A class of tubulin / AKT1 dual-targeting podophyllotoxin derivatives and their applications

Abstract

The present invention discloses a class of tubulin / AKT1 dual-targeted podophyllotoxin derivatives and their applications, belonging to the field of chemical pharmaceutical technology, and specifically relates to a class of podophyllotoxin ester derivative molecules and their applications in the preparation of anti-tumor drugs. The present invention connects podophyllotoxin and phenoxyacetic acid aniline carboxylic acid groups through ester bonds to obtain a class of podophyllotoxin ester derivative molecules by semi-synthesis, and in vitro anti-tumor activity studies show that this class of molecules has strong inhibitory activity on tumor cell lines and can simultaneously target tubulin and AKT1 proteins.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical pharmacy, and specifically relates to the preparation of a class of tubulin / AKT1 dual-targeting podophyllotoxin derivatives. Background Art

[0002] Podophyllotoxin (PPT) is a natural cyclolignan polysaccharide and the main component of podophyllotoxin resin. It shows strong cytotoxic activity against various cancer cell lines, but it has not been used in human clinical trials due to unacceptable toxic side effects. To overcome these limitations, researchers have focused on structural modifications at the 4-position of the cycloalkane (C ring) to generate derivatives with excellent pharmacological properties. Etoposide and teniposide are two semisynthetic epipodophyllotoxin derivatives that have been successfully used in clinical practice as antitumor drugs by inhibiting DNA-topoisomerase II. A new series of podophyllotoxin derivatives discovered in recent years can show antitumor activity by inhibiting the assembly of microtubules in cancer cells. However, their antitumor activity is still not strong (micromolar level), and targeting tubulin still has significant toxic side effects. Therefore, this study aims to design dual-targeted podophyllotoxin derivatives that simultaneously target tubulin and AKT1 based on the podophyllotoxin scaffold to discover some more effective antitumor drug molecules.

[0003] Summary of the invention

[0004] The purpose of the present invention is to provide a novel podophyllotoxin ester derivative and a synthesis method and application thereof.

[0005] The technical solution adopted by the present invention is as follows:

[0006] A class of podophyllotoxin ester derivatives has the following general formula:

[0007]

[0008] The range of substituents is: 1 , R 2 , R 3 , R 4 , R 5 Each is independently selected from H, Cl, F, CH(CH3)2, C(CH3)3, CH3, Br or OCH3; R 6 =H or CH3.

[0009] A method for preparing the above-mentioned podophyllotoxin ester derivatives comprises the following steps:

[0010] Step 1: Take phenoxyacetic acid (A) with different substitutions and HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate) in a round-bottom flask, add DMF solution, stir and dissolve at room temperature, add methyl p-aminophenylacetate (or methyl m-aminophenylacetate) and DIPEA (N,N-diisopropylethylamine), stir and react at room temperature for 6 hours, and detect the progress of the reaction by TCL. After the reaction is completed, the reaction solution is diluted with water and extracted with ethyl acetate, the organic phase is washed with saturated brine, dried over anhydrous sodium sulfate, and the organic solvent is removed by rotary evaporation to obtain a crude product of B1 (or B2), add a dichloromethane solution and silica gel powder, spin dry the sample, and separate the intermediate (B1 or B2) by column chromatography.

[0011]

[0012] Among them, the molar ratio of differently substituted phenoxyacetic acid (A), HATU, methyl p-aminophenylacetate (or methyl m-aminophenylacetate) and DIPEA (N, N-diisopropylethylamine) is 1:1.5:1:1.5.

[0013] Step 2: Dissolve the intermediate (B1 or B2) in a mixed solvent of tetrahydrofuran: methanol: water = 3:1:1 by volume, add LiOH·H2O, stir at room temperature for 3 hours, and monitor the progress of the reaction by TCL. After the reaction is completed, remove the organic solvent by rotary evaporation, acidify with hydrochloric acid to pH = 3, and solid precipitate. Filter and dry naturally to obtain the intermediate (C1 or C2).

[0014]

[0015] The molar ratio of the intermediate (B1 or B2) to LiOH·H2O is 1:4.

[0016] Step 3: The product C1 or C2 obtained in step 2 is mixed with podophyllotoxin in an ice bath and dissolved in dichloromethane. DCC (N,N'-dicyclohexylcarboximide) and DMAP (dimethylaminopyridine) are added and stirred for 30 minutes. After the reaction is completed, the target compound D1 or D2 is obtained by passing through a silica gel column.

[0017]

[0018] Wherein, the molar ratio of (C1 or C2) to podophyllotoxin is 1:1.

[0019] The molecular structural formula of the podophyllotoxin ester derivatives of the present invention is as follows:

[0020]

[0021]

[0022]

[0023] The present invention proves that the podophyllotoxin ester derivative molecules have significant antiproliferative activity against human non-small cell lung cancer cells H1975 and H1299 through in vitro tumor cell inhibition activity, and most of the compounds have a median lethal concentration (IC 50 The values ​​were all less than 10 μmol·L -1 , especially the IC of compound D1-1 on H1975 cells 50 The value is 0.102 μmol·L -1 , and its parent compound podophyllotoxin (IC 50 =12.56μmol·L -1 ) has an anti-tumor activity that is more than 100 times higher than that of Figure 1 , 2 From the structure-activity relationship comparison, podophyllotoxin can only bind to tubulin (colchicine binding site), while D1-1 can bind to the active site of podophyllotoxin and also has good affinity with the active site of AKT1. In addition, between the two targets, D1-1 is more inclined to bind to AKT1 protein, see Figure 3 .

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] In the present invention, the obtained podophyllotoxin ester derivatives have obvious tumor cell inhibitory activity compared with podophyllotoxin, and most of the novel compounds have significantly better inhibitory activity on human non-small cell lung cancer cell lines than their parent molecules. Previous studies on podophyllotoxin did not focus on the AKT1 target, but in this study, in order to further improve the anti-tumor activity of podophyllotoxin and reduce its toxic side effects, we simultaneously designed drug molecules for tubulin and AKT1 protein, aiming to obtain dual-target molecules that can simultaneously target the above two targets. This study intends to conduct an in-depth exploration of the drug mechanism of action by flow cytometry, western blot and laser confocal microscopy. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 , CCK8 assay was used to detect the in vitro proliferation inhibitory activity of podophyllotoxin ester derivatives D1-1 to D2-10 on human lung cancer cell lines H1975 and H1299;

[0027] Figure 2 , CCK8 assay was used to detect the in vitro proliferation inhibitory activity of podophyllotoxin ester derivatives D1-1 to D2-10 on the human lung epithelial cell line BEAS-2B;

[0028] Figure 3, EdU detection method was used to detect the in vitro proliferation inhibitory activity of podophyllotoxin ester derivative D1-1 on human lung cancer cell line H1975;

[0029] Figure 4 , biolayer interferometry (BLI) technology was used to detect the affinity of podophyllotoxin ester derivative D1-1 to tubulin and AKT1 protein. DETAILED DESCRIPTION

[0030] A method for preparing podophyllotoxin ester derivatives, comprising the following steps:

[0031] Step 1, take 1.5mmol of differently substituted phenoxyacetic acid (A) and 2.25mmol (1.5eq) HATU in a 25mL round-bottom flask, add 5mL DMF solution, stir and dissolve at room temperature, add 1.5mmol p-aminophenylacetic acid methyl ester (or 1.5mmol m-aminophenylacetic acid methyl ester) and 2.25mmol (1.5eq) DIPEA, stir and react at room temperature for 6h, and detect the reaction progress by TCL. After the reaction is completed, the reaction solution is diluted with 30mL water and extracted with ethyl acetate twice, the organic phase is washed twice with saturated brine, dried over anhydrous sodium sulfate, and the organic solvent is removed by rotary evaporation to obtain a crude product of B1 (or B2), add 10mL of dichloromethane solution and 930mg of silica gel powder, spin dry and prepare a sample, and separate by column chromatography to obtain an intermediate (B1 or B2).

[0032]

[0033] Dissolve 1 mmol of intermediate (B1 or B2) in a 3:1:1 mixed solvent (5 mL) of tetrahydrofuran: methanol: water, add 4 mmol of LiOH·H2O, stir at room temperature for 3 h, and monitor the reaction progress by TCL. After the reaction is completed, remove the organic solvent by rotary evaporation and use 0.1 mol﹒ L -1 Acidify with hydrochloric acid to pH=3, solid precipitates, filter and dry naturally to obtain the intermediate (C1 or C2).

[0034]

[0035] Under ice bath conditions, intermediate C1 (or C2) (0.5 mmol), DCC (1 mmol), purified dichloromethane (20 mL), catalyst DMAP (0.25 mmol) and podophyllotoxin (0.5 mmol) were added to a 50 mL round-bottom flask in sequence and stirred for 1 hour. The reaction was complete when detected by TLC. The mixture was separated by thin plate chromatography (V 乙酸乙酯 :V 石油醚 =1:1) to obtain the corresponding podophyllotoxin-like derivatives (D1 or D2).

[0036]

[0037] Example 1

[0038]

[0039] The preparation method of compound D1-1 is the same as that of compound D1, except that the differently substituted phenoxyacetic acid (A) is replaced with m-chlorophenoxyacetic acid, and other conditions remain unchanged to obtain compound D1-1 in the form of white powder with a yield of 84%. Compound D1-1: 1 H NMR (600MHz, CDCl3) δ8.31(s,1H,NH),7.59(d,J=8.3Hz,2H,Ar-H),7.31–7.24(m,3H,Ar-H),7.04(d,J=7.9Hz,1H,Ar-H),7.01(d,J=1.9Hz,1H,A r-H),6.88(d,J=8.3Hz,1H,Ar-H),6.66(s,1H,Ar-H),6.50(d,J=17.7Hz,1H,Ar-H),6.37(d,J=12.9Hz,2H,Ar-H),5.97(d,J=15.7Hz,2H,O-CH2-O ),5.86(d,J=9.3Hz,1H,CH-OC=O),4.58(dd,J=9.8,3.1Hz,3H,O-CH2-C=O,Ar-CH),4.24(t,J=8.1Hz,1H,CH2-OC=O),4.16–4.09(m,1H,CH2-OC=O ),3.80(s,3H,OCH3),3.73(s,6H,OCH3),3.71(d,J=1.6Hz,2H,Ar-CH2-C=O),2.89(dd,J=15.5,3.5Hz,1H,O=C-CH),2.82–2.73(m,1H,O-CH2-CH).

[0040] Example 2

[0041]

[0042] The preparation method of compound D1-2 is the same as that of compound D1, except that the differently substituted phenoxyacetic acid (A) is replaced with p-chlorophenoxyacetic acid, and other conditions remain unchanged to obtain compound D1-2 in the form of white powder with a yield of 82%. Compound D1-2: 1H NMR (600MHz, CDCl3) δ8.25 (s, 1H, NH), 7.58 (d, J = 8.5Hz, 2H, Ar-H), 7.30 (dd, J = 13.4, 8.7Hz, 4H, Ar-H), 6.94 (d, J = 9.0Hz, 2H, Ar-H), 6.66(s,1H,Ar-H),6.53(s,1H,Ar-H),6.36(s,2H,Ar-H),5.98(dd,J=16.3,1.2Hz,2H,O-CH2-O),5.87(d,J=9.3Hz,1H,CH-OC=O),4.5 8(d,J=4.7Hz,3H,O-CH2-C=O,Ar-CH),4.24(dd,J=9.2,7.2Hz,1H,CH2-OC=O),4.14(d,J=9.4Hz,1H,CH2-OC=O),3.81(s,3H,OCH3),3. 74(s,6H,OCH3),3.71(d,J=1.9Hz,2H,Ar-CH2-C=O),2.90(dd,J=14.6,4.5Hz,1H,O=C-CH),2.78(dt,J=16.6,10.0Hz,1H,O-CH2-CH).

[0043] Example 3

[0044]

[0045] The preparation method of compound D1-3 is the same as that of compound D1, except that the differently substituted phenoxyacetic acid (A) is replaced with p-fluorophenoxyacetic acid, and other conditions remain unchanged to obtain compound D1-3 in the form of white powder with a yield of 80%. Compound D1-3: 1H NMR (600MHz, CDCl3) δ8.36(s,1H,NH),7.57(d,J=8.4Hz,1H,Ar-H),7.29(s,2H,Ar-H),7.12(s,2H,Ar-H),7.03(t,J= 8.6Hz,1H,Ar-H),6.94(dd,J=7.9,5.4Hz,1H,Ar-H),6.68–6.34(m,5H,Ar-H),5.90(dd,J=53.8,9.7Hz,3H,O-CH2-O, CH-OC=O),4.56(d,J=3.4Hz,3H,O-CH2-C=O,Ar-CH),4.11(q,J=7.1Hz,1H,CH2-OC=O),4.01(t,J=9.1Hz,1H,CH2-OC= O),3.79(s,3H,OCH3),3.73(s,6H,OCH3),3.71(d,J=2.9Hz,2H,Ar-CH2-C=O),2.81–2.75(m,2H,O=C-CH,O-CH2-CH).

[0046] Example 4

[0047]

[0048] The preparation method of compound D1-4 is the same as that of compound D1, except that the differently substituted phenoxyacetic acid (A) is replaced with 2-methyl-4-chlorophenoxyacetic acid, and other conditions remain unchanged to obtain compound D1-4 in the form of white powder with a yield of 87%. Compound D1-4: 1HNMR (600MHz, CDCl3) δ8.29(s,1H,NH),7.57(d,J=8.5Hz,2H,Ar-H),7.29(d,J=8.5Hz,2H,Ar-H),7.19(d,J=2.3Hz,1H,Ar-H),7.15(dd,J=8.6,2.5Hz,1H ,Ar-H),6.77(d,J=8.7Hz,1H,Ar-H),6.66(s,1H,Ar-H),6.53(s,1H,Ar-H),6 .36(s,2H,Ar-H),5.98(dd,J=16.0,1.2Hz,2H,O-CH2-O),5.87(d,J=9.3Hz,1 H,CH-OC=O),4.58(d,J=4.2Hz,3H,O-CH2-C=O,Ar-CH),4.24(dd,J=9.2,7.2Hz,1H,CH2-OC=O),4.18–4.12(m,1H,CH2-OC=O),3.81(s,3H,OCH3),3.74(s,6 H,OCH3),3.72(d,J=2.8Hz,2H,Ar-CH2-C=O),2.90(dd,J=14.6,4.5Hz,1H,O=C-CH),2.78(ddd,J=20.1,12.5,8.4Hz,1H,O-CH2-CH),2.34(s,3H,Ar-CH3).

[0049] Example 5

[0050]

[0051] The preparation method of compound D1-5 is the same as that of compound D1, except that the differently substituted phenoxyacetic acid (A) is replaced with 2,5-dimethylphenoxyacetic acid, and other conditions remain unchanged to obtain compound D1-5 in the form of white powder with a yield of 79%. Compound D1-5: 1HNMR (600MHz, CDCl3) δ8.39 (s, 1H, NH), 7.59 (d, J = 8.5Hz, 2H, Ar-H), 7.29 (d, J = 8.4Hz, 2H, Ar-H), 7.08 (d, J = 7.6Hz, 1H, Ar-H), 6.78 (d, J = 7.5Hz, 1H,Ar-H),6.65(s,2H,Ar-H),6.52(s,1H,Ar-H),6.36(s,2H,Ar-H),5.96(dd,J=17.5,1.2Hz,2H,O-CH2-O),5.85(d,J=9.2Hz,1H,CH-OC=O),4.5 9(s,2H,O-CH2-C=O),4.57(d,J=4.4Hz,1H,Ar-CH),4.25(dd,J=9.2,7.1Hz,1H,CH2-OC=O),4.12(dt,J=14.1,8.6Hz,1H,CH2-OC=O),3.80(s,3H, OCH3),3.73(s,6H,OCH3),3.71(d,J=2.4Hz,2H,Ar-CH2-C=O),2.90–2.74(m,2H,O=C-CH,O-CH2-CH),2.32(s,3H,Ar-CH3),2.31(s,3H,Ar-CH3).

[0052] Example 6

[0053]

[0054] The preparation method of compound D1-6 is the same as that of compound D1, except that the differently substituted phenoxyacetic acid (A) is replaced with p-isopropylphenoxyacetic acid, and other conditions remain unchanged to obtain compound D1-6 in the form of white powder with a yield of 85%. Compound D1-6: 1HNMR(600MHz,CDCl3)δ8.33(s,1H,NH),7.59(d,J=8.5Hz,2H,Ar-H),7.28(d,J=8.5Hz,2H,Ar-H),7.20(d,J=8.6Hz,2H,Ar-H),6.92(d,J=8.7Hz,2H,Ar-H), 6.65(s,1H,Ar-H),6.52(s,1H,Ar-H),6.36(s,2H,Ar-H),5.97(dd,J=16.9,1. 2Hz,2H,O-CH2-O),5.86(d,J=9.3Hz,1H,CH-OC=O),4.59(s,2H,O-CH2-C=O),4 .58(d,J=4.5Hz,1H,Ar-CH),4.25(dd,J=9.2,7.1Hz,1H,CH2-OC=O),4.14(dd ,J=16.1,6.7Hz,1H,CH2-OC=O),3.81(s,3H,OCH3),3.74(s,6H,OCH3),3.71(d ,J=1.9Hz,2H,Ar-CH2-C=O),2.89(dt,J=20.8,6.5Hz,2H,O=C-CH,O-CH2-CH), 2.82–2.74(m,1H,CH(CH3)2),1.24(s,3H,CH(CH3)2),1.23(s,3H,CH(CH3)2).

[0055] Example 7

[0056]

[0057] The preparation method of compound D1-7 is the same as that of compound D1, except that the differently substituted phenoxyacetic acid (A) is replaced with p-isobutylphenoxyacetic acid, and other conditions remain unchanged to obtain compound D1-7 in the form of white powder with a yield of 82%. Compound D1-7: 1HNMR (600MHz, CDCl3) δ8.30 (s, 1H, NH), 7.51 (d, J = 8.4Hz, 1H, Ar-H), 7.28 (d, J = 8.7Hz, 2H, Ar-H), 7.20 (d, J = 7.2Hz, 2H, A r-H),7.06(s,1H,Ar-H),6.84(d,J=8.7Hz,1H,Ar-H),6.50(d,J=81.1Hz,1H,Ar-H),6.33(t,J=36.9Hz,4H,Ar-H),5.88( t,J=12.5Hz,3H,O-CH2-O,CH-OC=O),4.52–4.45(m,3H,O-CH2-C=O,Ar-CH),4.20–4.00(m,2H,CH2-OC=O),3.72(s,3H,OC H3),3.65(s,6H,OCH3),3.63(d,J=2.6Hz,2H,Ar-CH2-C=O),2.75–2.66(m,2H,O=C-CH,O-CH2-CH),1.23(s,9H,C(CH3)3).

[0058] Example 8

[0059]

[0060] The preparation method of compound D1-8 is the same as that of compound D1, except that the differently substituted phenoxyacetic acid (A) is replaced with p-methylphenoxyacetic acid, and other conditions remain unchanged to obtain compound D1-8 in the form of white powder with a yield of 78%. Compound D1-8: 1HNMR (600MHz, CDCl3) δ8.35(s,1H,NH),7.59(d,J=8.4Hz,2H,Ar-H),7.27(d,J=8.5Hz,2H,Ar-H),7.13(d,J=8.4Hz,2H,Ar-H),6.88(d,J= 8.5Hz,2H,Ar-H),6.65(s,1H,Ar-H),6.51(s,1H,Ar-H),6.36(s,2H,Ar-H),5.96(d,J=16.7Hz,2H,O-CH2-O),5.85(d,J=9.2Hz,1H,CH-OC= O),4.56(s,3H,O-CH2-C=O,Ar-CH),4.26–4.21(m,1H,CH2-OC=O),4.12(dt,J=11.5,8.5Hz,1H,CH2-OC=O),3.80(s,3H,OCH3),3.73(s,6H ,OCH3),3.70(s,2H,Ar-CH2-C=O),2.86(dd,J=14.6,4.4Hz,1H,O=C-CH),2.77(ddd,J=16.6,9.9,7.3Hz,1H,O-CH2-CH),2.30(s,3H,CH3).

[0061] Example 9

[0062]

[0063] The preparation method of compound D1-9 is the same as that of compound D1, except that the differently substituted phenoxyacetic acid (A) is replaced with 2,3-dimethylphenoxyacetic acid, and other conditions remain unchanged to obtain compound D1-9 in the form of white powder with a yield of 83%. Compound D1-9: 1HNMR (600MHz, CD2Cl2) δ8.36(s,1H,NH),7.59(d,J=8.4Hz,2H,Ar-H),7.29(d,J=8.4Hz,2H,Ar-H),7.09(t,J=7.9Hz,1H,Ar-H),6.89(d,J=7.6Hz,1H,Ar-H ),6.73(d,J=8.1Hz,1H,Ar-H),6.65(s,1H,Ar-H),6.52(s,1H,Ar-H),6.36(s ,2H,Ar-H),5.98(d,J=16.4Hz,2H,O-CH2-O),5.87(d,J=9.3Hz,1H,CH-OC=O) ,4.60(s,2H,O-CH2-C=O),4.58(d,J=4.5Hz,1H,Ar-CH),4.25(dd,J=9.1,7.3 Hz,1H,CH2-OC=O),4.15(t,J=8.6Hz,1H,CH2-OC=O),3.81(s,3H,OCH3),3.74 (s,6H,OCH3),3.71(d,J=2.5Hz,2H,Ar-CH2-C=O),2.90(dd,J=14.5,4.5Hz,1 H,O=C-CH),2.83–2.75(m,1H,O-CH2-CH),2.32(s,3H,CH3),2.28(s,3H,CH3).

[0064] Example 10

[0065]

[0066] The preparation method of compound D1-10 is the same as that of compound D1, except that the differently substituted phenoxyacetic acid (A) is replaced with o-chlorophenoxyacetic acid, and other conditions remain unchanged to obtain compound D1-10 in the form of white powder with a yield of 75%. Compound D1-10: 1H NMR (600MHz, CDCl3) δ8.70 (s, 1H, NH), 7.62 (d, J = 8.4Hz, 2H, Ar-H), 7.53 (d, J = 8.6Hz, 1H, Ar-H), 7.45 (dd, J = 7.9, 1.4Hz, 1H, Ar-H), 7.37–7.3 4(m,1H,Ar-H),7.30(d,J=8.5Hz,3H,Ar-H),6.65(s,1H,Ar-H),6.52(s,1H,Ar-H),6.36(s,2H,Ar-H),5.98(d,J=18.5Hz,2H,O-CH2-O),5.87 (d,J=9.3Hz,1H,CH-OC=O),4.66(s,2H,O-CH2-C=O),4.58(d,J=4.4Hz,1H,Ar-CH),4.27(dd,J=9.2,7.2Hz,1H,CH2-OC=O),4.18–4.13(m,1H, CH2-OC=O),3.81(s,3H,OCH3),3.74(s,6H,OCH3),3.72(d,J=2.1Hz,2H,Ar-CH2-C=O),2.90(dd,J=14.6,4.5Hz,1H,O=C-CH),2.85–2.73(m,1H O-CH2-CH).

[0067] Embodiment 11

[0068]

[0069] The preparation method of compound D2-1 is the same as that of compound D2, except that the differently substituted phenoxyacetic acid (A) is replaced with m-chlorophenoxyacetic acid, and other conditions remain unchanged to obtain compound D2-1 in the form of white powder with a yield of 85%. Compound D2-1: 1H NMR (600MHz, CDCl3) δ8.23(s,1H,NH),7.73(s,1H,Ar-H),7.42(d,J=9.2Hz,1H,Ar-H),7.36–7.29(m,2H,Ar-H),7.10(d,J=7.7Hz,1H,Ar-H),7.06(dd ,J=7.6,1.4Hz,1H,Ar-H),7.02(dd,J=4.7,2.6Hz,1H,Ar-H),6.89(dd,J=8.3,2.3Hz,1H,Ar-H),Ar-H,6.69(s,1H,Ar-H),6.53(s,1H,Ar-H),6.37(s, 2H,Ar-H),5.96(dd,J=11.1,1.3Hz,2H,O-CH2-O),5.88(d,J=9.2Hz,1H,CH-OC=O),4.59(s,3H,Ar-CH,O-CH2-C=O),4.30(dd,J=9.2,7.0Hz,1H,CH2-O C=O),4.19–4.14(m,1H,CH2-OC=O),3.81(s,3H,OCH3),3.75(d,J=4.4Hz,2H,Ar-CH2-C=O),3.74(s,6H,OCH3),2.93–2.78C(m,2H,O=C-CH,O-CH2-CH).

[0070] Example 12

[0071]

[0072] The preparation method of compound D2-2 is the same as that of compound D2, except that the differently substituted phenoxyacetic acid (A) is replaced with p-chlorophenoxyacetic acid, and other conditions remain unchanged to obtain compound D2-2 in the form of white powder with a yield of 81%. Compound D2-2: 1H NMR (600MHz, CDCl3) δ8.30(s,1H,NH),7.72(s,1H,Ar-H),7.42(d,J=7.1Hz,1H,Ar-H),7.35–7.28(m,3H,Ar-H),7.09(d,J=7.6Hz,1H,Ar-H ),6.93(d,J=9.0Hz,2H,Ar-H),6.68(s,1H,Ar-H),6.52(s,1H,Ar-H),6.36(s,2H,Ar-H),5.96(dd,J=11.2,1.2Hz,2H,O-CH2-O),5.87(d,J =9.2Hz,1H,CH-OC=O),4.58(d,J=4.9Hz,3H,Ar-CH,O-CH2-C=O),4.29(dd,J=9.2,7.1Hz,1H,CH2-OC=O),4.18–4.13(m,1H,CH2-OC=O),3.8 0(s,3H,OCH3),3.75(d,J=3.9Hz,2H,Ar-CH2-C=O),3.73(s,6H,OCH3),2.90(dd,J=14.6,4.5Hz,1H,O=C-CH),2.86–2.77(m,1H,O-CH2-CH).

[0073] Example 13

[0074]

[0075] The preparation method of compound D2-3 is the same as that of compound D2, except that the differently substituted phenoxyacetic acid (A) is replaced with p-fluorophenoxyacetic acid, and other conditions remain unchanged to obtain compound D2-3 in the form of white powder with a yield of 80%. Compound D2-3: 1H NMR (600MHz, CDCl3) δ8.30(s,1H,NH),7.73(s,1H,Ar-H),7.42(dd,J=8.1,1.1Hz,1H,Ar-H),7.33(t,J=7.8Hz,1H,Ar-H),7.09(d,J=7.6Hz,1H,Ar-H),7 .06–7.02(m,2H,Ar-H),6.95(dd,J=9.2,4.2Hz,2H,Ar-H),6.69(s,1H,Ar-H ),6.52(s,1H,Ar-H),6.37(s,2H,Ar-H),5.96(dd,J=11.3,1.2Hz,2H,O-CH2- O),5.88(d,J=9.2Hz,1H,CH-OC=O),4.58(d,J=4.4Hz,1H,Ar-CH),4.57(s,2H,O-CH2-C=O),4.30(dd,J=9.2,7.0Hz,1H,CH2-OC=O),4.19–4.14(m,1H,CH 2-OC=O),3.80(s,3H,OCH3),3.75(d,J=4.1Hz,2H,Ar-CH2-C=O),3.73(s,6H ,OCH3),3.49–3.43(m,1H,O=C-CH),2.91(dd,J=14.6,4.5Hz,1H,O-CH2-CH).

[0076] Embodiment 14

[0077]

[0078] The preparation method of compound D2-4 is the same as that of compound D2, except that the differently substituted phenoxyacetic acid (A) is replaced with 2-methyl-4-chlorophenoxyacetic acid, and other conditions remain unchanged to obtain compound D2-4 in the form of white powder with a yield of 75%. Compound D2-4: 1H NMR(600MHz,CDCl3)δ8.29(s,1H,NH),7.75(s,1H,Ar-H),7.38–7.34(m,2H,Ar-H),7.19(d,J=2.4Hz,1H,Ar-H),7.15(dd,J=8.6,2.5Hz,1H,Ar-H), 7.11–7.08(m,1H,Ar-H),6.77(d,J=8.7Hz,1H,Ar-H),6.69(s,1H,Ar-H),6.52(s,1H,Ar-H),6.37(s,2H,Ar-H),5.95(dd,J=8.5,1.2Hz,2H,O-CH2-O ),5.87(d,J=9.2Hz,1H,CH-OC=O),4.58(d,J=4.4Hz,1H,Ar-CH),4.57(s,2H,O-CH2-C=O),4.30(dd,J=9.2,7.0Hz,1H,CH2-OC=O),4.16(t,J=9.8Hz ,1H,CH2-OC=O),3.80(s,3H,OCH3),3.75(d,J=4.5Hz,2H,Ar-CH2-C=O),3.73(s,6H,OCH3),2.93–2.78(m,2H,O=C-CH,O-CH2-CH),2.33(s,3H,CH3).

[0079] Embodiment 15

[0080]

[0081] The preparation method of compound D2-5 is the same as that of compound D2, except that the differently substituted phenoxyacetic acid (A) is replaced with 2,5-dimethylphenoxyacetic acid, and other conditions remain unchanged to obtain compound D2-5 in the form of white powder with a yield of 78%. Compound D2-5: 1H NMR (600MHz, CDCl3) δ8.38(d,J=16.1Hz,1H,NH),7.77(s,1H,Ar-H),7.56(s,1H,Ar-H),7.42–7.30(m,2H,Ar-H),7.09(d,J=7.5Hz,2H,Ar-H),6.79(d,J =7.5Hz,1H,Ar-H),6.69(s,1H,Ar-H),6.67(d,J=4.2Hz,1H,Ar-H),6.53(s, 1H,Ar-H),6.37(s,1H,Ar-H),5.95(dd,J=5.3,1.2Hz,2H,O-CH2-O),5.88(d, J=9.2Hz,1H,CH-OC=O),4.59(s,3H,Ar-CH,O-CH2-C=O),4.33–4.15(m,1H,CH2-OC=O),4.13(q,J=7.1Hz,1H,CH2-OC=O),3.81(s,3H,OCH3),3.76(d,J=5. 6Hz,2H,Ar-CH2-C=O),3.74(s,6H,OCH3),3.20(td,J=9.6,4.4Hz,1H,O=C-CH),2.94–2.79(m,1H,O-CH2-CH),2.33(s,3H,Ar-CH3),2.32(s,3H,Ar-CH3).

[0082] Example 16

[0083]

[0084] The preparation method of compound D2-6 is the same as that of compound D2, except that the differently substituted phenoxyacetic acid (A) is replaced with p-isopropylphenoxyacetic acid, and other conditions remain unchanged to obtain compound D2-6 in the form of white powder with a yield of 73%. Compound D2-6: 1H NMR (600MHz, CDCl3) δ8.34 (s, 1H, NH), 7.74 (s, 1H, Ar-H), 7.43 (d, J = 9.1Hz, 1H ,Ar-H),7.32(t,J=7.8Hz,1H,Ar-H),7.20(d,J=8.6Hz,2H,Ar-H),7.08(d,J=7 .6Hz,1H,Ar-H),6.92(d,J=8.6Hz,2H,Ar-H),6.68(s,1H,Ar-H),6.52(s,1H,A r-H), 6.37 (s, 2H, Ar-H), 5.95 (dd, J=10.5, 1.0Hz, 2H, O-CH2-O), 5.87 (d, J=9. 1Hz,1H,CH-OC=O),4.58(d,J=3.4Hz,3H,Ar-CH,O-CH2-C=O),4.30(dd,J=9.3,7.0Hz,1H,CH2-OC=O),4.16(t,J=9.8Hz,1H,CH2-OC=O),3.80(s,3H,OCH3),3. 74(d,J=7.9Hz,8H,Ar-CH2-C=O,OCH3),2.92–2.87(m,2H,O=C-CH,CH(CH3)2), 2.87–2.78(m,1H,O-CH2-CH),1.24(s,3H,CH(CH3)2),1.23(s,3H,CH(CH3)2).

[0085] Embodiment 17

[0086]

[0087] The preparation method of compound D2-7 is the same as that of compound D2, except that the differently substituted phenoxyacetic acid (A) is replaced with p-bromophenoxyacetic acid, and other conditions remain unchanged to obtain compound D2-7 in the form of white powder with a yield of 76%. Compound D2-7: 1H NMR(600MHz,CDCl3)δ8.20(s,1H,NH),7.65(s,1H,Ar-H),7.37(d,J=8.8Hz,2H,Ar-H),7.29–7.23(m,2H,Ar-H),7.03–6.99(m,1H,Ar-H),6.81(d, J=9.0Hz,2H,Ar-H),6.61(s,1H,Ar-H),6.45(s,1H,Ar-H),6.29(s,2H,Ar-H),5.88(dd,J=11.3,1.2Hz,2H,O-CH2-O),5.80(d,J=9.2Hz,1H,CH-OC =O),4.51(d,J=4.4Hz,1H,Ar-CH),4.49(s,2H,O-CH2-C=O),4.22(dd,J=9.2,7.0Hz,1H,CH2-OC=O),4.11–4.05(m,1H,CH2-OC=O),3.73(s,3H,OCH 3), 3.67(d,J=3.8Hz,2H,Ar-CH2-C=O), 3.66(s,6H,OCH3), 2.82(dd,J=14.6,4.5Hz,1H,O=C-CH), 2.75(ddd,J=21.6,12.4,8.7Hz,1H,O-CH2-CH).

[0088] Embodiment 18

[0089]

[0090] The preparation method of compound D2-8 is the same as that of compound D2, except that the differently substituted phenoxyacetic acid (A) is replaced with p-methoxyphenoxyacetic acid, and other conditions remain unchanged to obtain compound D2-8 in the form of white powder with a yield of 77%. Compound D2-8: 1H NMR (600MHz, CDCl3) δ8.37(s,1H,NH),7.74(s,1H,Ar-H),7.42(d,J=8.3Hz,1H,Ar-H),7.32(t,J=7.9Hz,1H,Ar-H),7.08(d,J=7.6Hz,1H,Ar-H),6.92(d, J=9.1Hz,2H,Ar-H),6.86(d,J=9.0Hz,2H,Ar-H),6.68(s,1H,Ar-H),6.51(s ,1H,Ar-H),6.36(s,2H,Ar-H),5.95(d,J=10.9Hz,2H,O-CH2-O),5.86(d,J=9 .1Hz,1H,CH-OC=O),4.57(d,J=4.0Hz,1H,Ar-CH),4.53(s,2H,O-CH2-C=O),4.29(t,J=8.1Hz,1H,CH2-OC=O),4.15(t,J=9.5Hz,1H,CH2-OC=O),3.80(s,3 H,OCH3),3.77(s,3H,OCH3),3.74(d,J=4.9Hz,2H,Ar-CH2-C=O),3.73(s,6H,OCH3),2.88(dd,J=14.6,4.3Hz,1H,O=C-CH),2.85–2.77(m,1H,O-CH2-CH).

[0091] Embodiment 19

[0092]

[0093] The preparation method of compound D2-9 is the same as that of compound D2, except that the differently substituted phenoxyacetic acid (A) is replaced with m-methoxyphenoxyacetic acid, and other conditions remain unchanged to obtain compound D2-9 in the form of white powder with a yield of 81%. Compound D2-9: 1H NMR (600MHz, CDCl3) δ8.33 (s, 1H, NH), 7.73 (s, 1H, Ar-H), 7.43 (d, J = 9.1Hz, 1H ,Ar-H),7.32(t,J=7.8Hz,1H,Ar-H),7.23(t,J=8.2Hz,1H,Ar-H),7.08(d,J=7 .6Hz,1H,Ar-H),6.68(s,1H,Ar-H),6.61(dd,J=8.3,2.0Hz,1H,Ar-H),6.56(d t,J=4.4,2.1Hz,2H,Ar-H),6.51(s,1H,Ar-H),6.36(s,2H,Ar-H),5.94(d,J=1 0.6Hz,2H,O-CH2-O),5.85(d,J=9.1Hz,1H,CH-OC=O),4.57(d,J=6.2Hz,3H,Ar -CH,O-CH2-C=O),4.29(dd,J=9.1,7.0Hz,1H,CH2-OC=O),4.14(t,J=9.7Hz,1H ,CH2-OC=O),3.80(s,6H,OCH3),3.74(d,J=4.9Hz,2H,Ar-CH2-C=O),3.73(s,6 H,OCH3),2.88(dd,J=14.6,4.4Hz,1H,O=C-CH),2.85–2.77(m,1H,O-CH2-CH).

[0094] Embodiment 20

[0095]

[0096] The preparation method of compound D2-10 is the same as that of compound D2, except that the differently substituted phenoxyacetic acid (A) is replaced with phenoxyacetic acid, and other conditions remain unchanged to obtain compound D2-10 in the form of white powder with a yield of 88%. Compound D2-10: 1HNMR (600MHz, CDCl3) δ8.29 (s, 1H, NH), 7.71 (d, J = 8.3Hz, 1H, Ar-H), 7.36 (t, J = 9. 2Hz,1H,Ar-H),7.34–7.29(m,3H,Ar-H),7.04(dd,J=16.7,8.3Hz,2H,Ar-H),6.97( dd,J=8.0,3.9Hz,2H,Ar-H),6.68(d,J=1.9Hz,1H,Ar-H),6.52(d,J=1.9Hz,1H,Ar- H),6.37(d,J=2.3Hz,2H,Ar-H),5.98–5.92(m,2H,O-CH2-O),5.86(d,J=9.1Hz,1H, CH-OC=O),4.78(qd,J=6.7,1.5Hz,1H,O-CH(CH3)-C=O),4.57(d,J=4.3Hz,1H,Ar-C H),4.29(t,J=8.0Hz,1H,CH2-OC=O),4.14(t,J=8.4Hz,1H,CH2-OC=O),3.80(s,3H, OCH3),3.73(s,2H,Ar-CH2-C=O),3.73(s,6H,OCH3),2.89(dd,J=14.5,4.4Hz,1H,O =C-CH), 2.82(tdd,J=16.1,9.9,6.5Hz,1H,O-CH2-CH), 1.63(d,J=6.8Hz,3H,CH3).

[0097] Example 21: Application of podophyllotoxin ester derivative molecules of Formula I

[0098] Using human lung cancer cell lines H1975 and H1299 as test strains and CCK-8 colorimetry as the detection method, the in vitro tumor cell inhibition activity of podophyllotoxin ester derivatives of Formula I was studied and it was found that this type of novel structural derivatives has obvious in vitro tumor cell inhibition activity. The results are shown in the attached Figure 1 At the same time, the derivatives had no significant inhibitory effect on the proliferation of human lung epithelial cell line BASE-2B, and IC 50 The values ​​were all greater than 80 μmol·L -1 , indicating that the toxicity of this type of derivative to normal cells is significantly lower than that of its parent core structure podophyllotoxin. The results are shown in the attached Figure 2 .

[0099] Table 1. Antiproliferative activity of podophyllotoxin ester derivatives against human non-small cell lung cancer cells

[0100]

[0101] In addition, the best active compound D1-1 was subjected to EdU antiproliferation experiment. The results showed that D1-1 could significantly reduce the proportion of EdU positive cells and showed superior antiproliferative activity compared with podophyllotoxin. The results are shown in the attached Figure 3 .

[0102] Example 22: Compound D1-1 can simultaneously target tubulin and AKT1 protein

[0103] The biofilm interferometry (BLI) technique was used to detect the affinity of D1-1 to tubulin (colchicine binding site) and AKT1 (covalent allosteric site (G4N binding site)). The results showed that D1-1 had a strong affinity for both AKT1 and tubulin, with the affinity for AKT1 (K D =3.328×10 -6 M) is slightly stronger than its affinity for tubulin (K D =6.28×10 -5 M); its parent structure podophyllotoxin (PPT) has a high affinity for tubulin (K D =7.65×10 -5 M), while the affinity for AKT1 is almost zero. The results are shown in the attached Figure 4 .

[0104] According to the above conclusions, this type of compound has application prospects as microtubule protein and / or AKT1 inhibitors and can be prepared into anti-tumor drugs.

Claims

1. A class of tubulin / AKT1 dual-targeting podophyllotoxin derivatives, characterized by: The general structural formula of podophyllotoxin derivatives is as follows: The range of substituents is: 1 , R 2 , R 3 , R 4 , R 5 Each is independently selected from H, Cl, F, CH(CH3)2, C(CH3)3, CH3, Br or OCH3; R 6 =H or CH3.

2. The microtubule protein / AKT1 dual-targeting podophyllotoxin derivative according to claim 1, characterized in that: The podophyllotoxin derivative is one of the following structural formulas:

3. Use of a class of tubulin / AKT1 dual-targeting podophyllotoxin derivatives according to claim 1 or 2 in the preparation of drugs for treating lung cancer.

Images