Podophyllotoxin triazole ether derivative as well as preparation method and application thereof

Abstract

The invention discloses a podophyllotoxin triazole ether derivative as well as a preparation method and application thereof, belongs to the technical field of chemical pharmacy, and particularly relates to a podophyllotoxin triazole derivative and application of the podophyllotoxin triazole ether derivative in preparation of antitumor drugs. A series of novel 1, 2, 3-triazole podophyllotoxin derivatives are designed and synthesized by combining a triazole structure with a podophyllotoxin matrix through click chemistry reaction, the dynamic characteristics of podophyllotoxin are optimized by introducing the triazole structure, and the anti-cancer effect and druggability of podophyllotoxin are improved. Molecular docking shows that the series of compounds have relatively strong binding energy to AKT1 and tubulin and can inhibit tubulin / AKT1 in a targeted manner, and in-vitro anti-tumor activity research shows that the molecules have very strong inhibitory activity to proliferation of human colon cancer cell strains, and a new strategy can be provided for structural derivatization of podophyllotoxin. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of chemical pharmacy, and specifically relates to a podophyllotoxin triazole ether derivative and a preparation method and application thereof. Background Art

[0002] Podophyllotoxin (PPT) is a lignin anti-tumor component extracted from the roots and stems of Dysosma Versipelli (Hance) M. Cheng, a plant of the genus Podophyllotoxin in the family Botryaceae. It has strong cytotoxic activity against multiple tumor cell lines and can block the cell cycle at the G2 / M phase, mainly by interfering with the polymerization of microtubules and the assembly of spindles to prevent the division and proliferation of tumor cells. Although podophyllotoxin has a good inhibitory effect on a variety of tumor cells, its clinical application is limited by many factors, such as high toxicity, low selectivity, and low bioavailability. Therefore, researchers have tried to design its derivatives through structural modification, hoping to reduce its toxic side effects, improve its selectivity and bioavailability while maintaining its anti-tumor activity.

[0003] At present, the research on the derivatization of podophyllotoxin structure will mainly focus on the hydroxyl group at the 4th position of the cycloalkane (C ring). For example, etoposide and teniposide are two semi-synthetic epipodophyllotoxin derivatives, which have been successfully used as anti-tumor drugs in clinical practice by inhibiting DNA-topoisomerase II. The design of podophyllotoxin derivatives disclosed in recent years mainly exerts anti-tumor activity by enhancing microtubule targeting and inhibiting the assembly of microtubules in cancer cells, but has failed to effectively solve the problems of large toxic side effects and low bioavailability.

[0004] In cancer cells, the PI3K / AKT pathway is an important signaling pathway for tumor progression, which interacts with other signaling pathways to drive tumor progression and drug resistance. As a downstream effector of PI3K, AKT is an important kinase in the PI3K / AKT pathway that realizes the "upload and down" of the cell signaling cascade, and plays a vital role in cell growth, proliferation and metabolism. After abnormal activation, AKT in cancer cells can phosphorylate downstream transcription factors and promote cell proliferation. Therefore, AKT activation is considered to be an important signal for cancer cells to resist apoptosis. AKT has three subtypes, and AKT1 is highly expressed in cancer cells such as colorectal cancer, breast cancer, and lung cancer. AKT2 is overexpressed in breast cancer and ovarian cancer, and has been shown to be associated with cancer invasiveness and low survival rate. AKT3 is upregulated in some androgen-independent prostate cancer cells and estrogen receptor-deficient breast cancer cells.

[0005] AKT protein mainly contains three important domains: the amino-terminal homologous PH domain, the central kinase domain, and the carboxyl-terminal regulatory domain. There are three types of inhibitors designed for these three domains, namely competitive inhibitors, allosteric inhibitors, and covalent inhibitors. Among them, allosteric inhibitors have the greatest potential in anti-tumor applications. MK2206 is the most representative AKT allosteric inhibitor. It stabilizes the "PH-in" conformation by binding to a hydrophobic region at the junction of the PH domain and the central kinase domain and exerts an inhibitory effect. It can not only inhibit the kinase activity of AKT, but also inhibit non-kinase activity. However, MK2206 was stagnant in Phase I clinical trials due to limited efficacy.

[0006] The triazole structure has significant advantages in drug design, such as high chemical stability, wide biological activity, good metabolic stability, and convenient synthesis. These characteristics make it the core structure of many drug molecules and widely used in multiple therapeutic areas such as antibacterial, antifungal, antiviral and anticancer. By rationally designing and optimizing the triazole structure, new drugs with high efficiency, safety and good pharmacokinetic properties can be developed.

[0007] The synthesis methods of podophyllotoxin triazole derivatives synthesized in existing documents and public patents (CN101602769A) are all the first step of using podophyllotoxin and highly toxic, explosive and environmentally polluting sodium azide to react to generate 4-azidopodophyllotoxin. The synthesis of podophyllotoxin triazole ether derivatives mentioned in the present invention does not involve the use of sodium azide throughout the process, which reduces the risk that the synthesizer is susceptible to during operation and the synthesis method is more environmentally friendly. In addition, the podophyllotoxin triazole derivatives synthesized in the past have not paid attention to the application of anti-tumor targeting AKT1 targets. The patent of the present invention focuses on the application of podophyllotoxin triazole derivatives, especially the synthesized novel podophyllotoxin triazole ether derivatives in the anti-tumor effect of AKT1 protein, because the ether bond of the podophyllotoxin triazole ether compound as the connecting bond between the podophyllotoxin mother nucleus and the triazole structure makes the compound as a whole more flexible, thereby making it easier to adjust the spatial structure of the molecule and affect the conformation and stereochemical properties of the molecule. The novel podophyllotoxin triazole ether synthetic derivatives are more likely to act on the active cavity of the AKT1 protein, enhancing the targeting and binding energy of the synthetic derivatives to the AKT1 protein. In addition, due to the introduction of ether bonds, the novel podophyllotoxin triazole ether derivatives synthesized in this patent have greater polarity and solubility, which can improve the anti-tumor effect and facilitate drug design.

[0008] Summary of the invention

[0009] The purpose of the present invention is to provide a novel podophyllotoxin triazole ether derivative and its preparation method and application. The present invention connects triazole to the hydroxyl group at the C4 position of the cycloalkane of podophyllotoxin with an ether bond, designs and synthesizes a series of novel 4β-(1,2,3-triazole)-podophyllotoxin derivatives that simultaneously target tubulin and AKT1, and conducts preliminary in vitro antitumor activity studies.

[0010] The present invention first provides a class of podophyllotoxin triazole ether derivatives, the general structural formula of which is as follows:

[0011]

[0012] The substituents are selected from:

[0013]

[0014] The present invention also provides a method for preparing the compound of formula I, and the synthetic route is as follows:

[0015]

[0016] The preparation method of the compound of formula I comprises the following steps:

[0017] Step 1: Using podophyllotoxin and halogenated propargyl (bromopropargyl, chloropropargyl or fluoropropargyl) as raw materials, anhydrous N, N-dimethylacetamide (DMF), dimethyl sulfoxide (DMSO), or tetrahydrofuran (THF) as solvent, slowly add sodium hydride (NaH) to a dry reaction vessel under ice bath conditions, stir the reaction at room temperature for more than 12 hours, and monitor the entire reaction process with a thin layer chromatography silica gel plate. After the reaction is completed, saturated ammonium chloride water is added for quenching, and anhydrous ethyl acetate is added for extraction three times, the solution obtained by the combined extraction is washed with saturated brine, and then dehydrated with anhydrous sodium sulfate, filtered and spin-dried to obtain a podophyllotoxin acetylene intermediate A. The molar ratio of podophyllotoxin to halogenated propargyl and sodium hydride is 1:1.2:2 to 1:1.5:2.4. The halogenated propargyl is preferably bromopropargyl, and the solvent is preferably DMF.

[0018] Step 2: Add copper sulfate pentahydrate, sodium ascorbate and differently substituted azide compounds to a round-bottom flask containing podophyllotoxin acetylene intermediate A, and add N,N-dimethylformamide (DMF) to dissolve the solid reactant. Heat the reaction at 60°C-80°C (preferably 60°C) for 6-12 hours under nitrogen protection, and monitor the reaction progress with a thin layer chromatography silica gel plate. After the reaction is completed, distilled water is added to the reaction solution and extracted with ethyl acetate, the organic phases are combined, the water is removed with anhydrous sodium sulfite, and the solvent is dried using a rotary evaporator. Finally, it is separated and purified by column chromatography to obtain a podophyllotoxin triazole ether derivative. The molar ratio of compound A to differently substituted azide compounds, anhydrous copper sulfate, and sodium ascorbate is 1:0.8:0.2:0.6 to 1:1.2:0.4:0.8.

[0019] Specifically, the molecular structure of the podophyllotoxin triazole ether derivative prepared by the present invention is as follows:

[0020]

[0021] The present invention proves that the podophyllotoxin triazole ether derivative molecules have significant antiproliferative activity against human colon cancer cells HT-29 and HCT-116 through in vitro tumor cell inhibition activity, and most of the compounds have a median lethal concentration (IC 50 The IC values ​​of compound 2b on HT-29 and HCT-116 cells were all less than 10 μmol / L. 50 The values ​​were all less than 1 μmol / L, and the anti-tumor activity was increased by 3 to 4 times compared with its parent compound podophyllotoxin. Among them, the proliferation inhibition activity of podophyllotoxin derivatives on HT-29 cells was significantly better than that on HCT-116 cells. Figure 1 In addition, the cytotoxicity of compounds 1b-11b to human colon epithelial cells was significantly lower than that of their parent compound podophyllotoxin, indicating that the introduction of the triazole structure effectively reduced the toxic side effects of podophyllotoxin. Figure 2 Through the clone formation experiment, it was found that compound 2b can effectively inhibit the proliferation of human colon cancer cell lines HCT-116 and HT-29. Figure 3 , 4 From the structure-activity relationship comparison, podophyllotoxin can only bind to tubulin (colchicine binding site), while compound 2b can not only bind to the active site of podophyllotoxin, but also has good affinity with the active site of AKT1; in comparison, 2b has a better targeting effect on AKT1 protein than on tubulin, see Figure 5 , 6 , 7. The results of water solubility experiments showed that the introduction of triazole structure can effectively improve the water solubility of podophyllotoxin, among which compounds 2b and 10b have greatly improved water solubility compared with podophyllotoxin, achieving the best effect.

[0022] Based on the above experimental results, the present invention provides the use of a podophyllotoxin triazole ether derivative as shown in Formula I in the preparation of a microtubule protein inhibitor or an AKT inhibitor.

[0023] Specifically, the tubulin inhibitor interferes with the mitotic process of cells by inhibiting or promoting the polymerization of tubulin, interrupting cell mitosis and stagnating in the M phase, thereby causing apoptosis of tumor cells and exerting an anti-tumor effect. The AKT inhibitor can inhibit the activity of AKT1 protein. The podophyllotoxin triazole ether derivative shown in Formula I of the present invention can be used to prepare a dual-targeted inhibitor of tubulin and AKT1.

[0024] Furthermore, the present invention provides the use of the podophyllotoxin triazole ether derivative as shown in Formula I in the preparation of anti-tumor drugs.

[0025] Preferably, the tumor is a solid tumor, including but not limited to colorectal cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, Kaposi's sarcoma, etc.

[0026] The present invention also provides a pharmaceutical composition, comprising the podophyllotoxin triazole ether derivative shown in formula I as one of the main active ingredients, and a pharmaceutically acceptable carrier.

[0027] The innovation of the present invention is to synthesize a series of novel dual-targeted tubulin / AKT1 podophyllotoxin triazole derivatives. Compared with single-targeted podophyllotoxin, dual-targeted molecules have better efficacy or the potential to overcome drug resistance. The binding energy of the novel dual-targeted podophyllotoxin triazole derivatives to AKT1 is greater than that of the podophyllotoxin nucleus, which means that these derivatives have a stronger AKT1 inhibitory effect, which is a significant improvement in the structural derivatization of podophyllotoxin, and can produce stronger anti-tumor activity and better selectivity. At the same time, this structural modification changes the physicochemical properties of the podophyllotoxin molecule, improves its water solubility, and thus optimizes its pharmacokinetic properties. In addition, compared with previous podophyllotoxin ester derivatives, the podophyllotoxin triazole ether in the present invention is not easily hydrolyzed by esterases, and has stronger metabolic stability under physiological conditions, thereby prolonging its action time in tumor cells. The triazole ring has a high electronegativity and can enhance the binding of the drug to the target by forming hydrogen bonds or electrostatic interactions with the target; the nitrogen atom on the triazole ring can act as a hydrogen bond receptor to form hydrogen bonds with the amino acid residues of tumor-related target enzymes or receptors. This interaction can significantly enhance the selectivity and binding force of the drug. Therefore, the present invention is expected to lay a theoretical foundation for the development of podophyllotoxin triazole derivatives as candidate molecules for anticancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1This is a graph showing the in vitro proliferation inhibitory activity of podophyllotoxin triazole ether derivatives 1b-11b on human colon cancer cell lines HCT-116 and HT-29 detected by CCK8 assay;

[0029] Figure 2 The CCK8 assay was used to detect the in vitro proliferation inhibitory activity of podophyllotoxin triazole ether derivatives 1b-11b on the human colon epithelial cell line NCM460;

[0030] Figure 3 The figure shows the inhibitory activity of podophyllotoxin triazole ether derivative 2b on the proliferation of human colon cancer cell HCT-116 in vitro in a clone formation experiment (the upper figure is observed with the naked eye, and the lower figure is a 40-fold field of view under a microscope);

[0031] Figure 4 The figure shows the in vitro proliferation inhibition activity of podophyllotoxin triazole ether derivative 2b on human colon cancer cell HT-29 in a clone formation experiment (the upper figure is observed with the naked eye, and the lower figure is a 40-fold microscope view);

[0032] Figure 5 is a bar graph of the docking binding energy of the podophyllotoxin triazole ether derivative of Formula I with AKT1 and tubulin;

[0033] Figure 6 This is the docking binding mode diagram of podophyllotoxin triazole ether derivative 2b and target protein AKT1 (PDB: 6HHI);

[0034] Figure 7 This is the docking binding pattern diagram of podophyllotoxin triazole ether derivative 2b and target protein tubulin (PDB: 1SA0);

[0035] Figure 8 This is a graph showing the results of water solubility testing of podophyllotoxin triazole ether derivatives of formula I. DETAILED DESCRIPTION

[0036] The present invention is described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0037] The synthesis route of a novel podophyllotoxin triazole ether derivative (Formula I series compound) of the present invention is as follows:

[0038]

[0039] The preparation method of the podophyllotoxin triazole ether derivative of the present invention comprises the following specific steps:

[0040] Step 1: Podophyllotoxin (1mmol) and propargyl bromide (1mmol) are used as raw materials, and 10mL of anhydrous N,N-dimethylacetamide (DMF) is used as a solvent. 2.4mmol of sodium hydrogen (NaH) is slowly added to a dry reaction vessel under ice bath conditions, and the reaction is stirred for more than 12 hours at room temperature, and the entire reaction process is monitored by thin layer chromatography silica gel plate. After the reaction is completed, 5mL of saturated ammonium chloride water is added for quenching, and 20mL of anhydrous ethyl acetate is added for extraction three times, the solution obtained by the extraction is combined and washed with 12mL of saturated brine, and then dehydrated with anhydrous sodium sulfate, filtered and dried to obtain podophyllotoxin propargyl ether compound A.

[0041] Step 2: Add copper sulfate pentahydrate (0.2mmol), sodium ascorbate (0.8mmol) and differently substituted azide compounds (1mmol) to a round-bottom flask containing compound A (0.8mmol), and add 10mL N,N-dimethylformamide to dissolve the solid reactant. Heat the reaction at 60°C under nitrogen protection for more than 6 hours, and use a thin layer chromatography silica gel plate to detect the reaction progress. After the reaction is completed, wait for the oil bath to cool to room temperature, then add 10mL of distilled water to the reaction solution and extract it three times with 20mL of ethyl acetate, and wash the organic phase liquid collected by extraction with saturated brine. Add an appropriate amount of anhydrous sodium sulfite to the washed organic phase liquid to remove moisture, filter and spin dry the solvent with a rotary evaporator, and finally pass through column chromatography (V 石油醚 :V 乙酸乙酯 =3:1) to separate and purify the product to obtain a series of podophyllotoxin triazole derivatives.

[0042] Example 1

[0043]

[0044] Podophyllotoxin (1mmol) and propargyl bromide (1mmol) are used as raw materials, and 10mL of anhydrous N,N-dimethylacetamide (DMF) is used as a solvent. Under ice bath conditions, 2.4mmol of sodium hydrogen (NaH) is slowly added to a dry reaction vessel, and the reaction is stirred for more than 12 hours at room temperature, and the entire reaction process is monitored by a thin layer chromatography silica gel plate. After the reaction is completed, 5mL of saturated ammonium chloride water is added for quenching, and 20mL of anhydrous ethyl acetate is added for extraction three times, the solution obtained by the combined extraction is washed with 12mL of saturated brine, and then dehydrated with anhydrous sodium sulfate, filtered and spin-dried to obtain podophyllotoxin propargyl ether compound A, which is in the form of a light yellow solid, with a melting point of 192°C and a yield of 85%. 1H NMR (300MHz, CDCl3) δ6.98(d,J=50.7Hz,1H),6.45(d,J=6.8Hz,3H),5.93(dd,J=12.5,5.9Hz,2H),4.57-4.41(m,2H),4.36-4.22(m,1H),4.17( s,1H),3.84(s,3H),3.82(s,3H),3.81(s,3H),3.25(ddd,J=26.6,9.4,5.3Hz,1H),3.00(ddd,J=14.0,7.1,3.9Hz,1H),1.26(s,1H).HR-MS(ESI + )m / z:453.1538[M+H] + .Found:[M+H] + .453.1544.

[0045] Example 2

[0046]

[0047] Copper sulfate pentahydrate (0.2mmol), sodium ascorbate (0.8mmol) and 1mmol methyl azidoacetate (CAS No. 1816-92-8) were added to a round-bottom flask containing compound A (0.8mmol), and 10mL N,N-dimethylformamide was added to dissolve the solid reactant. The reaction was heated at 60°C under nitrogen protection for more than 6 hours, and the reaction progress was detected by thin layer chromatography silica gel plate. After the reaction was completed, the oil bath was cooled to room temperature, and then 10mL of distilled water was added to the reaction solution and extracted three times with 20mL of ethyl acetate, and the organic phase liquid collected by extraction was washed with saturated brine. An appropriate amount of anhydrous sodium sulfite was added to the organic phase liquid after washing to remove moisture, and the solvent was spin-dried with a rotary evaporator after suction filtration, and finally column chromatography (V 石油醚 :V 乙酸乙酯 =3:1) after separation and purification of the product, compound 1b was obtained, with a melting point of 124°C in the form of a white solid, and a yield of 61%. 1H NMR(400MHz,DMSO)δ8.14(s,1H),7.03(s,1H),6.61(s,2H),6.37(s,1H),5 .98(s,2H),5.44(s,2H),4.74(s,2H),4.52(d,J=6.6Hz,1H),4.42(t,J=8.1 Hz,1H),4.21(d,J=8.8Hz,1H),4.11(d,J=5.5Hz,1H),3.72(s,3H),3.67(s, 6H),3.64(s,3H),3.47(dd,J=9.6,5.7Hz,1H),2.98-2.84(m,1H)HR-MS(ESI + )m / z:568.1943[M+H] + .Found:568.1925[M+H] + .

[0048] Example 3

[0049]

[0050] The preparation method of compound 2b is the same as above, the azide compound is (R)-5-(azidomethyl)-3-[3-fluoro-4-(4-morpholinyl)phenyl]-2-oxazolidinone (CAS No. 168828-84-0), and other conditions remain unchanged to obtain compound 2b with a melting point of 146.9°C, a light yellow solid, and a yield of 57%. 1 H NMR (600MHz, CDCl3) δ7.35(s,1H),7.30(d,J=14.1Hz,1H),7.00(d,J=7.8Hz,1H),6.88(t,J=9.0Hz,1H),6 .81(s,1H),6.56(s,1H),6.36(s,2H),5.98(d,J=11.7Hz,2H),5.03(s,1H),4.71(s,2H),4.55(s,2H),4.40 (t,J=8.5Hz,1H),4.29(d,J=3.2Hz,1H),4.25(s,1H),4.11(t,J=7.3Hz,1H),3.99-3.90(m,2H),3.87-3.82 (m,4H),3.80(s,3H),3.72(s,6H),3.40(dd,J=9.4,3.2Hz,1H),3.11(s,1H),3.05-2.97(m,4H).HR-MS(ESI + )m / z:774.2780[M+H] + .Found:774.2780[M+H] + .

[0051] Example 4

[0052]

[0053] The preparation method of compound 3b is the same as above, the azide compound is 2-azidoethanol (CAS No. 1517-05-1), and other conditions remain unchanged to obtain compound 3b with a melting point of 124.5°C, a white solid form, and a yield of 43%. 1 H NMR (400MHz, CDCl3) δ7.27(s,1H),6.93(s,1H),6.60(s,1H),6.37(s,2H),6.00(d,J=9.2Hz,2H),4.54(s,2H),4.4 8-4.40(m,3H),4.31(s,2H),4.01(s,3H),3.81(s,3H),3.70(s,6H),3.42(d,J=6.3Hz,1H),3.15(s,1H).HR-MS(ESI + )m / z:540.1971[M+H] + .Found:540.1976[M+H] +

[0054] Example 5

[0055]

[0056] The preparation method of compound 4b is the same as above, the azide compound is benzyl azide (CAS No. 622-79-7), and other conditions remain unchanged to obtain compound 4b with a melting point of 139.5°C, a white solid form, and a yield of 70%. 1 H NMR (400MHz, CDCl3) δ7.37(d,J=6.6Hz,3H),7.28(d,J=6.9Hz,3H),6.93(s,1 H),6.49(s,1H),6.40(s,2H),5.95(d,J=6.3Hz,2H),5.52(s,2H),4.64(s,2H) ,4.37(dd,J=18.4,7.4Hz,2H),4.21(d,J=4.0Hz,1H),4.10(d,J=9.7Hz,1H),3 .83(s,3H),3.72(s,6H),3.31(dd,J=9.4,4.3Hz,1H),3.02(s,1H).HR-MS(ESI + )m / z:586.2179[M+H] + .Found:586.2183[M+H] + .

[0057] Example 6

[0058]

[0059] The preparation method of compound 5b is the same as above, the azide compound is 3′-azido-3′-deoxythymidine (CAS No. 30516-87-1), and other conditions remain unchanged to obtain compound 5b with a melting point of 143.8° C. in the form of a light yellow solid and a yield of 63%. 1 HNMR(400MHz,d6-DMSO)δ11.40(s,1H),8.28(s,1H),7.85(s,1H),7.03(s,1H),6.60(s,2H),6.44 (t,J=6.2Hz,1H),6.40(s,1H),5.99(s,2H),5.35(d,J=18.7Hz,2H),4.70(s,2H),4.51(d,J=6.4Hz ,1H),4.43(t,J=8.0Hz,1H),4.19(d,J=10.5Hz,2H),4.13(d,J=5.0Hz,1H),3.75(s,1H),3.67(s,6 H),3.65(s,3H),3.54-3.46(m,1H),2.96-2.87(m,1H),2.76-2.65(m,2H),2.51(s,3H).HR-MS(ESI + )m / z:720.2524[M+H] + .Found:720.2512[M+H] + .

[0060] Example 7

[0061]

[0062] The preparation method of compound 6b is the same as above, except that the azide compound is p-toluenesulfonyl azide (CAS No. 941-55-9), and other conditions remain unchanged to obtain compound 6b with a melting point of 140.9°C, a light yellow solid, and a yield of 65%. 1H NMR(400MHz, CDCl3) δ7.92(d,J=7.5Hz,2H),7.33(d,J=7.7Hz,2H),7.26(s,1H),6 .76(s,1H),6.61(s,1H),6.37(s,2H),5.99(d,J=13.9Hz,2H),4.42(t,J=8.5Hz,2H ),4.33(s,2H),4.12(d,J=4.3Hz,1H),3.98(d,J=9.4Hz,1H),3.82(s,3H),3.81(s ,6H),3.59(dd,J=14.1,8.9Hz,1H),3.44(d,J=9.1Hz,1H),2.43(s,3H).HR-MS(ESI + )m / z:650.1800[M+H] + .Found:650.1802[M+H] + .

[0063] Example 8

[0064]

[0065] The preparation method of compound 7b is the same as above, the azide compound is 1-azidomethyl-4-methoxybenzene (CAS No. 70978-37-9), and other conditions remain unchanged to obtain compound 7b with a melting point of 145.6°C, a white solid form, and a yield of 75%. 1 HNMR (600MHz, CDCl3) δ7.32-7.17(m,3H),6.96-6.86(m,3H),6.49(s,1H),6.41(s,2H),5.96(d,J=9.0Hz,2H),5.45(s,2H),4.62(s,2H ),4.37(d,J=17.3Hz,2H),4.21(s,1H),4.15-4.06(m,1H),3.83(s,3H),3.80(s,3H),3.73(s,6H),3.29(s,1H),3.03(s,1H).HR-MS(ESI + )m / z:616.2297[M+H] + .Found:616.2290[M+H] + .

[0066] Example 9

[0067]

[0068] The preparation method of compound 8b is the same as above, the azide compound is 4-methoxyphenoxycarbonyl azide (CAS No. 25474-85-5), and other conditions remain unchanged to obtain compound 8b with a melting point of 145.6°C, a white solid form, and a yield of 53%. 1 HNMR(600MHz, CDCl3)δ7.24(d,J=8.4Hz,2H),7.20(s,1H),6.91(d,J=6.8Hz,2H), 6.89(s,1H),6.49(s,1H),6.40(s,2H),5.96(d,J=9.2Hz,2H),5.45(s,2H),4.63(s ,2H),4.41-4.36(m,1H),4.34(d,J=5.9Hz,1H),4.15-4.07(m,2H),3.84(s,3H),3. 81(s,3H),3.73(s,6H),3.29(dd,J=9.6,4.5Hz,1H),3.04-2.98(m,1H).HR-MS(ESI + )m / z:660.2176[M+H] + .Found:660.2188[M+H] + .

[0069] Example 10

[0070]

[0071] The preparation method of compound 9b is the same as above, the azide compound is p-azidobenzoic acid (CAS No. 6427-66-3), and other conditions remain unchanged to obtain compound 9b with a melting point of 159.7°C, a white solid form, and a yield of 64%. 1 H NMR (600MHz, CDCl3) δ8.27(d,J=8.4Hz,2H),7.89(d,J=8.4Hz,2H),7.76(s,1H),6.98(s,1H), 6.62(s,1H),6.42(s,1H),6.00(d,J=13.8Hz,2H),4.66(dd,J=33.7,11.9Hz,2H),4.48(t,J=8 .7Hz,1H),4.42(d,J=4.8Hz,1H),4.35(d,J=3.3Hz,1H),4.09(dd,J=9.4,4.1Hz,1H),3.81(s, 3H), 3.74 (s, 6H), 3.41 (dd, J=9.8, 3.5Hz, 1H), 3.18 (ddd, J=13.0, 9.0, 4.4Hz, 1H). HR-MS (ESI + )m / z:616.1978[M+H] +.Found:616.1926[M+H] + .

[0072] Embodiment 11

[0073]

[0074] The preparation method of compound 10b is the same as above, the azide compound is triethylene glycol azide (CAS No. 86520-52-7), and other conditions remain unchanged to obtain compound 10b with a melting point of 137.7°C, a white solid form, and a yield of 47.3%. 1 H NMR (600MHz, CDCl3) δ7.72(s,1H),6.98(s,1H),6.47(s,2H),6.44(s,2H),5.95(d,J=10.0Hz,2H),4.56(s,2H),4.39(s,2H),4.17(dd,J=16.3,6.6H z,2H),3.90(s,2H),3.83(s,3H),3.76(s,6H),3.68(s,3H),3.63(s,5H),3 .57-3.54(m,2H),3.40(t,J=4.9Hz,1H),3.30(d,J=3.4Hz,1H).HR-MS(ESI + )m / z:628.2495[M+H] + .Found:628.2500[M+H] + .

[0075] Example 12

[0076]

[0077] The preparation method of compound 11b is the same as above, the azide compound is 4-acetylaminobenzenesulfonyl azide (CAS No. 2158-14-7), and other conditions remain unchanged to obtain compound 11b in the form of a light yellow solid with a melting point of 124°C and a yield of 50%. 1 HNMR(400MHz,DMSO)δ10.31(s,1H),7.75(s,1H),7.07(s,1H),6.61(s,3H),6.00(s,3H),5.93(s,3H),4.51( d,J=8.8Hz,2H),4.46-4.37(m,2H),3.91(d,J=7.3Hz,2H),3.75(s,9H),3.69(s,4H),2.09(s,1H).HR-MS(ESI + )m / z:693.1789[M+H] + .Found:693.1788[M+H]+ .

[0078] Example 13

[0079] Effects of podophyllotoxin triazole derivatives on cell proliferation activity

[0080] Cell proliferation activity was detected by CCK-8 colorimetric assay. Cells were grown in complete medium to the logarithmic growth phase and diluted to 2×10 4 / mL. Then, the cell suspension was inoculated into a 96-well flat-bottom plate, 100 μL per well, and incubated for 12 hours at 37°C and 5% CO2. The compound to be tested was diluted to a preset concentration (0.1, 1, 10, 100, 1000 μM) and added to a 96-well flat-bottom plate, with gefitinib as a positive control. Gefitinib is a selective epidermal growth factor receptor tyrosine kinase inhibitor that can be used to treat non-small cell lung cancer, colon cancer, breast cancer, prostate cancer, etc. After 48 hours of treatment, the supernatant in each well was aspirated, and 100 μL of fresh culture medium containing 10% CCK-8 solution was added again, and the incubation continued for about 2 hours. The absorbance was measured and recorded at a test wavelength of 450 nm using an ELIX800, BioTek, USA. Each sample was set up with three replicate wells, and each experiment was repeated three times. Finally, Origin 7.5 software was used to calculate the drug concentration required for the cell viability to reach 50%, i.e., IC 50 value.

[0081] Using human colon cancer cell lines HCT-116 and HT-29 as test strains, the in vitro tumor cell inhibition activity of the podophyllotoxin triazole derivatives of Formula I was studied and found that the derivatives had significant in vitro tumor cell inhibition activity. Among them, the proliferation inhibition activity of the podophyllotoxin derivatives on HCT-116 cells was significantly better than that of gefitinib. The results are shown in the attached Figure 1 At the same time, the half inhibitory concentration (IC 50 The values ​​were all greater than 50 μmol / L, indicating that the toxicity of this type of derivatives to normal cells was significantly lower than that of its parent structure podophyllotoxin (PPT). The results are shown in the attached Figure 2 .

[0082] In addition, the best active compound 2b was subjected to interference clone formation experiments. The results showed that 2b could significantly inhibit the number and size of HCT-116 and HT-29 cell clone formation, and showed superior anti-proliferative activity compared with podophyllotoxin. The results are shown in the attached Figure 3 , 4 .

[0083] Embodiment 14

[0084] Podophyllotoxin triazole derivatives can effectively target AKT1

[0085] The 1b~11b series of small molecules were energy minimized and optimized by the MM2 position of chem3D, saved as sdf files, and then imported into Autodock vina software for dehydration, hydrogenation, charge calculation, determination of ligand root, and selection of ligand small molecule torsional bonds. Pymol software was used to import AKT1 protein (PDB: 6HHI) and tubulin (PDB: 1SA0) to remove solvent and ligand small molecules, and then AKT1 protein and tubulin macromolecules were hydrogenated and charged in Autodock vina software, and AKT1 protein and tubulin were used as docking receptors and saved as Pdbqt files. The coordinates of the docking box were determined according to the original covalent conformational regulatory AKT inhibitor ligand small molecule coordinates in the AKT1 protein crystal structure (Grid center: X-1.96252Y4.46218 Z-10.6489; Grid size: X 26Y 26Z 26; Grid space: 0.375) and semi-flexible docking was performed. The coordinates of the docking box were determined according to the original tubulin inhibitor colchicine ligand small molecule in the microtubule protein crystal structure (Grid center: X52.3796Y 52.2827Z-7.67196; Grid size: X 69.2Y69.2Z 69.2; Grid space: 0.375) and semi-flexible docking was performed.

[0086] Depend on Figure 5 It can be seen that all podophyllotoxin triazole derivatives have lower binding energies to AKT1 than podophyllotoxin, indicating a better binding effect. The binding energies of most podophyllotoxin triazole derivatives to tubulin are not significantly different from those of podophyllotoxin, indicating that podophyllotoxin triazole derivatives further target AKT1 on the basis of targeting tubulin, retaining the effect on tubulin while inhibiting AKT1. Among the 1b to 11b series of compounds, compound 2b has the lowest binding energy to AKT1, which is -9.58 kcal / mol, significantly lower than the binding energy of podophyllotoxin to AKT1 (-5.21 kcal / mol). The triazole structure in compound 2b can form hydrogen bonds with TYRA:315, and other functional groups can also form hydrogen bonds with the three amino acids GLU A:85, THRA:82, and LEU A:295 of the AKT1 protein, significantly increasing the targeting of compound 2b to the AKT1 protein, see for details. Figure 6 The binding energy of compound 2b to tubulin is -6.421 kcal / mol, slightly higher than the binding energy of podophyllotoxin to tubulin (-8.688 kcal / mol). Figure 7The results showed that compound 2b formed multiple forces with the amino acids of microtubules, such as hydrogen bonds, van der Waals forces, carbon-hydrogen bonds, etc., but the number of hydrogen bonds formed was significantly less than that of AKT1, so the binding energy was reduced, and its microtubule targeting was weaker than that of AKT1.

[0087] Embodiment 15

[0088] Improved water solubility of podophyllotoxin triazole derivatives

[0089] like Figure 8 As shown in the figure, after the introduction of the triazole structure, the water solubility of compounds 1b, 2b, 3b, 5b, 8b, 10b, and 11b is improved compared with podophyllotoxin and etoposide. All the b-series compounds have higher water solubility than the AKT allosteric inhibitor drug MK2206 and the targeted drug gefitinib for the treatment of non-small cell lung cancer. In general, -6 <log(S w )<-4 indicates that the water solubility of the compound is low, <log(S w )<-2 indicates that the compound has good water solubility, -2 <log(S w )<0 indicates that the compound has the best water solubility. The water solubility of compounds 2b and 10b is greatly improved compared with podophyllotoxin, achieving the best effect. This suggests that this type of podophyllotoxin triazole derivative has more favorable physicochemical properties for drug formulation in pharmacy.

[0090] According to the above conclusions, this type of podophyllotoxin triazole derivative has application prospects as microtubule protein and / or AKT1 inhibitors and can be prepared into anti-tumor drugs.

[0091] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A podophyllotoxin triazole ether derivative, characterized in that: The structural formula is shown in Formula I: Wherein the substituent R is selected from Any one of .

2. The method for preparing the podophyllotoxin triazole ether derivative according to claim 1, characterized in that: The steps include: Step (1): using podophyllotoxin and halogenated propyne as raw materials and an anhydrous organic solvent as solvent, slowly adding sodium hydride to a dry reaction vessel under ice bath conditions, stirring the reaction at room temperature, and monitoring the entire reaction process with a thin layer chromatography silica gel plate; after the reaction is completed, saturated ammonium chloride water is added to quench, the organic phase is extracted, washed, dehydrated, and spin-dried to obtain a podophyllotoxin acetylene intermediate A; Step (2): dissolving the podophyllotoxin acetylene intermediate A, copper sulfate pentahydrate, sodium ascorbate and differently substituted azide compounds in N,N-dimethylformamide; reacting at 60°C-80°C for 6-12 hours under nitrogen protection, and monitoring the reaction progress with a thin layer chromatography silica gel plate; after the reaction is completed, extracting and combining the organic phases, removing water, spinning dryness, separating and purifying to obtain a podophyllotoxin triazole ether derivative.

3. The method for preparing the podophyllotoxin triazole ether derivative according to claim 2, characterized in that: In the step (1), the molar ratio of podophyllotoxin to halopropyne to sodium hydride is 1:1.2-1.5:2-2.

4.

4. The method for preparing a podophyllotoxin triazole ether derivative according to claim 2, characterized in that: In the step (1), the organic solvent is selected from one or more of N,N-dimethylacetamide, dimethyl sulfoxide, and tetrahydrofuran.

5. The method for preparing the podophyllotoxin triazole ether derivative according to claim 2, characterized in that: In the step (2), the molar ratio of the podophyllotoxin acetylene intermediate A to the differently substituted azide compounds, anhydrous copper sulfate, and sodium ascorbate is 1:0.8-1.2:0.2-0.4:0.6-0.

8.

6. Use of the podophyllotoxin triazole ether derivative according to claim 1 in the preparation of anti-tumor drugs.

7. The use according to claim 6, characterized in that: The anti-tumor drug is a microtubule protein inhibitor or an AKT inhibitor.

8. The use according to claim 6 or 7, characterized in that: The tumor includes a solid tumor.

9. The use according to claim 6 or 7, characterized in that: The tumors include colorectal cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, and Kaposi's sarcoma.

10. A pharmaceutical composition, characterized in that The invention comprises the podophyllotoxin triazole ether derivative as claimed in claim 1 as one of the main active ingredients, and a pharmaceutically acceptable carrier.

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