Preparation of a 10-trifluoromethoxycamptothecin derivative and its use in antitumor therapy

Abstract

This invention relates to the preparation of 10-trifluoromethoxycamptothecin compounds and their use in antitumor drugs. The structural formula of these compounds is shown below. In vitro cytotoxicity screening results show that 10-trifluoromethoxycamptothecin 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). Compounds 10-trifluoromethoxycamptothecin I and 7-ethyl-10-trifluoromethoxycamptothecin II showed strong inhibitory effects against all seven tested tumor cell lines, with IC50 values ​​exceeding 100%. 50 The concentrations were 0.913–0.0661 μM and 4.932–0.0578 μM, respectively, both significantly superior to the control drug topotecan. Among them, compounds I and II exhibited the strongest inhibitory activity against the BxPC-3 cell line, with IC50 values ​​of [missing value]. 50 The values ​​were 0.0661±0.0065μM and 0.0578±0.0043μM, respectively. Therefore, 10-trifluoromethoxycamptothecin compounds hold promise for development into a novel antitumor drug.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of natural medicinal chemistry, and relates to preparation of a 10-trifluoromethoxy substituted camptothecin compound and its use in anti-tumor. BACKGROUND

[0002] The focus of fluorine chemistry has been on a few functional group reactions such as trifluoromethyl, monofluorination, trifluoromethylthio, etc. for a long time, while many useful fluorine-containing functional groups are ignored, one of which is trifluoromethoxy. Trifluoromethoxy can enhance the metabolic stability and lipophilicity of drug molecules, and improve the bioavailability of drug molecules. In addition, the trifluoromethoxy group is perpendicular to the benzene ring, and this two-dimensional conformation can enhance the affinity of the drug molecule to the target. At present, there are more than 350000 physiologically active substances containing trifluoromethoxy functional groups. Therefore, trifluoromethoxy has potential development value in the field of medicinal chemistry.

[0003] Camptothecin is a natural topoisomerase I inhibitor chemotherapy drug isolated in 1966, which plays a role by binding to DNA-topoisomerase I complex, thereby inhibiting DNA cleavage, relaxation and reannealing process, and ultimately leading to cell death. At present, two small molecule camptothecin topotecan and irinotecan (a prodrug of SN-38) have been approved by FDA for the treatment of ovarian cancer, cervical cancer, small cell lung cancer and colon cancer patients. Another small molecule camptothecin drug belotecan has also been approved in Korea for the treatment of ovarian cancer patients. In recent years, the research on the post-modification of the optimized camptothecin skeleton has still attracted widespread attention, and the structure-activity relationship of camptothecin shows that the modification of A ring can significantly improve its anti-tumor activity, which is the best modification site.

[0004] Therefore, we introduce trifluoromethoxy at the 10 position of camptothecin as a lead structure, synthesize a 10-trifluoromethoxy camptothecin derivative, and test its inhibitory activity on 7 kinds of tumor cells. The experimental results show that the 10-trifluoromethoxy camptothecin derivative has broad-spectrum anti-tumor activity, and the in vitro inhibitory activity on various tumor cells is better than that of the clinical drug topotecan, which can be developed as a new type of anti-tumor drug. SUMMARY

[0005] The present application provides a preparation of a 10-trifluoromethoxy camptothecin compound and its use in anti-tumor

[0006] The 10-trifluoromethoxy camptothecin compound provided by the present application has the following structural formula:

[0007]

[0008] The preparation method of the 10-trifluoromethoxy camptothecin compound provided by the present application is carried out according to the following chemical formula 1:

[0009]

[0010] Starting with 2-amino-5-trifluoromethoxybenzoic acid, 2-amino-5-trifluoromethoxybenzyl alcohol (compound III) was obtained by reduction with lithium aluminum hydride. Compound III was oxidized to 2-amino-5-trifluoromethoxybenzaldehyde (compound IV) using manganese dioxide as an oxidant. Compound IV and compound V were heated under reflux overnight in toluene solution using p-toluenesulfonic acid as a catalyst to obtain the target product 10-trifluoromethoxycamptothecin I. Compound I was reacted with n-propanal under ice bath conditions to obtain the final product 7-ethyl-10-trifluoromethoxycamptothecin II.

[0011] This invention relates to a 10-trifluoromethoxycamptothecin 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 10-trifluoromethoxycamptothecin 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). Compounds 10-trifluoromethoxycamptothecin I and 7-ethyl-10-trifluoromethoxycamptothecin II showed strong inhibitory effects against all seven tested tumor cell lines, with IC50 values ​​of [missing data]. 50 The concentrations were 0.913–0.0661 μM and 4.932–0.0578 μM, respectively, both significantly superior to the control drug topotecan. Among them, compounds I and II exhibited the strongest inhibitory activity against the BxPC-3 cell line, with IC50 values ​​of [missing value]. 50 The values ​​were 0.0661±0.0065μM and 0.0578±0.0043μM, respectively.

[0012] Therefore, the 10-trifluoromethoxycamptothecin compounds of this 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 Compound III: 2-Amino-5-trifluoromethoxybenzoic 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 gray solid III (yield: 55%).

[0019] Synthesis of compound IV: Compound III (3.56 mmol) was dissolved in anhydrous dichloromethane (10 mL), and activated manganese dioxide (7.13 mmol) 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 yellow solid IV (yield: 65%).

[0020] Synthesis of target compound I: Compound IV (21.9 mmol) and compound V (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 mixture 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-trifluoromethoxycamptothecin I.

[0021] The reaction product test data are as follows: Yield: 50%; White solid; 1HNMR(400MHz,CHLOROFORM-D)δ8.37(s,1H),7.88(ddd,J=29.2,9.5,4.2Hz,2H),7.66(s,1H),7.44(td,J=8.8,2.5Hz,1H),5. 73(d,J=16.3Hz,1H),5.30(d,J=14.5Hz,3H),3.74(s,1H),1.97–1.81(m,2H),1.03(t,J=7.4Hz,3H); ESI-MSm / z:433.1[M+H] + .

[0022] Example 2: Synthesis of target compound II

[0023] The synthesis method of compound II described in this invention is carried out according to chemical formula 3:

[0024]

[0025] 10-Trifluoromethoxycamptothecin (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-trifluoromethoxycamptothecin 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-trifluoromethoxycamptothecin 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-trifluoromethoxycamptothecin II.

[0026] The detection data of the product obtained from the reaction are as follows: Yield: 40%; Yellow solid; 1 HNMR (400MHz, CDCl3) δ8.21(d,J=9.2Hz,1H),7.84(d,J=2.6Hz,1H),7.60(d,J=7.3Hz,2H),5.69(d,J=16.4Hz,1H),5.29–5.20(m, 3H),3.76(s,1H),3.11(q,J=7.7Hz,2H),1.91–1.73(m,2H),1.35(t,J=7.7Hz,3H),0.97(t,J=7.4Hz,3H); ESI-MSm / z:461.1[M+H] + .

[0027] Example 3: Test methods and results of the antitumor activity of compounds I and II

[0028] 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 and II 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 performed in triplicate or in triplicate. The results of the cytotoxic activity tests for compounds I and II are shown in Table 1.

[0029] Table 1. In vitro cytotoxic activities of compounds I and II

[0030]

[0031] Note: (1) Screening method: standard MTT colorimetric method; (2) Reaction time: 48 hours; (3) Compounds I and II are the products obtained in Examples 1-2 above.

[0032] In vitro cytotoxicity screening results showed that 10-trifluoromethoxycamptothecin 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). Compounds 10-trifluoromethoxycamptothecin I and 7-ethyl-10-trifluoromethoxycamptothecin II showed strong inhibitory effects against all seven tested tumor cell lines, with IC50 values ​​of [missing data].50 The concentrations were 0.913–0.0661 μM and 4.932–0.0578 μM, respectively, both significantly superior to the control drug topotecan. Among them, compounds I and II exhibited the strongest inhibitory activity against the BxPC-3 cell line, with IC50 values ​​of [missing value]. 50 The values ​​were 0.0661±0.0065μM and 0.0578±0.0043μM, respectively. Therefore, 10-trifluoromethoxycamptothecin compounds hold promise for development into novel antitumor drugs.

Claims

1. Use of 10-trifluoromethoxy camptothecin compounds I and II as shown in the formula in the manufacture of a medicament for the treatment of human pancreatic cancer cell PANC-1. 。 2. Use of 10-trifluoromethoxy camptothecin compounds I and II as shown in the formula in the manufacture of a medicament for the treatment of human pancreatic cancer cell BxPC-3. 。

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