Podophyllotoxin triazol ether derivatives, processes for their preparation and use thereof

Abstract

The application discloses a class of podophyllotoxin triazole ether derivatives, a preparation method and application thereof, belongs to the technical field of chemical pharmacy, and particularly relates to a class of podophyllotoxin triazole derivatives and application thereof in preparation of antitumor drugs. The application combines a triazole structure with a podophyllotoxin parent body by using a click chemistry reaction, designs and synthesizes a series of novel 1,2,3-triazole podophyllotoxin derivatives, optimizes the kinetic characteristics of the podophyllotoxin by introducing the triazole structure, and improves the anticancer effect and drug property. Molecular docking shows that the series of compounds have strong binding energy on AKT1 and tubulin, can target inhibit tubulin / AKT1, and in-vitro antitumor activity research shows that the molecules have strong inhibitory activity on the proliferation of human colon cancer cell strains, and can provide a new strategy for structural derivatization of podophyllotoxin.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of chemical pharmacy, and particularly relates to a class of podophyllotoxin triazole ether derivatives, a preparation method and application thereof. BACKGROUND

[0002] Podophyllotoxin (PPT) is a lignan anti-tumor component extracted from the roots and stems of Dysosma Versipelli (Hance) M. Cheng of the Berberidaceae family. It has strong cytotoxic activity on multiple tumor cell lines, can block the cell cycle at the G2 / M phase, and mainly interferes with the polymerization of microtubules and the assembly of the spindle to prevent tumor cell division and proliferation. Although podophyllotoxin has good inhibitory effect on various tumor cells, its clinical application is limited by many factors, such as greater toxicity, low selectivity, and low bioavailability. Therefore, researchers try to design derivatives by structural modification, hoping to maintain the anti-tumor activity while reducing the toxic side effects, improving the selectivity and bioavailability.

[0003] The current podophyllotoxin structural derivation research will mainly focus on the hydroxyl group at position 4 of the cycloalkane (C ring). For example, Etoposide and Teniposide are two semi-synthetic epipodophyllotoxin derivatives, which are successfully used as anti-tumor drugs in clinic by inhibiting DNA-topoisomerase II. The design of podophyllotoxin derivatives disclosed in recent years mainly aims to enhance the targeting of tubulin protein to inhibit the assembly of cancer cell microtubules to exert anti-tumor activity, but it fails to effectively solve the problems of high 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. AKT, as the downstream effector of PI3K, is an important kinase in the PI3K / AKT pathway that realizes the cell signal cascade "top-down" in cell growth, proliferation and metabolism. After abnormal activation in cancer cells, AKT can phosphorylate downstream transcription factors to promote cell proliferation. Therefore, AKT activation is considered an important signal for cancer cells to resist apoptosis. AKT has three subtypes, AKT1 is highly expressed in colorectal cancer, breast cancer, lung cancer and other cancer cells. AKT2 is overexpressed in breast cancer and ovarian cancer, and has been proven to be related to the invasiveness and low survival rate of cancer. AKT3 is up-regulated in some androgen-independent prostate cancer cells and estrogen receptor-deficient breast cancer cells.

[0005] AKT protein mainly contains three important domains, including the amino-terminal homologous PH domain, the central kinase domain and the carboxy-terminal regulatory domain. The inhibitors designed for the three domains are of three types, namely competitive inhibitors, allosteric inhibitors and covalent inhibitors. Among them, allosteric inhibitors have the most potential in anti-tumor applications. MK2206 is the most representative AKT allosteric inhibitor, which inhibits the activity by binding to a hydrophobic region between the PH domain and the central kinase domain, stabilizing the "PH-in" conformation. It can not only inhibit the kinase activity of AKT, but also inhibit the non-kinase activity. However, MK2206 was stalled in the clinical phase I experiment due to limited efficacy.

[0006] The triazole structure has the advantages of high chemical stability, wide biological activity, good metabolic stability and convenient synthesis in drug design. These characteristics make it a core structure of many drug molecules and are widely used in the treatment of antibacterial, antifungal, antiviral and anticancer and other fields. By rational design and optimization of the triazole structure, new drugs with high efficiency, safety and good pharmacokinetic properties can be developed.

[0007] The synthesis of the podophyllotoxin triazole derivatives in the existing literature and published patent (CN101602769A) is as follows: the first step is to react podophyllotoxin with sodium azide, which is highly toxic, explosive and easy to pollute the environment, to generate 4-azido podophyllotoxin. The synthesis of the podophyllotoxin triazole ether derivatives mentioned in the present application does not involve the use of sodium azide throughout the synthesis, reducing the risk of exposure to the synthesis personnel during operation and making the synthesis method more environmentally friendly. Moreover, the previously synthesized podophyllotoxin triazole derivatives have not been focused on the application of AKT1 target for anti-tumor. The present patent focuses on the application of the podophyllotoxin triazole derivatives, especially the newly synthesized podophyllotoxin triazole ether derivatives, in the AKT1 protein anti-tumor aspect. The ether bond of the podophyllotoxin triazole ether compound serves as the connecting bond between the podophyllotoxin parent nucleus and the triazole structure, making the compound more flexible as a whole, so as to more easily adjust the spatial structure of the molecule, affect the conformation and stereochemical properties of the molecule. The newly synthesized podophyllotoxin triazole ether derivatives are more likely to act on the active cavity of AKT1 protein, enhancing the targeting and binding energy of the synthesized derivatives to AKT1 protein. In addition, due to the introduction of the ether bond, the newly synthesized podophyllotoxin triazole ether derivatives of the present patent have greater polarity and solubility, which can improve the anti-tumor effect and facilitate the design of drugs.

[0008] SUMMARY

[0009] The present application aims to provide a new type of podophyllotoxin triazole ether derivative, a preparation method and application thereof. The present application connects triazole to the hydroxyl group at the C4 position of the cycloalkane of podophyllotoxin by an ether bond, designs and synthesizes a series of new 4beta-(1,2,3-triazole)-podophyllotoxin derivatives which simultaneously target microtubulin and AKT1, and performs preliminary in-vitro antitumor activity research.

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

[0011]

[0012] wherein the selection range of the substituent group is: R is selected from:

[0013]

[0014] The present application also provides a preparation method of 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 (bromine propargyl, chlorine propargyl or fluorine propargyl) as raw materials, anhydrous N,N-dimethylacetamide (DMF), dimethyl sulfoxide (DMSO) or tetrahydrofuran (THF) as a solvent, sodium hydride (NaH) is slowly added into a dry reaction container under ice bath conditions, stirring is performed at room temperature for more than 12 hours, and the whole reaction process is monitored by using 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 extracted three times, the obtained solution is combined, washed with saturated brine, and then dehydrated by using anhydrous sulfuric acid, filtered and dried by rotation, to obtain a podophyllotoxin alkyne intermediate A. The molar ratio of podophyllotoxin, halogenated propargyl and sodium hydride is 1:1.2:2-1:1.5:2.4. The halogenated propargyl is preferably bromine propargyl, and the solvent is preferably DMF.

[0018] Step 2: A round bottom flask containing the podophyllotoxin alkyne intermediate A was charged with copper sulfate pentahydrate, sodium ascorbate and different substituted azide compounds, and N,N-dimethylformamide (DMF) was added to dissolve the solid reactants. The reaction was heated at 60-80°C (preferably 60°C) for 6-12 hours under nitrogen protection, and the reaction progress was monitored by thin layer chromatography on silica gel plate. After the reaction was completed, distilled water was added to the reaction solution and extracted with ethyl acetate, and the organic phase was combined and dehydrated with anhydrous sodium sulfite, and the solvent was dried using a rotary evaporator. Finally, column chromatography was used for separation and purification to obtain the podophyllotoxin triazole ether derivatives. The molar ratio of compound A to different substituted azide compounds, anhydrous copper sulfate and sodium ascorbate was 1:0.8:0.2:0.6-1:1.2:0.4:0.8.

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

[0020]

[0021] The present application proves that the podophyllotoxin triazole ether derivative molecule has significant anti-proliferative activity on human colon cancer cells HT-29 and HCT-116 by in vitro tumor cell inhibition activity, and the half lethal concentration (IC 50 value) of most compounds is less than 10 μmol / L, and the IC 50 value of compound 2b on HT-29 and HCT-116 cells is less than 1 μmol / L, which is 3-4 times higher than that of the parent compound podophyllotoxin. Among them, the proliferation inhibition activity of the podophyllotoxin derivative on HT-29 cells is significantly better than that on HCT-116 cells, and the details are shown in Table 1 of the accompanying drawings. Figure 1 In addition, the cytotoxicity of compounds 1b-11b on human colon epithelial cells is significantly lower than that of the parent compound podophyllotoxin, which indicates that the introduction of the triazole structure effectively reduces the toxic side effects of podophyllotoxin, and the details are shown in Table 2 of the accompanying drawings. Figure 2 Through the clonal formation experiment, it can be known that compound 2b can effectively inhibit the proliferation ability of human colon cancer cell lines HCT-116 and HT-29, and the details are shown in Table 3 of the accompanying drawings. Figure 3 4 From the structure-activity relationship, 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 AKT1 active site; in comparison, the targeting of 2b to AKT1 protein is better than that to tubulin, and the details are shown in Table 4 of the accompanying drawings. Figure 5 6 7. The water solubility experiment results show that the introduction of the triazole structure can effectively improve the water solubility of podophyllotoxin, and the water solubility of compounds 2b and 10b is greatly improved compared with that of podophyllotoxin, achieving the best effect.​​

[0022] Based on the above experimental results, the present invention provides the application of podophyllotoxin triazole ether derivatives as shown in Formula I in the preparation of microtubule inhibitors or AKT inhibitors.

[0023] Specifically, the tubulin inhibitor interferes with cell mitosis by inhibiting or promoting tubulin polymerization, causing mitosis to stop and arrest in the M phase, thereby leading to apoptosis of tumor cells and exerting an anti-tumor effect. The AKT inhibitor inhibits AKT1 protein activity. The podophyllotoxin triazole ether derivative of Formula I of this invention can be used to prepare dual-targeting inhibitors of tubulin and AKT1.

[0024] Furthermore, the present invention provides the use of podophyllotoxin triazole ether derivatives as shown in Formula I in the preparation of antitumor drugs.

[0025] Preferably, the tumor is a solid tumor. Solid tumors include, but are 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, as one of the main active ingredients, a podophyllotoxin triazole ether derivative of Formula I, and a pharmaceutically acceptable carrier.

[0027] The innovation of this invention lies in the synthesis of a series of novel dual-targeting tubulin / AKT1 podophyllotoxin triazole derivatives. Compared to single-target podophyllotoxin, these dual-targeting molecules exhibit better efficacy or the potential to overcome drug resistance. The binding energies of these novel dual-targeting podophyllotoxin triazole derivatives to AKT1 are all greater than those to the podophyllotoxin core, meaning these derivatives possess stronger AKT1 inhibitory effects. This represents a significant improvement in the structural derivatization of podophyllotoxin, resulting in stronger antitumor activity and better selectivity. Simultaneously, this structural modification alters the physicochemical properties of the podophyllotoxin molecule, improving its water solubility and thus optimizing its pharmacokinetic characteristics. Furthermore, the podophyllotoxin triazole ethers in this invention are less susceptible to esterase hydrolysis than previous podophyllotoxin ester derivatives, exhibiting stronger metabolic stability under physiological conditions, thereby prolonging their duration of action within tumor cells. The triazole ring has high electronegativity, which can enhance drug binding to the target site through hydrogen bonding or electrostatic interactions. The nitrogen atom on the triazole ring can act as a hydrogen bond acceptor, forming hydrogen bonds with amino acid residues of tumor-associated target enzymes or receptors. This interaction can significantly enhance drug selectivity and binding affinity. Therefore, this invention is expected to lay a theoretical foundation for developing podophyllotoxin triazole derivatives as candidate molecules for anticancer drugs. Attached Figure Description

[0028] Figure 1Figure for CCK8 detection method to detect the in-vitro proliferation inhibition activity of podophyllotoxin triazol ether derivatives 1b-11b on human colon cancer cell lines HCT-116 and HT-29;

[0029] Figure 2 Figure for CCK8 detection method to detect the in-vitro proliferation inhibition activity of podophyllotoxin triazol ether derivatives 1b-11b on human colon epithelial cell line NCM460;

[0030] Figure 3 Figure for clone formation experiment to detect the in-vitro proliferation inhibition activity of podophyllotoxin triazol ether derivative 2b on human colon cancer cell HCT-116 (the upper figure is the naked eye observation, and the lower figure is the 40-fold field of view under a microscope);

[0031] Figure 4 Figure for clone formation experiment to detect the in-vitro proliferation inhibition activity of podophyllotoxin triazol ether derivative 2b on human colon cancer cell HT-29 (the upper figure is the naked eye observation, and the lower figure is the 40-fold field of view under a microscope);

[0032] Figure 5 Figure for the docking binding energy column chart of podophyllotoxin triazol ether derivative of formula I and AKT1 and tubulin;

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

[0034] Figure 7 Figure for the docking binding mode of podophyllotoxin triazol ether derivative 2b and target protein tubulin (PDB: 1SA0);

[0035] Figure 8 Figure for the water solubility detection result of podophyllotoxin triazol ether derivative of formula I. DETAILED DESCRIPTION

[0036] The present application will be described in detail below with reference to examples, but they should not be understood as limiting the scope of protection of the present application.

[0037] The synthetic route of the new type of podophyllotoxin triazol ether derivatives (formula I series of compounds) of the present application is as follows:

[0038]

[0039] The preparation method of the podophyllotoxin triazol ether derivatives of the present application is as follows:

[0040] Step 1: Using Podophyllotoxin (1 mmol) and bromo-propargyl (1 mmol) as starting materials, 10 mL of dry N,N-dimethylacetamide (DMF) as solvent. 2.4 mmol of sodium hydride (NaH) was slowly added into a dry reaction vessel under ice-bath condition, and the reaction was stirred at room temperature for more than 12 hours. The whole reaction progress was monitored by thin layer chromatography (TLC) on silica gel plate. After the reaction was completed, 5 mL of saturated ammonium chloride water was added to quench the reaction, and 20 mL of dry ethyl acetate was added to extract the reaction mixture three times. The combined organic solution was washed with 12 mL of saturated brine, and then dried over anhydrous sodium sulfate, filtered and concentrated to give compound A, podophyllotoxin propargyl ether, as a light yellow solid with a melting point of 192 °C and a yield of 85%.

[0041] Step 2: Compound A (0.8 mmol) was added into a round-bottom flask, followed by copper sulfate pentahydrate (0.2 mmol), sodium ascorbate (0.8 mmol) and different substituted azide compounds (1 mmol). The solid reactants were dissolved in 10 mL of N,N-dimethylformamide (DMF), and the reaction was heated at 60 °C under nitrogen protection for more than 6 hours. The reaction progress was monitored by thin layer chromatography (TLC) on silica gel plate. After the reaction was completed, the oil bath was cooled to room temperature, and then the reaction solution was added into 10 mL of distilled water and extracted with 20 mL of ethyl acetate three times. The combined organic solution was washed with saturated brine. After washing, an appropriate amount of anhydrous sodium sulfite was added to remove water from the organic phase, and then the solution was filtered and concentrated to give the product, podophyllotoxin triazole series derivatives, by column chromatography (V 石油醚 :V 乙酸乙酯 = 3:1).

[0042] Example 1

[0043]

[0044] Step 1: Using Podophyllotoxin (1 mmol) and bromo-propargyl (1 mmol) as starting materials, 10 mL of dry N,N-dimethylacetamide (DMF) as solvent. 2.4 mmol of sodium hydride (NaH) was slowly added into a dry reaction vessel under ice-bath condition, and the reaction was stirred at room temperature for more than 12 hours. The whole reaction progress was monitored by thin layer chromatography (TLC) on silica gel plate. After the reaction was completed, 5 mL of saturated ammonium chloride water was added to quench the reaction, and 20 mL of dry ethyl acetate was added to extract the reaction mixture three times. The combined organic solution was washed with 12 mL of saturated brine, and then dried over anhydrous sodium sulfate, filtered and concentrated to give compound A, podophyllotoxin propargyl ether, as 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] Add 0.2 mmol copper sulfate pentahydrate, 0.8 mmol sodium ascorbate, and 1 mmol methyl azidoacetate (CAS No. 1816-92-8) to a round-bottom flask containing compound A (0.8 mmol). Add 10 mL of N,N-dimethylformamide to dissolve the solid reactants. Heat the mixture at 60°C for at least 6 hours under nitrogen protection. Monitor the reaction progress using silica gel plate chromatography. After the reaction is complete, allow the oil bath to cool to room temperature. Then, add 10 mL of distilled water and extract three times with 20 mL of ethyl acetate. Wash the collected organic phase with saturated brine. Add an appropriate amount of anhydrous sodium sulfite to the washed organic phase to remove water. Filter the mixture and evaporate the solvent to dryness using a rotary evaporator. Finally, perform column chromatography (V... 石油醚 :V 乙酸乙酯 After separating and purifying the product (ratio 3:1), compound 1b was obtained, with a melting point of 124°C, as 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] Compound 2b was prepared using the same method as above, with the azide compound being (R)-5-(azidomethyl)-3-[3-fluoro-4-(4-morpholinyl)phenyl]-2-oxazolidinone (CAS No. 168828-84-0). All other conditions remained unchanged, yielding compound 2b with a melting point of 146.9℃, as a pale 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 azido compound is 2-azidoethanol (CAS No. 1517-05-1), and other conditions are the same, to obtain compound 3b, the melting point is 124.5°C, the form is white solid, and the yield is 43%. 1 H NMR (400 MHz, CDC13) δ 7.27 (s, 1H), 6.93 (s, 1H), 6.60 (s, 1H), 6.37 (s, 2H), 6.00 (d, J = 9.2 Hz, 2H), 4.54 (s, 2H), 4.48-4.40 (m, 3H), 4.31 (s, 2H), 4.01 (s, 3H), 3.81 (s, 3H), 3.70 (s, 6H), 3.42 (d, J = 6.3 Hz, 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 azido compound is benzyl azide (CAS No. 622-79-7), and other conditions are the same, to obtain compound 4b, the melting point is 139.5°C, the form is white solid, and the yield is 70%. 1 H NMR (400 MHz, CDC13) δ 7.37 (d, J = 6.6 Hz, 3H), 7.28 (d, J = 6.9 Hz, 3H), 6.93 (s, 1H), 6.49 (s, 1H), 6.40 (s, 2H), 5.95 (d, J = 6.3 Hz, 2H), 5.52 (s, 2H), 4.64 (s, 2H), 4.37 (dd, J = 18.4, 7.4 Hz, 2H), 4.21 (d, J = 4.0 Hz, 1H), 4.10 (d, J = 9.7 Hz, 1H), 3.83 (s, 3H), 3.72 (s, 6H), 3.31 (dd, J = 9.4, 4.3 Hz, 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 procedure was same as above for the preparation of compound 5b, except that the azido compound was 3'-azido-3'-deoxythymidine (CAS No. 30516-87-1), and other conditions were the same. Compound 5b was obtained as a light yellow solid with a melting point of 143.8 °C in a yield of 63%. 1 HNMR (400 MHz, d6-DMSO) δ 11.40 (s, 1H), 8.28 (s, 1H), 7.85 (s, 1H), 7.03 (s, 1H), 6.60 (s, 2H), 6.44 (t, J = 6.2 Hz, 1H), 6.40 (s, 1H), 5.99 (s, 2H), 5.35 (d, J = 18.7 Hz, 2H), 4.70 (s, 2H), 4.51 (d, J = 6.4 Hz, 1H), 4.43 (t, J = 8.0 Hz, 1H), 4.19 (d, J = 10.5 Hz, 2H), 4.13 (d, J = 5.0 Hz, 1H), 3.75 (s, 1H), 3.67 (s, 6H), 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 procedure was same as above for the preparation of compound 5b, except that the azido compound was 3'-azido-3'-deoxythymidine (CAS No. 30516-87-1), and other conditions were the same. Compound 5b was obtained as a light yellow solid with a melting point of 143.8 °C in a yield of 63%. 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] Compound 7b was prepared using the same method as above, with the azide compound being 1-azidomethyl-4-methoxybenzene (CAS No. 70978-37-9). All other conditions remained unchanged, yielding compound 7b with a melting point of 145.6℃, as a white solid, 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 compound 8b was prepared as above, using 4-methoxyphenyloxy carbonic azide (CAS No. 25474-85-5) as the azido compound, and otherwise under the same conditions to give the compound 8b as a white solid with a melting point of 145.6 °C in a yield of 53%. 1 HNMR (600 MHz, CDC13) δ 7.24 (d, J = 8.4 Hz, 2H), 7.20 (s, 1H), 6.91 (d, J = 6.8 Hz, 2H), 6.89 (s, 1H), 6.49 (s, 1H), 6.40 (s, 2H), 5.96 (d, J = 9.2 Hz, 2H), 5.45 (s, 2H), 4.63 (s, 2H), 4.41-4.36 (m, 1H), 4.34 (d, J = 5.9 Hz, 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, 4.5 Hz, 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 compound 9b was prepared as above, using p-azidobenzoic acid (CAS No. 6427-66-3) as the azido compound, and otherwise under the same conditions to give the compound 9b as a white solid with a melting point of 159.7 °C in a yield of 64%. 1 HNMR (600 MHz, CDC13) δ 7.24 (d, J = 8.4 Hz, 2H), 7.20 (s, 1H), 6.91 (d, J = 6.8 Hz, 2H), 6.89 (s, 1H), 6.49 (s, 1H), 6.40 (s, 2H), 5.96 (d, J = 9.2 Hz, 2H), 5.45 (s, 2H), 4.63 (s, 2H), 4.41-4.36 (m, 1H), 4.34 (d, J = 5.9 Hz, 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, 4.5 Hz, 1H), 3.04-2.98 (m, 1H). HR-MS (ESI + )m / z: 616.1978 [M+H] +Found: 616.1926 [M+H] + .

[0072] Example 11

[0073]

[0074] Compound 10b was prepared using the same method as above, with the azide compound being triethylene glycol azide (CAS No. 86520-52-7). All other conditions remained unchanged, yielding compound 10b with a melting point of 137.7℃, appearing as a white solid, 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-acetamidobenzenesulfonyl azide (CAS No. 2158-14-7). All other conditions remain unchanged. Compound 11b is obtained as a pale yellow solid with a melting point of 124℃ 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] CCK-8 colorimetric method was used to detect cell proliferation activity. Cells were grown in complete medium to the logarithmic growth phase and diluted to 2 x 10 4 / mL. Then, the cell suspension was inoculated into a 96-well flat-bottom plate at 100 μL per well, and incubated at 37°C, 5% CO2 for 12 hours. The test compound was diluted to the preset concentration (0.1, 1, 10, 100, 1000 μM) and added to the 96-well flat-bottom plate, with gefitinib as the positive control. Gefitinib is a selective epidermal growth factor receptor tyrosine kinase inhibitor, which 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 removed and 100 μL of fresh culture medium containing 10% CCK-8 solution was added, and incubation was continued for about 2 hours. The absorbance was measured and recorded using an enzyme label instrument (ELx800, BioTek, USA) at a test wavelength of 450 nm. Each sample had three repeated wells, and each experiment was repeated three times. Finally, the Origin 7.5 software was used to calculate the drug concentration required for 50% cell survival, i.e. IC 50 value.

[0081] Using human colon cancer cell lines HCT-116 and HT-29 as detection strains, it was found through in vitro tumor cell inhibition activity research of the podophyllotoxin triazole derivatives of formula I that the derivatives had obvious 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 (Gefitinib), and the results are shown in the attached Figure 1 At the same time, the half-inhibition concentration (IC 50 value) of the derivatives on human colon epithelial cell line NCM460 was greater than 50 μmol / L, indicating that the toxicity of the derivatives on normal cells was significantly lower than that of the parent structure podophyllotoxin (PPT), and the results are shown in the attached Figure 2 .

[0082] In addition, interference clone formation experiment was carried out on the best active compound 2b, and 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-proliferation activity compared with podophyllotoxin, and the results are shown in the attached Figure 3 、 4 .

[0083] Example 14

[0084] Podophyllotoxin triazole derivatives can effectively target AKT1

[0085] 1b~11b series of small molecules were optimized by MM2 station of chem3D to save as sdf file, then imported into Autodock vina software to remove water, hydrogen, calculate charge and determine the root of the ligand and select the ligand small molecule that can twist the bond. The imported AKT1 protein (PDB: 6HHI) and tubulin (PDB: 1SA0) were removed from the solvent and ligand small molecules using pymol software, and then hydrogen and charge were calculated in the Autodock vina software, and AKT1 protein and tubulin were used as the acceptor and saved as Pdbqt file. According to the original covalent conformation of AKT1 protein crystal structure, the coordinates of the ligand small molecule of AKT inhibitor were determined to determine the coordinates of the docking box (Grid center: X-1.96252 Y4.46218 Z-10.6489; Grid size: X 26 Y 26 Z 26; Grid space: 0.375) and semi-flexible docking was performed. According to the original tubulin inhibitor colchicine ligand small molecule in the tubulin crystal structure, the coordinates of the docking box were determined (Grid center: X 52.3796 Y 52.2827 Z-7.67196; Grid size: X 69.2 Y 69.2 Z 69.2; Grid space: 0.375) and semi-flexible docking was performed.

[0086] From Figure 5 It can be seen that all podophyllotoxin triazole derivatives have lower binding energy than podophyllotoxin to AKT1, indicating better binding effect. The binding energy of most podophyllotoxin triazole derivatives to tubulin is not significantly different from that 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~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 a hydrogen bond with TYRA:315, and other functional groups can also form hydrogen bonds with three amino acids GLU A:85, THRA:82 and LEU A:295 of AKT1 protein, significantly increasing the targeting of compound 2b to AKT1 protein, as shown in Figure 6 . The binding energy of compound 2b to tubulin is-6.421 kcal / mol, slightly higher than that of podophyllotoxin to tubulin (-8.688 kcal / mol). Figure 7The results showed that compound 2b formed various interactions with the amino acids of tubulin, such as hydrogen bonds, van der Waals forces, and carbon-hydrogen bonds. However, it formed significantly fewer hydrogen bonds than AKT1, resulting in a decrease in binding energy and weaker tubulin targeting compared to AKT1.

[0087] Example 15

[0088] Podophyllotoxin triazole derivatives have increased water solubility

[0089] like Figure 8 As shown, after introducing the triazole structure, the water solubility of compounds 1b, 2b, 3b, 5b, 8b, 10b, and 11b was improved compared to podophyllotoxin and etoposide. All the b-series compounds exhibited higher water solubility than the AKT allosteric inhibitor MK2206 and the targeted drug gefitinib for treating non-small cell lung cancer. Generally, -6 <log(S w -4 indicates that the compound has low water solubility. <log(S w -2 indicates that the compound is highly water-soluble. <log(S w A value less than 0 indicates optimal water solubility of the compound. Compounds 2b and 10b exhibit significantly improved water solubility compared to podophyllotoxin, achieving the best effect. This suggests that these podophyllotoxin triazole derivatives possess physicochemical properties that are more conducive to drug formulation.

[0090] Based on the above conclusions, this type of podophyllotoxin triazole derivative has the potential to be used as a microtubule and / or AKT1 inhibitor and can be prepared into an anti-tumor drug.

[0091] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A class of podophyllotoxin triazole ether derivatives, characterized in that, The structural formula of the podophyllotoxin triazole ether derivative is shown below: 。 2. The method for preparing the podophyllotoxin triazole ether derivative according to claim 1, characterized in that, Includes the following steps: Step (1): Using podophyllotoxin and halopropyne as raw materials and anhydrous organic solvent as solvent, sodium hydride was slowly added to a dry reaction vessel under ice bath conditions. The reaction was stirred at room temperature and the entire reaction process was monitored by silica gel plate by thin-layer chromatography. After the reaction was completed, saturated ammonium chloride water was added to quench the reaction, the organic phase was extracted, washed, dehydrated, and evaporated to dryness to obtain podophyllotoxin alkyne intermediate A. Step (2): Dissolve podophyllotoxin alkyne intermediate A, copper sulfate pentahydrate, sodium ascorbate, and different substituted azide compounds in N,N-dimethylformamide; react at 60℃-80℃ for 6-12 hours under nitrogen protection, and monitor the reaction progress with silica gel plate for thin-layer chromatography; after the reaction is completed, extract and combine the organic phases, remove water, evaporate to dryness, separate and purify to obtain podophyllotoxin triazole ether derivative.

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

4.

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

8.

6. The use of the podophyllotoxin triazole ether derivative according to claim 1 in the preparation of antitumor drugs.

7. The application according to claim 6, characterized in that, The antitumor drug is a microtubule inhibitor or an AKT inhibitor.

8. The application according to claim 6 or 7, characterized in that, The tumors include solid tumors.

9. The application 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, It includes the podophyllotoxin triazole ether derivative as described in claim 1 as one of the main active ingredients, and a pharmaceutically acceptable carrier.

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