CN111087301B - Synthesis method of ibuprofen caffeate and application of ibuprofen caffeate in preparation of immunosuppressive drugs - Google Patents

Abstract

_The invention discloses a method for synthesizing ibuprofen _caffeate and its application in the preparation of immunosuppressive drugs. Cl ₂ solution, the oil bath temperature was 55-65 ℃ and refluxed for 2 hours, spin-dried, and anhydrous dichloromethane was added to obtain a CH ₂ Cl ₂ solution of ibuprofenyl chloride; caffeic acid, DMF and anhydrous dichloromethane were mixed in an ice-water bath Cool to 5°C, add triethylamine dropwise, slowly add ibuprofen acyl chloride in CH ₂ Cl ₂ solution dropwise with stirring, stir in an ice-water bath for 2 h, and track the reaction by TLC to obtain ibuprofen caffeate. The cytotoxicity of the ibuprofen caffeate (CI) provided by the present invention is detected by MTT method, and the results show that the inhibition of the proliferation of MCF-7 cells and HepG2 cells by CI is very significant; Induces apoptosis and inhibits proliferation.

Description

Synthetic method of ibuprofen caffeate and its application in the preparation of immunosuppressive drugs

technical field

The invention belongs to the technical field of organic synthesis, and in particular relates to a method for synthesizing ibuprofen caffeate and its application in preparing immunosuppressive drugs.

Background technique

From the discovery of the structure of salicylic acid in willow bark, the clinical application of aspirin, ibuprofen and other non-steroidal anti-inflammatory drugs (NSAIDs) has been for many years. Their main mode of action is to inhibit cyclooxygenases (ie, COX-1 and COX-2) resulting in reduced synthesis of prostaglandins (messenger molecules in the inflammatory process) to achieve analgesic and anti-inflammatory effects. Ibuprofen has been used clinically for many years, and is the most widely used non-steroidal drug, which can be used to treat general antipyretic and analgesic, rheumatoid arthritis and neuritis.

Numerous reports have focused on the study of NSAIDs in tumors. Studies have shown that long-term use of ibuprofen can reduce the risk of cancer of the prostate, colon, bladder, liver, pancreas and other malignant tumors. Experiments have found that some non-steroidal drugs have the potential to inhibit the proliferation of ovarian cancer cells and induce their apoptosis. A secondary isoform of cyclooxygenase (COX-2) was found to play an important role in tumor progression and development. However, the antitumor mechanism of ibuprofen has not been elucidated. The purpose of this study was to investigate the effect of structurally modified ibuprofen on hepatocellular carcinoma cells HepG2 and breast cancer cells MCF-7, and to lay a theoretical foundation for the combined application of ibuprofen in the treatment of breast cancer.

SUMMARY OF THE INVENTION

In view of the problems existing in the prior art, the present invention provides a kind of inflammation which is an important part of tumor occurrence and progression, and non-steroidal anti-inflammatory drugs (NSAIDs) are effective in inhibiting early tumor progression and malignant transformation. For example, ibuprofen is effective in reducing the risk of various human cancers, including breast, lung, and prostate cancers. It has been reported that NSAIDs inhibit cancer through a cyclooxygenase (COX) mechanism, however there are also COX-independent targets including peroxisome proliferator-activated receptors, lipoxygenases and apoptosis signaling. Conventional NSAIDs are associated with toxicity, which makes their long-term use for cancer prevention problematic.

The present invention provides a new conventional NSAID chemical modification method to reduce its toxicity and enhance its efficacy. This modified ibuprofen caffeate (CI) is a derivative of ibuprofen by combining the active ingredients of traditional Chinese medicine phenolic acids. This paper aims to study the proliferation and apoptosis of two tumor cell lines after ibuprofen derivatives act on MCF-7 cells and HepG2 cells in vitro, and provide new ideas for clinical tumor treatment. The present invention firstly uses the MTT method to detect the proliferation inhibition of two tumor cell lines under the action of different concentrations of ibuprofen and CI. The experimental results showed that CI had better effect on the two kinds of tumor cells. Flow cytometry analysis showed that different doses of CI increased the apoptosis rate of MCF-7 cell line and affected the cell cycle progression. CI has a certain degree of anti-tumor effect, and its molecular mechanism needs to be further explored.

In order to solve the above-mentioned technical problems, the present invention adopts the following technical solutions:

A synthetic method of ibuprofen caffeate, comprising the following steps:

(1) Add ibuprofen, anhydrous dichloromethane and pyridine successively to the three-necked flask, slowly add SOCl ₂ CH ₂ Cl ₂ solution dropwise while stirring at room temperature, and keep the oil bath temperature at 55-65 ° C and reflux for 2 hours , then slowly cool the reaction solution, remove excess SOCl under reduced pressure, spin-dry to obtain a pale yellow solid, add anhydrous dichloromethane to obtain a _{CH 2} Cl solution of ibuprofen acid chloride, which is sealed for later use _; check whether the acid chloride is prepared by methanol-derivatized ester method success. Take a little methanol into a clean 1.5mL EP tube, add a small amount of ibuprofen acyl chloride dropwise and mix well. TLC spot plate confirms that the acyl chloride is successfully prepared;

(2) take another three-necked bottle and add caffeic acid, DMF and anhydrous dichloromethane, cool to 5°C in an ice-water bath, drip triethylamine, and slowly drip the _CH of the ibuprofen acid chloride obtained in step (1) under stirring The Cl ₂ solution was continuously stirred at low temperature for 2 h, followed by TLC to detect the reaction, suction filtration, and the filtrate was spin-dried to obtain an oily substance, which was subjected to silica gel column chromatography to obtain ibuprofen caffeate as a white solid. The reaction route is as follows:

Further, in the step (1), 15 mmol ibuprofen needs 20 mL of anhydrous dichloromethane, and 10 mmol ibuprofen needs 1 drop of pyridine.

Further, 15 mmol of ibuprofen in the step (1) requires 5 mL of SOCl ₂ in CH ₂ Cl ₂ solution, wherein SOCl ₂ is 0.5 mL.

Further, the molar ratio of ibuprofen to caffeic acid is 5:1.

Further, the mol ratio of caffeic acid and triethylamine in the step (2) is 3:5.

Further, in the step (2), the volume ratio of DMF and anhydrous dichloromethane is 1:10, and 3 mmol of caffeic acid requires 10 mL of anhydrous dichloromethane.

Further, the eluent used in the silica gel column chromatography in the step (2) is petroleum ether: ethyl acetate (V:V)=5:1.

The application of ibuprofen caffeate prepared by the synthetic method in the preparation of immunosuppressive drugs.

Beneficial effects of the present invention: the compound ibuprofen caffeate provided by the present invention is prepared by an acid chloride method; the raw material caffeic acid insoluble in a single solvent is dissolved in a mixed solvent; the method of the present invention is simple and convenient for industrial transformation. The cytotoxicity of the compound ibuprofen caffeate provided by the present invention is detected by MTT method, and the results show that the inhibition of the proliferation of MCF-7 cells and HepG2 cells by ibuprofen caffeate (CI) is very significant; The effect of CI on the apoptosis of MCF-7 cells. With the increase of CI concentration, the proportion of apoptotic cells in the cells increased continuously. It was observed that different concentrations of CI solution could induce apoptosis and inhibit proliferation of breast cancer cells. , and its growth inhibition rate is concentration-dependent.

Description of drawings

Figure 1 shows the inhibition of different concentrations of ibuprofen and CI on the proliferation of MCF-7 cells.

Figure 2 shows the inhibition of HepG2 cell proliferation by different concentrations of ibuprofen and CI.

Figure 3 is the result of flow cytometry to detect the effect of CI on apoptosis of MCF-7 cells.

Figure 4 is a hydrogen spectrum of CI.

Figure 5 is the carbon spectrum of CI.

Figure 6 is a mass spectrum of CI.

Detailed ways

The present invention will be further described below with reference to specific embodiments. It should be understood that the following examples are only used to illustrate the present invention rather than to limit the scope of the present invention, and those skilled in the art can make some non-essential improvements and adjustments according to the content of the above invention.

Example 1

The synthetic method of the ibuprofen caffeate of the present embodiment is as follows:

(1) 3.09 g (15 mmol) of ibuprofen, 20 mL of anhydrous dichloromethane and 1 drop of pyridine were sequentially added to the three-necked flask. 5 mL of SOCl ₂ (0.5 mL) in CH ₂ Cl ₂ was slowly added dropwise with stirring at room temperature. The oil bath temperature was maintained at 60°C up and down and refluxed for 2 hours. After that, the reaction solution was slowly cooled, excess SOCl ₂ was removed under reduced pressure, spin-dried to obtain a light yellow solid, 5 mL of anhydrous dichloromethane was added, and it was sealed for later use;

The methanol-derived ester method was used to check whether the acid chloride was successfully prepared. Take a little methanol into a clean 1.5mL EP tube, add a small amount of ibuprofen acyl chloride dropwise and mix well, TLC spot plate confirms that the acyl chloride is successfully prepared;

(2) Take another three-necked flask and add 0.54g (3mmol) of caffeic acid, 1mL DMF and 10mL anhydrous dichloromethane, cool to about 5°C in an ice-water bath, add triethylamine 5mmol dropwise, and slowly dropwise add one step of cloth under stirring A solution of profenyl chloride in CH ₂ Cl ₂ . The reaction was continued under low temperature stirring for 2h. The detection reaction was followed by TLC. Suction filtration, and the filtrate was spin-dried to obtain an oily substance. Silica gel column chromatography (petroleum ether:ethyl acetate=5:1) gave 427 mg of ibuprofen caffeate (CI) as a white solid.

The structure of CI

Ibuprofen caffeate, white solid, 79.1% yield, mp: 121.8-122.5°C. The molecular formula is C ₂₂ H ₂₄ O ₅ , M=368. Wherein MS (ESI, m/z): [MH]+ is 367; ¹ H NMR (500 MHz, DMSO-d ₆ ), δ/×10 ^-6 : 0.83 (s, 3H, CH ₃ ), 0.84 (s, 3H, CH ₃ ), 1.78-1.82 (m, 1H, CH), 1.40 (d, 3H, CH-CH ₃ ), 2.43 (d, 2H, CH-CH ₂ ), 3.72 (m, 1H, CH-CH ₃ ), 6.53(d, 1H, J=16Hz, CH=CH), 7.52(d, 1H, J=16Hz, CH=CH), 7.15(d, 2H, J=7.95Hz), 7.23(d, 2H , J=7.9Hz), 7.13 (d, 1H, J=8.55Hz), 7.16-7.19 (dd, 1H, J=2.9, 7.8Hz), 7.57 (d, 1H). ¹³ C NMR (500 MHz, DMSO-d ₆ ), δ/×10 ^-6 : 171.64, 167.25, 143.14, 142.22, 141.96, 140.17, 137.00, 133.20, 129.29, 127.23, 126.71, 123.76, 122.86, 4, 3.97.43 , 29.52, 22.10, 18.64, 18.38.

1. Effects of synthetic compounds on cancer cell proliferation

1. Cell culture

Human hepatoma cells HepG2 and breast cancer cells MCF-7 were cultured in DMEM complete medium (DMEM+10% fetal bovine serum+1% double antibody) in a 37°C, 5% CO ₂ and saturated humidity incubator. When the cells in the culture dish were observed to adhere to 80-85% under the microscope, they were digested and passaged with 1 mL of 0.25% trypsin.

2. MTT assay to detect inhibition rate

After the cells were passed for 3 to 4 generations, 5000-10000 cells/well were plated in a 96-well plate, and then placed in an incubator for 24 h after observation. After the cell density reached 85%, different concentrations (50, 100, 150, 200, 250, 300, 350, 400 μmol/L) of CI were added, and the normal group and the blank group were set up with 6 duplicate wells. Incubate in the incubator for 24h. After 24 hours, 20 μl of MTT (5 mg/ml) was added to each well to continue incubation at 37°C, and the incubation was terminated after 4 hours. Carefully remove the medium supernatant, add 150 μl DMSO to each well, incubate at 37°C for 10 min, shake at low speed with a flat shaker for 10 min to fully dissolve the crystals, adjust to zero with a blank control, and measure the absorbance of each well at a wavelength of 490 nm to find Mean values were calculated, and inhibition rate and IC50 were calculated. The results are shown in Figures 1 and 2.

From the results in Figures 1 and 2, it can be seen that CI has a strong inhibitory effect on MCF-7 cells and HepG2 cells in the range of 50-400 μmol/L, showing good activity. The 24-hour CI effect was calculated by Graph Pad Prism software. The IC 50 value for MCF-7 was 273.6 μmol/L, and the IC 50 value for HepG2 was 223.3 μmol/L.

3. Detection of apoptosis rate of breast cancer cells by flow cytometry

A 6-well plate was plated with 2×10 ⁵ cells/well, and the supernatant was discarded after culturing for 24 h, and new culture medium containing CI with concentrations of 200 μmol/L, 273.7 μmol/L, and 300 μmol/L was added, and a control group was set up. After culturing for 24 hours, 5×10 ⁵ cells were collected by centrifugation at each drug concentration. Dilute 5× binding buffer with double distilled water to make 1× working solution, take 500 μL of 1× binding buffer to resuspend cells, and add 5 μL Annexin V-CITC and 10 μL PI to each tube. After mixing by gentle vortexing, incubate at room temperature for 5 minutes in the dark. On a flow cytometer, Annexin V-CITC (Ex=488 nm; Em=530 nm) was detected by the CITC detection channel and PI was detected by the PE detection channel. The results are shown in Figure 3.

As can be seen from Figure 3, the apoptotic ratio in the control group only accounted for 0.19% of all cells, while with the increase of CI concentration, the proportion of apoptotic cells in the cells increased continuously. The percentage of apoptosis was as high as 82.17%. It was observed that different concentrations of CI solution can induce apoptosis and inhibit proliferation of breast cancer cells, and its growth inhibition rate is concentration-dependent.

The foregoing has shown and described the basic principles and main features of the present invention, as well as the advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above-mentioned embodiments. The above-mentioned embodiments and descriptions only illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will also have Various changes and modifications fall within the scope of the claimed invention. The claimed scope of the present invention is defined by the appended claims and their equivalents.

Claims (8)

1. a synthetic method of ibuprofen caffeate, is characterized in that comprising the following steps: (1) Add ibuprofen, anhydrous dichloromethane and pyridine in turn to the three-necked flask, slowly add SOCl ₂ CH ₂ Cl ₂ solution dropwise while stirring at room temperature, and keep the oil bath temperature at 55-65°C for 2 hours , then the reaction solution was slowly cooled, excess SOCl was removed under reduced pressure _, spin-dried to obtain a pale yellow solid, anhydrous dichloromethane was added to obtain a _{CH 2} Cl solution of ibuprofenyl chloride, which was sealed for later use; (2) Take another three-necked bottle, add caffeic acid, DMF and anhydrous dichloromethane, cool to 5°C in an ice-water bath, add triethylamine dropwise, and slowly add CH ₂ of ibuprofen acid chloride obtained in step (1) dropwise with stirring Cl ₂ solution, the ice-water bath continued to stir the reaction for 2h, TLC tracked the reaction, suction filtered, the filtrate was spin-dried to obtain an oily substance, and silica gel column chromatography was used to obtain ibuprofen caffeate as a white solid. 2. the synthetic method of ibuprofen caffeate according to claim 1, is characterized in that: in described step (1), 15mmol ibuprofen needs anhydrous dichloromethane 20mL, 10mmol ibuprofen needs 1 drop of pyridine . 3. the synthetic method of ibuprofen caffeate according to claim 1, is characterized in that: in the described step (1), 15mmol ibuprofen needs SOCl ₂ CH ₂ Cl ₂ solution 5mL, wherein SOCl ₂ is 0.5 mL. 4. the synthetic method of ibuprofen caffeic acid ester according to claim 1, is characterized in that: the mol ratio of described ibuprofen and caffeic acid is 5:1. 5. the synthetic method of ibuprofen caffeic acid ester according to claim 1, is characterized in that: in described step (2), the mol ratio of caffeic acid and triethylamine is 3:5. 6. the synthetic method of ibuprofen caffeic acid ester according to claim 1 is characterized in that: in the described step (2), the volume ratio of DMF and anhydrous dichloromethane is 1:10, and 3mmol caffeic acid needs no caffeic acid. Water dichloromethane 10mL. 7. the synthetic method of ibuprofen caffeate according to claim 1, is characterized in that: in described step (2), the used eluent of silica gel column chromatography is petroleum ether: ethyl acetate V: V= 5:1. 8. The application of ibuprofen caffeate prepared by the synthetic method according to any one of claims 1-7 in the preparation of medicines for the treatment of liver cancer and breast cancer.

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