Preparation of a trifluoromethyl camptothecin derivative and its use in the fight against tumors

Abstract

The present application relates to a kind of preparation of trifluoromethyl camptothecin compound and its use in antitumor drugs, the structural formula of the compound is shown as follows.In vitro cytotoxic activity screening results show that trifluoromethyl camptothecin compound has broad-spectrum antitumor activity, to human hepatocellular carcinoma cell (HepG2), human non-small cell lung cancer cell (A549), human colon cancer cell (SW480), human cholangiocarcinoma cell (QBC939), human breast cancer cell (MCF-7), human pancreatic cancer cell (PANC-1), human pancreatic cancer cell (BxPC-3) shows strong inhibitory activity.4 trifluoromethyl camptothecin compounds I, II, III, IV all show strong inhibition to the 7 tumor cell lines measured, IC 50 Value is 0.503-0.0112 μM, 4.8477-0.6516 μM, 1.825-0.0005 μM and 4.9043-0.4612 μM, all significantly better than control drug topotecan. Among them, the compound shows the strongest inhibitory activity to BxPC-3 cell line, IC 50 Value is in the range of 0.6516-0.0005 μM. Therefore, trifluoromethyl camptothecin compound is expected to be developed into a new antitumor drug.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry, and relates to the preparation of a trifluoromethyl-substituted camptothecin compound and its use in antitumor activity. Background Technology

[0002] Compounds containing trifluoromethyl (CF3) building blocks are a research hotspot in the biomedical field. Introducing fluorine atoms into compounds can alter their biological activity, with the main effects summarized in four aspects: 1. The introduction of fluorine can change the acidity or basicity of the compound; 2. Fluorine substituents can increase the affinity of drug molecules for proteases; 3. Fluorine atoms have a protective effect on the molecular biological active sites, capable of blocking or slowing down metabolism; 4. The introduction of fluorine atoms can alter the hydrophilicity or lipophilicity of the compound. Therefore, introducing trifluoromethyl molecular building blocks into the drug skeleton is helpful for the discovery of lead compounds and further structural optimization.

[0003] Camptothecin is a natural alkaloid isolated from *Camptotheca acuminata*, a plant endemic to my country. As a classic topoisomerase I inhibitor, it has received widespread attention in the field of anticancer treatment. To date, camptothecin-based drugs such as irinotecan, topotecan, and beloteccan have been approved for marketing in cancer treatment. The structure-activity relationship of camptothecin indicates that modification of the A ring can significantly enhance its antitumor activity, making it the optimal modification site for further development of novel, highly active molecules.

[0004] Therefore, we synthesized trifluoromethyl camptothecin derivatives by introducing trifluoromethyl groups into the A ring, using camptothecin as the lead structure, and tested their inhibitory activity against seven types of tumor cells. The experimental results showed that the trifluoromethyl camptothecin derivatives possess broad-spectrum antitumor activity, and their in vitro inhibitory activity against various tumor cells is superior to that of the clinical drug topotecan, suggesting their potential for development as a novel antitumor drug. Summary of the Invention

[0005] This invention provides a method for preparing trifluoromethylcamptothecin compounds and their use in antitumor applications.

[0006] The structural formula of the trifluoromethylcamptothecin-like compound described in this invention is shown below:

[0007]

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

[0009]

[0010] Starting with different an-aminobenzoic acids, the corresponding an-aminobenzyl alcohols were obtained by reduction with lithium aluminum hydride; an-aminobenzyl alcohol was oxidized to an-aminobenzaldehyde using manganese dioxide as an oxidant; an-aminobenzaldehyde and the key intermediate tricyclic ketone were refluxed overnight using p-toluenesulfonic acid as a catalyst to obtain the target products 10-trifluoromethylcamptothecin I or 11-trifluoromethylcamptothecin II; under ice bath conditions, compounds I and II were reacted with n-propanal to obtain the final products 7-ethyl-10-trifluoromethylcamptothecin III and 7-ethyl-11-trifluoromethylcamptothecin IV, respectively.

[0011] This invention relates to a trifluoromethyl camptothecin compound that can play a role in the preparation of antitumor drugs, and more specifically, its use in the preparation of drugs for treating human liver cancer, non-small cell lung cancer, colon cancer, cholangiocarcinoma, breast cancer, and pancreatic cancer. In vitro antitumor activity screening results show that the trifluoromethyl camptothecin compound has broad-spectrum antitumor activity, exhibiting 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). Four trifluoromethyl camptothecin compounds, I, II, III, and IV, all showed strong inhibitory effects against the seven tumor cell lines tested, with IC50 values ​​of [missing data]. 50 The concentrations were 0.503–0.0112 μM, 4.8477–0.6516 μM, 1.825–0.0005 μM, and 4.9043–0.4612 μM, respectively, all significantly superior to the control drug topotecan. Among these, the compound exhibited the strongest inhibitory activity against the BxPC-3 cell line, with an IC50 value of [missing value]. 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 antitumor drugs. They have novel structures, inexpensive and readily available raw materials, and high product purity. They also exhibit strong inhibitory effects on the proliferation of various tumor cell lines and have good application prospects.

[0013] The following detailed description of specific embodiments further illustrates the above-mentioned aspects of the present invention. However, this should not be construed as a limitation of the present invention. Detailed Implementation

[0014] To better understand the present invention, the following detailed description of the above-mentioned contents of the present invention is provided through specific embodiments. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods.

[0015] Example 1: Synthesis of target compound I

[0016] The synthesis method of compound I described in this 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 over 30 minutes. The reaction mixture was first stirred at 0 °C for 1 hour, 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 column chromatography to give a yellow solid (yield: 81%).

[0019] Synthesis of 2-amino-5-trifluoromethylbenzaldehyde: 3.56 mmol of 2-amino-5-trifluoromethylbenzyl alcohol was dissolved in 10 mL of anhydrous dichloromethane, and 7.13 mmol of activated manganese dioxide was added. The reaction mixture 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 column chromatography to give an orange-yellow solid (yield: 43%).

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

[0021] The reaction product test data 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 in Example 1, with 2-amino-4-trifluoromethylbenzoic acid as the starting material. The reaction product analysis data 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 described in this invention is carried out according to chemical formula 3:

[0026]

[0027] 10-Trifluoromethylcamptothecin (0.1 mmol) was dissolved in 10.41 mL of glacial acetic acid. 2.6 mL of 98% sulfuric acid was slowly added dropwise at 5–10 °C, stirring until a bright yellow solution was obtained, yielding a 10-trifluoromethylcamptothecin solution. In another reaction flask, 0.12 mmol of FeSO4·7H2O and 13 mL of deionized water were added and stirred until dissolved. The above 10-trifluoromethylcamptothecin solution was added at approximately 2 °C, and after stirring until homogeneous, 0.5 mmol of freshly distilled n-propanal was added dropwise. After stirring briefly, 0.5 mmol of 30% H2O2 was slowly added dropwise, and the reaction was stirred for 45 min. The solution was poured into an appropriate amount of ice water, filtered, and the filtrate was extracted with dichloromethane. The extract was concentrated to dryness under reduced pressure. The crude product was purified by column chromatography to obtain the final product, 7-ethyl-10-trifluoromethylcamptothecin III.

[0028] The reaction product test data 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 in Example 3, with 11-trifluoromethylcamptothecin II as the starting material. The reaction product detection data 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 methods and results of the antitumor activity of trifluoromethylcamptothecin compounds

[0032] In vitro antitumor assays were performed using the standard MTT assay. Topotecan was used as a positive control to test the inhibitory activity of target compounds I, II, III, and IV against human hepatocellular carcinoma 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 compounds were dissolved in DMSO to prepare a 10 mM stock solution, which was then diluted to appropriate concentrations using different culture media. The DMSO concentration in the diluent should be less than 0.01% (v / v) to reduce DMSO toxicity to cells and minimize testing errors. Tumor cells of different cell lines were cultured in RPMI-1640 or DMEM high-glucose medium containing 10% fetal bovine serum (FBS). Log-growing cancer cells were collected, digested with trypsin / EDTA digestion solution, and prepared into appropriate cell suspensions. 100 μL of cell suspension was added to 96-well plates (typically 5000 cells per well) and incubated at 37°C with 5% CO2 for 24 h. Then, different concentrations of the test compound solutions were added, and after 48 h of incubation, the old culture medium was discarded, and the cells were washed twice with PBS. 25 μL of fresh LMT (5 mg / mL) was added, and the cells were incubated for another 4 h. Afterward, the culture medium was discarded, and 200 μL of LDMSO was added. The plates were shaken for 15 min on a shaker until the formazan was completely dissolved. Finally, the absorbance at 490 nm was measured using a microplate reader, and the IC50 was calculated. 50 Values. All experiments were conducted in triplicate or in three replicates. The results of the cytotoxic activity assays for 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) reaction time: 48 hours; (3) Compound numbers I, II, III, and IV are the products obtained in Examples 1-4 above.

[0036] In vitro cytotoxicity screening results showed that trifluoromethylcamptothecin compounds possess broad-spectrum antitumor activity, exhibiting strong inhibitory activity against human hepatocellular carcinoma 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). Four trifluoromethylcamptothecin compounds (I, II, III, and IV) all showed strong inhibitory effects against the seven tumor cell lines tested, with IC50 values ​​of [missing data]. 50The concentrations were 0.503–0.0112 μM, 4.8477–0.6516 μM, 1.825–0.0005 μM, and 4.9043–0.4612 μM, respectively, all significantly superior to the control drug topotecan. Among these, the compound exhibited the strongest inhibitory activity against the BxPC-3 cell line, with an IC50 value of [missing value]. 50 The values ​​range from 0.6516 to 0.0005 μM. Therefore, trifluoromethylcamptothecin compounds hold promise for development as a novel antitumor drug.

Claims

1. Use of a trifluoromethyl camptothecin compound I, II, III, IV as shown in the formula in the manufacture of a medicament for treating human pancreatic cancer cell PANC-1; 。 2. Use of a trifluoromethyl camptothecin compound I, II, III, IV in the manufacture of a medicament for treating human pancreatic cancer cell BxPC-3; 。

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