A compound with anti-lung cancer activity, its preparation method and application

Abstract

The present invention relates to the fields of pharmaceutical engineering and medical technology, and particularly to a compound with anti-lung cancer activity, a preparation method thereof, and an application thereof. In the present invention, an ionic liquid is used as a solvent, and Z-2-chloromethyl-3-phenylacrylic acid, 3-p-chlorophenylacrolein, benzylamine, and cyclohexyl isocyanide are used as raw materials, and a compound with anti-lung cancer activity is obtained through a microwave-assisted one-pot reaction. The method of the present invention has the characteristics of simplicity, low cost, and greenness, and the prepared compound with anti-lung cancer activity has a good effect of inhibiting the proliferation and inducing apoptosis of lung cancer cells.

Description

Technical Field

[0001] The present invention relates to the fields of pharmaceutical engineering and medical technology, and particularly relates to a compound with anti-lung cancer activity, a preparation method thereof, and an application thereof. Background Art

[0002] Lung cancer is a cancer with a relatively high mortality rate, and both the incidence and mortality rates rank among the top. Therefore, the research on new anti-lung cancer chemotherapy drugs has good prospects. The marine alkaloids Rhopaladins A-D exist in marine tunicate organisms and have cytotoxicity against human tumor cell lines. Three important parts in the structure of Rhopaladins are: a five-membered heterocyclic center and two indole rings connected thereto by an alkene bond and a carbonyl group respectively. Therefore, it is very necessary to synthesize safe, low-toxic, and effective drugs using the marine alkaloid Rhopaladins as a lead compound for the treatment of diseases. However, the existing synthesis methods of Rhopaladins analogues are cumbersome, and there is no report on their anti-lung cancer activity. Summary of the Invention

[0003] The object of the present invention is to provide a compound with anti-lung cancer activity, a preparation method thereof, and an application thereof. The present invention can realize the preparation of the Rhopaladins analogue (2E,4E)-N-cyclohexyl-1-benzyl-2-p-chlorostyryl-4-p-chlorobenzylidene-5-pyrrolidinone-2-carboxamide (abbreviated as RPDPRQ) through a microwave-assisted one-pot method. The method has the characteristics of simplicity, low cost, and greenness, and the prepared Rhopaladins analogue RPDPRQ has a significant inhibitory effect on lung cancer cells.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] One of the technical solutions of the present invention is a compound (RPDPRQ) with anti-lung cancer activity, and the structural formula is shown in Formula I;

[0006]

[0007] Another technical solution of the present invention is a preparation method of the above-mentioned compound with anti-lung cancer activity, including the following steps:

[0008] Using an ionic liquid as a solvent, using Z-2-chloromethyl-3-phenylacrylic acid, 3-p-chlorophenylacrolein, benzylamine, and cyclohexyl isocyanide as raw materials, reacting through a microwave-assisted one-pot method, and dropping a basic solution during the reaction to obtain the compound with anti-lung cancer activity.

[0009] Further, it includes the following steps:

[0010] Dissolve Z-2-chloromethyl-3-phenylacrylic acid, 3-(4-chlorophenyl)acrolein, benzylamine and cyclohexyl isocyanide in an ionic liquid, stir for 15 - 45 min, then carry out a microwave-assisted reaction for 16 - 24 min (preferably 16 min), and then add an alkaline solution dropwise to the reaction system in 5 - 10 portions. After the addition is completed, continue the microwave-assisted reaction until the reaction is complete. Then freeze the reaction solution, filter by suction after solid precipitation, wash with water, and recrystallize to obtain the compound with anti-lung cancer activity;

[0011] During the microwave-assisted reaction process, set the power to 300 W, heat for 8 s, stop for 8 s, and repeat the steps of heating and stopping until the reaction ends;

[0012] The parameter setting of the microwave-assisted reaction is very important. Setting according to the above parameters can ensure the reaction temperature. If the duration is too long, such as the heating time exceeds 8 s and the stopping time is less than 8 s, it will lead to an increase in by-products and a decrease in yield; if the heating time is less than 8 s and the stopping time exceeds 8 s, the reaction temperature cannot be guaranteed, resulting in too long a reaction time and too low a microwave-assisted efficiency, and a decrease in yield.

[0013] The time interval between two adjacent dropwises of the alkaline solution is 96 s, and a microwave-assisted reaction is carried out during the interval.

[0014] Furthermore, the molar ratio of Z-2-chloromethyl-3-phenylacrylic acid, 3-(4-chlorophenyl)acrolein, benzylamine and cyclohexyl isocyanide is 1:1:1:1.

[0015] Furthermore, the molar volume ratio of Z-2-chloromethyl-3-phenylacrylic acid to the ionic liquid is 1 mmol: 2 - 10 mL; the ionic liquid is [BMIM][PF 6 .

[0016] Compared with the reaction in traditional organic solvents, microwave assistance is more efficient and convenient; the ionic liquid [BMIM][PF 6 as the reaction solvent can avoid the volatilization of organic reaction solvents and is also safer and greener.

[0017] If an ethanol aqueous solution is used to replace the ionic liquid [BMIM][PF 6 , the hydration rate of cyclohexyl isocyanide will be accelerated under microwave-assisted reaction, resulting in an increase in by-products and too low a yield.

[0018] Furthermore, the alkaline solution is an aqueous solution of potassium carbonate or sodium carbonate; the mass concentration of the alkaline solution is 10 - 30 wt%; the total dropwise addition amount of potassium carbonate or sodium carbonate in the alkaline solution and the molar ratio of Z-2-chloromethyl-3-phenylacrylic acid is 0.5:1.

[0019] The third technical solution of the present invention is the application of the above-mentioned compound with anti-lung cancer activity in the preparation of anti-lung cancer drugs.

[0020] The fourth technical solution of the present invention is an anti-lung cancer pharmaceutical composition, which comprises the above-mentioned compound with anti-lung cancer activity or a pharmaceutically acceptable salt thereof.

[0021] Further, by mass percentage, the compound with anti-lung cancer activity is 0.01%-5%, and the pharmaceutically acceptable excipients are 95%-99.99%.

[0022] Further, the pharmaceutically acceptable excipients include a pH regulator.

[0023] Further, the anti-lung cancer pharmaceutical composition is an injectable composition; preferably a freeze-dried powder injection.

[0024] Further, the pH regulator is sodium hydroxide, which converts the compound with anti-lung cancer activity (Rhopaladins analogue RPDPRQ) into the sodium salt form to prepare a freeze-dried powder injection; the conversion is partial or complete conversion.

[0025] The fifth technical solution of the present invention is a freeze-dried powder injection for anti-lung cancer. By mass percentage, the above-mentioned compound with anti-lung cancer activity is 0.01%-5%, and the pharmaceutically acceptable excipients are 95%-99.99%; wherein, the pharmaceutically acceptable excipients contain sodium hydroxide as a pH regulator, which can convert the compound with anti-lung cancer activity into the sodium salt form. The conversion is partial or complete conversion; the partial conversion is at least 20% conversion into the sodium salt form; specifically, it can be 20%, 30%, 40%, 50%, 60% or 80% conversion into the sodium salt form.

[0026] The present invention discloses the following technical effects:

[0027] Activity tests prove that the Rhopaladins analogue RPDPRQ prepared by the present invention is a suitable anti-tumor candidate drug, especially as a candidate drug for anti-lung cancer treatment; it has good effects on inhibiting the proliferation and inducing apoptosis of lung cancer cells A549. It is found that its IC 50 values for lung cancer cells A549 and PC9 are 9.48 μM and 18.76 μM respectively, and the effect is better than that of cisplatin.

[0028] The synthesis method of the Rhopaladins analogue RPDPRQ of the present invention is simple, the raw materials are easily available, the yield of the synthesis route is high, it is environmentally friendly, and the operation process is simple. The compounds of the present invention have broad prospects in the development and application of anti-tumor drugs. Description of the Drawings

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for use in the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained based on these drawings.

[0030] Figure 1 It is the synthetic route diagram of the Rhopaladins analogue RPDPRQ of the present invention;

[0031] Figure 2 It is the spectrogram of the Rhopaladins analogue RPDPRQ prepared in Example 1 of the present invention. Detailed implementation manners

[0032] Now, the various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0034] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0035] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and embodiments are only exemplary.

[0036] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0037] In the embodiments of the present invention, the raw materials used can be obtained through purchasing channels without special instructions.

[0038] The synthetic route of the Rhopaladins analog RPDPRQ of the present invention is as Figure 1 shown.

[0039] Example 1

[0040] Weigh Z-2-chloromethyl-3-phenylacrylic acid (10 mmol) and 3-p-chlorophenylacrolein (10 mmol) into a 250 mL round-bottom flask, add 50 mL of ionic liquid [BMIM][PF 6 and stir to dissolve. Then add benzylamine (10 mmol) and cyclohexyl isocyanide (10 mmol). After stirring at room temperature for 30 min, carry out a reaction under microwave assistance (set the power to 300 W, heat for 8 s, stop for 8 s, and repeat the above heating and stopping steps) for 16 min. Divide the prepared 20 mL aqueous potassium carbonate solution (5 mmol) into 10 portions and add them dropwise to the reaction system 10 times. The time interval between adjacent additions of the aqueous potassium carbonate solution is 96 s (during the interval, microwave-assisted heating is carried out for 8 s and stopping for 8 s, with a total of 6 cycles). Stop microwave assistance when adding the aqueous potassium carbonate solution, and continue microwave-assisted reaction after each single addition of the aqueous potassium carbonate solution. After all 10 portions of the aqueous potassium carbonate solution are added dropwise, continue the microwave-assisted reaction until the reaction is complete as monitored by TLC. Place the reaction solution in the refrigerator and freeze it overnight. After complete precipitation of the solid, filter it by suction, wash it with water, and recrystallize it with ethanol to obtain the Rhopaladins analog RPDPRQ, with a total yield of 91%.

[0041] The structural formula of the Rhopaladins analog prepared in this example is shown in Formula I. The obtained compound Rhopaladins analog RPDPRQ has the chemical name of (2E,4E)-N-cyclohexyl-1-benzyl-2-p-chlorostyryl-4-p-chlorobenzylidene-5-pyrrolidinone-2-carboxamide, and its spectral data are as follows: white solid (5.1 g, total yield: 91%). Melting point: 121 - 123 °C. 1 H NMR(CDCl 3 ,400MHz):δ7.50 - 7.00(m,14H,13Ar-H and=CH),7.64(d,J=16.2Hz,1H,=CH),6.43(d,J=16.2Hz,1H,=CH),5.61(s,1H,NH),4.77(d,J=15.2Hz,1H,CH 2 a ),4.45(d,J=15.2Hz,1H,CH 2 b), 3.58 - 3.50 (m, 1H, NCH), 3.44 (dd, J 1 = 12.9 Hz, J 2 = 33.0 Hz, 2H, CH 2 ), 1.77 - 0.68 (m, 10H, 5CH 2 ). MS (m / z, %) 558 (M + , 4), 467 (100), 432 (27), 341 (64), 204 (13), 126 (12), 111 (43), 91 (93), 76 (17). Anal. Calcd for C 33 H 32 Cl 2 N 2 O 2 : C, 70.84; H, 5.76; N, 5.01. Found: C, 70.12; H, 5.83; N, 5.09. Its spectrum is as shown in Figure 2 shown.

[0042] Example 2

[0043] Verify the effect of Rhopaladins analogue RPDPRQ prepared in Example 1 on the proliferation of lung cancer A549 and PC9 cells

[0044] Collect lung cancer cells A549 and PC9 in the logarithmic growth phase, digest the cells with 0.25% trypsin, and then centrifuge. Pour off the cell supernatant, and prepare a single cell suspension with DMEM medium containing 10% fetal bovine serum. Adjust the concentration of the cell suspension so that each milliliter contains 2×10 5 cells, transfer and culture them in a 96-well plate, add 100 μL of the cell suspension to each well, and fill the edge of the 96-well plate with sterile PBS. Incubate in a 5% CO 2 , 37 °C incubator for 24 h. Set up blanks and treat the cells in the logarithmic growth phase with different concentrations of Rhopaladins analogue RPDPRQ (using cisplatin as the positive control), namely 3.125, 6.25, 12.5, 25, 50, 100 μM, for 48 h. Then add MTT reagent, measure the absorbance value with an enzyme-linked immunosorbent assay reader (repeat three times for each concentration), and finally calculate the IC 50 value (the concentration that inhibits the growth of half of the cells). The results are shown in Table 1.

[0045] Table 1 In vitro anti-proliferative activity of Rhopaladins analogue RPDPRQ

[0046]

[0047] As can be seen from Table 1, the Rhopaladins analog RPDPRQ can inhibit the proliferation of lung cancer cells A549 and PC9, and has a better effect than cisplatin.

[0048] Example 3

[0049] Verify the effect of the Rhopaladins analog RPDPRQ prepared in Example 1 on the apoptosis of lung cancer A549 cells

[0050] Collect lung cancer cells A549 in the logarithmic growth phase, digest the cells with 0.25% trypsin, and then centrifuge. Pour off the cell supernatant, and prepare a single cell suspension with DMEM medium containing 10% fetal bovine serum. Adjust the concentration of the cell suspension so that each milliliter contains 2×10 5 cells, and add 2 mL of the cell suspension to each well of a 6-well plate. Incubate in a 5% CO 2 , 37 °C incubator for 48 h. Set up a blank control group, and treat the cells with low, medium, and high concentrations (5 μM, 10 μM, 20 μM) according to the IC 50 value of the Rhopaladins analog RPDPRQ on lung cancer A549 cells for 48 h. After 48 h, collect the cells. When digesting the cells, use 0.25% trypsin without EDTA, and then centrifuge. Operate according to the instructions of the rhAnnexin V-FITC cell apoptosis detection kit, and use a flow cytometer to determine the effect of the Rhopaladins analog RPDPRQ on the apoptosis of lung cancer A549 cells (repeated three times for each concentration). The apoptosis rate of the cells is shown in Table 2.

[0051] Table 2 Effect of Rhopaladins analog RPDPRQ on the apoptosis of lung cancer A549 cells

[0052]

[0053] As can be seen from Table 2, in the results identified by the flow cytometer, as the concentration of the Rhopaladins analog RPDPRQ increases, the apoptosis rate of A549 cells increases from 5.83% to 68.64%.

[0054] Example 4

[0055] Weigh (10 mmol) of Z-2-chloromethyl-3-phenylacrylic acid and (10 mmol) of 3-p-chlorophenylacrolein into a 250 mL round-bottom flask, and add the ionic liquid [BMIM][PF 650 mL, after stirring and dissolving, add benzylamine (10 mmol) and cyclohexyl isocyanide (10 mmol). After stirring at room temperature for 30 min, carry out a reaction for 16 min under microwave assistance (set the power to 300 W, heat for 8 s, stop for 8 s, and repeat the above heating and stopping steps); add dropwise 10 mL of an aqueous potassium carbonate (5 mmol) solution prepared into the reaction system. After the addition is complete, continue the reaction for 16 min (during the reaction process, heat for 8 s and stop for 8 s under microwave assistance, and repeat the heating and stopping steps). After monitoring the reaction to completion by TLC, place the reaction solution in the refrigerator and freeze overnight. After complete precipitation of the solid, carry out suction filtration, wash with water, and recrystallize with ethanol to obtain the Rhopaladins analog RPDPRQ, with an overall yield of 63%. (That is, compared with Example 1, the only difference in this example is that the concentration of the aqueous potassium carbonate solution is different, and the aqueous potassium carbonate solution is added dropwise all at once.)

[0056] Example 5

[0057] Weigh Z-2-chloromethyl-3-phenylacrylic acid (10 mmol) and 3-p-chlorophenylacrolein (10 mmol) into a 250 mL round-bottom flask, and add the ionic liquid [BMIM][PF 6 50 mL, after stirring and dissolving, add benzylamine (10 mmol) and cyclohexyl isocyanide (10 mmol). After stirring at room temperature for 30 min, carry out a reaction for 16 min under microwave assistance (set the power to 300 W, heat for 8 s, stop for 8 s, and repeat the above heating and stopping steps); divide the prepared 10 mL of an aqueous potassium carbonate (5 mmol) solution into 5 portions, and add the aqueous potassium carbonate solution to the reaction system in 5 portions. The time interval between adjacent additions of the aqueous potassium carbonate solution is 192 s (during the interval, heat for 8 s and stop for 8 s under microwave assistance, and cycle 12 times in total). After all 5 portions of the aqueous potassium carbonate solution have been added, continue the reaction under microwave assistance until the reaction is monitored to completion by TLC. Place the reaction solution in the refrigerator and freeze overnight. After complete precipitation of the solid, carry out suction filtration, wash with water, and recrystallize with ethanol to obtain the Rhopaladins analog RPDPRQ, with an overall yield of 74%. (That is, compared with Example 1, the only differences in this example are that the concentration of the aqueous potassium carbonate solution is different, the aqueous potassium carbonate solution is added dropwise in 5 portions, and the time interval between two additions of the aqueous potassium carbonate solution is different.)

[0058] Comparative Example 1

[0059] The compound is

[0060] Verify the effect of the compound shown in Formula II above on the proliferation of lung cancer A549 and PC9 cells, and the verification method is the same as in Example 2. The results show that the IC 50 of the compound shown in Formula II for lung cancer A549 is 14.82 μM, and the IC 50 for lung cancer PC9 cells is 29.58 μM.

[0061] The present invention is based on an ionic liquid [BMIM][PF 6 as a solvent for microwave-assisted one-pot synthesis of a new Rhopaladins analog RPDPRQ. Without the separation step of intermediates, the overall reaction yield is increased (the overall yield is 91%); through multi-component condensation, the reaction steps can be reduced to obtain relatively more functional group substitutions; microwave assistance is more efficient and convenient; the ionic liquid [BMIM][PF 6 as the reaction solvent can avoid the volatilization of organic reaction solvents, is more environmentally friendly, and the operation safety is also improved.

[0062] The embodiments described above are only descriptions of the preferred modes of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A compound with anti-lung cancer activity, characterized in that, its structural formula is as shown in Formula I; 2. A preparation method of the compound with anti-lung cancer activity according to claim 1, characterized in that, it comprises the following steps: Using an ionic liquid as a solvent, and using Z-2-chloromethyl-3-phenylacrylic acid, 3-p-chlorophenylacrolein, benzylamine and cyclohexyl isocyanide as raw materials, through a microwave-assisted one-pot reaction, and dropping a basic solution during the reaction to obtain the compound with anti-lung cancer activity.

3. According to the preparation method described in claim 2, characterized in that, it comprises the following steps: Dissolve Z-2-chloromethyl-3-phenylacrylic acid, 3-p-chlorophenylacrolein, benzylamine and cyclohexyl isocyanide in an ionic liquid and stir for 15 - 45 min, then carry out a microwave-assisted reaction for 16 - 24 min, and then add the basic solution to the reaction system in 5 - 10 portions. After the addition is completed, continue the microwave-assisted reaction until the reaction is complete. Then freeze the reaction solution. After the solid precipitates, filter by suction, wash with water, and recrystallize to obtain the compound with anti-lung cancer activity; During the microwave-assisted reaction process, set the power to 300 W, heat for 8 seconds, stop for 8 seconds, and repeat the steps of heating and stopping until the reaction ends; The time interval between two adjacent additions of the basic aqueous solution is 96 s, and a microwave-assisted reaction is carried out during the interval.

4. According to the preparation method described in claim 2 or 3, characterized in that, the molar ratio of Z-2-chloromethyl-3-phenylacrylic acid, 3-p-chlorophenylacrolein, benzylamine and cyclohexyl isocyanide is 1:1:1:

1.

5. According to the preparation method described in claim 2 or 3, characterized in that, The molar volume ratio of the Z-2-chloromethyl-3-phenylacrylic acid to the ionic liquid is 1 mmol: 2-10 mL; the ionic liquid is [BMIM][PF 6 .

6. According to the preparation method described in claim 2 or 3, characterized in that, the basic solution is an aqueous solution of potassium carbonate or an aqueous solution of sodium carbonate; the mass concentration of the basic solution is 10 - 30 wt%; the total dropwise addition amount of potassium carbonate or sodium carbonate in the basic solution and the molar ratio of Z-2-chloromethyl-3-phenylacrylic acid is 0.5:

1.

7. An application of the compound with anti-lung cancer activity according to claim 1 in the preparation of anti-lung cancer drugs.

8. An anti-lung cancer pharmaceutical composition, characterized in that, it contains the compound with anti-lung cancer activity according to claim 1 or a pharmaceutically acceptable salt thereof.

9. According to the anti-lung cancer pharmaceutical composition described in claim 8, characterized in that, by mass percentage, the compound with anti-lung cancer activity is 0.01% - 5%, and pharmaceutically acceptable excipients are 95% - 99.99%.

10. An anti-lung cancer freeze-dried powder injection, characterized in that, by mass percentage, the compound with anti-lung cancer activity according to claim 1 is 0.01% - 5%, and pharmaceutically acceptable excipients are 95% - 99.99%.

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