A podophyllotoxin spliced ​​quinine derivative and its application

Abstract

The invention discloses a podophyllotoxin spliced ​​quinine derivative and its application. The invention reacts podophyllotoxin and succinic anhydride in an organic solvent under the catalysis of a catalyst organic alkaline small molecule and the action of an acid-binding agent to generate an intermediate, and then places the anti-tumor active molecule and the intermediate in an organic solvent respectively, and performs an esterification reaction under the catalysis of the catalyst organic alkaline small molecule and the action of a condensing agent to obtain a podophyllotoxin spliced ​​anti-tumor active molecule compound. It has important application value for drug screening and the pharmaceutical industry, and it is found through in vitro anti-tumor activity screening that the compound has a very good inhibitory effect on human non-small cell lung cancer cells and human lung adenocarcinoma cisplatin-resistant strains, and the cytotoxicity to human lung adenocarcinoma cisplatin-resistant strains is more than 80 times that of the positive control drug etoposide, and it is very likely to be further developed into a new drug for preventing and treating tumors.

Description

Technical Field

[0001] The invention relates to the technical field of chemistry, in particular to a podophyllotoxin spliced ​​quinine derivative and application thereof. Background Art

[0002] Quinine, also known as cinchona alkaloids and cinchona cream, is the main alkaloid in the bark of the Cinchona quinine tree and its congener plants of the Rubiaceae family. It not only has significant antimalarial effects, but also has significant anti-tumor activity and can promote apoptosis of tumor cells. Camptothecin is extracted and separated from the Davidia involucrata plant and has significant anti-tumor activity. A series of compounds, such as irinotecan, topotecan, belotecan, BNP-1350, and Gimatecan, have been obtained by structural modification using camptothecin as a lead compound. These compounds are now on the market for clinical use or are in clinical trial research. Indole is an important class of active skeletons that exists in many natural active compounds. The most prominent biological activity is its anti-tumor activity. Through a large number of scientific studies, many lead compounds with anti-tumor activity, anti-tumor drugs that are in clinical trial research or on the market have been obtained, such as Selumetinib, Osimertinib, Strychnofoline, Ganetespib, etc.

[0003] Podophyllotoxin is an aromatic naphthalene lignan extracted and separated from the root and stem of Podophyllum chinensis of the family Botrytis cernua. It is mainly used in the treatment of genital condyloma acuminatum in clinic because it can inhibit the division and proliferation of epithelial cells with warty proliferation caused by human papillomavirus (HPV) infection. At the same time, the most prominent biological activity of podophyllotoxin is anti-tumor activity, but its application is limited by strong adverse reactions caused by high toxicity. Therefore, a large number of structural modifications have been carried out on podophyllotoxin, and a series of podophyllotoxin derivatives with anti-tumor activity have been obtained, such as etoposide (VP-16) and teniposide (VM-26), which have become widely used anticancer drugs in clinic. However, clinical applications have found that etoposide and teniposide have the disadvantages of narrow anti-tumor spectrum, poor water solubility, easy to produce multidrug resistance and severe gastrointestinal dysfunction, and further structural modification or splicing with other active molecules are needed to solve the above problems.

[0004] Therefore, we adopted the principle of drug splicing and spliced ​​podophyllotoxin with quinine, camptothecin, and 3-hydroxymethylindole respectively through succinic anhydride as a linker to obtain podophyllotoxin spliced ​​anti-tumor active molecular compounds, which have very good anti-tumor activity, improve the anti-tumor spectrum, and solve the problem of drug resistance. They can be further developed and studied, and can also provide a compound source for biological activity screening, which has important application value for drug screening and the pharmaceutical industry. Summary of the invention

[0005] The purpose of the present invention is to provide a podophyllotoxin spliced ​​quinine derivative and a preparation method and application thereof. The podophyllotoxin spliced ​​quinine derivative is an important class of pharmaceutical intermediate analogs and drug molecule analogs, which provides a compound source for in vitro anti-tumor activity, anti-viral activity and anti-malarial activity screening, and has important application value for drug screening and the pharmaceutical industry. The synthesis method thereof has the advantages of high yield, great economy and simplicity, simple post-processing and the like.

[0006] The present invention also finds the application of the compound in preparing medicines for preventing and treating tumor diseases.

[0007] The present invention is achieved by: a podophyllotoxin spliced ​​quinine derivative, the compound having the following structure:

[0008]

[0009] A podophyllotoxin spliced ​​quinine derivative and its preparation method:

[0010] Podophyllotoxin and succinic anhydride are placed in an organic solvent at a molar ratio of 1:2, and react to generate an intermediate under the catalysis of a catalyst organic alkaline small molecule and the action of an acid-binding agent. Then, an anti-tumor active molecule (quinine) and the above intermediate are placed in an organic solvent at a molar ratio of 1:1.1, and an esterification reaction is carried out under the catalysis of a catalyst organic alkaline small molecule and the action of a condensation agent to obtain a podophyllotoxin spliced ​​anti-tumor active molecule compound.

[0011] The synthetic route is exemplified as follows:

[0012]

[0013] The drug is quinine.

[0014] The organic small molecule alkaline catalyst is N,N,N,,N,-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate, 4-dimethylaminopyridine or other organic alkaloids, and examples of the organic small molecule alkaline catalyst are as follows (but it should be emphasized that the organic small molecule alkaline catalyst of the present invention is not limited to the contents shown below):

[0015]

[0016] The acid binding agent is pyridine, triethylamine or other organic alkaloids, and examples of the acid binding agent are as follows (but it should be emphasized that the acid binding agent of the present invention is not limited to the contents shown below):

[0017]

[0018] The condensing agent is N,N'-dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or other organic alkaloids, and the condensing agent is exemplified as follows (but it should be emphasized that the condensing agent of the present invention is not limited to the following contents):

[0019]

[0020] The organic solvent is N,N'-dimethylformamide, dichloromethane, toluene, chloroform, ether and the like.

[0021] The reaction temperature of podophyllotoxin and succinic anhydride, the intermediate and the anti-tumor active molecule (quinine) in an organic solvent is 0-60° C. and the reaction time is 0.5-12.0 hours.

[0022] The invention discloses an application of a podophyllotoxin spliced ​​quinine derivative in the preparation and prevention of tumor disease medicine.

[0023] By adopting the above technical scheme, podophyllotoxin and succinic anhydride are placed in an organic solvent at a molar ratio of 1:2, and react to generate an intermediate under the catalysis of a catalyst organic alkaline small molecule and the action of an acid-binding agent, and then the anti-tumor active molecule (quinine) and the above intermediate are placed in an organic solvent at a molar ratio of 1:1.1, and an esterification reaction is performed under the catalysis of the catalyst organic alkaline small molecule and the action of a condensing agent to obtain a podophyllotoxin splicing anti-tumor active molecular compound. This type of compound contains podophyllotoxin and quinine at the same time, and not only has potential biological activity in terms of anti-tumor activity, antiviral activity, and anti-malarial activity, but also has important significance in the research of multi-target drugs, and provides a compound source for biological activity screening, which has important application value for drug screening and the pharmaceutical industry, and through in vitro anti-tumor activity screening, it is found that this type of compound has a very good inhibitory effect on human non-small cell lung cancer cells (A549) and human lung adenocarcinoma cisplatin-resistant strains (A549 / DDP), which is better than positive control drugs etoposide and cisplatin, and is likely to be further developed into a new drug for preventing and treating tumors. The operation method of the present invention is very economical and simple, the product yield is high, the post-processing is simple, the raw materials are cheap and easy to obtain, and further research can be conducted to explore its anti-tumor mechanism and anti-tumor spectrum. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Attached Figure 1 The spectral data of compound 1a of Example 1 of the present invention;

[0025] Attached Figure 2 The spectral data of compound 2a in Example 1 of the present invention;

[0026] Attached Figure 3 The spectral data of compound 2b of Example 1 of the present invention; DETAILED DESCRIPTION

[0027] Embodiment of the present invention: 414.41 mg podophyllotoxin (1 mmol), 200.1 mg succinic anhydride (2 eq, 2.0 mmol), 101.2 mg acid-binding agent triethylamine, 122.1 mg catalyst 4-dimethylaminopyridine and 5 ml dichloromethane solution were added to the reaction tube in sequence, and the reaction was carried out at room temperature for 2 hours. The TLC detection showed that the basic reaction was complete, the solvent was spin-dried, and the sample was purified by silica gel column chromatography (eluent: V (dichloromethane): V (methanol) = 100:3) to obtain the intermediate compound 1a. White solid, melting point: 75.5-76.4 ° C; yield 99.2%; nuclear magnetic resonance and high-resolution mass spectrometry test results are as follows: 1 H NMR (CDCl 3 ,400MHz)δ:6.78(s,1H),6.53(s,1H),6.39(s,2H),5.98(dd,J=6.2,1.3Hz,2H),5.93(d,J=9.1Hz,1H),4.60(d,J=4.4Hz, 1H), 4.36 (dd, J=9.3, 6.9Hz, 1H), 4.17 (t, J=9.8Hz, 1H), 3.81 (s, 3H), 3.76 (s, 6H), 2.97–2.78 (m, 2H), 2.75–2.68 (m, 2H). 13 C NMR (CDCl 3 ,100MHz)δ:176.90,173.88,172.76,152.65,148.20,147.64,137.18,134.91,132.38,128.16,109.76,108.21, 107.03,101.65,74.00,71.40,60.80,56.24,45.55,43.74,38.62,29.05,28.77.;HRMS(ESI-TOF)m / z:Calcd.for C 26 H 26 NaO 11 [M+Na] + :537.1373;Found:537.1378.

[0028] Then 64.9 mg of quinine (0.2 mmol), 113.1 mg (0.22 mmol) of the intermediate 1a, 4.88 mg of the catalyst 4-dimethylaminopyridine (0.04 mmol), 76.5 mg of the condensing agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 5 ml of dichloromethane solution were added to a reaction tube and reacted under vacuum for 12 hours. After TLC detection, the reaction was basically complete. 20 ml of dichloromethane was added to dilute the mixture, and then 1 M hydrochloric acid solution was added to wash the mixture. The dichloromethane layer was taken, washed with saturated brine, dried over anhydrous sodium sulfate, and the dichloromethane layer was spin-dried and loaded with a sample and purified by silica gel column chromatography (eluent: V (dichloromethane): V (methanol) = 15:1 to 10:1) to obtain the final product 2a as a white solid with a melting point of 180.1-180.9° C. and a yield of 95.1%. The results of nuclear magnetic resonance and high-resolution mass spectrometry tests are as follows: 1 H NMR (CDCl 3 ,400MHz)δ:8.75(d,J=4.5Hz,1H),8.04(d,J=9.2Hz,1H),7.42(dd,J=9.2,2.6Hz,1H),7.35(d,J=4 .5Hz,1H),7.29(s,3H),6.77(s,1H),6.53(s,1H),6.38(s,2H),5.98(s,2H),5.87(d,J=9.2Hz,1H) ,5.76(s,1H),5.13–5.06(m,2H),4.60(d,J=4.4Hz,1H),4.09(s,3H),4.00(t,J=9.9Hz,1H),3.82( s,3H),3.76(s,6H),3.67(s,1H),3.47(s,1H),2.92–2.73(m,6H),2.02–1.72(m,8H),1.28(s,1H). 13 C NMR (CDCl 3 ,100MHz)δ:173.65,172.87,152.65,148.22,147.64,144.79,137.17,134.85,132.31,131.89,127.99,109.72,108.12,107.12,101.6 4,101.14,74.22,71.11,70.60,60.80,58.76,56.21,45.53,43.71,42.89,38.60,29.16,28.89,27.28.; HRMS(ESI-TOF)m / z:Calcd.for C 46 H 48 N 2 NaO 12 [M+Na] +:843.3105;Found:843.3101.

[0029] The preparation methods of compounds 2b to 2c are the same as those of compound 2a, and the feed ratio is the same as that of compound 2a, and compounds 2b and 2c can be obtained. The reaction yields are shown in Table 1, but it should be emphasized that the compounds of the present invention are not limited to those shown in Table 1.

[0030] Table 1 shows the chemical structure of a podophyllotoxin spliced ​​anti-tumor active molecular compound

[0031]

[0032] Compound 2b prepared in this example: white solid, melting point: 115.4-116.4°C; yield: 99.3%; nuclear magnetic resonance and high-resolution mass spectrometry test results are as follows: 1 H NMR (CDCl 3 ,400MHz)δ:8.07(d,J=8.2Hz,1H),7.52(dd,J=7.5,1.6Hz,1H),7.23(td,J=7.2,1.6Hz,2H ),6.76(s,1H),6.53(s,1H),6.38(s,2H),6.00–5.94(m,2H),5.80(d,J=8.9Hz,1H),5.31(d ,J=3.5Hz,2H),4.60(d,J=4.3Hz,1H),4.34(dd,J=9.3,6.7Hz,1H),4.14(t,J=9.7Hz,1H), 3.81(s,3H),3.75(s,6H),2.92–2.78(m,2H),2.74–2.66(m,4H),2.63(s,3H),1.68(s,9H). 13 C NMR (CDCl 3 ,100MHz)δ:173.79,172.87,172.25,152.68,150.53,148.17,147.62,137.48,137 .16,135.70,134.91,132.32,128.93,128.27,123.91,122.90,118.03,115.54,11 2.90,109.71,108.09,107.10,101.63,84.20,73.97,71.42,60.81,57.61,56.18, 45.59,43.77,38.65,29.29,29.13,28.28,14.04.;HRMS(ESI-TOF)m / z:Calcd.for C 41 H 43 NNaO 13[M+Na] + :780.2632;Found:780.2634.

[0033] Compound 2c prepared in this example: brown solid, melting point: 195.5-196.5°C; yield: 86.2%; nuclear magnetic resonance and high-resolution mass spectrometry test results are as follows: 1 H NMR (CDCl 3 ,400MHz)δ:8.34(s,1H),8.11(d,J=8.5Hz,1H),7.87(dd,J=8.3,1.4Hz,1H),7.75–7.71(m,1H),7.61–7 .57(m,1H),7.24(s,1H),6.67(s,1H),6.39(s,1H),6.20(s,2H),5.88(s,2H),5.71(d,J=9.3Hz,1H),5.6 0(d,J=17.2Hz,1H),5.30(d,J=17.3Hz,1H),4.36(d,J=4.5Hz,1H),3.80(dd,J=9.3,7.0Hz,1H),3.74–3. 69(m,5H),3.64(s,6H),3.60–3.55(m,1H),2.65–2.53(m,4H),2.23–1.94(m,4H),0.94(t,J=7.5Hz,3H). 13 C NMR (CDCl 3 ,100MHz)δ:173.4,172.9,171.5,167.5,162.6,157.3,152.6,152.4,148.8,148.1,14 7.6,146.2,145.9,137.1,134.7,132.2,131.3,130.7,129.5,128.8,128.4,128.3,128 .1,128.0,119.9,109.7,108.0,106.9,101.6,96.2,74.1,71.0,67.0,60.8,56.1,50. 1,45.2,43.7,38.3,36.5,31.7,31.5,28.9,28.8,7.6.;HRMS(ESI-TOF)m / z:Calcd.For C 46 H 40 N 2 NaO 14 [M+Na] + :867.2377;Found:867.2374.

[0034] The compound of formula (I) of the present invention has important biological activity. The cytotoxicity test of human non-small cell lung cancer cells (A549) and human lung adenocarcinoma cisplatin-resistant strain (A549 / DDP) in vitro by MTT method shows that: a podophyllotoxin spliced ​​anti-tumor active molecule compound of the structure shown in formula (I) has a very good inhibitory effect on tumor cell growth, which is significantly better than the positive control drugs etoposide and cisplatin. Among them, the cytotoxicity of compound 2a to human lung adenocarcinoma cisplatin-resistant strain (A549 / DDP) is more than 80 times that of the positive control drug etoposide. At the same time, compound 2a shows good anti-resistance. After a series of mechanism studies, it is very likely to be further developed into a new drug for preventing and treating tumors. However, it should be emphasized that the compound of the present invention is not limited to the cytotoxicity expressed by human non-small cell lung cancer cells (A549) and human lung adenocarcinoma cisplatin-resistant strain (A549 / DDP).

[0035] Pharmacological Example: The in vitro antitumor activity of compounds 2a-2c against human non-small cell lung cancer cells (A549) and human lung adenocarcinoma cisplatin-resistant strain (A549 / DDP) was tested using the modified MTT method, with A549 / DDP cell line as an example.

[0036] A549 / DDP cells in the logarithmic growth phase and in good growth condition were taken and prepared into a cell suspension in a medium containing 10% calf serum. 3 100 μL of MTT / well were inoculated in a 96-well plate, and a blank group was set up at the same time. The cells were cultured at 37°C overnight. The newly prepared dimethyl sulfoxide solutions of compounds 2a-2c were added to each well in a concentration gradient, so that the final concentrations of the compounds in the wells were 0.01 μmol / L, 0.1 μmol / L, 1 μmol / L, 10 μmol / L and 100 μmol / L, respectively. Three replicates were set up in each group. After 72 hours of cell culture, 10 μL of MTT phosphate buffer was added to each well, and then cultured at 37°C for 4 hours. The unconverted MTT was removed by centrifugation for 5 minutes. 150 μL of dimethyl sulfoxide was added to each well to dissolve the reduced MTT crystal formazan, and the absorbance value OD of each well was measured at a wavelength of 568 nm using an enzyme marker. Cell proliferation inhibition rate = 1-(OD value of the experimental group-OD value of the blank group) / (OD value of the control group-OD value of the blank group), A549 / DDP cell half-inhibitory concentration IC 50 Analyzed by spss software.

[0037] The in vitro antitumor activity tests of compounds 2a-2c are shown in Table 2, wherein the cytotoxicity of compound 2a to A549 / DDP (IC 50 =0.988±0.016μmol / L) is the positive control drug etoposide (IC 50=84.386±2.692μmol / L), more than 80 times that of compound 2a. Meanwhile, compound 2a showed good anti-resistance activity and its cytotoxicity to non-resistant strain A549 (IC 50 =0.085±0.003 μmol / L) Cytotoxicity of compound 2c to A549 cells (IC 50 =0.068±0.003μmol / L) is the positive control drug cisplatin (IC 50 =39.137±2.873μmol / L) is more than 500 times.

[0038] Table 2 In vitro antitumor activity test results of compounds 2a-2c

[0039]

[0040] a Data were expressed as mean IC 50 ±SD (μM), n=3

[0041] b RF was computed as IC 50 (A549 / DDP) / IC 50 (A549)

[0042] Experimental conclusion: Non-small cell lung cancer cells (A549) and human lung adenocarcinoma cisplatin-resistant strains (A549 / DDP) are effective tools and evaluation indicators for testing compound cytotoxicity to tumor cells in vitro, and are also important indicators for evaluating compound cytotoxicity to drug-resistant tumor cells. This experiment shows that a podophyllotoxin spliced ​​anti-tumor active molecule compound shown in formula (I) has very strong cytotoxicity to human non-small cell lung cancer cells (A549) and human lung adenocarcinoma cisplatin-resistant strains (A549 / DDP), which are significantly better than positive control drugs etoposide and cisplatin. Among them, the cytotoxicity of compound 2a to human lung adenocarcinoma cisplatin-resistant strains (A549 / DDP) is more than 80 times that of the positive control drug etoposide. At the same time, compound 2a shows good anti-resistance, and its activity to drug-resistant strains and non-resistant strains is consistent. After a series of mechanism studies in the later stage, it is very likely to be further developed into a new drug for preventing and treating tumors. It can be seen that the above compounds have the potential to be developed into anti-tumor drugs, and have certain economic and clinical value, which is worth our continued in-depth research.

Claims

1. Application of a podophyllotoxin spliced ​​quinine derivative in the preparation of a drug for preventing and treating tumor diseases, Features: The tumor disease is human lung adenocarcinoma resistant to cisplatin A549 / DDP, and the podophyllotoxin spliced ​​quinine derivative has the following structure: 。

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