Method for preparing chiral spiro phenol compound by catalyzing asymmetric dearomatization

Abstract

The invention discloses a method for preparing a chiral spiro phenol compound by catalyzing asymmetric dearomatization, and belongs to the technical field of organic synthesis. A compound 1 and a chiral catalyst are used as raw materials and react with NBS at low temperature in an organic solvent to obtain a compound 2; and carrying out ring closing reaction on the intermediate in an organic solvent under an acid condition to obtain the chiral spiro phenol compound 3. The synthesis method is simple and easy to implement, the compound 2 also shows good anti-cancer activity, and a new theme framework is provided for anti-cancer drug screening.

Description

Technical Field

[0001] The invention relates to a method for preparing chiral spirocyclic phenol compounds by catalytic asymmetric dearomatization, and belongs to the technical field of organic synthesis. Background Art

[0002] Phenolic substances are widely found in drugs, agrochemicals, catalysts / ligands, materials and fragrances. They are also useful basic materials for constructing other functional molecules through CH functionalization or dearomatization processes.

[0003] The spirocyclic system products obtained by enantioselective construction of catalytic asymmetric dearomatization reactions have a quaternary carbon stereocenter and a relatively rigid three-dimensional skeleton. Such product skeletons are widely present in natural products and bioactive compounds with biological activity. So far, in the field of organic chemistry, the study of catalytic asymmetric construction of spirocarbon rings in spirocyclic compounds has aroused sustained interest, and some synthetic (spirocarbon ring) strategies have been very mature, including alkylation, rearrangement, free radical cyclization, and cleavage of bridged systems.

[0004] To date, existing strategies include functionalization of phenolic CH bonds via electrophilic substitution reactions or metal-catalyzed CH activation, usually at the ortho- and para-positions. Subfunctionalization of phenols is particularly difficult due to the inherent orientation of the hydroxyl group to the phenyl group. Although some progress has been made, current CH functionalization at the meta-position generally gives chiral or racemic products.

[0005] Therefore, it is necessary to develop practical methods for phenol-catalyzed asymmetric meta-functionalization to obtain a variety of chiral spirocyclic phenolic compounds. Summary of the invention

[0006] In order to overcome the above technical defects, the present invention provides a method for preparing chiral spirocyclic phenol compounds by catalytic asymmetric dearomatization. Compound 1 and a chiral catalyst are used as raw materials, reacted with NBS at low temperature in an organic solvent to obtain compound 2; then the intermediate is subjected to a ring-closure reaction in an organic solvent under acidic conditions to obtain a chiral spirocyclic phenol compound 3. The synthesis method of the present invention is simple and easy, and compound 2 also exhibits good anticancer activity, providing a new theme skeleton for anticancer drug screening.

[0007] The present invention provides a chiral spirocyclic phenol compound, the general structural formula of which is as follows:

[0008]

[0009] Where: R 1 -R 4Each is independently selected from one or more of hydrogen, C1-C7 alkane, C1-C7 cycloalkane, C1-C6 alkoxy, halogen, nitrile, nitro, phenyl or substituted phenyl; the substituent in the substituted phenyl is C1-C4 alkyl, C1-C4 alkoxy, halogen.

[0010] The method for preparing chiral spirocyclic phenol compounds by catalytic asymmetric dearomatization of the present invention comprises the following steps:

[0011]

[0012] Where: R 1 -R 4 Each is independently selected from one or more of hydrogen, C1-C7 alkane, C1-C7 cycloalkane, C1-C6 alkoxy, halogen, nitrile, nitro, phenyl or substituted phenyl; the substituent in the substituted phenyl is C1-C4 alkyl, C1-C4 alkoxy, halogen.

[0013] The first step: using compound 1 and a chiral catalyst as raw materials, reacting with NBS in an organic solvent at low temperature to obtain compound 2;

[0014] Step 2: Compound 2 undergoes a ring-closure reaction with an acid in an organic solvent to obtain a chiral spirocyclic phenol compound 3.

[0015] Furthermore, in the above technical solution, the organic solvent is selected from tetrahydrofuran, dichloromethane, 1,2-dichloroethane, toluene, ether or acetone.

[0016] Furthermore, in the above technical solution, the low temperature in the first step is -40°C to -80°C.

[0017] Furthermore, in the above technical solution, the chiral catalyst in the first step is selected from the following structures:

[0018]

[0019] Furthermore, in the above technical solution, under preferred conditions, the chiral catalyst is selected from Catalyst I.

[0020] Furthermore, in the above technical solution, the molar ratio of compound 1, NBS and chiral catalyst is 1:1-1.2:0.05-0.20.

[0021] Furthermore, in the above technical solution, the acid in the second step is selected from trichloroacetic acid, trifluoroacetic acid or p-toluenesulfonic acid monohydrate. Preferably, it is selected from p-toluenesulfonic acid monohydrate.

[0022] Furthermore, in the above technical solution, the reaction temperature of the second step is 0°C to 30°C.

[0023] Furthermore, in the above technical solution, the molar ratio of compound 2 to acid is 1:1-1.2.

[0024] The present invention further provides the use of compound 2 in treating anticancer drugs.

[0025] Furthermore, in the above technical solution, the anticancer drugs refer to eight types of cancer cells, namely A375, Hela, HT29, HepG2, HCT116, 4T1, MCF-7 and MIAPaca-2.

[0026] Instruction Manual

[0027] Figure 1 The activity detection data of compound 2m in Example 5 is shown in FIG. 5 ; wherein:

[0028] (A) IC50 values ​​of 2m against different cancer cells, data are the average of three experiments;

[0029] (B) 2m induced A375 cell cycle arrest in a time-dependent (0, 12, 24, 36 h) and dose-dependent (0, 3, 6, 9 μM) manner;

[0030] (C) 2m induced apoptosis of A375 cells in a time-dependent (0, 12, 24, 36 h) and dose-dependent (0, 3, 6, 9 μM) manner;

[0031] (D) 2m induced an increase in ROS in A375 cells in a dose-dependent manner (0, 3, 6, and 9 μM);

[0032] (E) 2m induced a decrease in mitochondrial membrane potential in a dose-dependent manner (0, 3, 6, and 9 μM). DETAILED DESCRIPTION

[0033] Example 1

[0034] Condition optimization experiment

[0035]

[0036]

[0037]

[0038]

[0039] a1a (0.05mmol), catalysts AI (10mol%) in solvent at corresponding temperature for 10min, then NBS (0.05mmol) were added at corres pondingtemperature, unless otherwise specified. b Acid optimization. c Ee was determined by HPLC. d Isolated yield.

[0040] Compound 1a was used as the substrate to start the study. The reaction was carried out at -78°C in dichloromethane and NBS was added to test the catalytic activity of different catalysts AI. The dearomatization spirocyclization of phenol-substituted 2-ethynylnaphthol derivatives was achieved in the presence of an organic catalyst by activation of stoichiometric electrophilic halogen species.

[0041] First, the screening results of cinchona alkaloid catalysts showed that the catalyst skeleton had a significant effect on the reaction results. When catalyst AE was used, product 2a was obtained with moderate yield / poor to moderate enantioselectivity. However, when catalyst F was used to catalyze the reaction, product 2a was obtained with 78% ee and moderate yield; when cinchona alkaloid derivative amide catalysts G and H were used, only -6% ee and 57% ee were obtained.

[0042] Solvent screening showed that dichloromethane was superior to other solvents (toluene, acetone, ethyl acetate, tetrahydrofuran, ether). Studies have shown that low temperature conditions are very important for product formation, while high temperature will destroy the cyclization process. The optimal reaction temperature is -78°C, and the product 2a is obtained with a yield of 70% and 97% ee, while at -40°C, the yield and ee values ​​are significantly reduced to 32% and 86%, respectively.

[0043] In the re-aromatization reaction, a series of acids were used to screen the reaction conditions for the second step. The results showed that p-toluenesulfonic acid monohydrate was the optimal acid and the reaction proceeded smoothly at room temperature.

[0044] Example 2

[0045]

[0046] Typical operation steps:

[0047] Step 1: In a reaction tube, add substrate 1a (0.25 mmol) and chiral catalyst H (0.025 mmol) in sequence, add 5.0 mL of dichloromethane, stir the reaction system at -78°C for 10 min, add bromosuccinimide (0.25 mmol), and react at -78°C for 1 hour. Monitor the reaction progress by thin layer chromatography. After the reaction is completed, quench with ammonium chloride solution (5 mL), extract with dichloromethane and water three times (3*5 mL), extract with saturated brine, dry with anhydrous sodium sulfate, filter, wash, collect the filtrate, concentrate and distill under reduced pressure, separate and purify by silica gel column chromatography (petroleum ether / ethyl acetate volume ratio = 20 / 1) to obtain 77.3 mg of yellow solid 2a, with a yield of 70% and 97% ee. 1 H NMR (400 MHz, CDCl 3 )δ7.81(d,J=7.6Hz,1H),7.74(t,J=7.8Hz,2H),7.65(d,J=8.3Hz,1H),7.39-7.27(m,4H),7.19(d,J=8.9Hz,1H),7.14(d,J=7.1Hz,1H),6 .77(s,1H),6.58(dd,J=9.8,2.1Hz,1H),6.25(s,1H),5.81(d,J=9.8Hz,1H),3.64(d,J=15.6Hz,1H),3.17(d,J=15.6Hz,1H),1.72(s,3H). 13 CNMR (100MHz, CDCl 3 )δ204.21,150.31,146.34,137.27,132.61,132.17,131.92,131.76,130.42,130.17,129.49,129.27,128.98, 128.26,127.75,127.42,125.82,124.50,124.34,123.25,121.52,119.47,58.73,40.32,20.92.HRMS(ESI)m / z Calcd for C 26 H 19 BrNaO 2 + [M+Na] + :462.0568,Found:465.0573.Optical Rotation: (c=0.17, acetone). HPLC analysis: Chiralcel IB-H (Hexane / i-PrOH=85:15, flow rate=1.0mL / min, wave length=254nm), tR =7.430min(major),t R =10.117min(minor),97%ee.

[0048] Step 2: Add compound 2a (0.1 mmol) and 4.0 mL of dichloromethane to a reaction tube, then add p-toluenesulfonic acid monohydrate (0.1 mmol), stir the reaction system at room temperature for 1 h, monitor the reaction progress by thin layer chromatography, and after the reaction is completed, add 5 mL of water to quench. Extract with dichloromethane and water (3*5 mL), wash with saturated brine, dry with anhydrous sodium sulfate, filter, wash, collect the filtrate, concentrate and distill under reduced pressure, separate and purify by silica gel column chromatography (petroleum ether / ethyl acetate volume ratio = 10 / 1) to obtain 33.1 mg of white solid 3a, with a yield of 75% and 97% ee. 1 H NMR (400 MHz, CDCl 3 )δ8.26(d,J=8.5Hz,1H),7.83(d,J=8.1Hz,1H),7.78(dd,J=8.8,5.5Hz,2 H),7.65-7.58(m,1H),7.40(t,J=7.5Hz,1H),7.37-7.32(m,1H),7.27(dd, J=5.5,3.4Hz,2H),7.04(d,J=8.8Hz,1H),6.72-6.61(m,2H),4.87(s,1H), 4.81(s,1H),4.58(d,J=13.0Hz,1H),3.64(d,J=13.0Hz,1H),1.54(s,3H). 13 CNMR (100MHz, CDCl 3 )δ150.49,149.40,142.15,137.88,135.90,135.42,132.35,130.70,130.56,130.53,130.48,130.09,129.96, 129.36,128.62,127.27,126.29,126.04,125.71,123.28,122.68,118.27,115.29,31.31,22.16.HRMS(ESI)m / z Calcd for C 26 H 19 BrNaO 2 + [M+Na] + :465.0568,Found:465.0572.Optical Rotation: (c=0.05, acetone). HPLC analysis: Chiralcel IB-H (Hexane / i-PrOH=85:15, flow rate=1.0mL / min, wavelength=254nm), t R =8.473min(minor),t R =9.970min(major),97%ee.

[0049] Example 3

[0050] The typical operation steps in Example 2 were used to obtain series compound 2. The results are as follows:

[0051] The characterization data of representative compounds are as follows:

[0052] Compound 2b: yellow solid, yield: 70% (79.8 mg, 0.25 mmol scale); 1 H NMR (400 MHz, CDCl 3 )δ7.81(d,J=7.1Hz,1H),7.73(t,J=8.0Hz,2H),7.66(d,J=8.3Hz,1H),7.32(dq, J=22.9,7.0Hz,4H),7.17(d,J=8.9Hz,1H),7.12(d,J=7.1Hz,1H),6.79(s,1H),6. 65(dd,J=9.8,2.2Hz,1H),6.23(s,1H),5.84(d,J=9.8Hz,1H),3.58(d,J=15.6Hz, 1H), 3.18 (d, J=15.6Hz, 1H), 1.99 (tt, J=15.2, 7.3Hz, 2H), 0.80 (t, J=7.5Hz, 3H). 13 C NMR (100 MHz, CDCl 3 )δ204.34,150.32,145.49,136.14,134.64,132.69,132.11,131.94,130.43,130.09,129.44,128.95,128.24 ,127.72,127.34,125.90,124.65,124.39,123.21,121.53,119.41,58.68,40.46,27.75,12.17.HRMS(ESI)m / z Calcd for C 27 H 21 BrNaO 2 +[M+Na] + :479.0724,Found:479.0729.Optical Rotation: (c=0.3, acetone). HPLC analysis: Chiralcel IB-H (Hexane / i-PrOH=85:15, flow rate=1.0mL / min, wave length=254nm), t R =7.130min(major),t R =11.327min(minor),96%ee.

[0053] Compound 2c: yellow solid, yield: 67% (78.7 mg, 0.25 mmol scale); 1 H NMR (400 MHz, CDCl 3 )δ7.81(d,J=7.6Hz,1H),7.72(t,J=8.7Hz,2H),7.67(d,J=8.3Hz,1H),7.39-7.25(m ,4H),7.17(d,J=8.8Hz,1H),7.11(d,J=7.1Hz,1H),6.79(s,1H),6.73(dd,J=9.9,2. 3Hz, 1H), 6.22 (s, 1H), 5.87 (d, J = 9.9Hz, 1H), 3.53 (d, J = 15.6Hz, 1H), 3.18 (d, J = 15. 6Hz, 1H), 2.21 (dt, J=13.6, 6.8Hz, 1H), 0.81 (d, J=6.8Hz, 3H), 0.76 (d, J=6.8Hz, 3H). 13 C NMR (100 MHz, CDCl 3 )δ204.46,150.29,144.43,138.72,135.18,132.71,132.06,131.93,130.45,130.04,129.44,128.92,128.25,12 7.71,127.30,126.03,124.78,124.40,123.20,121.50,119.36,58.53,40.51,32.69,20.98,20.85.HRMS(ESI)m / z Calcd forC 28 H 23 BrNaO 2 + [M+Na] +: 493.0881, Found: 493.0887. Optical Rotation: (c = 0.45, acetone). HPLC analysis: Chiralcel IB-H (Hexane / i-PrOH = 85:15, flow rate = 1.0 mL / min, wavelength = 254 nm), t R = 6.303 min (major), t R = 12.117 min (minor), 94% ee.

[0054] Compound 2e: Yellow solid, yield: 68% (79.9 mg, 0.25 mmol scale); 1 H NMR (400 MHz, CDCl 3 ) δ 7.81 (d, J = 7.5 Hz, 1H), 7.78 - 7.66 (m, 3H), 7.41 - 7.26 (m, 4H), 7.16 (d, J = 8.9 Hz, 1H), 7.11 (d, J = 7.1 Hz, 1H), 6.65 (d, J = 11.1 Hz, 1H), 6.22 (s, 1H), 5.86 (d, J = 9.8 Hz, 1H), 3.50 (d, J = 15.6 Hz, 1H), 3.26 (d, J = 15.6 Hz, 1H), 1.92 (t, J = 7.3 Hz, 2H), 1.21 (dq, J = 13.6, 6.9 Hz, 2H), 0.57 (t, J = 7.3 Hz, 3H). 13 C NMR (100 MHz, CDCl 3 ) δ 204.22, 150.24, 145.57, 137.62, 133.12, 132.80, 131.93, 130.58, 130.10, 129.46, 128.98, 128.29, 128.21, 127.75, 127.33, 126.08, 124.55, 124.24, 123.22, 121.44, 119.28, 58.91, 40.45, 36.68, 20.97, 12.95. HRMS (ESI) m / z Calcd for C 28 H 23 BNarO 2 + [M + Na] + : 493.0881, Found: 493.0889. Optical Rotation: (c=0.07, acetone). HPLC analysis: Chiralcel IB-H (Hexane / i-PrOH=85:15, flow rate=1.0mL / min, wave length=254nm), t R =6.443min(major),t R =10.323min (minor), 93% ee. Compound 2g: yellow solid, yield: 66% (84.1 mg, 0.25 mmol scale); 1 H NMR (400 MHz, CDCl 3 )δ7.81(d,J=7.5Hz,1H),7.70(dt,J=17.5,8.2Hz,3H),7.39-7.26(m,4H),7.17(d,J=8.8Hz,1H ),7.11(d,J=7.1Hz,1H),6.72(d,J=11.8Hz,1H),6.22(s,1H),5.85(d,J=9.9Hz,1H),3.55(d,J= 15.6Hz,1H),3.15(d,J=15.6Hz,1H),1.82(t,J=11.7Hz,1H),1.63(s,3H),1.52(d,J=12.9Hz,1 H),1.39(d,J=12.8Hz,1H),1.22-1.09(m,2H),1.08-0.98(m,1H),0.84(dd,J=18.2,9.9Hz,2H). 13 C NMR (100 MHz, CDCl 3 )δ204.62,150.35,145.09,138.04,135.73,132.70,132.15,131.96,130.36,130.06,129.45,128.94,128.26,127.71,1 27.31,126.00,124.64,124.52,123.23,121.63,119.44,58.62,42.67,40.72,31.40,31.17,26.06,25.81.HRMS(ESI)m / z Calcdfor C 31 H 27 BrNaO 2 + [M+Na] + :533.1094,Found:534.1099.Optical Rotation: (c = 0.1, acetone). HPLC analysis: Chiralcel IB-H (Hexane / i-PrOH = 85:15, flow rate = 1.0 mL / min, wave length = 254 nm), t R = 6.390 min (major), t R = 11.020 min (minor), 90% ee.

[0055] Compound 2m: Yellow solid, yield: 87% (100.5 mg, 0.25 mmol scale); 1 1H NMR (400 MHz, CDCl 3 ) δ 7.82 (d, J = 7.6 Hz, 1H), 7.76 (dd, J = 8.2, 5.9 Hz, 2H), 7.61 (d, J = 8.4 Hz, 1H), 7.45 - 7.29 (m, 4H), 7.17 (t, J = 7.2 Hz, 2H), 6.57 (dd, J = 10.0, 2.6 Hz, 1H), 6.51 (d, J = 2.4 Hz, 1H), 6.37 (s, 1H), 5.81 (d, J = 10.0 Hz, 1H), 3.69 (d, J = 15.7 Hz, 1H), 3.21 (d, J = 15.7 Hz, 1H). 13 13C NMR (100 MHz, CDCl 3 ) δ 201.28, 150.23, 143.66, 137.01, 132.30, 131.66, 131.36, 131.21, 130.55, 130.42, 129.85, 129.00, 128.47, 128.39, 128.06, 127.58, 126.91, 126.39, 126.15, 123.97, 123.54, 120.63, 119.17, 59.78, 39.78. HRMS (ESI) m / z Calcd for C 25 1 16 H 2 + [M + Na] + : 485.0022, Found: 485.0029. Optical Rotation: (c = 0.45, acetone). HPLC analysis: Chiralcel IB-H (Hexane / i-PrOH = 85:15, flow rate = 1.0 mL / min, wave length = 254 nm), tR =8.163min(major),t R =14.447min(minor),95%ee.

[0056] Compound 2q: yellow solid, yield: 87% (103.5 mg, 0.25 mmol scale); 1 H NMR (400 MHz, CDCl 3 )δ7.81(d,J=7.5Hz,1H),7.74(t,J=8.9Hz,2H),7.61(d,J=8.3Hz,1H),7.33(ddt,J=19.9,14.1,7.4Hz,4H),7.16(dd,J=12. 4,8.1Hz,2H),6.69(d,J=1.9Hz,1H),6.23(s,1H),6.18(s,1H),3.71(d,J=15.7Hz,1H),3.17(d,J=15.7Hz,1H),1.68(s,3H). 13 C NMR (100 MHz, CDCl 3 )δ196.40,150.14,143.08,135.98,132.34,131.86,131.77,131.01,130.85,130.37,129.69,129.03,128.92, 128.38,128.33,127.89,127.56,125.80,124.35,123.33,120.63,119.20,59.98,40.05,20.90.HRMS(ESI)m / z Calcd for C 26 H 18 BrClNaO 2 + [M+Na] + :499.0178,Foun d:477.0183.Optical Rotation: (c=0.06, acetone). HPLC analysis: Chiralcel IA-H (Hexane / i-PrOH=85:15, flow rate=1.0mL / min, wave length=254nm), t R =9.843min(minor),t R =20.513min(major),94%ee.

[0057] Compound 2s: yellow solid, yield: 67% (77 mg, 0.25 mmol scale);1 H NMR (400 MHz, CDCl 3 ) δ 7.74 (t, J = 8.0 Hz, 2H), 7.63 (d, J = 8.1 Hz, 1H), 7.55 (d, J = 9.3 Hz, 1H), 7.41 - 7.33 (m, 1H), 7.34 - 7.27 (m, 1H), 7.17 (d, J = 8.8 Hz, 1H), 7.13 - 7.04 (m, 1H), 6.99 (t, J = 7.0 Hz, 1H), 6.71 (s, 1H), 6.58 (d, J = 9.5 Hz, 1H), 6.21 (s, 1H), 5.81 (d, J = 9.7 Hz, 1H), 3.56 (d, J = 15.5 Hz, 1H), 3.17 (d, J = 15.5 Hz, 1H), 1.70 (s, 3H). 13 C NMR (100 MHz, CDCl 3 ) δ 203.85, 162.19 (d, J = 244 Hz), 150.20, 146.32, 136.97, 134.69 (d, J = 8 Hz), 133.48, 131.75, 130.31, 129.51, 129.16, 128.98, 128.81 (d, J = 8 Hz), 128.33, 127.59 (d, J = 3 Hz), 125.99, 124.43, 124.15, 123.34, 121.24, 119.46, 115.78, 115.76 (d, J = 46 Hz), 58.84, 39.51, 20.91. 19 F NMR (564 MHz, CDCl 3 ) δ -113.77. HRMS (ESI) m / z Calcd for C 26 H 18 BrFNaO 2 + [M + Na] + : 483.0474, Found: 483.0477. Optical Rotation: (c = 0.125, acetone). HPLC analysis: Chiralcel IB-H (Hexane / i-PrOH = 90:10, flow rate = 1.0 mL / min, wavelength = 254 nm), t R = 8.609 min (major), t R = 9.881 min (minor), 97% ee.

[0058] Example 4

[0059] The typical operation steps in Example 2 were used to obtain series compound 3, and the results were as follows:

[0060]

[0061] The characterization data of representative compounds are as follows:

[0062] Compound 3b: white solid; yield: 77% (35 mg, 0.1 mmol scale); 1 H NMR (400 MHz, CDCl 3 )δ8.30(d,J=8.5Hz,1H),7.82(d,J=8.1Hz,1H),7.76(d,J=8.8Hz,2H),7.6 3(t,J=7.3Hz,1H),7.41(t,J=7.2Hz,1H),7.36-7.32(m,1H),7.29-7.24(m, 2H),7.02(d,J=8.8Hz,1H),6.78-6.67(m,2H),4.90(s,2H),4.57(d,J=13. 0Hz, 1H), 3.70 (d, J = 13.0Hz, 1H), 2.17-1.99 (m, 2H), 0.25 (t, J = 7.4Hz, 3H). 13 C NMR (100 MHz, CDCl 3 )δ150.91,149.18,142.44,137.98,137.09,135.63,135.40,131.93,130.74,130.55,130.06,129.84,129.26,128.85, 128.51,128.45,127.06,126.33,126.09,125.76,123.51,123.29,118.35,115.69,31.28,26.82,14.79.HRMS(ESI)m / z Calcd for C 27 H 21 BrNaO 2 + [M+Na] + :479.0725,Found:479.0729.Optical Rotation: (c=0.07, acetone). HPLC analysis: Chiralcel IB-H (Hexane / i-PrOH=85:15, flow rate=1.0mL / min, wave length=254nm), t R =7.923min(minor),tR =8.830min(major),94%ee.

[0063] Compound 3c: white solid; yield: 76% (35.7 mg, 0.1 mmol scale); 1 H NMR (400 MHz, CDCl 3 )δ8.29(d,J=8.5Hz,1H),7.82(d,J=8.1Hz,1H),7.79-7.73(m,2H),7.65(t,J=7.6Hz,1 H),7.41(t,J=7.5Hz,1H),7.37-7.32(m,1H),7.31-7.25(m,2H),7.01(d,J=8.7Hz,1H), 6.86(d,J=8.4Hz,1H),6.73(d,J=8.4Hz,1H),4.95(s,2H),4.57(d,J=13.3Hz,1H),3.72 (d,J=13.2Hz,1H),2.90(p,J=6.7Hz,1H),0.79(d,J=6.8Hz,3H),0.01(d,J=6.7Hz,3H). 13 CNMR (100MHz, CDCl 3 )δ150.97,149.09,142.59,142.20,138.12,135.46,135.22,131.80,130.58,130.09,129.56,129.22,128.93,128.50 ,126.91,126.39,126.12,125.90,125.33,123.93,123.26,118.30,115.98,31.31,29.90,24.76,22.27.HRMS(ESI)m / z Calcd forC 28 H 23 BrNaO 2 + [M+Na] + :493.0881,Found:493.0889.Optical Rotation: (c=0.065, acetone). HPLC analysis: Chiralcel IB-H (Hexane / i-PrOH=85:15, flow rate=1.0mL / min, wave length=254nm), t R =8.763min(minor),t R =10.273min(major),91%ee.

[0064] Compound 3d: white solid; yield: 78% (37 mg, 0.1 mmol scale); 1 H NMR (400 MHz, Acetone-d 6 )δ8.38(d,J=8.5Hz,2H),8.22(s,1H),7.85(d,J=8.1Hz,1H),7.78(d,J=8.8Hz,1H),7.73(dd,J=5.3,3.6Hz,1H),7 .61(t,J=7.6Hz,1H),7.40-7.31(m,2H),7.24(dd,J=5.6,3.4Hz,2H),7.14(d,J=8.8Hz,1H),6.73(d,J=8.2Hz,1H) ,6.58(d,J=8.2Hz,1H),4.53(d,J=12.3Hz,1H),3.88(d,J=12.3Hz,1H),2.12(td,J=12.6,11.9,4.5Hz,1H),1.93( ddt,J=22.6,14.6,8.4Hz,1H),0.86(tt,J=11.8,6.1Hz,1H),0.06(dq,J=12.5,6.6Hz,1H),-0.05(t,J=7.1Hz,3H). 13 CNMR(100MHz,Acetone-d 6 )δ151.96,149.97,144.23,138.93,138.15,136.71,133.02,132.62,132.23,129.91,129.75,128.73,128.61,128.42,127 .77,127.20,126.79,125.85,125.70,125.51,124.08,122.53,118.00,114.35,35.84,31.03,24.72,12.87.HRMS(ESI)m / z Calcd for C 28 H 23 BrNaO 2 + [M+Na] + :493.0881,Found:493.0889.Optical Rotation: (c=0.25, acetone). HPLC analysis: Chiralcel IA-H (Hex ane / i-PrOH=80:20, flow rate=1.0mL / min, wave length=254nm), tR =5.858min(major),t R =7.792min(minor),93%ee.

[0065] Compound 3e: white solid; yield: 80% (41 mg, 0.1 mmol scale); 1 H NMR (400 MHz, Acetone-d 6 )δ8.41(d,J=8.5Hz,1H),8.34(s,1H),8.20(s,1H),7.85(d,J=8.1Hz,1H),7.76(d,J=8.8Hz,1H),7.75-7.71(m,1H),7.66(t,J=7.6Hz,1 H),7.43-7.30(m,2H),7.23(dd,J=5.5,3.4Hz,2H),7.11(d,J=8.8Hz,1H),6.77(d,J=8.4Hz,1H),6.69(d,J=8.4Hz,1H),4.54(d,J=12.4H z,1H),3.88(d,J=12.4Hz,1H),2.69(tt,J=11.6,2.8Hz,1H),1.44(d,J=15.0Hz,1H),1.27(d,J=12.5Hz,1H),1.20-1.12(m,1H),1.07(t d,J=12.3,3.0Hz,1H),0.98-0.88(m,1H),0.84-0.73(m,2H),0.65(qd,J=12.6,3.3Hz,1H),0.02(d,J=12.0Hz,1H),-0.70--0.85(m,1H). 13 C NMR (100 MHz, Acetone-d 6 )δ151.87,149.80,144.30,138.97,138.44,138.28,136.39,132.54,132.21,129.95,129.73,128.73,128.49,127.75,126.61, 125.95,125.79,125.57,124.30,122.57,117.94,114.56,38.84,34.74,33.76,31.12,26.40,25.76,24.98.HRMS(ESI)m / zCalcd for C 31 H 27 BrNaO 2 + [M+Na] +: 533.1194, Found: 533.1199. Optical Rotation: (c = 0.125, acetone). HPLC analysis: Chiralcel IA-H (Hexane / i-PrOH = 80:20, flow rate = 1.0 mL / min, wavelength = 254 nm), t R = 5.006 min (major), t R = 6.656 min (minor), 89% ee. Compound 3g: White solid; Yield: 77% (35.5 mg, 0.1 mmol scale); 1 H NMR (400 MHz, Acetone-d 6 ) δ 8.60 (s, 1H), 8.31 (d, J = 8.5 Hz, 1H), 7.98 (s, 1H), 7.78 (dd, J = 15.9, 8.4 Hz, 3H), 7.54 (t, J = 7.6 Hz, 1H), 7.41 - 7.35 (m, 1H), 7.34 - 7.25 (m, 3H), 7.15 (d, J = 8.8 Hz, 1H), 6.79 (q, J = 8.5 Hz, 2H), 4.55 (d, J = 12.4 Hz, 1H), 3.91 (d, J = 12.4 Hz, 1H). 13 C NMR (100 MHz, Acetone-d 6 ) δ 151.20, 150.98, 143.39, 137.70, 137.33, 136.08, 134.56, 133.79, 130.18, 129.90, 129.17, 128.67, 128.29, 128.13, 127.98, 127.37, 125.29, 123.57, 122.68, 122.40, 117.72, 115.27, 31.17. HRMS (ESI) m / z Calcd for C 25 H 16 BrClNaO 2 + [M + Na] + : 485.0022, Found: 485.0029. Optical Rotation: (c = 0.5, acetone). HPLC analysis: Chiralcel IA-H (Hexane / i-PrOH = 80:20, flow rate = 1.0 mL / min, wavelength = 254 nm), t R=8.526min(major),t R =13.162min(minor),93%ee.

[0066] Compound 3f: white solid; yield: 77% (37 mg, 0.1 mmol scale); 1 H NMR (400 MHz, Acetone-d 6 )δ8.54(s,1H),8.32(d,J=8.5Hz,1H),7.90(s,1H),7.85(d,J=8.1Hz,1H),7.80(d,J=8.8Hz,1H),7.78-7.72(m,1H),7.60(t,J=7.5Hz,1H),7.3 6(dt,J=11.9,6.7Hz,2H),7.31-7.21(m,2H),7.18(d,J=8.8Hz,1H),6.70(s,1H),4.59(d,J=12.4Hz,1H),3.91(d,J=12.4Hz,1H),1.50(s,3H). 13 C NMR (100 MHz, CDCl 3 )δ150.33,145.23,141.88,137.76,135.14,134.99,132.35,131.60,130.73,130.65,130.62,130.11,129.50,128.91 ,128.69,128.63,127.14,126.44,126.24,125.85,123.39,122.46,119.75,118.14,32.05,22.10.HRMS(ESI)m / zCalcd for C 26 H 18 BrClNaO 2 + [M+Na] + :499.0178,Found:499.0183.Opti cal Rotation: (c=0.115, acetone). HPLC analysis: Chiralcel IA-H (Hexane / i-PrOH=80:20, flow rate=1.0mL / min, wave length=254nm), t R =5.314min(major),t R =11.969min(minor),92%ee.

[0067] Compound 3j: white solid; yield: 78% (36 mg, 0.1 mmol scale); 1 H NMR (400 MHz, Acetone-d 6 )δ8.50(s,1H),8.32(d,J=7.9Hz,2H),7.85(d,J=8.1Hz,1H),7.80(d,J=8.8Hz ,1H),7.60(t,J=7.6Hz,1H),7.47(dd,J=10.0,2.6Hz,1H),7.41-7.31(m,2H),7 .16(d,J=8.8Hz,1H),7.05(td,J=8.5,2.6Hz,1H),6.71(d,J=8.1Hz,1H),6.54 (d,J=8.1Hz,1H),4.55(d,J=12.5Hz,1H),3.78(d,J=12.5Hz,1H),1.50(s,3H). 13 CNMR (100MHz, CDCl 3 )δ160.91(d,J=242Hz),150.39,149.41,139.27(d,J=8Hz),138.24(d,J=3Hz),136.65,135.84,132.20,130.74,130.70,130.39,130. 02,128.71,128.60,127.24,127.18,126.47,123.42,122.54,118.26,116.76(d,J=23Hz),116.49(d,J=22Hz),115.43,30.50,22.22. 19 F NMR (564MHz, CDCl 3 )δ-116.45.HRMS(ESI)m / zCalcd for C 26 H 18 BrFNaO 2 + [M+Na] + :483.0474,Found:483.0479.Optic al Rotation: (c=0.125, acetone). HPLC analysis: Chir alcel IA-H (Hexane / i-PrOH=80:20, flow rate=1.0mL / min, wavelengt h=254nm),t R =6.870min(major),t R =14.304min(minor),96%ee.

[0068] Example 5

[0069] 1. Cell culture This study used eight cancer cell lines: A375, Hela, HT29, HepG2, HCT116, 4T1, MCF-7, and MIAPaca-2. All cell lines were grown in Dulbecco's modified Eagle's medium (DMEM, Hyclone), except for the A375 cell line, which was grown in DMEM 12430 (Invitrogen) supplemented with sodium pyruvate (Invitrogen). All culture media were supplemented with 10% fetal bovine serum (FBS, BI) and 100 units / ml penicillin-streptomycin (Sigma-Aldrich) and incubated at 37°C / 5% CO 2 Cultivate in a humid environment.

[0070] 2. Antiproliferation Assay The antiproliferative activity of the prepared compounds on the above cell lines was evaluated using a standard (MTT) colorimetric method. The cell lines were grown to the logarithmic phase in DMEM supplemented with 10% fetal bovine serum. A cell suspension was prepared and dispensed into a 96-well plate at a dose of 100 μL / well to obtain 2×10 3 After 24 h of culture, cells were treated with 1.56, 3.125, 6.25, 12.5, 25, and 50 μM of the target compound and then incubated at 37°C / 5% CO 2 Cultured in a humidified environment for 48 hours. Then, the cell viability was assessed by the conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction method strictly following the manufacturer's instructions (Sigma). The absorbance (OD570) was read on a Spectramax microplate reader (Molecular Devices, USA). In all experiments, three replicate wells were used for each drug concentration. Each test was performed at least three times.

[0071] 3. Cell cycle analysis The cells were then cultured with different concentrations (0, 3, 6, and 9 μM) of compound 2m for 36 hours, or with 16 μM compound 2m for different times (0, 12, 24, and 36 hours). After treatment, the cells were centrifuged at 4°C, 1500 rpm for 5 minutes, fixed in 70% ethanol at 4°C for at least 12 hours, and then resuspended in phosphate-buffered saline (PBS) containing 0.1 mg / mL RNaseA and 5 mg / mL propidium iodide (PI). Cellular DNA content was measured by flow cytometry, and cell cycle distribution analysis was performed, with at least 10,000 events plotted for each sample. Flowjo 10 software was used to determine the percentage of cells in the sub-G0 / G1, G0 / G1, S, and G2 / M phases of the cell cycle.

[0072] 4. Apoptosis analysis Approximately 105 cells / well A375 cells were cultured in a 12-well plate and allowed to adhere. Subsequently, the culture medium was replaced with fresh culture medium containing different concentrations (0, 3, 6 and 9 μM) of compound 2m for 24 hours, or with fresh culture medium containing 16 μM compound 2m for different times (0, 12, 24 and 36 hours). Untreated wells received an equal volume of ethanol (<0.1%). After treatment, the cells were trypsinized, rinsed with PBS and centrifuged at 2000 rpm for 5 minutes. The precipitate was resuspended in 500 μL staining solution (binding buffer containing 5 μL Annexin V-FITC and 5 μL PI (5 mg / mL)), gently mixed and incubated in the dark at room temperature for 15 minutes. The samples were then analyzed using a FACSCalibur flow cytometer (Becton Dickinson, USA).

[0073] 5. ROS measurement 2,7-dichlorofluorescein diacetate (DCFH-DA) was used to detect intracellular ROS levels. A375 cells were seeded into 6-well plates (106 cells / well) for 24 hours and then treated with compound 2m (0, 3, 6 and 9 μM) for 12 hours. After treatment, 400 mL of 10 μm / L DCFH-DA was added to each well and incubated for 20 minutes according to the manufacturer's instructions (Thermo Fisher Scientific, USA). The stained cells were washed three times with serum-free culture medium and then analyzed by flow cytometry. ROS expression levels are expressed as fluorescence intensity.

[0074] 6. Mitochondrial membrane potential assessment The mitochondrial transmembrane potential (ΔΨm) was measured using the JC-1 mitochondrial membrane potential detection kit (Beyotime Biotech). After 2 minutes of treatment, A375 cells were incubated with 5 μg / mL JC-1 (5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide) at 37°C for 20 minutes, then rinsed twice with PBS and placed in fresh medium without serum. Samples were analyzed using a FACSCalibur cell counter (Becton Dickinson) and a confocal laser scanning microscope (LeicaTCS SP8, Solms, Germany).

[0075] 7. Protein immunoblot analysis After treatment with compound 2m (0, 3, 6 and 9 μM) for 48 hours, A375 cells were harvested and lysed with RIPA buffer in an ice bath for 30 minutes, and then centrifuged at 12000 rpm for 10 minutes at 4°C. The supernatant was taken and the protein concentration was determined using a BCA detection kit, and then boiled at 100°C for 10 minutes. Equal amounts of protein (20 μg) were separated on a 10% or 15% SDS-PAGE gel and then transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% BSA, and then the primary antibody (1:500 dilution) was gently shaken overnight at 4°C. The immunoreactive bands were visualized using an ECL detection kit (Invitrogen, USA) according to the manufacturer's instructions. The intensity of the protein bands was quantified using Image Lab analysis software. The relative optical density value represents the protein expression level.

[0076] IC of the aromatization product 3g of compound 2m on cancer cells in 2m 50 More than 10μM.

[0077] Figure 1 In vitro MTT assays revealed that compound (Rc, aSc-c axis) 2m was the most effective. Overall, (Rc, aSc-c axis) 2m had significant antiproliferative effects on cancer cells including A375, Hela, HT29, HepG2, HCT116, 4T1, MCF-7, and MIAPaca-2, with single micromolar IC50 values ​​ranging from 1.46 to 5.04 μM. To clarify its mechanism of action, systematic flow cytometry analysis was performed. The study found that (Rc, aSc-c axis) 2m treatment induced cell cycle arrest and apoptosis in A375 in a dose-dependent and time-dependent manner. In addition, it also caused an increase in ROS levels in A375 cells. The percentage change from red to green fluorescence indicated that (Rc, aSc-c axis) 2m induced a dose-dependent decrease in mitochondrial membrane potential (0, 3, 6, and 9 μM), which was a result of ROS accumulation. In summary, (Rc, aSc-c axis) 2m has been shown to effectively inhibit the proliferation of various cancer cells and is associated with ROS-related apoptosis mechanisms.

Claims

1. A chiral spirocyclic phenol compound, characterized in that: The general structure is as follows: Where: R 1 -R 4 Each is independently selected from one or more of hydrogen, C1-C7 alkane, C1-C7 cycloalkane, C1-C6 alkoxy, halogen, nitrile, nitro, phenyl or substituted phenyl; the substituent in the substituted phenyl is C1-C4 alkyl, C1-C4 alkoxy, halogen.

2. A method for preparing chiral spirocyclic phenolic compounds by catalytic asymmetric dearomatization, characterized in that: The steps include: The first step: using compound 1 and a chiral catalyst as raw materials, reacting with NBS in an organic solvent at low temperature to obtain compound 2; Step 2: Compound 2 reacts with an acid in an organic solvent to undergo a ring-closing reaction to obtain a chiral spirocyclic phenol compound 3; The reaction equation is shown as follows: Where: R 1 -R 4 Each is independently selected from one or more of hydrogen, C1-C7 alkane, C1-C7 cycloalkane, C1-C6 alkoxy, halogen, nitrile, nitro, phenyl or substituted phenyl; the substituent in the substituted phenyl is C1-C4 alkyl, C1-C4 alkoxy, halogen.

3. The method for preparing chiral spirocyclic phenolic compounds by catalytic asymmetric dearomatization according to claim 2, characterized in that: The organic solvent is selected from tetrahydrofuran, dichloromethane, 1,2-dichloroethane, toluene, ether or acetone.

4. The method for preparing chiral spirocyclic phenolic compounds by catalytic asymmetric dearomatization according to claim 2, characterized in that: In the first step, the low temperature is -40°C to -80°C; the molar ratio of compound 1, NBS and chiral catalyst is 1:1-1.2:0.05-0.

20.

5. The method for preparing chiral spirocyclic phenolic compounds by catalytic asymmetric dearomatization according to claim 2, characterized in that: In the first step, the chiral catalyst is selected from the following structures:

6. The method for preparing chiral spirocyclic phenolic compounds by catalytic asymmetric dearomatization according to claim 2, characterized in that: In the second step, the acid is selected from trichloroacetic acid, trifluoroacetic acid or p-toluenesulfonic acid monohydrate.

7. The method for preparing chiral spirocyclic phenolic compounds by catalytic asymmetric dearomatization according to claim 2, characterized in that: In the second step, the reaction temperature is 0°C to 30°C, and the molar ratio of compound 2 to acid is 1:1-1.

2.

8. Use of compound 2 as claimed in claim 1 in the preparation of anticancer drugs.

9. The use of compound 2 according to claim 7 in the preparation of anticancer drugs, characterized in that: The anticancer drugs refer to eight types of cancer cells: A375, Hela, HT29, HepG2, HCT116, 4T1, MCF-7 and MIAPaca-2.

Images