A podophyllotoxin derivative and its preparation method and application

Abstract

The present application relates to the field of pharmaceutical chemistry technology, and specifically discloses a podophyllotoxin derivative, its preparation method, and application. The general structural formula of the podophyllotoxin derivative is: wherein the general structural formula of R is: R1 is selected from one of CH3, (CH2)4CH3, citronellol, and piperonyl alcohol; X is selected from one of S, S-S, Se, O, C, and C-C; and n is 1 or 2. The podophyllotoxin derivatives in this application have good in vitro antitumor activity, specifically releasing or accumulating drugs in the tumor environment to achieve low-toxicity, high-efficiency, targeted tumor treatment.

Description

Technical Field

[0001] The present application relates to the technical field of pharmaceutical chemistry, and in particular to a podophyllotoxin derivative and a preparation method and application thereof. Background Art

[0002] Cancer is one of the most difficult diseases to cure worldwide, with a large number of people dying from cancer and its complications every year. Over the past century, tremendous efforts have been made to combat cancer. While some success has been achieved, it remains difficult to detect and eliminate all cancers in a timely and effective manner. Currently, there are limited options for treating cancer. Conventional treatments include radiotherapy, surgery, and chemotherapy, but these approaches have significant limitations and side effects. To effectively address this issue, scientists have proposed drug delivery methods that target the tumor microenvironment.

[0003] Podophyllotoxin is a natural product extracted from plants that has significant anti-tumor activity. However, podophyllotoxin is also highly cytotoxic. Currently, podophyllotoxin-based anticancer drugs include etoposide and teniposide. However, as the number of patients using these drugs increases, it has been discovered that these two drugs also have significant side effects. Therefore, existing technologies need to be improved. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the purpose of the present application is to provide a method to solve the problem of strong toxic and side effects of existing podophyllotoxin anticancer drugs.

[0005] The technical solution of this application is as follows:

[0006] In the first aspect of the present application, a podophyllotoxin derivative is provided, the general structural formula of which is:

[0007]

[0008] Among them, the general structural formula of R is:

[0009]

[0010] R1 is selected from one of CH3, (CH2)4CH3, citronellol, and piperonyl alcohol; X is selected from one of S, SS, Se, O, C, and CC; and n is 1 or 2.

[0011] Through experimental verification, it was found that the podophyllotoxin derivatives of the first aspect of the present application have excellent anti-tumor activity. While being anti-tumor, they can target the reactive oxygen species (ROS) environment or glutathione (GSH) environment in the tumor microenvironment, and specifically release or accumulate drugs in the tumor environment to achieve low-toxicity and high-efficiency targeted treatment of tumors.

[0012] Optionally, X is selected from one of S, SS, and Se.

[0013] When sulfide-containing podophyllotoxin derivatives enter a reactive oxygen species (ROS) environment, the monosulfide bond will be oxidatively broken to release the original drug; when disulfide-containing podophyllotoxin derivatives enter a strongly reduced glutathione (GSH) environment, the disulfide bond can be broken to release the original drug, thereby achieving the specific release or accumulation of drugs in the tumor environment to achieve low-toxicity, high-efficiency, targeted treatment of tumors.

[0014] Optionally, R is selected from one of the following structures:

[0015]

[0016]

[0017]

[0018] In a second aspect of the present application, a method for preparing a podophyllotoxin derivative is provided, comprising the steps of:

[0019] Podophyllotoxin, a first compound, an activator, an acid-binding agent, and a solvent are mixed and reacted to obtain the podophyllotoxin derivative; wherein the first compound has the general structural formula:

[0020]

[0021] R2 is selected from one of CH3, (CH2)4CH3, citronellol, and piperonyl alcohol; X is selected from one of S, SS, Se, O, C, and CC; n is 1 or 2;

[0022]

[0023] The second aspect of the present application provides a method for preparing a podophyllotoxin derivative, which can obtain the target product in only one step, has mild reaction conditions, low cost, high reaction efficiency, high yield, and can effectively reduce the three wastes.

[0024] Optionally, the molar ratio of the podophyllotoxin to the first compound is 1:1.2-1:2. For example, the molar ratio of the podophyllotoxin to the first compound is 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9 or 1:2, etc. Preferably, the molar ratio of the podophyllotoxin to the first compound is 1:1.5.

[0025] Optionally, the molar ratio of the podophyllotoxin to the activator is 1:1.5-1:3. For example, the molar ratio of the podophyllotoxin to the activator is 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9 or 1:3, etc. Preferably, the molar ratio of the podophyllotoxin to the activator is 1:2.5. The activator includes at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), N,N'-dicyclohexylcarbodiimide (DCC), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), and N,N'-carbonyldiimidazole (CDI).

[0026] Optionally, the molar ratio of the podophyllotoxin to the acid binding agent is 1:1.5-1:2.5. For example, the molar ratio of the podophyllotoxin to the acid binding agent is 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4 or 1:2.5. Preferably, the molar ratio of the podophyllotoxin to the acid binding agent is 1:2. The acid binding agent includes at least one of 4-dimethylaminopyridine (DMAP), pyridine (Py), triethylamine (TEA), N,N-diisopropylethylamine (DIPEA), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and lithium diisopropylamide (LDA).

[0027] Optionally, in the mixture obtained by mixing, the concentration of the solvent is 0.01-0.1M. For example, the concentration of the solvent is 0.01M, 0.02M, 0.03M, 0.04M, 0.05M, 0.06M, 0.07M, 0.08M, 0.09M or 0.1M. The solvent includes at least one of dichloromethane, chloroform, dichloroethane, dichloropropane, and carbon tetrachloride. The reaction temperature is 20-30°C, for example, the reaction temperature is 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C. Preferably, the reaction temperature is 25°C. The reaction time is 12-16h, for example, the reaction time is 12h, 13h, 14h, 15h or 16h.

[0028] The third aspect of the present application provides a use of the preparation method of the podophyllotoxin derivative of the first aspect and the podophyllotoxin derivative of the second aspect in the preparation of anti-tumor drugs.

[0029] Optionally, the tumor includes at least one of oral cancer, brain tumor, pharyngeal cancer, esophageal cancer, lung cancer, stomach cancer, liver cancer, kidney cancer, prostate cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer, bladder cancer, skin cancer, rectal cancer, sarcoma, and keratinoma.

[0030] Beneficial effects of the present application: The podophyllotoxin derivatives described in the present application not only have good anti-tumor activity, but also have been found through experimental verification to have significantly reduced toxicity to normal liver cells, laying a material foundation for the future development and research of new low-toxic and highly effective podophyllotoxin-based anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for describing the embodiments.

[0032] Figure 1 This is a graph showing the inhibition of HepG2 cell viability provided in the examples of this application;

[0033] Figure 2 Cell scratch test analysis diagram provided in the examples of this application:

[0034] A is a cell scratch assay; B is a cell migration rate analysis diagram;

[0035] Figure 3 Transwell assay analysis diagram provided for the examples of this application:

[0036] A is an optical microscope observation of HepG2 cells; B is a quantitative analysis of the number of migrating cells;

[0037] Figure 4 This is a graph showing the weight measurement of mice provided in the examples of this application;

[0038] Figure 5 This is a tumor tissue volume observation diagram provided in the examples of this application. DETAILED DESCRIPTION

[0039] To make the purpose, technical solutions and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the drawings and embodiments of this application. Obviously, the described embodiments are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application. In the absence of conflict, the features in the following embodiments and embodiments can be combined with each other.

[0040] It should be noted that if there are descriptions involving "first", "second", etc. in the implementation of this application, the descriptions such as "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or suggesting their relative importance and implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of such features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the fact that ordinary technicians in this field can implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by this application.

[0041] The present invention will be further described in detail below with reference to the examples. The experimental methods in the examples are conventional methods unless otherwise specified. Where specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0042] Example 1 Synthesis of Podophyllotoxin Derivatives G1-G23

[0043] In a round-bottom flask, 150 mg (0.36 mmol) of podophyllotoxin and the first compound (0.54 mmol) were added, followed by 10 mL of dichloromethane (DCM), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 278 mg, 1.44 mmol), and 4-dimethylaminopyridine (DMAP, 48.6 mg, 0.40 mmol). The mixture was reacted overnight at room temperature. After completion of the reaction, the organic layer was washed with water and collected, dried over anhydrous NaSO4, filtered, and dried by spin drying. Podophyllotoxin G1-G23 was obtained after column chromatography (DCM:MeOH=100-200:1). The structures of podophyllotoxin G1-G23 are as follows:

[0044]

[0045] The structures of the R groups in podophyllotoxins G1-G23 are shown in Table 1.

[0046] Table 1 Chemical structures of R groups in podophyllotoxins G1-G23

[0047]

[0048]

[0049] Compounds G1-G23 were subjected to H NMR analysis, and the results were as follows:

[0050] G1. 1H NMR(600MHz,DMSO-d6)δ7.02(s,1H),6.61(s,1H),6.33(s,2H),6.03(d,J=6.0Hz,2H),5.90(d,J=9.3Hz,1H),4.57(d,J=4.7Hz,1H),4.35(dd,J=8.4,7.1Hz,1H),4.19(dd,J=10.6,8.5Hz,1H),3.65(s,6H),3.61(d,J=4.0Hz,6H),3.58(d,J=7.0Hz,2H),3.52(d,J=2.4Hz,2H),3.40(dd,J=14.6,4.8Hz,1H),2.77-2.70(m,1H). 13 C NMR(150MHz,DMSO-d6)δ171.35,171.28,153.24,148.58,148.10,136.76,133.49,129.64,110.47,109.20,108.26,102.60,74.77,71.82,61.11,56.93,53.25,45.25,43.99,39.48,34.97,34.53.HRMS(ESI-TOF)m / z:[M+Na] + Calcd for C 27 H 28 O 11 SNa 583.1250;Found 583.1240。

[0051] G2. 1 H NMR(600MHz,DMSO-d6)δ7.02(s,1H),6.61(s,1H),6.33(s,2H),6.03(d,J=6.7Hz,2H),5.91(d,J=9.3Hz,1H),4.57(d,J=4.7Hz,1H),4.38-4.32(m,1H),4.23-4.16(m,1H),4.02(t,J=6.7Hz,2H),3.63(d,J=17.3Hz,9H),3.58(d,J=7.2Hz,2H),3.50(d,J=1.6Hz,2H),3.40(dd,J=14.6,4.8Hz,1H),2.74(dd,J=12.4,9.6,5.1Hz,1H),1.54(p,J=6.8Hz,2H),1.26-1.23(m,4H),0.85-0.80(m,3H). 13C NMR(150MHz,DMSO-d6)δ174.21,170.52,170.04,152.41,136.70,135.94,128.80,109.65,108.27,107.50,101.79,73.92,71.03,65.16,60.27,56.05,55.30,44.42,43.15,38.68,34.07,33.80,28.07,27.79,22.07,14.16.HRMS(ESI-TOF)m / z:[M+Na] + Calcd forC 31 H 36 O 11 SNa 639.1876;Found639.1880。

[0052] G3. 1 H NMR(600 MHz,DMSO-d6)δ7.02(s,1H),6.62(d,J=2.2 Hz,1H),

[0053] 6.33(s,2H),6.03(d,J=8.1 Hz,2H),5.91(d,J=9.3 Hz,1H),5.06(t,J=7.3Hz,1H),4.57(d,J=4.6 Hz,1H),4.34(t,J=7.8 Hz,1H),4.19(dd,J=10.6,8.5 Hz,1H),4.09-4.03(m,2H),3.65(d,J=2.6 Hz,6H),3.62(d,J=2.8 Hz,3H),3.58(d,J=6.5 Hz,2H),3.49(d,J=2.2 Hz,2H),3.42-3.37(m,1H),2.74(dd,J=15.1,7.8 Hz,1H),1.92(dt,J=28.7,7.5 Hz,2H),1.62(s,3H),1.60-1.56(m,1H),1.54(s,3H),1.48(q,J=6.9 Hz,1H),1.37(dd,J=13.7,6.9Hz,1H),1.31-1.24(m,1H),1.15-1.08(m,1H),0.84(dd,J=6.7,31.9Hz,3H). 13C NMR(150 MHz,DMSO-d6)δ170.52,170.02,152.40,147.75,147.27,135.94,132.67,130.96,128.78,124.82,109.65,108.26,107.50,101.79,73.92,71.03,63.51,60.26,56.05,44.42,43.15,38.67,36.78,35.18,34.07,33.83,29.14,25.87,25.20,19.46,17.86.HRMS(ESI-TOF)m / z:[M+Na] + Calcd forC 36 H 44 O 11 SNa 707.2502;Found707.2508。

[0054] G4. 1 H NMR(600 MHz,DMSO-d6)δ7.02(s,1H),6.93(t,J=1.0 Hz,1H),

[0055] 6.85(t,J=1.1 Hz,2H),6.61(s,1H),6.33(s,2H),6.03(dd,J=9.5,1.0 Hz,2H),5.99(s,2H),5.89(d,J=9.3 Hz,1H),5.00(d,J=3.0 Hz,2H),4.57(d,J=4.7Hz,1H),4.31(dd,J=8.4,7.1 Hz,1H),4.18(dd,J=10.6,8.5 Hz,1H),3.64(s,6H),3.61(s,3H),3.59(d,J=9.1 Hz,2H),3.55(d,J=3.6 Hz,2H),3.39(dd,J=14.5,4.7 Hz,1H),2.77-2.69(m,1H). 13C NMR(150 MHz,DMSO-d6)δ173.78,170.10,169.48,152.02,147.27,147.16,136.41,135.54,132.26,129.31,128.41,122.14,109.24,108.79,108.03,108.00,107.06,101.38,101.04,73.57,66.30,59.87,55.71,54.87,44.04,42.78,38.25,33.67,33.43.HRMS(ESI-TOF)m / z:[M+Na] + Calcd for C 34 H 32 O 13 SNa 703.1461;Found 703.1458。

[0056] G5. 1 H NMR(600 MHz,CDCl3-d)δ6.80(s,1H),6.53(s,1H),6.39(s,2H),5.98(dd,J=8.1,1.3 Hz,2H),5.93(d,J=9.1 Hz,1H),4.60(d,J=4.4 Hz,1H),4.44-4.39(m,1H),4.22-4.17(m,1H),3.81(s,3H),3.76(s,6H),3.68(s,3H),2.92(dd,J=14.5,4.5 Hz,1H),2.90-2.79(m,5H),2.77-2.70(m,2H),2.62(t,J=7.2 Hz,2H). 13 C NMR(150 MHz,CDCl3-d)δ173.52,172.14,152.56,148.08,137.13,134.70,132.28,128.01,109.65,108.08,106.96,101.52,73.89,71.27,60.66,56.11,51.78,45.47,43.67,38.60,34.63,34.45,29.60,26.88.HRMS(ESI-TOF)m / z:[M+Na] + Calcd for C 29 H 32 NaO 11 S 611.1563;Found 611.1569。

[0057] G6. 1H NMR(600 MHz,CDCl3-d)δ6.83(s,1H),6.56(s,1H),6.41(s,2H),6.01(d,J=6.9 Hz,2H),5.96(d,J=9.2 Hz,1H),4.63(d,J=4.5 Hz,1H),4.44(dd,J=9.3,7.0 Hz,1H),4.26-4.21(m,1H),4.10(t,J=6.8 Hz,2H),3.84(s,3H),3.79(s,6H),2.96(dd,J=14.5,4.5 Hz,1H),2.90-2.73(m,7H),2.64(t,J=7.3 Hz,2H),1.65(t,J=7.0 Hz,2H),1.35(q,J=5.1,4.2 Hz,4H),0.92(t,J=6.8 Hz,3H). 13 C NMR(150 MHz,CDCl3-d)δ173.57,172.20,152.54,137.00,132.24,127.99,109.65,107.95,106.97,101.53,73.88,71.30,64.94,60.69,56.08,45.46,43.65,38.59,34.64,34.59,28.17,27.93,27.11,26.88,22.22,13.90.HRMS(ESI-TOF)m / z:[M+Na] + Calcd for C 33 H 40 NaO 11 S 667.2189;Found667.2192。

[0058] G7. 1H NMR(600 MHz,CDCl3-d)δ6.74(s,1H),6.47(s,1H),6.32(s,2H),5.92(d,J=7.1 Hz,2H),5.86(d,J=9.2 Hz,1H),5.01(dd,J=8.6,7.1,1.5 Hz,1H),4.54(d,J=4.5Hz,1H),4.35(dd,J=9.3,7.0 Hz,1H),4.14(dd,J=10.4,9.3 Hz,1H),4.10-4.02(m,2H),3.74(s,3H),3.69(s,6H),2.86(dd,J=14.5,4.5Hz,1H),2.83-2.72(m,5H),2.68(dt,J=12.3,6.9 Hz,2H),2.55(t,J=7.3 Hz,2H),1.97-1.85(m,2H),1.61(d,J=1.5 Hz,3H),1.60-1.57(m,1H),1.54-1.52(m,3H),1.47(q,J=6.5 Hz,1H),1.40-1.34(m,1H),1.27(dd,J=13.5,9.7,5.9Hz,1H),1.11(dd,J=13.5,9.6,7.8,5.9 Hz,1H),0.84(d,J=6.6 Hz,3H). 13 CNMR(150 MHz,CDCl3-d)δ173.57,172.19,152.54,148.08,134.72,132.24,131.33,124.41,109.65,107.96,106.97,101.53,73.88,71.30,63.36,60.70,56.09,45.46,43.65,38.59,36.87,35.29,29.38,25.67,25.29,19.30,17.60.HRMS(ESI-TOF)m / z:[M+Na] + Calcdfor C 38 H 48 NaO 11 S 735.2815;Found735.2817。

[0059] G8. 1H NMR(600 MHz,CDCl3-d)δ6.85-6.76(m,4H),6.54(s,1H),6.38(s,2H),5.98(dd,J=5.7,1.3 Hz,2H),5.95(s,2H),5.92(d,J=9.2 Hz,1H),5.02(s,2H),4.60(d,J=4.4 Hz,1H),4.40(dd,J=9.3,7.0 Hz,1H),4.19(t,J=9.8 Hz,1H),3.80(s,3H),3.75(s,6H),2.92(dd,J=14.5,4.5 Hz,1H),2.87-2.81(m,5H),2.73(dt,J=11.5,6.9 Hz,2H),2.65(t,J=7.3 Hz,2H). 13 CNMR(150 MHz,CDCl3-d)δ173.58,172.17,171.47,152.54,148.07,147.51,137.00,134.72,132.24,129.24,127.98,122.31,109.64,108.99,108.18,107.95,106.98,101.54,101.13,73.89,71.30,66.55,60.69,56.08,45.45,43.64,38.57,34.66,34.58,27.12,26.83.HRMS(ESI-TOF)m / z:[M+Na] + Calcd forC 36 H 36 NaO 13 S 731.1774;Found 731.1783。

[0060] G9. 1 H NMR(600 MHz,CDCl3-d)δ6.79(s,1H),6.54(s,1H),6.39(s,2H),5.99(d,J=8.2 Hz,2H),5.93(d,J=9.2 Hz,1H),4.60(d,J=4.4 Hz,1H),4.40(dd,J=9.3,7.0 Hz,1H),4.20(t,J=9.8 Hz,1H),3.81(s,3H),3.76(s,6H),3.69(s,3H),2.99-2.84(m,8H),2.74(t,J=7.1 Hz,2H). 13C NMR(150 MHz,CDCl3-d)δ172.05,152.57,148.11,147.53,134.66,132.31,127.94,109.67,108.07,106.94,101.54,74.01,71.25,60.67,56.12,51.84,45.48,43.66,38.60,33.94,33.79,33.07,32.87.HRMS(ESI-TOF)m / z:[M+Na] + CalcdforC 29 H 32 NaO 11 S2 643.1284;Found 643.1281。

[0061] G10. 1 H NMR(600 MHz,CDCl3-d)δ6.78(s,1H),6.54(s,1H),6.39(s,2H),5.98(d,J=9.4 Hz,2H),5.92(d,J=9.1 Hz,1H),4.60(d,J=4.4 Hz,1H),4.40(dd,J=9.2,7.0 Hz,1H),4.19(t,J=9.8 Hz,1H),4.09(t,J=6.8 Hz,2H),3.81(s,3H),3.76(s,6H),3.01-2.82(m,9H),2.73(t,J=7.1 Hz,2H),1.63(t,J=6.9 Hz,2H),1.32(p,J=3.9 Hz,4H),0.94-0.86(m,3H). 13 C NMR(150 MHz,CDCl3-d)δ173.48,172.05,152.57,148.11,147.53,134.66,127.95,109.66,108.06,106.94,101.53,71.25,64.97,60.66,56.11,45.48,43.66,38.60,34.04,33.94,33.15,32.87,28.17,27.94,22.20,13.86.HRMS(ESI-TOF)m / z:[M+Na] + Calcdfor C 33 H 40 NaO 11 S2 699.1910;Found 699.1913。

[0062] G11. 1H NMR(600 MHz,CDCl3-d)δ6.79(s,1H),6.54(s,1H),6.39(s,2H),6.00-5.97(m,2H),5.93(d,J=9.2 Hz,1H),5.08(t,J=1.4 Hz,1H),4.60(d,J=4.5 Hz,1H),4.40(dd,J=9.2,6.9 Hz,1H),4.22-4.10(m,3H),3.81(s,3H),3.76(s,6H),3.00-2.83(m,9H),2.72(t,J=7.2 Hz,2H),1.97(dd,J=22.4,7.5 Hz,2H),1.71-1.65(m,4H),1.62-1.58(m,3H),1.54(q,J=6.5 Hz,1H),1.45(ddd,J=11.8,7.5,6.0 Hz,1H),1.38-1.31(m,1H),1.22-1.16(m,1H),0.91(d,J=6.7 Hz,3H). 13 C NMR(150 MHz,CDCl3-d)δ173.47,172.05,171.52,152.58,134.66,127.95,124.41,109.67,108.07,106.94,101.53,71.25,63.38,60.67,56.12,45.49,43.67,38.60,36.86,35.32,34.06,33.95,33.12,32.86,29.61,29.39,25.63,25.29,19.30,17.58.HRMS(ESI-TOF)m / z:[M+Na] + Calcdfor C 38 H 48 NaO 11 S2767.2536;Found 767.2541。

[0063] G12. 1 H NMR(600 MHz,CDCl3-d)δ6.85-6.81(m,2H),6.78(d,J=8.5

[0064] Hz,2H),6.54(s,1H),6.39(s,2H),5.98(dd,J=8.5,1.3 Hz,2H),5.96(s,2H),5.92(d,J=9.1 Hz,1H),5.03(s,2H),4.60(d,J=4.3 Hz,1H),4.39(dd,J=9.2,6.9 Hz,1H),4.19(s,1H),3.81(s,3H),3.76(s,6H),3.00-2.81(m,8H),2.77(t,J=7.1 Hz,2H). 13 CNMR(150 MHz,CDCl3-d)δ173.48,172.04,171.30,152.58,148.11,147.74,147.53,134.66,132.31,122.31,109.67,108.99,108.17,108.07,106.94,101.53,101.12,71.25,66.59,60.67,56.12,45.49,43.66,38.59,34.06,33.92,33.01,32.90.HRMS(ESI-TOF)m / z:[M+Na] + Calcd for C 36 H 36 NaO 13 S2763.1495;Found 763.1493。

[0065] G13. 1 H NMR(600 MHz,CDCl3-d)δ6.87(s,1H),6.54(s,1H),6.39(s,2H),5.99(d,J=8.7 Hz,2H),5.91(d,J=8.9 Hz,1H),4.61(d,J=4.4 Hz,1H),4.42(dd,J=9.3,6.8 Hz,1H),4.21(t,J=9.7 Hz,1H),3.81(s,3H),3.76(s,6H),3.73(s,3H),3.43(d,J=28.5 Hz,4H),2.96-2.86(m,2H). 13C NMR(150 MHz,CDCl3-d)δ173.42,171.32,152.59,148.16,147.57,134.64,132.32,127.92,109.67,108.08,106.88,101.54,71.14,60.66,56.12,52.50,45.44,43.66,38.48,23.48,23.36.HRMS(ESI-TOF)m / z:[M+Na] + Calcd forC27H28NaO11Se631.0695;Found 631.0699。

[0066] G14. 1 H NMR(600 MHz,CDCl3-d)δ6.88(s,1H),6.54(s,1H),6.39(s,2H),5.99(dd,J=9.7,1.3 Hz,2H),5.91(d,J=8.9 Hz,1H),4.61(d,J=4.3 Hz,1H),4.44-4.40(m,1H),4.21(d,J=9.9 Hz,1H),4.12(t,J=6.8 Hz,2H),3.81(s,3H),3.76(s,6H),3.46(s,2H),3.39(s,2H),2.96-2.84(m,2H),1.65(s,2H),1.35-1.31(m,4H),0.90(s,3H). 13 C NMR(150MHz,CDCl3-d)δ173.43,171.36,170.54,152.59,148.16,147.57,134.64,127.94,109.66,108.07,106.89,101.53,71.15,65.68,60.67,56.12,45.44,43.66,38.48,29.61,28.11,27.85,23.82,23.26,22.18,13.85.HRMS(ESI-TOF)m / z:[M+Na] + Calcd forC 31 H 36 NaO 11 Se687.1321;Found 687.1324。

[0067] G15. 1<h2 style=";text-align:left;direction:ltr">H NMR(600 MHz,CDCl3-d)δ6.88(s,1H),6.54(s,1H),6.39(s,2H),5.98(dd,J=9.9,1.3 Hz,2H),5.91(d,J=8.9 Hz,1H),5.08(d,J=7.2 Hz,1H),4.61(d,J=4.3 Hz,1H),4.42(dd,J=9.3,6.7 Hz,1H),4.23-4.13(m,3H),3.81(s,3H),3.76(s,7H),3.45(s,2H),3.39(s,2H),2.96-2.85(m,2H),1.97(dd,J=22.7,7.4 Hz,2H),1.79(d,J=11.0 Hz,0H),1.68(s,5H),1.60(s,3H),1.55(p,J=6.6 Hz,1H),1.46(dt,J=13.7,6.9 Hz,1H),0.91(d,J=6.6 Hz,3H).<h2 style=";text-align:left;direction:ltr"> 13 <h2 style=";text-align:left;direction:ltr"> CNMR(150 MHz,CDCl3-d)δ173.43,171.36,170.53,152.58,137.16,134.64,132.30,127.94,124.37,109.66,108.07,106.89,101.53,71.15,6 HRMS(ESI-TOF)m / z:[M+Na]<h2 style=";text-align:left;direction:ltr"> + <h2 style=";text-align:left;direction:ltr"> Calcd for C<h2 style=";text-align:left;direction:ltr"> 36 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 44 <h2 style=";text-align:left;direction:ltr"> NaO<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> Se755.1947;Found755.1947。<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0068] <h2 style=";text-align:left;direction:ltr"> G16.<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> H NMR(600 MHz,CDCl3-d)δ6.88-6.82(m,3H),6.77(d,J=7.9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0069] Hz,1H),6.54(s,1H),6.38(s,2H),5.99(d,J=6.4 Hz,2H),5.96-5.94(m,2H),5.87(d,J=9.1 Hz,1H),5.05(d,J=1.7 Hz,2H),4.61(s,1H),4.39(dd,J=9.3,6.8 Hz,1H),4.18(t,J=9.8 Hz,1H),3.81(s,3H),3.75(s,6H),3.45-3.40(m,4H),2.91(d,J=4.4Hz,2H). 13 C NMR(150 MHz,CDCl3-d)δ171.27,170.26,152.59,147.75,134.64,132.28,127.94,122.39,109.64,108.99,108.18,108.08,106.89,101.53,101.15,71.14,67.24,60.67,56.12,45.43,43.66,38.46,23.75,23.31.HRMS(ESI-TOF)m / z:[M+Na] + Calcd forC 34 H 32 NaO 13 Se 751.0906;Found 751.0911。

[0070] G17. 1 H NMR(600 MHz,DMSO-d6)δ6.98(s,1H),6.61(s,1H),6.33(s,2H),6.03(dd,J=5.3,1.0 Hz,2H),5.93(d,J=9.3 Hz,1H),4.57(d,J=4.7 Hz,1H),4.34(dd,J=8.5,7.1Hz,1H),4.18(dd,J=10.7,8.5 Hz,1H),3.65(s,6H),3.62(s,3H),3.56(s,3H),3.39(dd,J=14.6,4.8 Hz,1H),2.89-2.71(m,9H). 13C NMR(150 MHz,DMSO-d6)δ174.27,172.74,172.60,152.40,147.71,136.73,136.01,132.60,128.98,109.68,108.33,107.41,101.78,73.12,71.04,60.28,56.07,51.74,44.40,43.20,38.75,35.63,35.43,18.08,17.81.HRMS(ESI-TOF)m / z:[M+Na] + Calcd for C 29 H 32 NaO 11 Se 659.1008;Found 659.1014。

[0071] G18. 1 H NMR(600 MHz,CDCl3-d)δ6.80(s,1H),6.54(s,1H),6.39(s,2H),6.00-5.96(m,2H),5.93(d,J=9.2 Hz,1H),5.08(t,J=7.2 Hz,1H),4.60(d,J=4.3 Hz,1H),4.42(dd,J=9.3,7.0 Hz,1H),4.20(t,J=9.8 Hz,1H),4.13(q,J=7.6 Hz,2H),3.81(d,J=1.6Hz,3H),3.76(d,J=1.6 Hz,7H),2.95-2.80(m,9H),2.72(t,J=7.4 Hz,2H),1.67(s,4H),1.60(s,3H),1.54(q,J=6.6 Hz,1H),1.44(dt,J=13.9,7.0 Hz,1H),1.37-1.30(m,1H),1.18(dd,J=13.0,7.7,4.5 Hz,1H),0.91(d,J=6.6 Hz,3H). 13 C NMR(150 MHz,DMSO-d6)δ168.37,167.34,147.40,142.92,142.36,127.11,122.86,119.26,104.50,102.89,101.82,96.36,68.73,66.13,58.17,55.52,50.95,40.32,38.51,33.44,31.71,30.44,30.33,30.15,24.24,20.47,20.13,12.82,12.67.HRMS(ESI-TOF)m / z:[M+Na]+ Calcd forC 33 H 40 NaO 11 Se 715.1634;Found 715.1636。

[0072] G19. 1 H NMR(600 MHz,DMSO-d6)δ6.98(s,1H),6.60(s,1H),6.33(s,2H),6.02(d,J=5.3 Hz,2H),5.93(d,J=9.3 Hz,1H),4.57(d,J=4.7 Hz,1H),4.33(d,J=7.1 Hz,1H),4.18(dd,J=10.6,8.5 Hz,1H),3.98(t,J=6.7 Hz,2H),3.65(s,6H),3.62(s,3H),3.39(dd,J=14.6,4.8 Hz,1H),2.86(t,J=6.7 Hz,2H),2.81-2.70(m,7H),1.56-1.50(m,2H),1.26(h,J=4.1,3.6 Hz,4H),0.86-0.82(m,3H). 13 C NMR(150 MHz,DMSO-d6)δ174.23,172.73,152.41,147.71,147.29,136.75,135.99,132.61,128.98,109.67,108.35,107.40,101.77,73.13,71.03,64.37,60.27,56.06,55.29,44.40,43.21,38.76,35.67,35.61,28.13,27.86,22.09,18.06,17.94,14.18.HRMS(ESI-TOF)m / z:[M+Na] + CalcdforC 38 H 48 NaO 11 Se 783.2260;Found 783.2260。

[0073] G20. 1H NMR(600 MHz,CDCl3-d)δ6.86-6.79(m,3H),6.79-6.74(m,1H),6.54(s,1H),6.39(s,2H),6.00-5.90(m,5H),5.02(s,2H),4.64-4.59(m,1H),4.41(t,J=8.3 Hz,1H),4.19(t,J=9.9 Hz,1H),3.80(d,J=2.1 Hz,3H),3.75(d,J=2.1 Hz,6H),2.96-2.73(m,10H). 13 C NMR(150 MHz,DMSO-d6)δ168.78,167.73,167.04,147.80,143.33,142.88,142.76,129.96,127.52,124.53,123.26,117.54,104.90,104.22,103.41,103.29,102.23,96.77,96.36,69.14,66.54,61.77,55.92,51.36,40.72,38.91,33.84,30.76,13.12.HRMS(ESI-TOF)m / z:[M+Na] + Calcd for C 36 H 36 NaO 13 Se 779.1219;Found 779.1227。

[0074] G21. 1 H NMR(600 MHz,DMSO-d6)δ7.04(s,1H),6.61(s,1H),6.32(s,2H),6.02(d,J=8.4 Hz,2H),5.92(d,J=9.2 Hz,1H),4.56(d,J=4.7 Hz,1H),4.42-4.36(m,2H),4.28(d,J=16.7 Hz,1H),4.26(s,2H),4.22-4.16(m,1H),4.07(t,J=6.7 Hz,2H),3.62(s,3H),3.38(dd,J=14.6,4.7 Hz,1H),1.61-1.55(m,2H),1.29(h,J=3.7,3.2 Hz,4H),0.86(s,2H),0.86(d,J=13.4 Hz,1H). 13C NMR(150 MHz,DMSO-d6)δ173.84,170.41,152.00,146.88,136.38,135.53,132.31,128.38,109.20,107.96,107.33,101.36,73.04,70.74,67.40,64.14,59.87,55.67,44.08,42.77,38.29,27.72,27.44,21.69,13.77.HRMS(ESI-TOF)m / z:[M+Na] + Calcd for C 31 H 36 NaO 12 623.2104;Found 623.2105。

[0075] G22. 1 H NMR(600 MHz,CDCl3-d)δ6.85(s,1H),6.54(s,1H),6.38(s,2H),5.98(d,J=8.8 Hz,2H),5.93(d,J=8.6 Hz,1H),4.60(d,J=4.0 Hz,1H),4.43(dd,J=9.3,6.5 Hz,1H),4.21(t,J=9.6 Hz,1H),4.13(t,J=6.9 Hz,2H),3.80(s,3H),3.75(s,6H),3.69(d,J=6.8 Hz,2H),3.57(s,2H),2.92(d,J=3.8Hz,2H),1.64(t,J=6.9 Hz,2H),1.35-1.30(m,4H),0.89(d,J=6.6 Hz,3H). 13 C NMR(150 MHz,DMSO-d6)δ168.26,164.61,164.10,147.41,143.02,142.40,131.92,129.44,127.22,122.52,104.47,102.83,101.92,96.39,69.68,66.09,60.78,55.50,50.94,40.30,38.49,36.19,35.80,33.37,22.94,22.69,17.02.HRMS(ESI-TOF)m / z:[M+Na] + Calcd for C 31 H 36 NaO 11 S2 671.1597;Found 671.1606。

[0076] G23. 1 H NMR (600 MHz, DMSO-d6) δ7.04 (s, 1H), 6.95 (d, J = 1.4 Hz,

[0077] 1H),6.88(d,J=3.1 Hz,2H),6.61(s,1H),6.32(s,2H),6.04-6.00(m,4H),5.92(d,J=9.3 Hz,1H),5.05(s,2H),4.56(d,J=4.7 Hz,1H),4.42-4.35(m,2H),4.30(s,3H),4.18(dd,J=10.7,8.5 Hz,1H),3.63(s,6H),3.61(s,3H),3.38(dd,J=14.6,4.7 Hz, 1H), 2.73 (dd, J = 14.9, 7.6 Hz, 1H). 13 C NMR(150 MHz, DMSO-d6)δ174.27,170.82,170.08,152.39,147.57,147.27,136.71,132.69,129.75,122.65,109.59,109.29,108.48,108.30,10 7.76,101.77,101.47,73.43,71.16,67.81,66.10,60.27,56.05,55.30,48.97,44.47,43.16,40.42,38.67.HRMS(ESI-TOF)m / z:[M+Na] + Calcd for C34H32NaO14 687.1690; Found687.1684.

[0078] Example 2 Evaluation of the in vitro antitumor biological activity of podophyllotoxin derivatives G1-G23

[0079] Liver cancer cells (BEL-7402, HepG2), human gastric cancer cell line (AGS), and control normal human liver cells (L02) were selected to determine the in vitro antitumor bioactivity of podophyllotoxin derivatives using MTT assay, CCK8 assay, cell scratch assay, and Transwell assay. Specific implementation methods are as follows:

[0080] (1) MTT method: cells in the logarithmic growth phase were inoculated into 96-well plates (the inoculation standard was 1×10 cells per well). 4After incubation for 24 hours, the culture medium was removed. Each sample was prepared into four different concentrations (1, 10, 50, and 100 μmol / L) and added to a 96-well plate with three replicates of cells at each concentration. After 48 hours of incubation, the culture medium was discarded, 10 μL of MTT (5 mg / mL) was added, and the cells were incubated for another 4 hours. The culture medium was then discarded, 150 μL of DMSO was added, and the solution was shaken for 10 minutes to fully dissolve. The absorbance was measured at 492 nm using a microplate reader. A control group was treated with 0.1% DMSO in a normal culture medium. The in vitro antitumor activities of the podophyllotoxin derivatives are shown in Table 2.

[0081] Table 2 In vitro antitumor activity of podophyllotoxin and its derivatives

[0082]

[0083] As can be seen from Table 2, the IC of podophyllotoxin derivative G22 on BEL-7402 cells 50 The value was 2.84 μM, and the IC value for HepG2 cells 50 The value was 21.84 μM, showing good in vitro inhibitory activity on liver cancer cells; the IC of podophyllotoxin derivative G18 on AGS cells 50 The value was 42.19 μM, showing good in vitro inhibitory activity against gastric cancer cells.

[0084] (2) CCK8 method: HepG2 cells were seeded into 96-well culture plates. After the cells were completely attached and treated with drugs for 24 hours, the original culture medium was discarded and washed with PBS. 100 μL of serum-free DMEM high-glucose culture medium and 10 μL of CCK-8 solution were added to each well. A blank well without cells was set up. The cells were cultured in the dark in an incubator for 1-4 hours. The absorbance at 450 nm was measured using a microplate reader. The results were as follows. Figure 1 shown.

[0085] from Figure 1 It can be seen that compared with the control group (Ctrl), the podophyllotoxin derivative G22 has a significant inhibitory effect on HepG2 cells, and its inhibitory effect is comparable to that of the podophyllotoxin parent drug (PPT) and the positive drug etoposide (CDDP), showing good in vitro activity in inhibiting liver cancer cells.

[0086] (3) Cell scratch test: HepG2 cells were seeded into 6-well plates and cultured in a 37°C, 5% CO incubator. After ensuring that the cells reached 90%-100% confluence, the culture medium was replaced to remove dead cells and residual culture medium components. A straight line was gently scratched on the HepG2 cells using a yellow pipette tip. The cells were immediately washed with sterile PBS to remove the scratched cells and cell fragments. The culture medium treated with drugs was replaced. The scratched area was observed and photographed using a microscope at 0 h and 24 h after the scratch. The migration ability of HepG2 cells in each group was observed. The results are shown in Figure 2. Figure 2 shown.

[0087] from Figure 2 As shown in Figures A and B, the width of the scratch in the control group (Ctrl) gradually narrowed over time. However, after 24 hours, the wound healing in the drug-treated group was significantly inhibited with increasing concentrations, in a dose-dependent manner. Quantitative analysis showed that at a G22 concentration of 20 μM, HepG2 cells showed a statistically significant difference compared to the control group (P<0.01 vs Ctrl). Furthermore, 20 μM G22 was significantly more effective than the parent podophyllotoxin drug, PPT, at the same concentration. These results demonstrate that the podophyllotoxin derivative G22 significantly inhibits the migration of HepG2 cells.

[0088] (4) Transwell experiment: The Transwell chamber was placed in a 24-well plate. An appropriate amount of HepG2 cell suspension was added to the upper chamber. After the cells were completely attached to the wall, the upper chamber was cultured with drugs. DMEM culture medium containing 10% FBS was added to the lower chamber. The 24-well plate was placed in an incubator at 37°C and 5% CO for 24 hours. The culture medium in the upper chamber was removed and the cells were fixed with 4% paraformaldehyde for 30 minutes. The cells were washed three times with PBS for 5 minutes each. 0.1% crystal violet solution was added for staining for 10-15 minutes. After washing with PBS three times, the HepG2 cells that did not migrate in the Transwell chamber were carefully wiped off with a cotton swab and observed and recorded under an optical microscope. The results are shown in the figure below. Figure 3 shown.

[0089] from Figure 3 As can be seen in Figures A and B, the control group (Ctrl) had significantly more purple-positive cells than the other groups. Over 24 hours, the number of positive cells in the drug-treated groups decreased significantly with increasing concentrations, showing a dose-dependent pattern. Quantitative analysis showed that when the G22 concentration was 20 μM, HepG2 cells showed a statistically significant difference compared to the control group (P<0.001 vs Ctrl). Furthermore, the effect of 20 μM G22 was significantly superior to the same concentration of the parent podophyllotoxin drug PPT and the positive drug CDDP at 10 μM. These results demonstrate that compound G22 significantly inhibits the migration of HepG2 cells.

[0090] Example 3 In vivo antitumor biological activity and safety evaluation of podophyllotoxin derivative G22

[0091] (1) Safety evaluation: median lethal dose (LD) 50 C57BL / 6 mice were randomly divided into five groups of six mice each and administered different doses (50 mg / kg, 100 mg / kg, 200 mg / kg, 500 mg / kg, and 1000 mg / kg) of the podophyllotoxin derivative G22. After administration, the survival of mice in each group was observed for 48 hours, and the number of deaths in each group was recorded. The results are shown in Table 3.

[0092] Table 3 Mouse median lethal dose

[0093]

[0094] The median lethal dose (LD) was calculated using the modified Reed-Muench method. 50 : 359.329 mg / kg, confidence interval (34.719-683.939 mg / kg).

[0095] (2) Evaluation of antitumor activity: The antitumor effect of podophyllotoxin derivative G22 was evaluated using a nude mouse subcutaneous transplant tumor model and HepG2 tumors in female BALB / c nude mice as an in vivo model. BALB / c nude mice were subcutaneously injected with 100 μL of HepG2 cell suspension (5×10 7 A tumor-bearing model was established using 500 mg / mL of G22. When tumor volume reached a certain level, mice were randomly divided into five groups of eight: a control group (normal saline), a G22 5 mg / kg group, a G22 10 mg / kg group, a PPT 10 mg / kg group, and a CDDP 10 mg / kg group. Dosing was repeated every three days for a total of five doses. Tumor volume and body weight were monitored during each dose. Mice were sacrificed after the final treatment, and tumor tissue was harvested.

[0096] During the administration process, the mortality rate of each group is shown in Table 4, and the weight measurement results of mice are shown in Table 4. Figure 4 The tumor tissue volume observation results are shown in Figure 5 shown.

[0097] Table 4 Mouse mortality

[0098]

[0099] Combined with Table 4, Figure 4 as well as Figure 5As can be seen, the podophyllotoxin derivative G22 groups and the parent podophyllotoxin drug PPT showed comparable, moderate tumor growth delays. The parent podophyllotoxin drug PPT had a survival rate of only 37.5%, demonstrating significant systemic toxicity. This suggests that the podophyllotoxin derivative G22 can largely avoid the off-target toxicity of podophyllotoxin and improve treatment efficiency. Furthermore, compared to the control group, the changes in mouse body weight revealed that none of the podophyllotoxin derivative G22 groups caused a significant decrease in mouse body weight after treatment, demonstrating good safety during treatment.

[0100] In summary, the podophyllotoxin derivatives described in this application not only exhibit excellent in vitro antitumor activity but also target the reactive oxygen species (ROS) or glutathione (GSH) environment in the tumor microenvironment while simultaneously combating tumors, specifically releasing or accumulating drugs in the tumor environment to achieve low-toxicity, high-efficiency, targeted tumor treatment. Experimental verification also demonstrated that the podophyllotoxin derivatives significantly reduced their toxicity to normal liver cells, laying a foundation for the future development of new, low-toxic, and highly effective podophyllotoxin-based antitumor drugs.

[0101] It should be understood that the application of this application is not limited to the above examples. For ordinary technicians in this field, they can make improvements or changes based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to this application.

Claims

1. A podophyllotoxin derivative, characterized in that: Its structural formula is: ； Among them, the structural formula of R is: 。 2. A method for preparing a podophyllotoxin derivative according to claim 1, characterized in that: Including steps: mixing podophyllotoxin, a first compound, an activator, an acid-binding agent and a solvent, and reacting to obtain the podophyllotoxin derivative; The structural formula of the first compound is: 。 3. The preparation method according to claim 2, characterized in that The molar ratio of the podophyllotoxin to the first compound is 1:1.2-1:

2.

4. The preparation method according to claim 2, characterized in that The molar ratio of the podophyllotoxin to the activator is 1:1.5-1:3; The activator includes at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, and N,N'-carbonyldiimidazole.

5. The preparation method according to claim 2, characterized in that The molar ratio of the podophyllotoxin to the acid-binding agent is 1:1.5-1:2.5; The acid binding agent includes at least one of 4-dimethylaminopyridine, pyridine, triethylamine, N,N-diisopropylethylamine, 1,8-diazabicyclo[5.4.0]undec-7-ene, and lithium diisopropylamide.

6. The preparation method according to claim 2, characterized in that In the mixture obtained by mixing, the solvent includes at least one of dichloromethane, chloroform, dichloroethane, dichloropropane, and carbon tetrachloride; The reaction temperature is 20-30° C., and the reaction time is 12-16 h.

7. Use of the podophyllotoxin derivative according to claim 1 in the preparation of antitumor drugs.

8. The use according to claim 7, characterized in that The tumor includes at least one of oral cancer, brain tumor, pharyngeal cancer, esophageal cancer, lung cancer, stomach cancer, liver cancer, kidney cancer, prostate cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer, bladder cancer, skin cancer, rectal cancer, sarcoma, and keratinoma.

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