Preparation of trifluoromethyl camptothecin derivative and application of trifluoromethyl camptothecin derivative in anti-tumor aspect

Abstract

The invention relates to preparation of trifluoromethyl camptothecin compounds and application of the trifluoromethyl camptothecin compounds in antitumor drugs. The structural formula of the compounds is shown in the specification. In-vitro cytotoxic activity screening results show that the trifluoromethyl camptothecin compound has broad-spectrum anti-tumor activity, and shows relatively strong inhibitory activity on human liver cancer cells (HepG2), human non-small cell lung cancer cells (A549), human colon cancer cells (SW480), human bile duct cancer cells (QBC939), human breast cancer cells (MCF-7), human pancreatic cancer cells (PANC-1) and human pancreatic cancer cells (BxPC-3). The four trifluoromethyl camptothecin compounds I, II, III and IV show a relatively strong inhibition effect on the seven detected tumor cell lines, and the IC50 values of the four trifluoromethyl camptothecin compounds I, II, III and IV are respectively 0.503 to 0.0112 [mu] M, 4.8477 to 0.6516 [mu] M, 1.825 to 0.0005 [mu] M and 4.9043 to 0.4612 [mu] M, which are obviously superior to those of a control drug topotecan. Wherein the compound shows the strongest inhibitory activity on a BxPC-3 cell line, and the IC50 (half maximal inhibitory concentration) value is in a range of 0.6516 to 0.0005 mu M. Therefore, the trifluoromethyl camptothecin compound is expected to be developed into a novel antitumor drug. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of natural drug chemistry and relates to the preparation of a trifluoromethyl-substituted camptothecin compound and its use in anti-tumor. Background Art

[0002] Compounds containing trifluoromethyl (CF3) building blocks are a hot topic in the field of biomedicine. The introduction of fluorine atoms in compounds can change biological activity, and its main effects can be summarized in four aspects: 1. The introduction of fluorine can change the acidity and alkalinity of the compound; 2. The fluorine substitution group can increase the affinity of the drug molecule with proteases; 3. Fluorine atoms have the function of protecting the biologically active sites of the molecule, and can block or slow down the metabolism of substances; 4. The introduction of fluorine atoms can change the hydrophilicity and lipophilicity of the compound. Therefore, the introduction of trifluoromethyl molecular building blocks in the drug skeleton is helpful for the discovery of lead compounds and further structural optimization.

[0003] Camptothecin is a natural alkaloid isolated from the Chinese plant Camptotheca acuminata, which is unique to my country. As a classic topoisomerase I inhibitor, it has always attracted widespread attention in the field of anti-cancer. So far, camptothecin drugs such as irinotecan, topotecan, and belotecan have been approved for marketing for tumor treatment. The structure-activity relationship of camptothecin shows that modification of the A ring can significantly improve its anti-tumor activity and is the best modification site for the in-depth development of new highly active molecules.

[0004] Therefore, we used camptothecin as the lead structure, introduced a trifluoromethyl group into the A ring, synthesized trifluoromethyl camptothecin derivatives, and tested their inhibitory activity against 7 types of tumor cells. The experimental results showed that trifluoromethyl camptothecin derivatives have a broad spectrum of anti-tumor activity, and their in vitro inhibitory activity against a variety of tumor cells is better than that of the clinical drug topotecan, and can be developed as a new type of anti-tumor drug. Summary of the invention

[0005] The present invention provides a preparation method of trifluoromethylcamptothecin compounds and their use in anti-tumor applications

[0006] The structural formula of the trifluoromethylcamptothecin compound of the present invention is as follows:

[0007]

[0008] The preparation method of trifluoromethylcamptothecin compounds of the present invention is carried out according to the following chemical formula 1:

[0009]

[0010] Different o-aminobenzoic acids are used as starting materials, and the corresponding o-aminobenzyl alcohols are obtained by reduction with lithium aluminum hydride; o-aminobenzyl alcohol is oxidized to the corresponding o-aminobenzaldehyde using manganese dioxide as an oxidant; o-aminobenzyl alcohol and a key intermediate tricyclic ketone are subjected to reflux reaction overnight using p-toluenesulfonic acid as a catalyst to obtain the target product 10-trifluoromethylcamptothecin I or 11-trifluoromethylcamptothecin II; under ice bath conditions, compounds I and II are reacted with n-propionaldehyde to obtain the final products 7-ethyl-10-trifluoromethylcamptothecin III and 7-ethyl-11-trifluoromethylcamptothecin IV.

[0011] The trifluoromethylcamptothecin compound of the present invention can play a role in the preparation of anti-tumor drugs, and more specifically, can be used in the preparation of drugs for treating human liver cancer, non-small cell lung cancer, colon cancer, bile duct cancer, breast cancer, and pancreatic cancer. The results of in vitro anti-tumor activity screening show that trifluoromethylcamptothecin compounds have a broad spectrum of anti-tumor activity, and show strong inhibitory activity against human liver cancer cells (HepG2), human non-small cell lung cancer cells (A549), human colon cancer cells (SW480), human bile duct cancer cells (QBC939), human breast cancer cells (MCF-7), human pancreatic cancer cells (PANC-1), and human pancreatic cancer cells (BxPC-3). The four trifluoromethylcamptothecin compounds I, II, III, and IV all showed strong inhibitory effects on the seven tumor cell lines tested, and the IC 50 The values ​​were 0.503-0.0112 μM, 4.8477-0.6516 μM, 1.825-0.0005 μM and 4.9043-0.4612 μM, respectively, which were significantly better than the control drug topotecan. Among them, the compound showed the strongest inhibitory activity against the BxPC-3 cell line, IC 50 The values ​​are in the range of 0.6516 to 0.0005 μM.

[0012] Therefore, the trifluoromethylcamptothecin compounds of the present invention can be used to prepare anti-tumor drugs, and have novel structures, cheap and available raw materials, high product purity, and exhibit strong inhibitory effects on the proliferation of various tumor cell lines, and have good application prospects.

[0013] The above contents of the present invention are further described in detail below through specific implementation modes, but this should not be construed as limiting the present invention. DETAILED DESCRIPTION

[0014] In order to better understand the present invention, the above contents of the present invention are further described in detail below through specific implementation methods. The experimental methods described in the following examples are conventional methods unless otherwise specified.

[0015] Example 1: Synthesis of target compound I

[0016] The synthesis method of compound I of the present invention is carried out according to chemical formula 2:

[0017]

[0018] Synthesis of 2-amino-5-trifluoromethylbenzyl alcohol: 2-amino-5-trifluoromethylbenzoic acid (5.78 mmol) was dissolved in tetrahydrofuran (30 ml), cooled to 0 ° C under nitrogen protection, and lithium aluminum hydride (8.7 mmol) was slowly added dropwise within 30 minutes. The reaction solution was first stirred at 0 ° C for 1 hour, and then stirred at room temperature for 18 hours. After the reaction was complete, it was transferred to an ice bath, quenched with ice water, and filtered through diatomaceous earth. The filtrate was extracted with ethyl acetate and saturated brine, and the organic phase was dried over anhydrous sodium sulfate and concentrated. The crude product was purified by chromatographic column to obtain a yellow solid (yield: 81%).

[0019] Synthesis of 2-amino-5-trifluoromethylbenzaldehyde: 2-amino-5-trifluoromethylbenzyl alcohol (3.56 mmol) was dissolved in anhydrous dichloromethane (10 ml), and active manganese dioxide (7.13 mmol) was added. The reaction solution was stirred at 80°C for 4 hours. After the reaction was complete, it was cooled to room temperature and filtered through diatomaceous earth. The filtrate was concentrated and the crude product was purified by chromatographic column to obtain an orange-yellow solid (yield: 43%).

[0020] Synthesis of target compound I: 2-amino-5-trifluoromethylbenzaldehyde (21.9mmol) and tricyclic ketone (35.1mmol) were dissolved in toluene solution (10ml), and water was removed by a water separator under heating reflux for 30min, and then P-TSA (6.6mmol) was added, and the reaction was continued overnight. After the reaction was completed, the solvent was removed by vacuum concentration, and the organic layer was extracted with dichloromethane and saturated brine, and the organic layer was concentrated to dryness under reduced pressure. The crude product was purified by chromatographic column to obtain the target compound 10-trifluoromethylcamptothecin I.

[0021] The test data of the product obtained by the reaction are as follows: yield: 66%; yellow solid; 1 HNMR (400MHz, CDCl3) δ8.49(s,1H),8.36(d,J=8.9Hz,1H),8.25(s,1H),8.00(dd,J=9.0,2.0Hz,1H),7.72(s,1H),5.7 7(d,J=16.5Hz,1H),5.40–5.27(m,3H),3.74(s,1H),1.98–1.78(m,2H),1.06(t,J=7.4Hz,3H); ESI-MSm / z:417.1[M+H] + .

[0022] Example 2: Synthesis of target compound II

[0023] Synthesis of target compound II: The specific steps are the same as those in Example 1, and the starting material is 2-amino-4-trifluoromethylbenzoic acid. The test data of the product obtained by the reaction are as follows: Yield: 59%; white solid; 1 HNMR (600MHz, CDCl3) δ8.42(s,1H),8.28(d,J=8.9Hz,1H),8.18(s,1H),7.92(dd,J=9.0,2.0Hz,1H),7.65(s,1H),5.6 9(d,J=16.3Hz,1H),5.30–5.15(m,3H),3.74(s,1H),1.91–1.76(m,2H),0.98(t,J=7.4Hz,3H); ESI-MSm / z:417.1[M+H] + .

[0024] Example 3: Synthesis of target compound III

[0025] The synthesis method of compound III of the present invention is carried out according to chemical formula 3:

[0026]

[0027] Dissolve 10-trifluoromethylcamptothecin (0.1mmol) in 10.41mL of glacial acetic acid, slowly add 2.6mL of 98% sulfuric acid at 5-10℃, stir until bright yellow, and obtain 10-trifluoromethylcamptothecin solution. Add FeSO4·7H2O (0.12mmol) and 13mL of deionized water to another reaction bottle, stir to dissolve; add the above 10-trifluoromethylcamptothecin solution at about 2℃, stir evenly, then add freshly distilled propionaldehyde (0.5mmol), stir for a while, slowly add 30% H2O2 (0.5mmol), stir and react for 45min. Pour into appropriate amount of ice water, filter, extract the filtrate with dichloromethane, concentrate the extract under reduced pressure to dryness, and purify the crude product by chromatographic column to obtain the final product 7-ethyl-10-trifluoromethylcamptothecin III.

[0028] The test data of the product obtained by the reaction are as follows: yield: 41%; yellow solid; 1HNMR (400MHz, CDCl3) δ8.34(s,1H),8.28(d,J=8.8Hz,1H),7.91(d,J=9.7Hz,1H),7.62(s,1H),5.70(d,J=16.4Hz,1H),5.26(d,J=14. 7Hz,3H),3.66(s,1H),3.19(q,J=7.7Hz,2H),1.91–1.75(m,2H),1.37(t,J=7.6Hz,3H),0.98(t,J=7.4Hz,3H); ESI-MSm / z:445.1[M+H] + .

[0029] Example 4: Synthesis of target compound IV

[0030] Synthesis of target compound IV: The specific steps are the same as those in Example 3, and the starting material is 11-trifluoromethylcamptothecin II. The test data of the product obtained by the reaction are as follows: Yield: 70%; yellow solid; 1 HNMR (400MHz, CDCl3) δ8.55(s,1H),8.26(d,J=8.8Hz,1H),7.88–7.81(m,1H),7.68(s,1H),5.77(d,J=16.4Hz,1H),5.31(d,J=3.1H z,3H),3.73(s,1H),3.25(q,J=7.7Hz,2H),2.01–1.82(m,2H),1.43(t,J=7.7Hz,3H),1.05(t,J=7.4Hz,3H); ESI-MSm / z:445.1[M+H] + .

[0031] Example 5: Test method and results of antitumor activity of trifluoromethylcamptothecin compounds

[0032] The standard MTT method was used for in vitro antitumor test. Topotecan was used as the positive control drug to test the inhibitory activity of target compounds I, II, III, and IV against human liver cancer cells (HepG2), human non-small cell lung cancer cells (A549), human colon cancer cells (SW480), human cholangiocarcinoma cells (QBC939), human breast cancer cells (MCF-7), human pancreatic cancer cells (PANC-1), and human pancreatic cancer cells (BxPC-3). The compound was dissolved in DMSO to prepare a mother solution with a concentration of 10mM, and diluted to a suitable concentration with different culture media. The DMSO concentration in the dilution should be less than 0.01% (v / v) to reduce the toxicity of DMSO to cells and reduce the test error. Tumor cells of different cell lines were cultured in RPMI-1640 or DMEM high-glucose medium containing 10% fetal bovine serum (FBS), and various cancer cells growing logarithmically were collected, digested with trypsin / EDTA digestion solution and prepared into a suitable cell suspension. Add 100uL of cell suspension to a 96-well plate (usually 5000 cells per well) and place in a 37°C incubator containing 5% CO2 for 24 hours. Then add solutions of the compound to be tested at different concentrations. After 48 hours of culture, discard the old culture medium and wash the cells twice with PBS. Add 25uLMTT (5mg / mL) of fresh culture medium and continue to culture for 4 hours. Thereafter, discard the culture medium and add 200uLDMSO, shake on a shaker for 15 minutes, and wait for the formazan to completely dissolve. Finally, measure the absorbance at 490nm using an enzyme reader and calculate the IC 50 All experiments were performed in three parallel groups or repeated three times. The results of the cytotoxic activity test of trifluoromethylcamptothecin compounds are shown in Table 1.

[0033] Table 1 In vitro cytotoxic activity of trifluoromethylcamptothecin compounds

[0034]

[0035] Note: (1) Screening method: standard MTT colorimetric method; (2) Action time: 48 hours; (3) Compound Nos. I, II, III, and IV are the products obtained in Examples 1-4 above.

[0036] The results of in vitro cytotoxic activity screening showed that trifluoromethylcamptothecin compounds have a broad spectrum of anti-tumor activity, and showed strong inhibitory activity against human liver cancer cells (HepG2), human non-small cell lung cancer cells (A549), human colon cancer cells (SW480), human cholangiocarcinoma cells (QBC939), human breast cancer cells (MCF-7), human pancreatic cancer cells (PANC-1), and human pancreatic cancer cells (BxPC-3). The four trifluoromethylcamptothecin compounds I, II, III, and IV all showed strong inhibitory effects on the seven tumor cell lines tested, with IC 50The values ​​were 0.503-0.0112 μM, 4.8477-0.6516 μM, 1.825-0.0005 μM and 4.9043-0.4612 μM, respectively, which were significantly better than the control drug topotecan. Among them, the compound showed the strongest inhibitory activity against the BxPC-3 cell line, IC 50 The values ​​are in the range of 0.6516 to 0.0005 μM. Therefore, trifluoromethylcamptothecin compounds are expected to be developed into a new type of anti-tumor drug.

Claims

1. A camptothecin compound, such as trifluoromethyl camptothecin compounds I, II, III, and IV shown in the formula.

2. A method for preparing a trifluoromethylcamptothecin compound according to claim 1: Different o-aminobenzoic acids are used as starting materials, and the corresponding o-aminobenzyl alcohols are obtained by reduction with lithium aluminum hydride; o-aminobenzyl alcohol is oxidized to the corresponding o-aminobenzaldehyde using manganese dioxide as an oxidant; o-aminobenzyl alcohol and a key intermediate tricyclic ketone are subjected to reflux reaction overnight using p-toluenesulfonic acid as a catalyst to obtain the target product 10-trifluoromethylcamptothecin I or 11-trifluoromethylcamptothecin II; under ice bath conditions, compounds I and II react with n-propionaldehyde to obtain the final products 7-ethyl-10-trifluoromethylcamptothecin III and 7-ethyl-11-trifluoromethylcamptothecin IV.

3. Use of a trifluoromethylcamptothecin compound according to claim 1 in the preparation of a drug for treating human liver cancer (HepG2).

4. Use of a trifluoromethylcamptothecin compound according to claim 1 in the preparation of a medicament for treating human non-small cell lung cancer (A549).

5. Use of a trifluoromethylcamptothecin compound according to claim 1 in the preparation of a drug for treating human colon cancer (SW480).

6. Use of a trifluoromethylcamptothecin compound according to claim 1 in the preparation of a drug for treating human cholangiocarcinoma (QBC939).

7. Use of a trifluoromethylcamptothecin compound according to claim 1 in the preparation of a drug for treating human breast cancer (MCF-7).

8. Use of a trifluoromethylcamptothecin compound according to claim 1 in the preparation of a drug for treating human pancreatic cancer (PANC-1).

9. Use of a trifluoromethylcamptothecin compound according to claim 1 in the preparation of a drug for treating human pancreatic cancer (BxPC-3).

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