mL of a bis(trimethylsilylamine)-lithium solution (1 min THF, 2.0 equiv) was injected using a syringe, followed by two more degassed cycles. The mixture was then heated in an oil bath at 85 °C for 6 h to allow for complete reaction. Then, 468 mg of (E)-3-oxohex-4-enoic acid ethyl ester reagent (3.0 mmol, 1.5 equiv) was added, and the reaction was continued at 85 °C in an oil bath for 5 h. It is important to ensure that the entire reaction is carried out under a high-purity nitrogen atmosphere. Post-treatment: 15 mL of saturated ammonium chloride solution was added, followed by extraction with ethyl acetate (3 × 40 mL), and then dried with anhydrous sodium sulfate. The filtrate was placed on a rotary evaporator and concentrated under reduced pressure. Then, silica gel column chromatography (petroleum ether: ethyl acetate = 1:1, v / v) was performed to finally obtain 1.06 g of pale yellow viscous liquid compound 5 (82%).

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

[0050] 1 H NMR (400MHz, CDCl3) 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),3.24(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). 13C NMR (100MHz, CDCl3) δ201.76,201.72,201.25,178.07,177.86,166.88,166.72,158.95,142.69,136.88,136.85,129.82,128.92,128.87,1 28.81,128.58,128.55,128.49,128.45,128.40,128.19,128.10,128. 02,127.88,127.85,127.45,127.37,123.15,123.09,123.00,122.67, 122.57,114.02,109.04,108.99,61.27,61.23,58.18,57.99,57.96, 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] + calcd for C 37 H 47 N2O6S: 647.3150, found 647.3151.

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

[0052] In a 50 mL single-necked round-bottom flask, 646 mg (1.0 mmol) of compound 5 was weighed and dissolved in 10 mL of anhydrous dichloromethane / methanol (1:1, v / v). The reaction flask was then placed at room temperature, and 1.0 mL (3.0 equiv) of 3 mmol / mL dilute hydrochloric acid was added. The mixture was stirred for 5 h. Post-treatment: Saturated sodium bicarbonate solution was added until the pH of the solution was >7. The solution was then extracted with ethyl acetate (3 × 40 mL), dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure using a rotary evaporator. The concentrate was then subjected to silica gel column chromatography (petroleum ether:ethyl acetate = 2:1, v / v) to obtain 456 mg of a pale yellow viscous liquid compound 6 (87%).

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

[0054] 1H NMR (400MHz, CDCl3) 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=8.4Hz,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=16.5 Hz,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=15.6,5.6H z,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 (100MHz, CDCl3) 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] + calcd for C 33 H 37 N2O4: 525.2748, found 525.2750.

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

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

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

[0058] 1 H NMR (400MHz, CDCl3) 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),4.0 1(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(dd,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 (100MHz, CDCl3) δ181.04,169.57,166.65,139.84,136.74,135.20,128.90,127.94,127.47,126.82,122.88, 122.46,109.35,84.06,58.60,55.79,51.64,46.14,39.59,37.35,29.03,18.21,14.79.HRMS(ESI-TOF)m / z[M+H] + calcd for C25 H 29 N2O3:405.2173, found 405.2171.

[0059] The X-ray single-crystal diffraction structure of compound 7 is shown in Figure 1. Figure 7 .

[0060] X-ray single-crystal data for compound 7: Crystal data for ptf-21:C 25 H 28 N₂O₃, 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 finalR1 values ​​were 0.0751(I>2σ(I)).The final wR(F 2 )values ​​were 0.0786(I>2σ(I)).Thegoodness offit onF 2 It was 1.194.

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

[0062] Compound 7 is a seven-membered spirocyclic indole compound. This compound has significant 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 using the MTS method.

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

[0065] A concentration gradient of seven-membered spirocyclic indole compound 7 was set up, namely 2.5, 5, 10, 20, 40, 50, 60 and 80 (μM), with three duplicate wells for each concentration gradient.

[0066] 1. Cell Culture: A549 / H1299 cells in logarithmic growth phase were digested and centrifuged, washed three times with PBS, and resuspended in PBS. Cells were counted using a cell counter, and the average value of the three counts was taken. After counting, 100 μL of complete culture medium was added to each well of a 96-well plate. Control and experimental wells were seeded with 1000 cells per well. The plates were shaken thoroughly to ensure even cell distribution. No cells were seeded in the blank wells. After standing for a few minutes, the plates were incubated overnight to allow cell adhesion.

[0067] 2. Drug addition: After the cells in the experimental wells have adhered, mix the drug with the culture medium thoroughly. Add the drug in sequence from low to high concentration, gently shake, let stand for a few minutes, and then place in an incubator for culture.

[0068] 3. MTS treatment: 72 h after drug intervention, add 20 μL of MTS to each of the blank well, control well and experimental well, and continue to incubate in the incubator for 4 h to ensure that the MTS reagent is reduced to the colored product.

[0069] 4. Microplate reader detection: After incubation, place the 96-well plate in a microplate reader and detect the absorbance of each well at a wavelength of 490 nm. Save the detection results.

[0070] 5. Data processing: Cell viability = (Absorbance of experimental wells - Absorbance of blank wells) / (Absorbance of control wells - Absorbance of blank wells) * 100%.

[0071] 6. Calculate IC50 using GraphpadPrism based on cell viability and plot the result.

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

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

[0074] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for synthesizing a seven-membered spirocyclic indole compound, characterized in that, Includes the following steps: A chiral sulfinamide compound with an amino-protected 4-methoxybenzyl-protected amino group was prepared by reducing the chiral sulfinamide compound. The amino-protected chiral sulfinamide compound was then reacted with an inorganic base and a nitrogen-alkylating agent in a solvent to prepare an N-alkylated amino-protected chiral sulfinamide compound. Under a protective atmosphere, the N-alkylated amino-protected chiral sulfinamide compound was further reacted with cuprous iodide and lithium bis(trimethylsilylamino)amine in a solvent under heating. Then, ethyl (E)-3-oxohexyl-4-enoate was added to continue the reaction and prepare an indole oxide compound with continuous quaternary and tertiary carbon stereocenters. The indole oxide compound with continuous quaternary and tertiary carbon stereocenters was converted into an amino-protected seven-membered spirocyclic indole oxide compound under acidic conditions. The 4-methoxybenzyl group on the nitrogen atom of the indole ring in the amino-protected seven-membered spirocyclic indole compound was removed to obtain the seven-membered spirocyclic indole oxide compound. The structural formula of the chiral sulfinylimide compound containing a 4-methoxybenzyl protected amino group is shown in Formula 1: ； The structural formula of the amino-protected chiral sulfinamide compound is shown in Formula 2: ； The structural formula of the N-alkylated amino-protected chiral sulfinamide compound is shown in Formula 3: ； The structural formula of the indole oxide compound with continuous quaternary and tertiary carbon stereocenters is shown in Formula 5: ； The structural formula of the amino-protected seven-membered spirocyclic indole oxide compound is shown in Formula 6: ； The structural formula of the seven-membered spirocyclic indole oxide compound is shown in Formula 7: 。 2. The method for synthesizing the seven-membered spirocyclic indole compound according to claim 1, characterized in that, When reducing chiral sulfinimide compounds containing a 4-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; the reaction temperature of the amino-protected chiral sulfinamide compound with 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 transformation under acidic conditions specifically involves dissolving the indole oxide compound with continuous quaternary and tertiary carbon stereocenters in an anhydrous solvent and adding hydrochloric acid solution, thereby transforming the indole oxide compound with continuous quaternary and tertiary carbon stereocenters into an amino-protected seven-membered spirocyclic indole oxide compound.

5. The method for synthesizing the seven-membered spirocyclic indole compound according to claim 1, characterized in that, The removal of 4-methoxybenzyl is specifically achieved by dissolving an amino-protected seven-membered spirocyclic indole compound in an anhydrous solvent, adding trifluoromethanesulfonic acid under ice-water bath conditions, reacting for 2 hours, then restoring to room temperature, and continuing the reaction to complete the removal of methoxybenzyl.

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

7. The use of the seven-membered spirocyclic indole compound of claim 6 in the preparation of a drug for treating lung cancer.

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