An indazole-substituted podophyllotoxin derivative, and a preparation method and application thereof

Abstract

The application discloses an indazole-nitrogen-substituted podophyllotoxin derivative, a synthesis method and application thereof, and belongs to the field of medicine. Different substituents, such as methyl, ethyl, propyl, benzyl, fluorine atom, chlorine atom, bromine atom, iodine atom and the like, are introduced into 1 and 3 positions of indazole podophyllotoxin to obtain a podophyllotoxin derivative shown in formula (V) with significantly improved antitumor activity. In vitro tumor cell activity inhibition experiments show that the antitumor activity of the compound shown in formula (V) is significantly improved compared with that of podophyllotoxin.

Description

Technical Field

[0001] This invention belongs to the field of podophyllotoxin derivatives, specifically relating to indazole-substituted podophyllotoxin derivatives and their preparation methods. This invention also relates to the use of the indazole-substituted podophyllotoxin derivatives in the preparation of antitumor drugs. Background Technology

[0002] Podophyllotoxin has the following structure: Figure 1 As shown in Chinese formula (Ⅰ); podophyllotoxin is a natural active lead compound with unique antitumor activity extracted from podophyllotoxin-containing plants such as *Podophyllum hexandrum*, *Polygonum hydropiper*, and *Podophyllum hexandrum*. However, its clinical application is limited by its strong toxic side effects and poor bioavailability. Previously, Tang et al. provided an amino-substituted podophyllotoxin derivative in patent CN108285455A. This derivative improved the toxic side effects, but the drug still suffers from insufficient stability in the human body. Summary of the Invention

[0003] To address the problems of poor antitumor activity, significant toxic side effects, and insufficient stability in the human body associated with podophyllotoxin derivatives, this invention provides a class of indazole nitrogen-substituted podophyllotoxin derivatives with good antitumor activity. A second objective of this invention is to provide a method for preparing the aforementioned indazole-substituted podophyllotoxin derivatives. A third objective of this invention is to apply the aforementioned indazole-substituted podophyllotoxin derivatives to the preparation of clinical antitumor drugs.

[0004] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0005] This invention provides an indazole-substituted podophyllotoxin derivative or a pharmaceutically acceptable salt thereof with antitumor activity, wherein the structural formula of the podophyllotoxin derivative is shown in (V):

[0006]

[0007] Wherein: R1 is selected from H, methyl, ethyl, propyl, One of them

[0008] R2 is selected from one of H, F, I, Cl, Br or -CH3;

[0009] * indicates the attachment point between R1 and the nitrogen atom.

[0010] The reaction monomers used in this invention are 3-methylindazole-5-amino, 3-fluoroindazole-5-amino, 3-chloroindazole-5-amino, 3-bromoindazole-5-amino, 3-iodoindazole-5-amino, 1-methylindazole-5-amino, 1-ethylindazole-5-amino, 1-propylindazole-5-amino, 1-cyclohexaneindazole-5-amino, 1-cyclopentaneindazole-5-amino, 1-cyclopropaneindazole-5-amino, 1-benzylindazole-5-amino, 1-methyl-3-chloroindazole-5-amino, 1-methyl-3-bromoindazole-5-amino, 1-ethyl-3-methylindazole-5-amino, or 1-methyl-3-methylindazole-5-amino.

[0011] The structural formulas of the above-mentioned reactive monomers are shown in [reference needed]. Figure 2 As shown, using it as a substituent group is beneficial to the formation of the β configuration at the 4-position of the C ring of podophyllotoxin.

[0012] The method for preparing the compound shown in formula (V) provided by the present invention includes the following steps:

[0013] A reactive monomer is introduced into the 4-position of the C ring of podophyllotoxin via a nucleophilic substitution reaction. The reactive monomer can be 3-methylindazole-5-amino, 3-fluoroindazole-5-amino, 3-chloroindazole-5-amino, 3-bromoindazole-5-amino, 3-iodoindazole-5-amino, 1-methylindazole-5-amino, 1-ethylindazole-5-amino, 1-propylindazole-5-amino, 1-cyclohexaneindazole-5-amino, 1-cyclopentaneindazole-5-amino, 1-cyclopropaneindazole-5-amino, 1-benzylindazole-5-amino, 1-methyl-3-chloroindazole-5-amino, 1-methyl-3-bromoindazole-5-amino, 1-ethyl-3-methylindazole-5-amino, or 1-methyl-3-methylindazole-5-amino.

[0014] In this invention, the nucleophilic substitution reaction can be carried out under the following conditions: podophyllotoxin is dissolved in an organic solvent, and then the reaction monomer is added and stirred; the organic solvent can be triethylamine.

[0015] In this invention, the molar ratio of podophyllotoxin to the reactant monomer in the nucleophilic substitution reaction can be 1:1 to 10.

[0016] In this invention, the nucleophilic substitution reaction temperature can be -20 to 30°C, preferably -20 to 20°C.

[0017] In this invention, the nucleophilic substitution reaction temperature can also be -10 to 10°C, and more preferably 0 to 10°C.

[0018] The above preparation method also includes: pouring the reaction solution after nucleophilic substitution reaction into 20 to 50 times the volume of deionized water, filtering after precipitation, and drying the filter cake to obtain crude products of indazole-substituted podophyllotoxin derivatives.

[0019] Furthermore, the method includes separating the crude product sequentially using silica gel column chromatography and gel column chromatography to obtain purified indazole-substituted podophyllotoxin derivative products.

[0020] Preferably, the silica gel column chromatography separation method includes: (1) the silica gel column chromatography is normal phase silica gel column chromatography or reverse phase silica gel column chromatography. The normal phase silica gel is packed into the column after being mixed with a low polarity organic solvent and equilibrated with an eluent. The eluent is preferably composed of petroleum ether and ethyl acetate in a volume ratio of 2:1. The reverse phase silica gel is packed into the column after being mixed with methanol and equilibrated with an eluent. The eluent is preferably composed of methanol and water in a volume ratio of 60:1. (2) the sample to be separated and purified is dissolved in the eluent, loaded for adsorption, then eluted with the eluent, the eluent is collected, the sample is evaporated and recrystallized.

[0021] Preferably, the gel column chromatography separation method includes: (1) soaking the gel in methanol; packing the treated gel into a column and equilibrating it with methanol; (2) dissolving the sample after preliminary separation by silica gel column chromatography in petroleum ether, loading the sample for adsorption, then eluting with methanol, collecting the eluent, evaporating the solvent in the sample and recrystallizing it.

[0022] The obtained product was used to test the inhibitory activity of A549, AsPC-1, HPAC-1, MIA-Paca-2, HepG2, HeLa, and HGC27 cells in vitro. The test results showed that the antitumor activity of the compound of formula (V) prepared in this invention was significantly improved compared with that of podophyllotoxin. The experimental results indicate that the compound of formula (V) of this invention can be prepared into an antitumor drug for clinical application in antitumor therapy. Furthermore, it is demonstrated that the compound of formula (V) of this invention not only has excellent antitumor properties against various hepatocellular carcinomas but also against pancreatic tumors, thus broadening the therapeutic spectrum.

[0023] The antitumor pharmaceutical composition provided by this invention comprises an effective amount of a compound of formula (V) or a salt thereof and a pharmaceutically acceptable carrier, that is, a pharmaceutically acceptable amount of the compound of formula (V) is combined with a pharmaceutically acceptable carrier and prepared into any suitable pharmaceutical composition according to conventional formulation methods in the art. This pharmaceutical composition is generally suitable for oral and injectable administration, and is also suitable for other administration methods, such as transdermal administration. The pharmaceutical composition can be in the form of tablets, capsules, powders, granules, lozenges, suppositories, or liquid formulations such as oral solutions or sterile parenteral suspensions. The composition can also be in the form of large or small volume injections, lyophilized powder for injection, sterile powder repackaging, etc. To achieve consistent dosing, the pharmaceutical composition of this invention is preferably in single-dose form. Single-dose forms for oral administration may be tablets and capsules, and may contain conventional excipients such as binders, such as syrup, gum arabic, gelatin, sorbitol, astragalus gum, or polyvinylpyrrolidone; fillers, such as lactose, sugar, corn starch, calcium phosphate, sorbitol, or glycine; tableting lubricants, such as magnesium stearate; disintegrants, such as starch, polyvinylpyrrolidone, sodium starch glycolate, or microcrystalline cellulose, or pharmaceutically acceptable wetting agents, such as sodium dodecyl sulfate.

[0024] The indazole-substituted podophyllotoxin derivatives obtained in this invention have excellent antitumor properties, and their antitumor activity against HepG2, HeLa, A549, and HGC27 cell lines is significantly improved.

[0025] The indazole-substituted podophyllotoxin derivatives obtained in this invention have excellent antitumor properties and good stability in the human body, maintaining a suitable concentration for a long time. Attached Figure Description

[0026] Figure 1 It is the structural formula of podophyllotoxin.

[0027] Figure 2 It is 3-methylindazole-5-amino, 3-fluoroindazole-5-amino, 3-chloroindazole-5-amino, 3-bromoindazole-5-amino, 3-iodoindazole-5-amino, 1-methylindazole-5-amino, 1-ethylindazole-5-amino, 1-propylindazole-5-amino, 1-cyclohexaneindazole-5-amino, 1-cyclopentaneindazole-5-amino, 1-cyclopropaneindazole-5-amino, 1-benzylindazole-5-amino, 1-methyl-3-chloroindazole-5-amino, 1-methyl-3-bromoindazole-5-amino, 1-ethyl-3-methylindazole-5-amino, or 1-methyl-3-methylindazole-5-amino.

[0028] Figure 3 This is the structural formula of compound (V) of the present invention.

[0029] Figure 4A. Stability diagram of the compound in SD rat plasma; Figure 4 B. Stability diagram of the compound in human liver microsomes. Detailed Implementation

[0030] The following specific embodiments, in conjunction with the appendix to the specification, illustrate this point. Figure 1-4 The technical solution of the present invention will be further described below.

[0031] Unless otherwise specified, the raw materials and equipment used in this invention can be purchased from the market or are commonly used in the field. Unless otherwise specified, the methods in the embodiments are conventional methods in the field.

[0032] Test materials

[0033] 1. Podophyllotoxin: Purchased from Xi'an Helin Bioengineering Co., Ltd.;

[0034] 2. 3-Methylindazole-5-amino, 3-fluoroindazole-5-amino, 3-chloroindazole-5-amino, 3-bromoindazole-5-amino, 3-iodoindazole-5-amino, 1-methylindazole-5-amino, 1-ethylindazole-5-amino, 1-propylindazole-5-amino, 1-cyclohexaneindazole-5-amino, 1-cyclopentaneindazole-5-amino, 1-cyclopropaneindazole-5-amino, 1-benzylindazole-5-amino, 1-methyl-3-chloroindazole-5-amino, 1-methyl-3-bromoindazole-5-amino, 1-ethyl-3-methylindazole-5-amino, or 1-methyl-3-methylindazole-5-amino were all purchased from Bid Pharmaceutical.

[0035] Example 14 Synthesis and purification of β-NH-(3-methylindazole-5-amino)-podophyllotoxin (compound 1)

[0036] (1) Synthesis of 4β-NH-(3-methylindazole-5-amino)-podophyllotoxin:

[0037] Starting with podophyllotoxin, the hydroxyl group at the fourth position of the C-ring is de-hydroxylated under the Lewis acid catalysis of boron trifluoride ether to form a carbocation. Sodium iodide then undergoes nucleophilic substitution to form the iodopodophyllotoxin intermediate (I-PTOX).

[0038] 150 mg (1 mmol) of 3-methylindazole-5-amino was added to 4 mL of tetrahydrofuran solvent. After dissolution, the mixture was pipetted and transferred to I-PTOX after rotary evaporation, mixed well, and placed in an ice bath. The reaction was carried out in an ice bath for 4 h. After the reaction was completed, the mixture was distilled under reduced pressure to give 85 mg of 4β-NH-(3-methylindazole-5-amino)-podophyllotoxin, with a yield of 53.5%.

[0039] (2) Separation and purification of 4β-NH-(3-methylindazole-5-amino)-podophyllotoxin:

[0040] Separation and purification were performed using silica gel column chromatography and gel column chromatography:

[0041] (A) Separation was performed using a normal-phase silica gel column (normal-phase silica gel: Qingdao Ocean Chemical Co., Ltd., HG / T2354-92; separation system: Buchi isocratic rapid chromatography system; chromatographic column: Buchi glass column C-690, 460 mm long, 15 mm inner diameter) or a similar polar column; a petroleum ether:ethyl acetate = 2:1 system was used as the eluent, with a sample loading volume of 2 ml and a constant flow rate of 1.0 ml / min; each 2 ml eluent was collected as a fraction. Each fraction was examined using normal-phase silica gel thin-layer chromatography (Mokers high-performance silica gel thin-layer chromatography, Germany) or a similar polar thin-layer chromatography system; a petroleum ether:ethyl acetate 2:1 system was used as the developing solvent, and fractions with an Rf value of 0.5 were combined; the combined sample was vacuum dried and stored in a refrigerator at 4°C under light-protected conditions as a sample for purification.

[0042] (B) Separation was performed using gel column chromatography (gel: Sephadex LH-20; separation column: glass column, 480 mm long, 30 mm inner diameter); the prepared Sephadex LH-20 gel was wet-packed into a column and equilibrated with methanol. The sample to be purified was dissolved in 6 ml of methanol and loaded for adsorption at a flow rate of 0.6 ml / min, followed by elution with 600 ml of methanol at a flow rate of 0.6 ml / min. Each 10 ml eluent was collected in one bottle, and each fraction was examined using normal-phase silica gel thin-layer chromatography (Mokk high-performance silica gel thin-layer chromatography, Germany) or similar polarity thin-layer chromatography; a chloroform:acetone = 2:1 system was used as the developing solvent, and fractions with an Rf value of 0.5 were combined; the white powder sample obtained after vacuum drying was 4β-NH-(3-methylindazole-5-amino)-podophyllotoxin.

[0043] 4β-NH-(3-methylindazole-5-amino)-podophyllotoxin: white powder, C 30 H 29 N3O7

[0044] 1HNMR(600MHz,DMSO-d6)δ12.24(s,1H),7.28–7.24(m,1H),6.92(dd,J=8.9,2.1Hz,1H),6.84( s,1H),6.81(d,J=2.0Hz,1H),6.60(s,1H),6.37(s,2H),6.02(dd,J=11.3,0.9Hz,2H),5.83(d ,J=8.4Hz,1H),4.93(dd,J=8.3,4.3Hz,1H),4.61(d,J=5.2Hz,1H),4.47–4.42(m,1H),3.84–3 .80(m,1H),3.71(s,6H),3.68(s,3H),3.10(dddd,J=14.7,10.8,7.4,4.3Hz,1H),2.45(s,3H). 13 CNMR(151MHz,DMSO-d6)δ175.31,170.83,147.55,142.87,136.51,131.68,111.15,109.97,109.56 ,108.66,101.61,96.86,69.27,60.39,60.23,56.29,51.70,43.50,39.09.MS-ESI:544.2098[M+H] +

[0045] Example 24: Synthesis and purification of β-NH-(3-chloroindazole-5-amino)-podophyllotoxin (compound 2)

[0046] (1) Synthesis of 4β-NH-(3-chloroindazole-5-amino)-podophyllotoxin:

[0047] Take 0.15 g (1 mmol) of 3-chloroindazole-5-amino and add 2 ml of tetrahydrofuran solvent. After dissolving, pipette the mixture and transfer it to I-PTOX after rotary evaporation. Mix well and place in an ice bath. React in an ice bath for 4 h. After the reaction is complete, distill under reduced pressure to obtain the product.

[0048] (2) Separation and purification of 4β-NH-(3-chloroindazole-5-amino)-podophyllotoxin:

[0049] Separation and purification were performed using silica gel column chromatography and gel column chromatography, in the same manner as in Example 1.

[0050] 4β-NH-(3-chloroindazole-5-amino)-podophyllotoxin: white powder, C 29 H 26 ClN3O7

[0051] 1HNMR(600MHz,Chloroform-d)δ10.28(s,1H),7.24(t,J=8.0Hz,1H),6.86–6.82 (m,2H),6.57(s,1H),6.36(s,2H),6.11(d,J=7.7Hz,1H),5.99(dd,J=6.0,1.3H z,2H),5.66(d,J=6.2Hz,1H),4.86(dd,J=6.2,4.2Hz,1H),4.68(d,J=4.9Hz,1H ),4.42(dd,J=8.7,7.4Hz,1H),3.83(s,3H),3.78(s,6H),3.21(dd,J=14.1,5.0 Hz,1H),3.08(dddd,J=14.0,10.8,7.4,4.2Hz,1H).13CNMR(151MHz,Chlorofor m-d)δ174.59,152.66,148.46,147.78,143.08,141.41,137.34,135.09,131.8 9,129.86,129.76,119.21,110.26,110.02,109.18,108.39,101.63,99.59,99 .08,68.91,60.79,56.34,52.14,43.67,42.11,38.67.MS-ESI:564.1507[M+H] +

[0052] Example 34: Synthesis and purification of β-NH-(3-bromoindazole-5-amino)-podophyllotoxin (compound 3)

[0053] (1) Synthesis of 4β-NH-(3-bromoindazole-5-amino)-podophyllotoxin: 0.15 g (1 mmol) of 3-bromoindazole-5-amino was added to 2 ml of tetrahydrofuran solvent. After dissolution, the mixture was pipetted and transferred to I-PTOX after rotary evaporation, mixed well, and placed in an ice bath. The reaction was carried out in an ice bath for 4 h. After the reaction was completed, the product was obtained by vacuum distillation.

[0054] (2) Separation and purification of 4β-NH-(3-bromoindazole-5-amino)-podophyllotoxin:

[0055] Separation and purification were performed using silica gel column chromatography and gel column chromatography, in the same manner as in Example 1.

[0056] 4β-NH-(3-bromoindazole-5-amino)-podophyllotoxin: white powder, C 29 H 26 BrN3O7

[0057] 1HNMR(600MHz,Chloroform-d)δ10.33(s,1H),7.24(t,J=8.0Hz,1H),6.86–6.82(m,2H),6.57(s,1H),6.36(s,2H),6.11(d,J=7.7Hz,1H),5.99(dd,J=6.0 ,1.3Hz,2H),5.66(d,J=6.2Hz,1H),4.86(dd,J=6.2,4.2Hz,1H),4.68(d,J= 4.9Hz,1H),4.42(dd,J=8.7,7.4Hz,1H),3.83(s,3H),3.78(s,6H),3.21(dd, J=14.1,5.0Hz,1H),3.12–3.04(m,1H).13CNMR(151MHz,Chloroform-d)δ174.60,152.66,148.46,147.78,143.09,141.40,137.34,135.09,131.90,129 .85,129.77,119.19,110.26,110.02,109.18,108.39,101.63,99.61,99.0 8,68.92,60.79,56.34,52.14,43.67,42.11,38.67.MS-ESI:608.1025[M+H] +

[0058] Example 44: Synthesis and purification of β-NH-(1-methylindazole-5-amino)-podophyllotoxin (compound 4)

[0059] (1) Synthesis of 4β-NH-(1-methylindazole-5-amino)-podophyllotoxin: 0.15 g (1 mmol) of 1-methylindazole-5-amino was added to 2 ml of tetrahydrofuran solvent. After dissolution, the mixture was pipetted and transferred to I-PTOX after rotary evaporation, mixed well, and placed in an ice bath. The reaction was carried out in an ice bath for 4 h. After the reaction was completed, the product was obtained by vacuum distillation.

[0060] (2) Separation and purification of 4β-NH-(1-methylindazole-5-amino)-podophyllotoxin:

[0061] Separation and purification were performed using silica gel column chromatography and gel column chromatography, in the same manner as in Example 1.

[0062] 4β-NH-(1-methylindazole-5-amino)-podophyllotoxin: white powder, C 30 H 29 N3O7

[0063] 1HNMR(600MHz,DMSO-d6)δ7.74(d,J=0.9Hz,1H),7.40(dt,J=9.0,0.8Hz,1H),6.98(dd,J=9.0, 2.2Hz,1H),6.85(d,J=2.1Hz,1H),6.80(s,1H),6.57(s,1H),6.35(s,2H),5.99(dd,J=7.5,0.9 Hz,2H),4.88(d,J=4.3Hz,1H),4.59(d,J=5.2Hz,1H),4.39(t,J=7.8Hz,1H),3.96(s,3H),3.7 8(dd,J=10.8,8.3Hz,1H),3.69(s,6H),3.65(s,3H),3.63(d,J=1.5Hz,1H),3.11–3.03(m,1H). 13 CNMR(151MHz,DMSO-d6)δ175.22,170.81,152.50,152.46,152.45,147.63,147.60,147. 00,143.56,136.90,136.43,134.94,132.28,131.71,130.87,124.95,117.93,110.74,10 9.96,109.56,108.69,108.58,101.63,101.59,97.46,69.16,60.38,60.35,60.22,56.32 ,56.27,56.21,51.77,43.51,41.46,39.01,35.74,21.18,14.51.MS-ESI:544.2098[M+H] +

[0064] Example 54: Synthesis and purification of β-NH-(1-ethylindazole-5-amino)-podophyllotoxin (compound 5)

[0065] (1) Synthesis of 4β-NH-(1-ethylindazole-5-amino)podophyllotoxin: 0.15 g (1 mmol) of 1-ethylindazole-5-amino was added to 2 ml of tetrahydrofuran solvent. After dissolution, the mixture was pipetted and transferred to I-PTOX after rotary evaporation, mixed well, and placed in an ice bath. The reaction was carried out in an ice bath for 4 h. After the reaction was completed, the product was obtained by vacuum distillation.

[0066] (2) Separation and purification of 4β-NH-(1-ethylindazole-5-amino)-podophyllotoxin:

[0067] Separation and purification were performed using silica gel column chromatography and gel column chromatography, in the same manner as in Example 1.

[0068] 4β-NH-(1-ethylindazole-5-amino)-podophyllotoxin: white powder, C 31 H 31 N3O7

[0069] 1 HNMR(400MHz, CDCl3)δ7.34-7.29m,1H),7.10-7.04(m,2H),6.95-6.92(m,1 H),6.94(s,1H),6.46(s,1H),6.27(s,2H),5.98(d,J=1.3Hz,1H),5.96(d,J =1.3Hz,1H),4.85(d,J=4.2Hz,1H),4.58(d,J=5.1Hz,1H),4.18-4.09(m,2H ),3.78(s,3H),3.74(s,6H)3.33(dd,J=13.7,5.1Hz,1H),3.24-3.21(m,1H). 13 CNMR(101MHz,CDCl3)δ174.26,162.93(d, 1 J=248Hz),152.54(2C),148.15,147.32,138.35(d, 3 J=5Hz),137.22,135.39,132.07,130.85,127.90,124.22,115.55(d, 2 J=23Hz), 114.01(d, 2 J=19Hz),109.97(2C),108.24(2C),101.57,69.40,60.37,56.22(2C),49.37,43.56,41.88,37.73.MS-ESI:558.2249[M+H] +

[0070] Example 64: Synthesis and purification of β-NH-(1-methyl-3-methylindazole-5-amino)-podophyllotoxin (compound 6)

[0071] (1) Synthesis of 4β-NH-(1-methyl-3-methylindazole-5-amino)podophyllotoxin: 0.15 g (1 mmol) of 1-methyl-3-methylindazole-5-amino was added to 2 ml of tetrahydrofuran solvent. After dissolution, the mixture was pipetted and transferred to I-PTOX after rotary evaporation, mixed well, and placed in an ice bath. The reaction was carried out in an ice bath for 4 h. After the reaction was completed, the product was obtained by vacuum distillation.

[0072] (2) Separation and purification of 4β-NH-(1-methyl-3-methylindazole-5-amino)-podophyllotoxin:

[0073] Separation and purification were performed using silica gel column chromatography and gel column chromatography, in the same manner as in Example 1.

[0074] 4β-NH-(1-methyl-3-methylindazole-5-amino)-podophyllotoxin: white powder, C 31 H 31 N3O7

[0075] 1 HNMR(600MHz,DMSO-d6)δ7.34(d,J=9.3Hz,1H),6.83(s,1H),6.60–6.57(m,3H),6.36–6.33(m,3H),6.00(dd,J=13.4,0.9Hz,2H),4.98(dd,J=8.4,4 .4Hz,1H),4.60(d,J=5.2Hz,1H),4.47–4.43(m,1H),3.82(s,3H),3.68(s, 6H), 3.64 (s, 3H), 3.05 (dddd, J=14.7, 10.9, 7.4, 4.4Hz, 1H), 2.34 (s, 3H). 13 CNMR(151MHz,DMSO-d6)δ175.18,152.50,148.22,147.68,147.06,143.09,140.19,136.92,136.44,131.86,131.83,121.00,115.76,11 1.32,109.93,109.64,108.67,101.67,86.94,69.19,60.41,56.31,50.98,43.46,41.45,39.00,35.04,11.96.MS-ESI:558.2190[M+H]+

[0076] Example 74: Synthesis and purification of β-NH-(1-methyl-3-chloroindazole-5-amino)-podophyllotoxin (compound 7)

[0077] (1) Synthesis of 4β-NH-(1-methyl-3-chloroindazole 5-amino)-podophyllotoxin: 0.15 g (1 mmol) of 1-methyl-3-chloroindazole 5-amino was added to 2 ml of tetrahydrofuran solvent. After dissolution, the mixture was pipetted and transferred to I-PTOX after rotary evaporation, mixed well, and placed in an ice bath. The reaction was carried out in an ice bath for 4 h. After the reaction was completed, the product was obtained by vacuum distillation.

[0078] (2) Separation and purification of 4β-NH-(1-methyl-3-chloroindazole-5-amino)-podophyllotoxin:

[0079] Separation and purification were performed using silica gel column chromatography and gel column chromatography, in the same manner as in Example 1.

[0080] 4β-NH-(1-methyl-3-chloroindazole-5-amino)-podophyllotoxin: white powder, C 30 H 28 ClN3O7

[0081] 1 HNMR (400MHz, CDCl3) δ7.76 (dd, J=8.9, 4.7Hz, 1H), 7.45 (dd, J=7.9, 2.5Hz, 1H), 7. 15(td,J=8.9,2.5Hz,1H),6.98(s,1H),6.47(s,1H),6.32(s,2H),5.96(d,J=1.0Hz, 1H),5.94(d,J=1.0Hz,1H),5.70(d,J=4.3Hz,1H),4.59(d,J=5.2Hz,1H),4.44-3.97 (m,2H),3.79(s,3H),3.75(s,6H),3.43–3.27(m,1H),3.18(dd,J=13.7,5.2Hz,1H). 13 CNMR(101MHz, CDCl3)δ174.27,164.86(d,J=2.9Hz),161.25,158.81,152.58,149.27 (d,J=1.7Hz),148.47,147.51,137.23,136.15(d,J=11.1Hz),135.26,132.70,126.7 9,122.17(d,J=9.3Hz),114.81,114.56,110.04(d,J=10.3Hz),108.27,107.92,107. 65,101.68,70.73,60.74,56.25,49.86,43.69,42.55,37.11.MS-ESI:578.1694[M+H] +

[0082] Example 84: Synthesis and purification of β-NH-(1-methyl-3-bromoindazole-5-amino)-podophyllotoxin (compound 8)

[0083] (1) Synthesis of 4β-NH-(1-methyl-3-bromoindazole 5-amino)-podophyllotoxin: 0.15 g (1 mmol) of 1-methyl-3-bromoindazole 5-amino was added to 2 ml of tetrahydrofuran solvent. After dissolution, the mixture was pipetted and transferred to I-PTOX after rotary evaporation, mixed well, and placed in an ice bath. The reaction was carried out in an ice bath for 4 h. After the reaction was completed, the product was obtained by vacuum distillation.

[0084] (2) Separation and purification of 4β-NH-(1-methyl-3-bromoindazole-5-amino)-podophyllotoxin:

[0085] Separation and purification were performed using silica gel column chromatography and gel column chromatography, in the same manner as in Example 1.

[0086] 4β-NH-(1-methyl-3-bromoindazole-5-amino)-podophyllotoxin: white powder, C 30 H 28 BrN3O7

[0087] 1 HNMR (400MHz, CDCl3) δ7.77 (dd, J=8.9, 4.7Hz, 1H), 7.46 (dd, J=8.0, 2.5Hz, 1H), 7. 17(td,J=8.9,2.6Hz,1H),6.98(s,1H),6.48(s,1H),6.32(s,2H),5.97(d,J=1.2Hz, 1H),5.95(d,J=1.2Hz,1H),5.71(d,J=4.3Hz,1H),5.50(s,1H),4.60(d,J=5.1Hz,1H ),4.43-3.88(m,2H),3.78(s,6H),3.42–3.28(m,1H),3.17(dd,J=13.7,5.2Hz,1H). 13 CNMR(101MHz, CDCl3)δ174.31,164.90(d,J=3.0Hz),161.27,158.82,149.30,14 8.46,147.46,146.45,136.16(d,J=11.1Hz),134.15,132.91,130.72,126.81,12 2.17(d,J=9.3Hz),114.81,114.57,110.03(d,J=2.6Hz),107.94(d,J=4.7Hz),10 7.65,101.65,70.70,56.48,49.87,43.53,42.66,37.04.MS-ESI:622.1189[M+H] +

[0088] Example 94 Synthesis and purification of β-NH-(1-ethyl-3-methylindazole 5-amino)-podophyllotoxin (compound 9)

[0089] (1) Synthesis of 4β-NH-(1-ethyl-3-methylindazole 5-amino)-podophyllotoxin: 0.15 g (1 mmol) of 1-ethyl-3-methylindazole 5-amino was added to 2 ml of tetrahydrofuran solvent. After dissolution, the mixture was pipetted and transferred to I-PTOX after rotary evaporation, mixed well, and placed in an ice bath. The reaction was carried out in an ice bath for 4 h. After the reaction was completed, the product was obtained by vacuum distillation.

[0090] (2) Separation and purification of 4β-NH-(1-ethyl-3-methylindazole-5-amino)-podophyllotoxin:

[0091] Separation and purification were performed using silica gel column chromatography and gel column chromatography, in the same manner as in Example 1.

[0092] 4β-NH-(1-ethyl-3-methylindazole-5-amino)-podophyllotoxin: white powder, C 32 H 33 N3O7

[0093] 1 HNMR(600MHz,DMSO-d6)δ7.34(d,J=8.9Hz,1H),6.94(ddd,J=9.0,2.1,1.0Hz,1H),6.83(s,1H),6.80( dd,J=2.3,1.3Hz,1H),6.58(s,1H),6.37(d,J=1.3Hz,2H),6.00(dd,J=10.1,0.9Hz,2H),5.84(d,J=8.3 Hz,1H),4.92(dd,J=8.4,4.3Hz,1H),4.60(d,J=5.2Hz,1H),4.43(t,J=7.8Hz,1H),4.25(q,J=7.2Hz,2H ),3.69(s,6H),3.66(s,3H),3.43–3.38(m,1H),3.12–3.03(m,1H),2.42(s,3H),1.33(t,J=7.2Hz,3H). 13CNMR(151MHz,DMSO-d6)δ175.29,152.51,147.59,147.01,143.02,142.30,138.61,138.02,1 36.91,136.50,134.83,134.78,132.39,131.70,124.20,124.07,118.24,117.72,110.52,10 9.99,109.96,109.58,108.69,101.63,100.83,97.15,69.26,60.39,60.22,56.28,51.75,43 .52,43.05,42.98,41.49,21.18,15.49,15.41,14.52,12.20,12.10.MS-ESI:572.2397[M+H] +

[0094] Example 104: Synthesis and purification of β-NH-(1-benzylindazole-5-amino)-podophyllotoxin (compound 10)

[0095] (1) Synthesis of 4β-NH-(1-benzylindazole-5-amino)podophyllotoxin: 0.15 g (1 mmol) of 1-benzylindazole-5-amino was added to 2 ml of tetrahydrofuran solvent. After dissolution, the mixture was pipetted and transferred to I-PTOX after rotary evaporation, mixed well, and placed in an ice bath. The reaction was carried out in an ice bath for 4 h. After the reaction was completed, the product was obtained by vacuum distillation.

[0096] (2) Separation and purification of 4β-NH-(1-benzylindazole-5-amino)-podophyllotoxin:

[0097] Separation and purification were performed using silica gel column chromatography and gel column chromatography, in the same manner as in Example 1.

[0098] 4β-NH-(1-Benzylindazole-5-amino)-podophyllotoxin: white powder, C 36 H 34 N3O7

[0099] 1HNMR(600MHz,DMSO-d6)δ7.81(d,J=0.9Hz,1H),7.44(dt,J=9.1,0.8Hz,1H),7.33–7.22(m,6H),6.94(d d,J=9.0,2.2Hz,1H),6.85(d,J=2.1Hz,1H),6.80(d,J=1.8Hz,1H),6.56(s,1H),6.33(s,2H),5.99(dd, J=9.2,0.9Hz,2H),5.91(d,J=8.2Hz,1H),5.56(s,2H),5.52(s,1H),4.86(dd,J=8.1,4.3Hz,1H),4.57( d,J=5.2Hz,1H),4.41–4.37(m,1H),3.68(s,6H),3.64(s,3H),3.37(d,J=5.3Hz,1H),3.08–3.02(m,1H). 13 CNMR(151MHz,DMSO-d6)δ175.21,152.48,147.59,147.00,143.72,143.15,138.42,138.33,1 36.88,136.43,134.47,134.44,132.23,131.71,131.69,131.37,128.92,128.88,127.85,12 7.78,127.68,125.46,125.31,118.57,118.04,110.98,110.45,110.00,109.55,108.67,101 .62,101.11,97.42,69.18,60.39,56.28,52.34,52.25,51.69,43.47.MS-ESI:620.2397[M+H] +

[0100] Example 114 Synthesis and purification of β-NH-(1-cyclohexaneinazole-5-amino)-podophyllotoxin (compound 11)

[0101] (1) Synthesis of 4β-NH-(1-cyclohexaneindazole-5-amino)podophyllotoxin: 0.15 g (1 mmol) of 1-cyclohexaneindazole-5-amino was added to 2 ml of tetrahydrofuran solvent. After dissolution, the mixture was pipetted and transferred to I-PTOX after rotary evaporation, mixed well, and placed in an ice bath. The reaction was carried out in an ice bath for 4 h. After the reaction was completed, the product was obtained by vacuum distillation.

[0102] (2) Separation and purification of 4β-NH-(1-cyclohexaneinazole-5-amino)-podophyllotoxin:

[0103] Separation and purification were performed using silica gel column chromatography and gel column chromatography, in the same manner as in Example 1.

[0104] 4β-NH-(1-cyclohexaneinazole-5-amino)-podophyllotoxin: white powder, C 36 H 39 N3O7

[0105] 1 HNMR(600MHz,Chloroform-d)δ7.80(d,J=0.9Hz,1H),7.26(dt,J=8.9,0.8Hz,1H),6.79(s,1H),6.75(dd,J=8.9,2 .2Hz,1H),6.68(d,J=2.2Hz,1H),6.53(s,1H),6.34(s,2H),5.95(dd,J=10.9,1.3Hz,2H),4.70(t,J=4.0Hz,1H),4. 60(d,J=5.0Hz,1H),4.44(dd,J=8.6,7.4Hz,1H),4.05(dd,J=10.8,8.6Hz,1H),3.82(s,3H),3.77(s,6H),3.22(dd ,J=14.0,5.0Hz,1H),3.09–3.00(m,1H),1.99(dqd,J=11.1,7.4,3.7Hz,1H),1.76–1.57(m,6H),1.24–0.98(m,6H). 13 CNMR(151MHz,Chloroform-d)δ174.92,152.62,148.20,147.61,141.94,13 7.26,135.35,135.23,131.72,131.11,130.94,124.46,117.45,110.66,10 9.90,109.14,108.31,101.52,98.28,69.11,60.75,56.27,56.23,55.34,5 3.44,43.62,41.99,38.90,38.77,30.97,26.29,25.71.MS(ESI)calcd.forC 36 H 39 N3O7[M+H] + 626.2861, found; 626.2864.

[0106] Example 12: Synthesis and purification of 4β-NH-(1-cyclopentaneindazole-5-amino)-podophyllotoxin (compound 12)

[0107] (1) Synthesis of 4β-NH-(1-cyclopentaneindazole-5-amino)podophyllotoxin: 0.15 g (1 mmol) of 1-cyclopentaneindazole-5-amino was added to 2 ml of tetrahydrofuran solvent. After dissolution, the mixture was pipetted and transferred to I-PTOX after rotary evaporation, mixed well, and placed in an ice bath. The reaction was carried out in an ice bath for 4 h. After the reaction was completed, the product was obtained by vacuum distillation.

[0108] (2) Separation and purification of 4β-NH-(1-cyclopentaneindazole-5-amino)-podophyllotoxin:

[0109] Separation and purification were performed using silica gel column chromatography and gel column chromatography, in the same manner as in Example 1.

[0110] 4β-NH-(1-cyclopentane-5-amino-indazole)-podophyllotoxin: white powder, C 35 H 37 N3O7

[0111] 1 HNMR (600MHz, DMSO-d6) δ7.71(d,J=0.9Hz,1H),7.39(d,J=9.0Hz,1H),6.94(dd,J=9.0,2.1Hz,1H),6.81( d,J=2.1Hz,1H),6.79(s,1H),6.54(s,1H),6.34(s,2H),5.98–5.93(m,2H),4.85(dd,J=7.8,4.2Hz,1H),4. 56(d,J=5.1Hz,1H),4.37(t,J=7.8Hz,1H),4.17(d,J=7.4Hz,2H),3.78(dd,J=10.8,8.3Hz,1H),3.66(s,6 H),3.62(s,3H),3.06(dddd,J=14.6,11.1,7.4,4.3Hz,1H),2.39(hept,J=7.4Hz,1H),1.61–1.17(m,10H). 13 CNMR(151MHz,DMSO-d6)δ175.24,170.79,152.53,147.62,147.03,143.47,13 6.95,136.44,134.65,132.30,131.74,130.92,124.84,117.85,110.78,109. 98,109.56,108.72,101.64,97.35,69.20,60.36,60.21,56.25,53.05,51.79 ,43.54,41.49,40.72,39.05,30.20,24.93,21.13,14.48.MS(ESI)calcd.forC35 H 37 N3O7[M+H] + 612.2704, found; 612.2697.

[0112] Example 13: Synthesis and purification of β-NH-(1-cyclopropane-5-amino-indazole)-podophyllotoxin (compound 13)

[0113] (1) Synthesis of 4β-NH-(1-cyclopropane-5-amino-indazole)-podophyllotoxin: 0.15 g (1 mmol) of 1-cyclopropane-indazole-5-amino was added to 2 ml of tetrahydrofuran solvent. After dissolution, the mixture was pipetted and transferred to I-PTOX after rotary evaporation, mixed well, and placed in an ice bath. The reaction was carried out in an ice bath for 4 h. After the reaction was completed, the product was obtained by vacuum distillation.

[0114] (2) Separation and purification of 4β-NH-(1-cyclopropane-5-amino-indazole)-podophyllotoxin:

[0115] Separation and purification were performed using silica gel column chromatography and gel column chromatography, in the same manner as in Example 1.

[0116] 4β-NH-(1-cyclopropane-5-amino-indazole)-podophyllotoxin: white powder, C 33 H 33 N3O7

[0117] 1 HNMR(600MHz,DMSO-d6)δ7.75(d,J=0.9Hz,1H),7.46(d,J=9.0Hz,1H),6.96(dd,J=8.9,2.2Hz,1H),6.84(d,J=2.1Hz,1H), 6.81(s,1H),6.57(s,1H),6.35(s,2H),5.99(dd,J=7.5,0.9Hz,2H),5.89(d,J=8.2Hz,1H),4.88(dd,J=8.2,4.3Hz,1H),4. 59(d,J=5.2Hz,1H),4.40(t,J=7.8Hz,1H),4.20(d,J=6.8Hz,2H),3.79(dd,J=10.8,8.3Hz,1H),3.69(s,6H),3.65(s,3H), 3.19(d,J=5.1Hz,1H),3.07(dddd,J=14.8,11.2,7.4,4.3Hz,1H),1.25–1.21(m,1H),0.50–0.44(m,2H),0.41–0.34(m,2H). 13CNMR(151MHz,DMSO-d6)δ175.24,152.50,147.60,147.01,143.52,136.89,136.44,134.41,132.30,131.71,131.01,125.03,117.88,110.9 4,109.98,109.56,108.69,101.63,97.36,60.39,56.28,52.96,51.78,49.07,43.50,41.48,39.01,11.96,4.07,4.04.MS(ESI)calcd.forC 33 H 33 N3O7[M+H] + 584.2391, found; 584.2400.

[0118] Example 144 Synthesis and purification of β-NH-(3-iodoindazole-5-amino)-podophyllotoxin (compound 14)

[0119] (1) Synthesis of 4β-NH-(3-iodoindazole-5-amino)-podophyllotoxin: 0.15 g (1 mmol) of 3-iodoindazole-5-amino was added to 2 ml of tetrahydrofuran solvent. After dissolution, the mixture was pipetted and transferred to I-PTOX after rotary evaporation, mixed well, and placed in an ice bath. The reaction was carried out in an ice bath for 4 h. After the reaction was completed, the product was obtained by vacuum distillation.

[0120] (2) Separation and purification of 4β-NH-(3-iodoindazole-5-amino)-podophyllotoxin:

[0121] Separation and purification were performed using silica gel column chromatography and gel column chromatography, in the same manner as in Example 1.

[0122] 4β-NH-(3-iodoindazole-5-amino)-podophyllotoxin: white powder, C 33 H 33 N3O7

[0123] 1HNMR(600MHz,DMSO-d6)δ7.75(d,J=0.9Hz,1H),7.46(d,J=9.0Hz,1H),6.96(dd,J=8.9,2.2Hz,1H),6.84(d,J=2.1Hz,1H), 6.81(s,1H),6.57(s,1H),6.35(s,2H),5.99(dd,J=7.5,0.9Hz,2H),5.89(d,J=8.2Hz,1H),4.88(dd,J=8.2,4.3Hz,1H),4. 59(d,J=5.2Hz,1H),4.40(t,J=7.8Hz,1H),4.20(d,J=6.8Hz,2H),3.79(dd,J=10.8,8.3Hz,1H),3.69(s,6H),3.65(s,3H), 3.19(d,J=5.1Hz,1H),3.07(dddd,J=14.8,11.2,7.4,4.3Hz,1H),1.25–1.21(m,1H),0.50–0.44(m,2H),0.41–0.34(m,2H). 13 CNMR(151MHz,DMSO-d6)δ175.24,152.50,147.60,147.01,143.52,136.89,136.44,134.41,132.30,131.71,131.01,125.03,117.88,110.9 4,109.98,109.56,108.69,101.63,97.36,60.39,56.28,52.96,51.78,49.07,43.50,41.48,39.01,11.96,4.07,4.04.MS(ESI)calcd.forC 33 H 33 N3O7[M+H] + 584.2391, found; 584.2400.

[0124] Example 154 Synthesis and purification of β-NH-(3-fluoroindazole-5-amino)-podophyllotoxin (compound 15)

[0125] (1) Synthesis of 4β-NH-(3-fluoroindazole-5-amino)-podophyllotoxin: 0.15 g (1 mmol) of 3-fluoroindazole-5-amino was added to 2 ml of tetrahydrofuran solvent. After dissolution, the mixture was pipetted and transferred to I-PTOX after rotary evaporation, mixed well, and placed in an ice bath. The reaction was carried out in an ice bath for 4 h. After the reaction was completed, the product was obtained by vacuum distillation.

[0126] (2) Separation and purification of 4β-NH-(3-fluoroindazole-5-amino)-podophyllotoxin:

[0127] Separation and purification were performed using silica gel column chromatography and gel column chromatography, in the same manner as in Example 1.

[0128] 4β-NH-(3-Fluorinazole-5-amino)-podophyllotoxin: White powder, C 29 H 26 FN3O7

[0129] 1 HNMR(600MHz,DMSO-d6)δ12.79(s,1H),7.85(s,1H),7.30(d,J=11.3Hz,1H),7.10(d ,J=8.1Hz,1H),6.80(s,1H),6.57(s,1H),6.34(s,2H),5.99(dd,J=5.1,0.9Hz,2H), 5.43(dd,J=8.7,2.7Hz,1H),4.92(dd,J=8.6,4.5Hz,1H),4.55(d,J=5.2Hz,1H),4.4 4(t,J=7.8Hz,1H),3.68(s,6H),3.65(s,3H),3.63–3.59(m,1H),3.12–3.03(m,1H). 13 CNMR(151MHz,DMSO-d6)δ175.33,152.49,147.63,146.99,136.86,136.48,132.06,131.94,120.07,1 09.78,109.59,108.65,101.62,69.05,60.40,56.28,51.74,43.56,41.18,38.71.MS(ESI)calcd.for C 29 H 26 FN3O7[M+H] + 548.1833, found; 548.1834.

[0130] Example 16: Synthesis and purification of β-NH-(1-propylindazole-5-amino)-podophyllotoxin (compound 16)

[0131] (1) Synthesis of 4β-NH-(1-propylindazole-5-amino)podophyllotoxin: 0.15 g (1 mmol) of 1-propylindazole-5-amino was added to 2 ml of tetrahydrofuran solvent. After dissolution, the mixture was pipetted and transferred to I-PTOX after rotary evaporation, mixed well, and placed in an ice bath. The reaction was carried out in an ice bath for 4 h. After the reaction was completed, the product was obtained by vacuum distillation.

[0132] (2) Separation and purification of 4β-NH-(1-propylindazole-5-amino)-podophyllotoxin:

[0133] Separation and purification were performed using silica gel column chromatography and gel column chromatography, in the same manner as in Example 1.

[0134] 4β-NH-(1-propylindazole-5-amino)-podophyllotoxin: white powder, C 31 H 31 N3O7

[0135] 1 HNMR (600MHz, DMSO-d6) δ7.75(d,J=1.0Hz,1H),7.43(d,J=9.0Hz,1H),6.96(dd,J=9.0,2.1Hz,1H),6.84(d,J=2. 2Hz,1H),6.80(s,1H),6.57(s,1H),6.34(s,2H),5.99(dd,J=8.5,0.9Hz,2H),5.88(d,J=8.2Hz,1H),4.88(dd,J=8 .2,4.3Hz,1H),4.58(d,J=5.2Hz,1H),4.39(t,J=7.8Hz,1H),3.78(dd,J=10.8,8.3Hz,1H),3.68(s,6H),3.65(s, 3H), 3.38 (dd, J=14.3, 5.2Hz, 1H), 3.34 (s, 2H), 3.06 (dddd, J=14.7, 11.2, 7.4, 4.3Hz, 1H), 1.37 (t, J=7.2Hz, 3H). 13 CNMR(151MHz,DMSO-d6)δ175.22,170.80,152.49,147.59,147.00,143.56,13 6.89,136.43,133.98,132.30,131.70,130.97,125.04,117.83,110.69,109.9 7,109.56,108.68,108.57,101.63,97.47,69.18,60.39,60.22,56.28,56.23 ,51.77,43.49,43.45,41.47,39.00,21.21,15.46,14.54.MS(ESI)calcd.forC 31 H 31 N3O7[M+H] + 558.2240, found; 558.2249.

[0136] Experimental Example 1: Activity test of the compound prepared in the embodiments of the present invention to inhibit tumor cells.

[0137] I. Experimental Materials

[0138] 1. Test compounds: The compounds prepared in Examples 1 to 16 are numbered as Compound 1 to 16 respectively;

[0139] 2. Control compound: Podophyllotoxin; purchased from Xi'an Helin Biotechnology Co., Ltd., purity 98%;

[0140] 3. Cell lines: HepG2, HeLa, A549, HGC27 and other cell lines were purchased from Wuhan Pronosai Biotechnology Co., Ltd.

[0141] II. Test Methods

[0142] HepG2, HeLa, A549, and HGC27 cell lines in logarithmic growth phase were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in an appropriate amount of culture medium to adjust the cell concentration to 3.5 × 10⁻⁶. 4 Cells were seeded per well in 96-well culture plates, and the following experimental groups were set up:

[0143] One negative control group; 10 experimental groups with the same concentration (i.e., compound 1-compound 10 groups); 2 control groups: paclitaxel group and colchicine group.

[0144] 0.10 mL of cells were added to each well, and RPMI 1640 containing 10% fetal bovine serum was used as the culture medium. After 24 h of culture at 37°C, 5% CO2 and saturated humidity, the cells were discarded when they were nearly confluent. 0.10 M of RPMI 1640 culture medium containing the same amount of compounds (1) and (10) in 10% fetal bovine serum was added to each of the 12 experimental groups. 0.10 M of RPMI 1640 culture medium containing paclitaxel and colchicine in 10% fetal bovine serum was added to the paclitaxel group and colchicine group, respectively. The amounts of paclitaxel and colchicine were exactly the same as those of compounds 1-10. The negative control group was added with a final concentration of 0.5% DMSO. Each group was divided into 3 replicates and cultured for another 48 h. 10 μL of 5 mg / ml MTT was added to each well and the cells were incubated at 37°C for 4 h. Add 100 μl DMSO to each well, shake on a shaker at 37°C for 30 min, and measure the absorbance (OD) at 490 nm. Calculate the MTT ratio as: OD value of the drug group / OD value of the negative control group.

[0145] III. Test Results

[0146] The experimental results are shown in Table 1. As can be seen from Table 1, compounds 1-16 significantly enhanced the antitumor activity of HepG2, HeLa, A549, and HGC27 cell lines. Among them, compound 2 showed higher activity against HepG2 cell line than paclitaxel and colchicine.

[0147] Table 1. IC50 of nitrogen-substituted podophyllotoxin derivatives against in vitro tumor lines. 50 value

[0148]

[0149] Table 2. IC50 of nitrogen-substituted podophyllotoxin derivatives on in vitro hepatocytes. 50 value

[0150]

[0151] Experimental Example 2: Stability test of the compound prepared in plasma according to the embodiments of the present invention.

[0152] I. Experimental Materials

[0153] 1. Test compounds: Compounds 1 and 2 prepared in the examples;

[0154] 2. Control compounds: 6-aminoindole-podophyllotoxin; 5-aminoindazole-podophyllotoxin 3. Test samples: SD rat plasma, human liver microsomes;

[0155] II. Test Methods

[0156] Plasma stability: The test compound and the control compound were placed in the plasma of SD rats and incubated for 0, 4 and 12 hours, respectively, and the stability of the compound in plasma was detected.

[0157] Liver metabolic stability: The test compound and the control compound were placed in human liver microsomal suspension and incubated for 0, 0.5, 1, 2, 4 and 6 hours, respectively, and the liver metabolic stability of the compounds was detected.

[0158] The relative percentage of compounds in plasma and liver microsomes was calculated by using liquid chromatography to detect the compounds.

[0159] III. Test Results

[0160] The test results are shown in Figure 4 A and Figure 4 B. The results showed that compounds 1 and 2 had good plasma stability and stability in liver microsomes, respectively, which were significantly enhanced compared with 6-aminoindole-podophyllotoxin and 5-aminoindazole-podophyllotoxin.

[0161] The experimental data above demonstrate that the compound of formula (V) provided by this invention can further enhance the antitumor activity of podophyllotoxin. The best compound showed an IC50 increase of one order of magnitude, as shown in Table 1. Compound 2 had an IC50 of 39 nM in MIA-Paca-2 cells, representing a two-order-of-magnitude improvement in antitumor activity compared to podophyllotoxin's IC50 of 6.2 μM. The data in Tables 1-2 also indicate that the compounds provided by this invention broaden the antitumor spectrum and have a significant inhibitory effect on pancreatic cancer. (See attached specification.) Figure 4 Data show that the compounds provided by this invention improve stability in plasma.

[0162] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications and variations will be apparent to those skilled in the art. These modifications or variations are included within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are incorporated herein by reference for all purposes.

Claims

1. A compound of formula (V) or a pharmaceutically acceptable salt thereof, , in: R1 is H, and R2 is selected from Cl or -CH3.

2. A method for preparing the compound of claim 1, characterized in that, Includes the following steps: A reactive monomer is introduced into the 4-position of the C ring of podophyllotoxin via a nucleophilic substitution reaction. The reactive monomer is either 3-methylindazole-5-amino or 3-chloroindazole-5-amino.

3. The method according to claim 2, characterized in that, The nucleophilic substitution reaction is carried out under the following conditions: iodopodophyllotoxin is dissolved in an organic solvent, and then the reaction monomer is added and stirred. The organic solvent may be triethylamine.

4. The method according to claim 3, characterized in that, The molar ratio of iodopodophyllotoxin to the reactant monomer is 1:1 to 10.

5. The method according to claim 3, characterized in that, The nucleophilic substitution reaction temperature is -20~20℃.

6. The method according to claim 5, characterized in that, The nucleophilic substitution reaction temperature is 0~10℃.

7. The method according to any one of claims 3-6, characterized in that, The preparation method further includes: pouring the reaction solution after nucleophilic substitution reaction into 20 to 50 times the volume of deionized water, precipitating and filtering, and drying the filter cake to obtain crude product of indazole-substituted podophyllotoxin derivative.

8. The method according to claim 7, characterized in that, The preparation method further includes the following steps: separating the crude product sequentially using silica gel column chromatography and gel column chromatography to obtain the purified indazole-substituted podophyllotoxin derivative product.

9. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of an antitumor drug.

10. An antitumor drug composition, characterized in that, This includes the compound of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

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