Podophyllotoxin derivative as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicinal chemistry, and particularly discloses a podophyllotoxin derivative as well as a preparation method and application thereof. The structural general formula of the podophyllotoxin derivative is # imgabs0, wherein the structural general formula of R is # imgabs1 # R1 is selected from one of CH3, (CH2) 4CH3, citronellol and piperonol; x is selected from one of S, S-S, Se, O, C and C-C; and n is 1 or 2. The podophyllotoxin derivative disclosed by the invention has good in-vitro anti-tumor activity, and the podophyllotoxin derivative can specifically release or accumulate drugs in a tumor environment to achieve the effect of low-toxicity and high-efficiency targeted treatment of tumors.

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 in the world. Every year, a large number of people around the world die from cancer and its complications. In the past 100 years, people have made great efforts to defeat cancer. Although they have achieved certain successes, they still cannot effectively detect and eliminate all cancers in a timely manner. Currently, there are limited means of treating cancer. Conventional treatments include radiotherapy, surgery, and chemotherapy, but these treatments have great limitations and side effects. In order to effectively solve the problem of cancer treatment, scientists have proposed a drug delivery method based on the tumor microenvironment as a target.

[0003] Podophyllotoxin is a natural product extracted from plants, which has significant anti-tumor activity, but the cytotoxicity of podophyllotoxin is relatively strong. At present, the podophyllotoxin anticancer drugs include etoposide and teniposide, but with the increase in the number of patients using the drugs, it is found that these two drugs also have strong side effects. Therefore, the existing technology needs 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 for solving 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 piperonol; X is selected from one of S, SS, Se, O, C and CC; 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-toxic 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 bonds will be oxidatively broken to release the original drug; when disulfide-containing podophyllotoxin derivatives enter a strongly reduced glutathione (GSH) environment, the disulfide bonds can be broken to release the original drug, thereby achieving specific release or accumulation of drugs in the tumor environment to achieve low-toxic, high-efficiency, targeted treatment of tumors.

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

[0015]

[0016]

[0017]

[0018] The second aspect of the present application provides a method for preparing a podophyllotoxin derivative, 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 piperonol; 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, etc. 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, etc. The solvent includes at least one of dichloromethane, chloroform, dichloroethane, dichloropropane, and carbon tetrachloride. The temperature of the reaction is 20-30°C, for example, the temperature of the reaction 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 temperature of the reaction is 25°C. The reaction time is 12-16h, for example, the reaction time is 12h, 13h, 14h, 15h or 16h, etc.

[0028] The third aspect of the present application provides an application of the preparation method of the podophyllotoxin derivative of the first aspect and the podophyllotoxin derivative of the second aspect of the present application 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 in the present application not only have good anti-tumor activity, but also have been found through experimental verification that the toxicity of the podophyllotoxin derivatives to normal liver cells is significantly reduced, laying a material foundation for the future development and research of new low-toxic and highly effective podophyllotoxin 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 drawings required for describing the embodiments are briefly introduced below.

[0032] Figure 1 This is a HepG2 cell viability inhibition analysis diagram 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 analysis; B is a cell migration analysis diagram;

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

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

[0037] Figure 4 This is a graph of mouse weight measurement provided in the examples of the present application;

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

[0039] In order to make the purpose, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings and embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments of the present application, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the protection scope of the present application. In the absence of conflict, the following embodiments and features in the 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 of "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 technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the 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 the field can implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by this application.

[0041] The present invention is further described in detail below in conjunction with the examples. The experimental methods in the examples are conventional methods unless otherwise specified. If no specific conditions are specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

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

[0043] Add podophyllotoxin 150mg (0.36mmol) and the first compound (0.54mmol) to a round-bottom flask, then add dichloromethane (DCM) 10mL, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl, 278mg, 1.44mmol), 4-dimethylaminopyridine (DMAP, 48.6mg, 0.40mmol), react at room temperature overnight, wash with water after the reaction is completed, collect the organic layer, dry with anhydrous NaSO4, filter, spin dry, and treat with column chromatography (DCM: MeOH = 100-200: 1) to obtain podophyllotoxin G1-G23. Among them, the structure of podophyllotoxin G1-G23 is as follows:

[0044]

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

[0046] Table 1 Chemical structures of R groups in podophyllotoxin 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 in vitro antitumor biological activity of podophyllotoxin derivatives G1-G23

[0079] Hepatoma 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 biological activity of podophyllotoxin derivatives by MTT method, CCK8 method, cell scratch test and Transwell test. The specific implementation method is:

[0080] (1) MTT method: Cells in the logarithmic growth phase were inoculated into a 96-well plate (the inoculation standard was 1×10 cells per well). 4After 24 hours of incubation, the culture medium was removed. Each sample was prepared into 4 different concentrations of culture solution (1, 10, 50, 100 μmol / L, respectively). The culture solution containing the sample was added to a 96-well plate, and each concentration had three replicate wells of cells. After 48 hours of incubation, the culture solution was discarded, 10 μL of MTT (5 mg / mL) was added, and the culture was continued for 4 hours. The culture solution was discarded, 150 μL of DMSO was added, and the shake was continued for 10 minutes to fully dissolve it. The absorbance was measured at 492 nm using an ELISA reader. The control group was treated with 0.1% DMSO to treat normal culture medium cells. The in vitro antitumor activity of the measured podophyllotoxin derivatives is 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 is 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 50 The value was 42.19 μM, showing good in vitro inhibitory activity on gastric cancer cells.

[0084] (2) CCK8 method: HepG2 cells were inoculated into 96-well culture plates. After the cells were completely attached to the wall and treated with drugs for 24 hours, the original culture medium was discarded, PBS was washed, 100 μL of serum-free DMEM high-glucose culture medium and 10 μL of CCK-8 solution were added to each well, and a blank well without cells was set up. The wells were cultured in an incubator in the dark for 1-4 hours, and the absorbance at 450 nm was measured using an ELISA reader. The results are shown in Figure 2. 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 inoculated into 6-well plates and cultured in a constant temperature incubator at 37°C and 5% CO. 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 gun tip, and 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 images of the scratched area were observed and recorded using a microscope at 0 h and 24 h after the scratch, and the migration ability of each group of HepG2 cells was observed. The results are shown in Table 2. Figure 2 shown.

[0087] from Figure 2 As can be seen in A and B, the width of the scratch in the control group (Ctrl) gradually narrowed with time, while under the condition of 24h, the healing of the scratch was significantly inhibited with the increase of concentration in the drug-added group, which was dose-dependent. The quantitative analysis results showed that when the concentration of G22 was 20μM, HepG2 cells had a significant statistical difference compared with the control group (P<0.01vsCtrl). In addition, the effect of 20μM G22 was significantly better than the parent drug PPT of podophyllotoxin at the same concentration. The results showed that the podophyllotoxin derivative G22 can significantly inhibit the migration of HepG2 cells.

[0088] (4) Transwell experiment: The Transwell chamber was placed in a 24-well plate, and 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 treated with empty culture and drug addition. 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, and then the culture medium in the upper chamber was removed. The cells were fixed with 4% paraformaldehyde for 30 minutes, washed with PBS three times for 5 minutes each, and stained with 0.1% crystal violet solution for 10-15 minutes. After washing with PBS three times, the HepG2 cells that did not migrate in the Transwell chamber were carefully wiped with a cotton swab and observed and recorded under an optical microscope. The results are shown in the figure. Figure 3 shown.

[0089] from Figure 3 As can be seen in A and B, the purple positive cells in the control group (Ctrl) are significantly more than those in other groups, and under the condition of 24h, the positive cells in the drug-added group decreased significantly with the increasing concentration, which was dose-dependent. The quantitative analysis results showed that when the concentration of G22 was 20μM, HepG2 cells had significant statistical differences compared with the control group (P<0.001vsCtrl). In addition, the effect of 20μM G22 was significantly better than the same concentration of podophyllotoxin parent drug PPT and 10μM positive drug CDDP. The results show that compound G22 can significantly inhibit 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 5 groups, 6 mice in each group, and different doses (50 mg / kg, 100 mg / kg, 200 mg / kg, 500 mg / kg, 1000 mg / kg) of podophyllotoxin derivative G22 were administered. 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: A nude mouse subcutaneous transplant tumor model was used to evaluate the antitumor effect of podophyllotoxin derivative G22 using the HepG2 tumor of 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 / mL) to establish a tumor-bearing model. When the tumor volume reached a certain volume, the mice were randomly divided into 5 groups, 8 mice in each group, divided into: control group (normal saline), G22 5mg / kg group, G22 10mg / kg group, PPT 10mg / kg group and CDDP 10mg / kg group. The drug was administered once every three days for a total of 5 times. The tumor volume and body weight were monitored at each administration, and the mice were killed after the last treatment to obtain tumor tissue.

[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 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 5It can be seen that the groups of podophyllotoxin derivative G22 and the podophyllotoxin parent drug PPT showed comparable and moderate tumor growth delays. The survival rate of the podophyllotoxin parent drug PPT was only 37.5%, showing obvious systemic toxicity. This shows that the podophyllotoxin derivative G22 can largely avoid the off-target toxicity of podophyllotoxin and improve the treatment efficiency. In addition, compared with the control group, it can be judged by the weight changes of mice that after the end of the drug treatment, the podophyllotoxin derivative G22 groups did not cause a significant decrease in the weight of mice, indicating that it showed good safety during the treatment.

[0100] In summary, the podophyllotoxin derivatives in this application not only have good in vitro antitumor activity, but also can target the reactive oxygen species (ROS) environment or glutathione (GSH) environment in the tumor microenvironment while fighting tumors, and specifically release or accumulate drugs in the tumor environment to achieve low-toxicity, high-efficiency, targeted treatment of tumors. At the same time, experimental verification found that the toxicity of podophyllotoxin derivatives to normal liver cells was significantly reduced, laying a material foundation for the future development and research of new low-toxic, high-efficiency podophyllotoxin-based antitumor drugs.

[0101] It should be understood that the application of the present application is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made 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 general structural formula is: Among them, the general structural formula of R is: R1 is selected from one of CH3, (CH2)4CH3, citronellol and piperonol; X is selected from one of S, SS, Se, O, C, and CC; n is 1 or 2.

2. The podophyllotoxin derivative according to claim 1, characterized in that X is selected from one of S, SS and Se.

3. The podophyllotoxin derivative according to claim 1, characterized in that R is selected from one of the following structures:

4. A method for preparing a podophyllotoxin derivative according to any one of claims 1 to 3, characterized in that: Includes steps: Mixing podophyllotoxin, the first compound, an activator, an acid-binding agent and a solvent, and reacting to obtain the podophyllotoxin derivative; The general structural formula of the first compound is: R2 is selected from one of CH3, (CH2)4CH3, citronellol and piperonol; X is selected from one of S, SS, Se, O, C, and CC; n is 1 or 2.

5. The preparation method according to claim 4, characterized in that: The molar ratio of the podophyllotoxin to the first compound is 1:1.2-1:

2.

6. The preparation method according to claim 4, 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.

7. The preparation method according to claim 4, 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.

8. The preparation method according to claim 4, characterized in that: In the mixture obtained by mixing, the concentration of the solvent is 0.01-0.1M; 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 12-16h.

9. Use of the podophyllotoxin derivative according to any one of claims 1 to 3 in the preparation of anti-tumor drugs.

10. The use according to claim 9, 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 keratin cancer.

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