Tubulin / EGFR (epidermal growth factor receptor) dual-targeting podophyllotoxin derivative and application thereof

Abstract

The invention discloses a podophyllotoxin structure-based tubulin / EGFR (epidermal growth factor receptor) dual-targeting podophyllotoxin triazole derivative and application thereof in antitumor drugs, and belongs to the technical field of medicinal chemistry. A triazole group is introduced into a podophyllotoxin mother nucleus through click chemistry, and a series of novel derivatives are designed and synthesized. Molecular docking studies show that the compound can target tubulin and EGFR kinase structural domains at the same time, and tumor cell proliferation is inhibited through a double-target synergistic effect. In-vitro experiments prove that the derivative has significant inhibitory activity on non-small cell lung cancer cells and has low toxicity on normal pulmonary epithelial cells. The dual-targeting strategy can overcome the drug resistance problem of the EGFR inhibitor, optimizes the pharmacokinetic characteristics, has the advantages of high oral availability, good in-vivo stability and the like, and is suitable for treatment of solid tumors such as non-small cell lung cancer and the like.

Description

Technical Field

[0001] This invention belongs to the field of chemical pharmaceutical technology, specifically relating to the synthesis of a class of microtubule / EGFR dual-targeting podophyllotoxin derivatives. Background Technology

[0002] Non-small cell lung cancer (NSCLC), a major subtype of lung cancer, accounts for approximately 80%-85% of all lung cancer cases. Its clinical treatment faces multiple severe challenges, including high drug resistance, high recurrence and metastasis rates, and poor prognosis. In current clinical treatment strategies, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are the first-line treatment for patients with driver gene positivity (such as EGFR L858R mutations and exon 19 deletion mutations). However, approximately 50% of patients develop drug resistance within 1-2 years due to secondary T790M / C797S mutations or bypass signaling activation (such as MET amplification), ultimately leading to treatment failure. Furthermore, wild-type EGFR patients have a response rate of less than 40% to traditional chemotherapy regimens (such as paclitaxel combined with platinum-based drugs), and treatment is often accompanied by severe myelosuppression and other toxic side effects.

[0003] Podophyllotoxin (PT) is a natural cyclophenol polysaccharide that, while exhibiting potent cytotoxicity against various tumor cells, including NSCLC, has had its clinical application significantly hampered by severe toxic side effects (such as neurotoxicity) and poor water solubility. Existing semi-synthetic derivatives (such as etoposide and teniposide) exert antitumor effects by inhibiting topoisomerase II, but suffer from insufficient targeting and a tendency to induce drug resistance. Recent studies have found that some podophyllotoxin derivatives can exert antitumor activity by inhibiting microtubule polymerization; other studies have confirmed that podophyllotoxin analogs can target and inhibit EGFR, thereby inhibiting the growth of gefitinib-resistant NSCLC cells and inducing apoptosis. However, because single-target action cannot overcome compensatory activation of the EGFR signaling pathway, their inhibitory activity against NSCLC cells is significantly limited. This is mainly because abnormal EGFR activation can enhance microtubule stability through the PI3K / AKT / mTOR pathway, and microtubule damage can in turn activate downstream pro-survival signals of EGFR, forming a resistance cycle.

[0004] 1,2,3-triazole is a highly attractive functional group with excellent aromatic and metabolic stability. This invention proposes introducing the 1,2,3-triazole group into the 4β position of the podophyllotoxin nucleus via click chemistry, aiming to enhance its inhibitory activity against tubulin and EGFR, overcoming the limitations of single-target inhibition in NSCLC treatment. This strategy addresses the problems of EGFR-TKI resistance and high toxicity of chemotherapy drugs, providing a novel treatment option for advanced NSCLC. Summary of the Invention

[0005] The purpose of this invention is to provide a novel class of tubulin / EGFR dual-targeting podophyllotoxin derivatives and their applications. One objective of this invention is to provide a novel class of tubulin / EGFR dual-targeting podophyllotoxin derivatives. A second objective is to provide a method for synthesizing a novel class of tubulin / EGFR dual-targeting podophyllotoxin derivatives. A third objective is to provide the application of a novel class of tubulin / EGFR dual-targeting podophyllotoxin derivatives in the preparation of antitumor drugs.

[0006] This invention first provides a class of triazole derivatives that target both tubulin and EGFR, specifically podophyllotoxin, with the following general formula:

[0007] The selection range of substituents is as follows: R is independently selected from: , , , , , .

[0008] The present invention further provides a method for preparing the above-mentioned tubulin / EGFR dual-targeting podophyllotoxin triazole derivative, the synthetic route of which is as follows:

[0009] Specifically, the above preparation method includes the following steps: Step 1: Weigh propynic acid into a round-bottom flask and dissolve it in anhydrous dichloromethane. Add the condensing agent N,N'-dicyclohexylcarbodiimide (DCC) and the catalyst 4-dimethylaminopyridine (DMAP) to the flask. Stir the reaction under ice bath conditions for 30 min to generate an O-acylisourea intermediate. Then, slowly add podophyllotoxin (PPT), gradually raise the temperature to room temperature, and continue stirring for 12 h (TLC monitoring the reaction progress). After the reaction is complete, add dilute hydrochloric acid dropwise under ice bath conditions and stir for 15 min to precipitate dicyclohexylurea (DCU). Filter through a Buchner funnel to remove the precipitate. Extract the filtrate with ethyl acetate, wash successively with dilute hydrochloric acid, saturated sodium bicarbonate, and saturated brine, and then dry with anhydrous sodium sulfate and rotary evaporate. Separate the crude product by column chromatography to obtain compound B. The molar ratio of podophyllotoxin, propynic acid, DCC, and DMAP is 1:1.2:2.0:0.1-1:1.5:2.0:0.1.

[0010] Step 2: Weigh intermediate B into a three-necked flask, add anhydrous N,N-dimethylformamide (DMF) to dissolve it, purge with nitrogen three times, and then inject copper sulfate pentahydrate (CuSO4). An aqueous solution of 5H₂O and sodium ascorbate (Na-L-ascorbate) was prepared. The azide compound was dissolved in DMF and slowly added dropwise to the reaction system under nitrogen protection, with stirring for 12 h (TLC monitoring of the reaction progress). After cooling, the reaction solution was diluted with water, extracted with ethyl acetate, quenched with saturated sodium thiosulfate solution, washed with brine, dried over anhydrous sodium sulfate, and then rotary evaporated. The crude product was purified by silica gel column chromatography to obtain the target product 1c–6c. The molar ratio of compound B to different substituted azide compounds, copper sulfate pentahydrate, and sodium ascorbate was 1:0.8:0.4:1–1:1.2:0.4:1.

[0011] The molecular structural formula of the podophyllotoxin triazole derivative synthesized in a specific embodiment of the present invention is shown below:

[0012] In vitro experiments have confirmed that the dual-targeting effect, by synergistically inhibiting EGFR signaling and microtubule polymerization, increases the tumor cell apoptosis rate to 2.3 times that of monotherapy, and reduces the dosage by 40%-60% while achieving the same efficacy, providing a safer and more efficient new strategy for the treatment of advanced lung cancer.

[0013] The tubulin / EGFR dual-targeting podophyllotoxin triazole ester synthesized in this invention exhibits stronger proliferative inhibitory activity against human lung cancer cells compared to the previously studied tubulin / AKT1 dual-targeting podophyllotoxin triazole ether.

[0014] Through molecular structure optimization, the derivatives can simultaneously target the EGFR kinase ATP-binding pocket (such as Lys745, Ser720, Gly719, and Cys797 residues) and the colchicine site of the tubulin β subunit (such as Ser140 and Phe141 residues), blocking the compensatory regulation of downstream pro-survival signals (such as the PI3K / AKT / mTOR pathway) and microtubule dynamics, significantly overcoming EGFR-TKI resistance (such as the T790M / C797S mutation); suggesting that the compounds of this invention have a dual-targeting synergistic mechanism. Molecular docking also showed that the podophyllotoxin triazole ester derivatives significantly enhanced the binding ability of EGFR kinase and tubulin compared to the parent podophyllotoxin.

[0015] The tubulin / EGFR dual-targeting podophyllotoxin derivative provided by this invention also includes pharmaceutically acceptable salts, isotopic variants, tautomers, stereoisomers, prodrugs, polymorphs, hydrates, or solvates thereof.

[0016] The present invention further provides a pharmaceutical composition in which one of the active ingredients comprises the above-mentioned tubulin / EGFR dual-targeting podophyllotoxin derivative, or a pharmaceutically acceptable salt, isotope variant, tautomer, stereoisomer, prodrug, polymorph, hydrate or solvate thereof; and a pharmaceutically acceptable excipient.

[0017] The present invention also provides the use of the above-mentioned podophyllotoxin derivative in the preparation of medicaments for treating and / or preventing diseases mediated by EGFR protein and its mutants.

[0018] The EGFR mutants described in this invention are preferably one or more of the T790M mutant and C797S mutant of the EGFR protein.

[0019] The diseases mediated by the EGFR protein and its mutants described in this invention are selected from lung cancer, colon cancer, urothelial carcinoma, breast cancer, prostate cancer, brain cancer, ovarian cancer, gastric cancer, pancreatic cancer, head and neck cancer, bladder cancer, and mesothelioma. Lung cancer is preferred, non-small cell lung cancer is more preferred, especially advanced non-small cell lung cancer.

[0020] The advantages of this invention compared to existing technologies are as follows: The innovation of this invention lies in the synthesis of a series of novel dual-targeting tubulin / EGFR podophyllotoxin triazole ester derivatives. Compared to single-target podophyllotoxin, dual-targeting molecules have the potential for better efficacy or overcoming drug resistance. In this invention, the obtained podophyllotoxin triazole ester derivatives exhibit less toxicity to normal lung epithelial cells, resulting in higher drug safety. Compared to previously selected tubulin and AKT1 targets, EGFR, one of the targets in this invention, is an upstream target protein of AKT1. Inhibitors designed targeting the upstream EGFR have a broader and stronger anti-tumor spectrum. This invention is expected to lay a theoretical foundation for developing podophyllotoxin triazole ester derivatives as candidate molecules for anticancer drugs. Attached Figure Description

[0021] Figure 1 The antiproliferative activities of podophyllotoxin ester derivatives, podophyllotoxin ether derivatives, podophyllotoxin, and gefitinib against different cancer cell lines; Figure 2 The proliferative activity of podophyllotoxin ester derivatives, podophyllotoxin, and gefitinib on normal lung cells; Figure 3 Flow cytometry analysis for detecting EdU 488 labeling in A549 cell proliferation; Figure 4 The effect of podophyllotoxin ester derivative 3c on apoptosis in A549 cells; Figure 5 The effect of podophyllotoxin ester derivative 3c on the migration ability of A549 cells; Figure 6 Computer simulation of the binding mode of podophyllotoxin ester derivatives 3c, PPT and microtubules; Figure 7 Molecular dynamics simulation of the binding of podophyllotoxin ester derivatives 3c and PPT to tubulin; Figure 8 The effect of podophyllotoxin ester derivative 3c on the microtubule cytoskeleton structure of A549 cells; Figure 9 To simulate the binding mode of podophyllotoxin ester derivatives 3c, PPT and EGFR protein using computer simulation; Figure 10 Molecular dynamics simulations of the binding of podophyllotoxin ester derivatives 3c, PPT, and EGFR; Figure 11 The effect of podophyllotoxin ester derivative 3c on the protein expression of AKT, p-AKT, EGFR and p-EGFR in A549 cells. Detailed Implementation

[0022] The present invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0023] The following specific embodiments of the present invention provide a method for preparing a class of microtubule / EGFR dual-targeting podophyllotoxin derivatives, the specific steps of which are as follows:

[0024] Step 1: Weigh propynic acid into a dry round-bottom flask, dissolve it in anhydrous dichloromethane, and add the condensing agent dicyclohexylcarbodiimide (DCC) and the catalyst 4-dimethylaminopyridine (DMAP). Stir in an ice bath (0°C) for 30 min to generate the O-acylisourea intermediate. Then, slowly add podophyllotoxin, gradually raise the temperature to room temperature, and stir for 6-12 h (TLC monitoring). After the reaction is complete, add dilute hydrochloric acid (1 mol / L HCl) dropwise in an ice bath and stir for 15-30 min to precipitate DCU. Filter through a Buchner funnel, extract the filtrate with ethyl acetate, wash successively with dilute hydrochloric acid, saturated sodium bicarbonate, and brine, dry with anhydrous sodium sulfate, and then rotary evaporate. Purify the crude product by silica gel column chromatography to obtain intermediate B.

[0025] Step 2: Weigh intermediate B into a three-necked flask, dissolve it in anhydrous DMF, purge the mixture three times with nitrogen, and then inject an aqueous solution of copper sulfate pentahydrate and sodium ascorbate. Dissolve the azide compound in DMF and slowly add it dropwise under nitrogen protection, stirring at 60°C for 12 hours (monitored by TLC). After cooling the reaction solution, dilute with water, extract with ethyl acetate, quench the organic phase with saturated sodium thiosulfate solution, wash with brine, dry with anhydrous sodium sulfate, and then rotary evaporate. The crude product is subjected to silica gel column chromatography (V... 石油醚 V 乙酸乙酯 Purification at a ratio of 3:1 yielded the target product 1c–6c.

[0026] Example 1

[0027] Step 1: Weigh propargyl acid (101 mg, 1.44 mmol, 1.2 eq) into a dry round-bottom flask, dissolve it in anhydrous dichloromethane, add condensing agent DCC (495 mg, 2.4 mmol, 2.0 eq) and catalyst DMAP (15 mg, 0.12 mmol, 0.1 eq). Stir in an ice bath (0℃) for 30 min to generate O-acylisourea intermediate, then slowly add podophyllotoxin (500 mg, 1.2 mmol, 1.0 eq), gradually raise the temperature to room temperature and stir for 6-12 h (TLC monitoring). After the reaction is complete, add dilute hydrochloric acid (1 mol / L HCl) dropwise under ice bath conditions and stir for 15-30 min to precipitate DCU. Filter through a Buchner funnel, extract the filtrate with ethyl acetate, wash successively with dilute hydrochloric acid, saturated sodium bicarbonate, and brine, dry with anhydrous sodium sulfate, and then rotary evaporate. The crude product was purified by silica gel column chromatography to obtain intermediate B in 80% yield. 1 H NMR (300 MHz, Chloroform- d )δ 6.98 (d, J = 50.7 Hz, 1H), 6.45 (d, J = 6.8 Hz, 3H), 5.93 (dd, J = 12.5,5.9 Hz, 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, 5.3 Hz, 1H), 3.00(ddd, J = 14.0, 7.1, 3.9 Hz, 1H), 1.26 (s, 1H).HR-MS (ESI + m / z: 453.1538 [M+H] + Found: 453.1544 [M+H] + .

[0028] Step 2: Weigh intermediate B (50 mg, 0.11 mmol, 1.0 eq) into a three-necked flask, dissolve it in anhydrous DMF (10 mL), purge with nitrogen three times, and then add 1 mL each of aqueous solutions of copper sulfate pentahydrate (2.75 mg, 0.011 mmol, 0.1 eq) and sodium ascorbate (32.7 mg, 0.165 mmol, 1.5 eq). Dissolve 0.132 mmol, 1.2 eq in DMF (5 mL) and add slowly dropwise under nitrogen protection, stirring at 60 °C for 12 h (monitored by TLC). After cooling, dilute with water, extract with ethyl acetate, quench the organic phase with saturated sodium thiosulfate solution, wash with brine, dry with anhydrous sodium sulfate, and then rotary evaporate. The crude product is subjected to silica gel column chromatography (V... 石油醚 V 乙酸乙酯 The mixture was purified by a ratio of 3:1 to give compound 1c, which has a melting point of 124.5-125.5℃ and is a white solid with a yield of 68%. 1 H NMR (300 MHz, Chloroform- d ) δ 8.50 (s,1H), 6.89 (s, 1H), 6.56 (s, 1H), 6.44 (s, 2H), 6.15 (d, J = 8.3 Hz, 1H), 5.96(s, 2H), 4.62 (t, J = 4.3 Hz, 4H), 4.45 (dd, J = 9.1, 6.2 Hz, 1H), 4.28 (t, J= 9.4 Hz, 1H), 3.89 (t, J = 4.8 Hz, 2H), 3.75 (d, J = 13.2 Hz, 10H), 3.60 (d,J = 20.7 Hz, 5H), 3.00 (s, 1H). 13 C NMR (151 MHz, Chloroform- d ) δ 173.73,161.90, 152.81, 148.43, 147.86, 137.29, 134.85, 132.64, 129.88, 128.04,109.88, 108.26, 107.49, 101.75, 74.78, 72.70, 71.45, 70.57, 70.28, 68.94,61.81, 60.87, 56.41, 50.67, 45.85, 43.96, 38.82. HR-MS (ESI+)m / z: 642.2293[M+H] +Found: 642.2294 [M+H] + .

[0029] Example 2

[0030] Compound 2c was prepared by the same method as above, except that the azide compound was replaced with 4-acetamidobenzenesulfonyl azide, and all other conditions remained unchanged. Compound 2c was obtained as a white solid with a melting point of 119.5-120.5 °C and a yield of 65%. 1 H NMR(300 MHz, DMSO-d6) δ 10.28 (s, 1H), 7.80 – 7.61 (m, 3H), 7.24 (s, 1H), 7.07(s, 1H), 6.68 – 6.56 (m, 1H), 6.32 (d, J = 10.9 Hz, 2H), 6.12 – 5.71 (m, 4H), 4.68 – 4.55 (m, 1H), 4.48 (t, J = 7.0 Hz, 1H), 4.16 – 3.96 (m, 1H), 3.64 (s, 3H), 3.62 (s, 6H), 3.32 (s, 1H), 2.78 – 2.56 (m, 1H), 2.08 (s, 3H). 13 C NMR(75 MHz, DMSO-d6) δ 174.78, 168.96, 151.99, 146.66, 146.51, 142.23, 138.11,136.61, 136.44, 135.07, 126.71, 118.47, 108.43, 71.10, 70.63, 59.95, 55.89,44.14, 43.42, 40.21, 24.13. HR-MS (ESI+) m / z: 586.2111 [M+H] + Found: 586.2179 [M+H] + .

[0031] Example 3

[0032] Compound 3c was prepared by the same method as above, except that the azide compound was replaced with 2-azidoethanol, and all other conditions remained unchanged. Compound 3c was obtained as a white solid with a melting point of 129.2-130.6 °C and a yield of 43%. 1 H NMR (600 MHz, Chloroform- d) δ 8.30 (s, 1H), 7.27 (s, 1H), 6.45 (s, 3H), 5.97 (s, 3H), 4.64(d, J = 3.4 Hz, 1H), 4.61 – 4.54 (m, 3H), 4.45 (dd, J = 9.2, 6.2 Hz, 1H), 4.28 (d, J = 9.1 Hz, 2H), 4.06 (d, J = 9.8 Hz, 1H), 3.78 (s, 9H), 3.43 (ddt, J = 11.0, 7.7, 3.4 Hz, 1H), 3.02 (d, J = 20.8 Hz, 1H). 13 C NMR (151 MHz, Chloroform- d ) δ 173.76, 161.28, 152.75, 148.41, 147.81, 139.06, 137.20,134.90, 132.58, 129.28, 127.99, 109.86, 108.24, 107.35, 101.75, 74.65, 71.43,60.83, 56.34, 53.02, 45.74, 43.89, 38.78.HR-MS (ESI+) m / z: 554.1768 [M+H] + Found: 554.1775 [M+H] + .

[0033] Example 4

[0034] Compound 4c was prepared by the same method as above, except that the azide compound was replaced with 3′-azido-3′-deoxythymidine, and all other conditions remained unchanged. Compound 4c was obtained with a melting point of 119.5-120.6 °C, in the form of a white powder, and a yield of 70%. 1H NMR (600MHz, DMSO-d6) δ 8.63 (s, 1H), 7.85 (s, 2H), 6.95 (s, 1H), 6.62 (s, 1H), 6.55(s, 2H), 6.44 (s, 1H), 6.00 (s, 2H), 5.97 (s, 1H), 4.51 (t, J = 8.5 Hz, 1H), 4.41 – 4.34 (m, 3H), 4.23 (s, 2H), 3.65 (s, 11H), 2.75 (dt, J = 15.7, 7.7 Hz, 2H), 2.71 – 2.63 (m, 2H), 1.70 (s, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 177.63,164.09, 160.04, 152.87, 150.70, 147.86, 146.52, 138.61, 138.53, 136.47,136.18, 132.59, 129.10, 126.47, 109.92, 109.30, 108.58, 105.73, 101.52,84.45, 84.09, 73.39, 70.80, 60.76, 60.18, 60.02, 56.05, 55.97, 43.91, 43.67,40.05, 37.36, 12.41. HR-MS (ESI+) m / z: 554.1768 [M+H] + Found: 554.1775 [M+H] + .

[0035] Example 5

[0036] Compound 5c was prepared by the same method as above, except that the azide compound was replaced with para-azidobenzoic acid, and all other conditions remained unchanged. Compound 5c was obtained with a melting point of 140.9℃, in the form of a white powder, and a yield of 65%. 1 H NMR (600 MHz, Chloroform- d ) δ 8.62 (s, 1H), 8.27 (s, 2H), 7.84 (s, 2H), 6.91 (s, 1H), 6.58 (s, 1H), 6.46 (s, 2H), 6.19 (d, J = 8.8 Hz, 1H), 5.97 (s, 2H), 4.65 (d,J =4.2 Hz, 1H), 4.48 (t, J = 8.0 Hz, 1H), 4.31 (t, J = 9.8 Hz, 1H), 3.79 (s,9H), 3.07 (dd, J = 15.1, 7.4 Hz, 1H), 3.01 (dd, J = 14.7, 4.3 Hz, 1H). 13 C NMR (151 MHz, Chloroform- d ) δ 179.80, 174.76, 173.79, 160.90, 157.92, 152.64,152.51, 148.36, 147.74, 140.08, 137.14, 134.91, 132.56, 131.78, 131.07,127.84, 120.01, 109.81, 109.66, 108.47, 108.30, 101.71, 75.06, 71.39, 60.72,56.29, 45.59, 43.80, 38.66.HR-MS (ESI + m / z: 630.1718 [M+H] + Found: 630.1730 [M+H] + .

[0037] Example 6

[0038] Compound 6c was prepared by the same method as above, except that the azide compound was replaced with (R)-5-(azidomethyl)-3-[3-fluoro-4-(4-morpholinyl)phenyl]-2-oxazolidinone, and all other conditions remained unchanged, to obtain compound 6c with a melting point of 116.5-117.5℃, in the form of a white powder, and a yield of 75%. 1 H NMR (300 MHz, Chloroform- d) δ 8.38 (s, 1H), 7.30(d, J = 2.6 Hz, 1H), 7.00 (dd, J = 8.8, 2.6 Hz, 1H), 6.93 (s, 0H), 6.89 (d, J= 9.1 Hz, 1H), 6.84 (s, 1H), 6.55 (s, 1H), 6.42 (s, 2H), 6.12 (d, J = 8.2 Hz,1H), 5.96 (s, 2H), 5.06 (dtd, J = 9.7, 6.1, 3.5 Hz, 1H), 4.92 – 4.68 (m, 2H),4.61 (d, J = 3.8 Hz, 1H), 4.42 (dd, J = 9.1, 6.2 Hz, 1H), 4.17 (tt, J = 23.6,8.1 Hz, 3H), 3.84 (d, J = 4.5 Hz, 4H), 3.76 (s, 9H), 3.06 – 2.97 (m, 6H). 13 CNMR (151 MHz, Chloroform- d ) δ 173.68, 160.82, 156.25, 154.62, 153.26, 152.74,148.42, 147.83, 139.81, 137.15, 137.10, 137.04, 134.86, 132.55, 132.21,132.15, 129.62, 127.86, 118.95, 118.93, 114.30, 114.28, 109.88, 108.16,107.83, 107.66, 107.27, 101.74, 74.86, 71.30, 70.28, 66.98, 60.80, 56.33,52.65, 50.96, 50.93, 47.45, 45.64, 43.90, 38.69.HR-MS (ESI + m / z: 788.2573 [M+H] + Found: 788.2578 [M+H] + .

[0039] Example 7

[0040] This embodiment investigates the cell proliferation activity of podophyllotoxin triazole ester derivatives against human lung cancer cells and normal lung cells, using compounds 2b, 10b, and 11b disclosed in CN 119528929 A as controls.

[0041]

[0042] The antiproliferative activity of podophyllotoxin ester derivatives 1c-6c against three lung cancer cell lines H1975, A549, and H460 was detected using the CCK-8 assay, with podophyllotoxin and the positive control drug gefitinib as controls. Results are as follows: Figure 1 As shown, compounds 1c-6c have an IC50 response against non-small cell lung cancer cells H1975, A549, and H460. 50 The values ​​were all significantly lower than those of the positive control drug gefitinib, indicating that the podophyllotoxin derivative designed in this study generally exhibited strong anti-tumor proliferation activity. Notably, in a non-small cell lung cancer cell model, compound 3c demonstrated potent and universal inhibitory activity across cell lines: IC50 values ​​for H1975 cells... 50 The concentration was 1.08 μmol / L, which was 4.8-fold and 48.7-fold higher than PPT (5.21 μmol / L) and gefitinib (52.63 μmol / L), respectively. In A549 cells, the activity of 3c (0.62 μmol / L) was significantly better than that of podophyllotoxin (3.06 μmol / L) and gefitinib (48.36 μmol / L), respectively, by 4.9-fold and 78-fold. In H460 cells, the IC50 of 3c was [missing value]. 50 The concentration was 2.05 μmol / L, which was 3.5-fold and 28.4-fold higher than PPT (7.23 μmol / L) and gefitinib (58.22 μmol / L), respectively. Among other compounds, 5c showed the second-best inhibitory activity against A549 (0.88 μmol / L), and 2c showed the second-best inhibitory activity against A549 (1.79 μmol / L). Furthermore, comparisons were made with previously synthesized podophyllotoxin ether derivatives (2b, 10b, 11b) with good anti-colon cancer activity to further clarify the superior anti-lung cancer activity of this class of compounds. The results showed that compounds 1c-6c had significantly better anti-lung cancer activity than the ether derivatives (2b, 10b, 11b), with compound 3c exhibiting the strongest activity, showing more than 10-fold increased inhibitory activity against the proliferation of all three lung cancer cell lines compared to compound 2b.

[0043] Meanwhile, the toxic side effects of the compound were detected using normal human lung epithelial cells (BEAS-2B), and the results were as follows: Figure 2 As shown, the toxicity of all derivatives to normal lung epithelial cells BEAS-2B (>100 μmol / L) was much lower than that of PPT (5.53 μmol / L) and gefitinib (68.24 μmol / L), with the safety window of 3c (102 μmol / L) being 18.4 times larger than that of the parent compound PPT.

[0044] Example 8

[0045] To further verify the antiproliferative effect of compound 3c during DNA synthesis, an EdU-labeled assay was used to evaluate the effects of 3c and PPT on the proliferation of A549 cells. Figure 3 As shown, the control group exhibited vigorous cell proliferation, with approximately 63% of cells being EdU-positive. In contrast, both 3c and PPT significantly reduced the proportion of EdU-positive cells, indicating that both have a certain inhibitory effect on cell proliferation. However, 3c showed the most significant inhibitory effect, which was clearly dose-dependent: at a concentration of only 0.4 µmol / L, 3c significantly reduced the proportion of EdU-positive cells, and was superior to the higher concentration of PPT (2 µmol / L).

[0046] Cancer cells in early and late apoptosis stages were labeled using Annexin V-FITC and PI double staining, and apoptosis was analyzed by flow cytometry. Figure 4 As shown, compound 3c significantly induced apoptosis in A549 cells in a concentration-dependent manner, particularly at a concentration of 0.4 µmol / L, where it induced apoptosis in approximately 50% of cells. In contrast, 2 µmol / L PPT and gefitinib only induced apoptosis in 20% and 30% of cells, respectively. Compound 3c at 0.4 µmol / L and PPT at 2 µmol / L both induced apoptosis to varying degrees, with apoptosis rates of 27.9% and 17.8%, respectively. This suggests that podophyllotoxin-triazole derivatives can effectively induce apoptosis in A549 cells.

[0047] Podophyllotoxin triazole derivatives effectively inhibited the migration ability of A549 cells: The Transwell assay was used to detect the inhibitory effect of drug treatment on the migration of A549 cells. Results are as follows... Figure 5 As shown, the number of migrating A549 cells decreased to varying degrees after drug treatment. Compound 3c significantly inhibited cell migration at a concentration of 0.1 µmol / L, and the inhibitory effect gradually increased with increasing concentration. At a concentration of 0.2 µmol / L, the anti-migration effect of compound 3c was significantly better than that of the 1 µmol / L PPT treatment group, indicating that 3c significantly inhibits cell migration and is far superior to the parent compound PPT.

[0048] Example 9

[0049] Podophyllotoxin triazole ester derivatives can target and promote microtubule depolymerization.

[0050] The 1c-6c series small molecules were optimized for energy minimization using the MM2 field in Chem3D and saved as SDF files. These were then imported into Autodock Vina software for dehydration, hydrogenation, charge calculation, ligand root determination, and selection of torsion-resistant bonds in the ligand small molecules. PyMOL software was used to remove solvent and ligand small molecules from the imported tubulin (PDB: 1SA0). Then, in Autodock Vina, the tubulin macromolecule was hydrogenated and its charge calculated. Tubulin was then used as the docking acceptor and saved as a PDBQT file. Based on the existing colchicine ligand small molecule, a tubulin inhibitor, in the 1SA0 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. Table 1 shows the binding energies of compounds 1c-6c, PPT, and tubulin. The binding energies of compounds 1c-6c with tubulin are all lower than those of PPT. The docking results of compound 3c, which has the lowest binding energy, with tubulin are visualized as follows: Figure 6 As shown in Figure A, compound 3c can form two hydrogen bonds with Ser140 and Phe141 in tubulin, with a binding free energy of -11.428 kcal / mol. The docking results of the parent compound PPT with tubulin are visualized as follows: Figure 6 As shown in B, PPT forms two hydrogen bonds with Tyr224 and Gln11 in tubulin, with a binding free energy of -8.081 kcal / mol. In contrast, compound 3c has a lower binding energy and stronger binding stability with tubulin.

[0051] Table 1. Molecular docking binding energies of compounds 1c-6c, PPT, and tubulin

[0052] Molecular dynamics simulation results of compounds 3c and PPT with tubulin are as follows: Figure 7 As shown. The RMSD changes of compounds 3c, tubulin, and the 3c-tubulin complex are shown in the figure. Figure 7 As shown in Figure A, after 20 ns, the RMSD curves of the tubulin and 3c-tubulin complex gradually stabilized, indicating that tubulin operated stably during the simulation. In particular, the 3c-tubulin complex stabilized at around 0.2 nm, maintaining a relatively stable structure within 100 ns, indicating that the binding of compound 3c to tubulin was relatively stable and did not undergo drastic structural changes, suggesting that compound 3c can dynamically bind to tubulin and maintain good stability. Figure 7B shows the RMSD changes of PPT, tubulin, and PPT-tubulin. The complex of tubulin and PPT showed small RMSD fluctuations, stabilizing at 0.2 nm after 40 ns, indicating that the complex was relatively stable overall, and the conformational change after drug binding to the receptor was small, suggesting that the binding process of PPT and tubulin was relatively stable. The RMSF distribution of compound 3c-tubulin complex shows ( Figure 7 C), most residues showed low fluctuations, mainly between 0.1-0.3 nm, with a few regions (such as residues at positions 380 and 450) showing slightly higher fluctuations, but the highest values ​​did not exceed 0.6 nm. These results indicate that the 3c-tubulin complex has a small fluctuation range and good overall structural stability. The RMSF distribution of the PPT-tubulin complex shows ( Figure 7 D), with relatively small overall fluctuations; the RMSF values ​​of most residues are between 0.1 and 0.5 nm, while some regions (such as terminal residues) show slightly higher fluctuations, exceeding 0.8 nm. This indicates that the PPT-tubulin complex has a moderate fluctuation range and strong structural stability. The 3c-tubulin complex exhibits a stable Gibbs free energy distribution (FEL) ( Figure 7 The lowest energy region is between 0 and 12 kcal / mol, indicating high binding stability. The MM / GBSA binding energy is -39.65 kcal / mol. The PPT-Tubulin complex ( Figure 7 F) also exhibits a relatively stable free energy landscape, with its lowest energy region ranging from 0 to 14 kcal / mol. Although the energy range is slightly wide, it still indicates relatively stable binding, with an MM / GBSA binding energy of -45.68 kcal / mol. The above molecular dynamics simulation results indicate that compound 3c retains the stability of the PPT backbone for targeted binding to tubulin.

[0053] The effects of compound 3c on the microtubule cytoskeleton of A549 cells were observed using laser confocal microscopy to clarify the rationality and effectiveness of its microtubule-inhibiting effect. Figure 8 As shown, microtubules in the control group cells exhibited a normal network structure; after treatment with paclitaxel, microtubules significantly polymerized to form a dense microtubule bundle structure, exhibiting typical characteristics of a microtubule stabilizer; while treatment with the microtubule destabilizer colchicine significantly disrupted the microtubule network, showing obvious breakage and depolymerization. After treatment with compound 3c, the microtubule framework exhibited significant depolymerization and breakage effects similar to colchicine, indicating that compound 3c, like colchicine, possesses typical characteristics of a microtubule destabilizing inhibitor, confirming that the designed and synthesized podophyllotoxin derivative 3c has the ability to target microtubules and significantly interfere with microtubule structure.

[0054] Example 10

[0055] Podophyllotoxin triazole ester derivatives can target and inhibit EGFR and its downstream signaling pathways.

[0056] The 1c-6c series small molecules were optimized for energy minimization using the MM2 field in Chem3D and saved as SDF files. These were then imported into Autodock Vina software for dehydration, hydrogenation, charge calculation, ligand root determination, and selection of torsion-resistant bonds in the ligand small molecules. PyMOL software was used to remove solvent and ligand small molecules from the imported EGFR protein (PDB: 1XKK). Then, in Autodock Vina, hydrogenation and charge calculation were performed on the EGFR protein molecule, and the EGFR protein was designated as the docking acceptor and saved as a PDBQT file. The coordinates of the docking box (Grid center: X -1.96252 Y 4.46218 Z -10.6489; Grid size: X 26 Y 26 Z 26; Grid space: 0.375) were determined based on the original coordinates of the EGFR inhibitor ligand small molecules in the 1XKK crystal structure, and semi-flexible docking was performed. Table 2 shows the binding energies of compounds 1c-6c, PPT, and EGFR. The binding energies of compounds 1c-6c to EGFR are all lower than those of PPT, with compounds 2c-5c being even lower than gefitinib. The docking results of compound 3c, which has the lowest binding energy, with EGFR are visualized as follows: Figure 9 As shown in Figure A, compound 3c can form five hydrogen bonds with LYS745, GLY719, CYS797, and SER720 in EGFR, with a binding free energy of -10.602 kcal / mol. The docking results of the parent compound PPT with EGFR are visualized as follows: Figure 9 As shown in B, PPT forms three hydrogen bonds with Cys797, Asn842, and Arg841 in EGFR, with a binding free energy of -6.515 kcal / mol. In contrast, compound 3c has a lower binding energy with EGFR and exhibits significantly better binding stability than PPT.

[0057] Table 2. Molecular docking binding energies of compounds 1c-6c, PPT, gefitinib, and EGFR

[0058] Molecular dynamics simulation results of compounds 3c and PPT with EGFR are as follows: Figure 10 As shown. The RMSD changes of compounds 3c, EGFR, and the 3c-EGFR complex are as follows. Figure 10As shown in Figure A, after 30 ns, the RMSD curves of compounds EGFR and the 3c-EGFR complex gradually stabilized, indicating that EGFR operated stably during the simulation. In particular, the 3c-EGFR complex stabilized at around 0.3 nm, maintaining a relatively stable structure within 100 ns, indicating that the binding of compound 3c to EGFR was relatively stable and did not undergo drastic structural changes, suggesting that compound 3c can dynamically bind to EGFR and maintain good stability. Figure 10 B shows the RMSD changes of PPT, EGFR, and the PPT-EGFR complex. In the early stage (approximately 10–37 ns), the RMSD of EGFR exhibits some stability. However, after PPT binds to EGFR, the RMSD fluctuation of the complex increases significantly, especially between 37 and 60 ns, indicating that PPT binding leads to a conformational change in EGFR. Until 80 ns, the RMSD of the complex and EGFR remains highly volatile, indicating poor stability of the EGFR-PPT complex. This suggests that the binding of PPT to EGFR is unstable and a relatively loose process, affecting the overall stability of the complex. The RMSF distribution of the 3c-EGFR complex shows (…). Figure 10 C), most residues show low fluctuations, mainly between 0.1-0.2 nm, indicating that these regions are relatively stable. However, a few regions (such as residues at positions 700-750, 875, 975, and 1025) exhibit higher fluctuations, with the highest value being approximately 0.5 nm. Nevertheless, overall, the 3c-EGFR complex has a relatively stable structure, indicating good binding stability. In contrast, the EGFR-PPT complex ( Figure 10 The RMSF values ​​of D) are generally high, with most regions fluctuating between 0.1 and 0.3 nm, while some regions show dramatic fluctuations, with the highest value approaching 0.7 nm, and the fluctuations are even greater at the terminal residues. These data indicate that the overall fluctuation of the EGFR-PPT complex is large, especially in some key regions, suggesting poor structural stability. The Gibbs free energy landscape of the 3c-EGFR complex ( Figure 10 E) shows a relatively narrow lowest energy region, with Gibbs free energy ranging from 0 to approximately 14 kcal / mol, indicating that the complex has a stable conformation in the lower energy region, although some conformational fluctuations still exist. The MM / GBSA binding energy is -48.70 kcal / mol. EGFR-PPT complex ( Figure 10 F) exhibits the most dispersed free energy distribution, with the highest proportion of high-energy regions, indicating the worst binding stability; the MM / GBSA binding energy is -31.35 kcal / mol. The above molecular dynamics simulation results demonstrate that compound 3c exhibits higher targeted binding stability to EGFR compared to its parent PPT backbone.

[0059] Western blotting analysis showed that... Figure 11 As shown, compound 3c significantly downregulated the phosphorylation level of AKT kinase (p-AKT) in tumor cells A549, and this inhibitory effect was concentration-dependent. EGFR, as a key upstream regulator of the PI3K / AKT signaling pathway, can initiate downstream signal transduction after its own phosphorylation activation (p-EGFR). With increasing 3c concentration, the gray intensity of the p-EGFR band gradually decreased, and the expression level of p-AKT also showed a downward trend, indicating that while the activity of the EGFR signaling pathway was effectively inhibited, AKT activation was also interfered with, further demonstrating that 3c can block signaling pathway transmission at multiple nodes. Statistical analysis showed that at a concentration of 0.2 µmol / L, the inhibitory effects of 3c on both p-AKT and p-EGFR were significantly different (p<0.05), proving that it has a strong inhibitory effect on the EGFR signaling pathway and the activation of downstream AKT. This result supports the mechanism of action of 3c as an EGFR inhibitor, namely, interfering with the proliferation and survival of tumor cells by reducing the phosphorylation activation level of EGFR. It also suggests that 3c may interfere with the biological behavior of tumor cells by affecting the phosphorylation of AKT and blocking signal transduction from upstream.

[0060] 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 derivatives, or pharmaceutically acceptable salts, isotopic variants, tautomers, stereoisomers, prodrugs, polymorphs, hydrates, or solvates thereof; the general structural formula of the podophyllotoxin derivatives is as follows: R is , , , , , ; The podophyllotoxin derivative targets tubulin and EGFR proteins.

2. A method for preparing the podophyllotoxin derivative as described in claim 1, characterized in that: Includes the following steps: Step 1: Dissolve propynic acid in anhydrous dichloromethane, add condensing agent N,N'-dicyclohexylcarbodiimide and catalyst 4-dimethylaminopyridine, and stir under ice bath conditions to generate O-acylisourea intermediate. Then, slowly add podophyllotoxin, gradually raise the temperature to room temperature, and continue stirring. Monitor the reaction progress by TLC. After the reaction is completed, add dilute hydrochloric acid dropwise under ice bath conditions and stir to precipitate dicyclohexylurea. Filter and collect the filtrate, and after separation and purification, obtain intermediate B. Step 2: Dissolve intermediate B in anhydrous N,N-dimethylformamide, replace with nitrogen, and then inject an aqueous solution of copper sulfate pentahydrate and sodium ascorbate. Dissolve the azide compound in DMF and slowly add it dropwise to the reaction system under nitrogen protection. Stir the reaction and monitor the reaction progress by TLC. After cooling the reaction solution, separate and purify it to obtain the target product.

3. The method for preparing the podophyllotoxin derivative according to claim 2, characterized in that: In step one, the molar ratio of podophyllotoxin, propionic acid, DCC, and DMAP is 1:1.2:2.0:0.1-1:1.5:2.0:0.1; in step two, the molar ratio of intermediate B to azide compound, copper sulfate pentahydrate, and sodium ascorbate is 1:0.8:0.4:1-1:1.2:0.4:

1.

4. A pharmaceutical composition, characterized in that, One of its active ingredients includes the podophyllotoxin derivative of claim 1, or a pharmaceutically acceptable salt, isotope variant, tautomer, stereoisomer, prodrug, polymorph, hydrate or solvate thereof.

5. The use of the podophyllotoxin derivative of claim 1, or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer, prodrug, polymorph, hydrate, or solvate thereof, in the preparation of a medicament for the treatment and / or prevention of diseases mediated by EGFR protein and its mutants.

6. The application according to claim 5, characterized in that, The EGFR mutant is selected from one or more of the T790M mutant and C797S mutant.

7. The application according to claim 5, characterized in that, The diseases mediated by the EGFR protein and its mutants are selected from lung cancer, colon cancer, urothelial carcinoma, breast cancer, prostate cancer, brain cancer, ovarian cancer, gastric cancer, pancreatic cancer, head and neck cancer, bladder cancer, and mesothelioma.

8. The application according to claim 7, characterized in that, The disease mediated by the EGFR protein and its mutants is lung cancer.

9. The application according to claim 8, characterized in that, The disease mediated by the EGFR protein and its mutants is non-small cell lung cancer.

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