A tetrachloroperylene diimide compound PDIC-NLC, a preparation method and application thereof

Abstract

This invention provides a tetrachloroperylene diimide compound PDIC-NLC, which is a compound with tetrachloroperylene diimide as the core skeleton and 4-(dimethylaminomethyl)phenyl substituents attached to both imide positions. This invention also provides a method for preparing PDIC-NLC, which involves reacting 1,6,7,12-tetrachloro-3,4,9,10-perylenetetracarboxylic dianhydride with 4-(dimethylaminomethyl)aniline in an inert gas atmosphere. After the reaction, the product is obtained by hydrochloric acid treatment, precipitation with anhydrous ethanol, and column chromatography purification. This invention also provides the application of PDIC-NLC in drugs that inhibit tumor cell proliferation. This invention provides a structurally well-defined tetrachloroperylene diimide compound PDIC-NLC and a feasible preparation method.

Description

Technical Field

[0001] This invention belongs to the field of organic compound synthesis and bioactive molecule technology, specifically relating to a tetrachloroperylene diimide compound PDIC-NLC, its preparation method, and its application. Background Technology

[0002] Perylene diimides possess favorable conjugated structural features, optical response properties, and chemical stability, thus attracting attention in the field of functional molecule design. By substituting the imide sites of the perylene diimide skeleton, derivatives with well-defined structures and tunable properties can be obtained. Current research on tetrachloroperylene diimides largely focuses on core modification and performance evaluation; however, systematic disclosure is still lacking regarding the structure, preparation methods, and applications in inhibiting tumor cell proliferation of PDIC-NLCs with 4-(dimethylaminomethyl)phenyl substituents at both imide sites.

[0003] Therefore, it is necessary to provide a tetrachloroperylene diimide compound PDIC-NLC with a well-defined structure, an feasible preparation method, and an inhibitory effect on tumor cell proliferation, as well as its preparation method. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the lack of disclosure in the prior art regarding the structure, preparation method and application of tetrachloroperylene diimide compound PDIC-NLC in tumor cell proliferation, and to provide a tetrachloroperylene diimide compound PDIC-NLC, its preparation method and application.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a tetrachloroperylene diimide compound PDIC-NLC, with the following structural formula:

[0006] .

[0007] Preferably, the tetrachloroperylene diimide compound PDIC-NLC is a compound with tetrachloroperylene diimide as the core skeleton and 4-(dimethylaminomethyl)phenyl substituents attached to both imide positions.

[0008] This invention also provides a method for preparing the above-mentioned tetrachloroperylene diimide compound PDIC-NLC, the preparation method comprising the following steps:

[0009] S1. 1,6,7,12-tetrachloro-3,4,9,10-perylenetetracarboxylic acid dianhydride and 4-(dimethylaminomethyl)aniline are added to the reaction system and reacted under inert gas protection to obtain a reaction solution.

[0010] S2. Add hydrochloric acid to the reaction solution obtained in S1 and stir to obtain a mixture;

[0011] S3. Add anhydrous ethanol to the mixture obtained in S2 while stirring. After standing overnight, filter and dry under vacuum to obtain a crude product containing salt. Purify the crude product containing salt by column chromatography to obtain tetrachloroperylene diimide compound PDIC-NLC.

[0012] Preferably, the reaction system in S1 is NMP / glacial acetic acid, and the volume ratio of NMP to glacial acetic acid in the reaction system is 4:5; the inert gas is N2; the reaction temperature is 120 °C and the reaction time is 24 h.

[0013] Preferably, the mass-to-volume ratio of 1,6,7,12-tetrachloro-3,4,9,10-perylenetetracarboxylic acid dianhydride, 4-(dimethylaminomethyl)aniline, and the reaction system is 200 mg: 0.6 mL: 18 mL.

[0014] Preferably, the concentration of hydrochloric acid in S2 is 2 mol / L, the volume ratio of the reaction solution to the hydrochloric acid is 6:5, and the stirring time is 20 min.

[0015] Preferably, the volume ratio of the mixture and anhydrous ethanol in S3 is 1:8; the vacuum drying temperature is 50-80 °C and the time is 1-12 h; the column chromatography purification uses CH2Cl2 / CH3OH as the eluent, and the volume ratio of CH2Cl2 to CH3OH is 8:1.

[0016] The present invention also provides the application of the above-mentioned tetrachloroperylene diimide compound PDIC-NLC in inhibiting tumor cell proliferation.

[0017] Preferably, the tumor cells are lung cancer cells.

[0018] Preferably, the lung cancer cells are A549 cells.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] 1. This invention provides a structurally well-defined tetrachloroperylene diimide compound PDIC-NLC and gives an implementable preparation method.

[0021] 2. Under the experimental conditions of 72 h in A549 cells, the IC50 of PDIC-NLC in this invention... 50 The effective concentration was 11.07 μM, which was superior to the comparative compounds PDIC-NFC (18.13 μM) and PDIC-NOC (17.80 μM).

[0022] 3. The experimental results in the embodiments of the present invention show that PDIC-NLC treatment is accompanied by changes in reactive oxygen species levels, Annexin V / 7-AAD clustering, and changes in Cleaved Caspase-1 and GSDMD-N related protein bands. The above results suggest that the treatment is accompanied by changes in cell state and can be used as supplementary technical effect data.

[0023] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the synthesis routes of PDIC-NLC obtained in Example 1 of the present invention, PDIC-NOC obtained in Comparative Example 1, and PDIC-NFC obtained in Comparative Example 2.

[0025] Figure 2 This is the mass spectrum of PDIC-NOC obtained in Comparative Example 1 of this invention.

[0026] Figure 3 This is the 1H NMR spectrum of PDIC-NOC obtained in Comparative Example 1 of this invention.

[0027] Figure 4 This is a graph showing the ultraviolet absorption and fluorescence emission results of PDIC-NOC obtained in Comparative Example 1 of this invention.

[0028] Figure 5 This is the mass spectrum of PDIC-NFC obtained in Comparative Example 2 of this invention.

[0029] Figure 6 This is the 1H NMR spectrum of the PDIC-NFC obtained in Comparative Example 2 of this invention.

[0030] Figure 7 This is a graph showing the ultraviolet absorption and fluorescence emission results of the PDIC-NFC obtained in Comparative Example 2 of this invention.

[0031] Figure 8 This is the mass spectrum of the PDIC-NLC obtained in Example 1 of the present invention.

[0032] Figure 9 This is the 1H NMR spectrum of the PDIC-NLC obtained in Example 1 of this invention.

[0033] Figure 10 This is a graph showing the ultraviolet absorption and fluorescence emission results of the PDIC-NLC obtained in Example 1 of this invention.

[0034] Figure 11The proliferation inhibition curves and IC50 values ​​of PDIC-NOC obtained in Comparative Example 1, PDIC-NFC obtained in Comparative Example 2, and PDIC-NLC obtained in Example 1 under A549 (72 h) conditions are shown. 50 Result image.

[0035] Figure 12 The figures show the clone formation results of PDIC-NOC obtained in Comparative Example 1, PDIC-NFC obtained in Comparative Example 2, and PDIC-NLC obtained in Example 1 at different concentrations.

[0036] Figure 13 This is a graph showing the time-dependent uptake results of PDIC-NLC cells obtained in Example 1 of this invention.

[0037] Figure 14 This is a graph showing the changes in reactive oxygen species after PDIC-NLC treatment obtained in Example 1 of this invention.

[0038] Figure 15 This is a flow cytometry result of Annexin V / 7-AAD after PDIC-NLC processing obtained in Embodiment 1 of the present invention.

[0039] Figure 16 This is a WB result image obtained after PDIC-NLC processing in Embodiment 1 of the present invention. Detailed Implementation

[0040] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments and comparative examples. The described embodiments are used to illustrate the present invention, and the comparative examples are used to illustrate the preparation and effect comparison of the comparative compounds, and do not imply that the present invention seeks protection for PDIC-NOC or PDIC-NFC.

[0041] Example 1

[0042] This embodiment provides a method for preparing the tetrachloroperylene diimide compound PDIC-NLC, including the following steps:

[0043] S1. 200 mg of 1,6,7,12-tetrachloro-3,4,9,10-perylenetetracarboxylic acid dianhydride, 0.6 mL of 4-(dimethylaminomethyl)aniline, 10 mL of glacial acetic acid and 8 mL of N-methylpyrrolidone (NMP) were added sequentially to a three-necked flask. The mixture was reacted at 120 °C for 24 h under N2 protection. After cooling to room temperature (24 °C), the reaction solution was obtained.

[0044] S2. Add 15 mL of 2 mol / L hydrochloric acid (HCl) to 18 mL of the reaction solution obtained in S1 and stir for 20 min to obtain a mixed solution;

[0045] S3. Add 250 mL of anhydrous ethanol to the mixture obtained in S2 while stirring for 1 h. After standing overnight, filter and then vacuum dry at 60 ℃ for 10 h to obtain a crude product containing salt. Purify the crude product containing salt by column chromatography using CH2Cl2 / CH3OH (V / V = 8:1) as the eluent and vacuum dry at 60 ℃ for 10 h to obtain tetrachloroperylene diimide compound PDIC-NLC, hereinafter referred to as PDIC-NLC, with a yield of 71.60%.

[0046] Figure 8 This is the mass spectrum of the PDIC-NLC obtained in this embodiment. Figure 9 This is the 1H NMR spectrum of the PDIC-NLC obtained in this embodiment. Figure 10 This is a graph showing the ultraviolet absorption and fluorescence emission results of the PDIC-NLC obtained in this embodiment. Figure 8-10 The characterization data are as follows:

[0047] 1H NMR (400 MHz, CF3COOD): δ 9.26 (s, 4H), 8.26–7.90 (m, 8H), 4.90 (s, 4H), 3.49 (s, 12H);

[0048] ESI-MS: m / z calcd 796.52; found 398.2 [(M 2+ / 2)];

[0049] The maximum ultraviolet absorption wavelength is 521 nm, and the maximum fluorescence emission wavelength is 562 nm.

[0050] Example 2

[0051] This embodiment provides the application of the tetrachloroperylene diimide compound PDIC-NLC in a drug for inhibiting tumor cell proliferation.

[0052] Comparative Example 1

[0053] This comparative example provides a method for preparing the comparative compound PDIC-NOC, which includes the following steps:

[0054] In a 100 mL dry three-necked flask, 0.3000 g (0.57 mmol) of 1,6,7,12-tetrachloro-3,4,9,10-perylenetetracarboxylic acid dianhydride and 30 mL of NMP were added sequentially, and the mixture was stirred at room temperature (24 °C) for 1 h. Subsequently, 0.24 mL (1.14 mmol) of N,N-dimethyl-p-phenylenediamine and 3.01 mL (52.6 mmol) of glacial acetic acid were added, and the mixture was heated to 120 °C under N2 protection and reacted for 24 h.

[0055] After the reaction was completed, the mixture was cooled to room temperature (24 °C), stirred for 2 h, treated with 2 mol / L HCl, and reacted for another 24 h at room temperature (24 °C). Then, 250 mL of anhydrous ethanol was added, and the mixture was allowed to stand overnight. The mixture was filtered and dried under vacuum to obtain crude hydrochloride. The crude hydrochloride was purified by column chromatography using CH2Cl2 as the eluent and dried under vacuum at 60 °C for 10 h to obtain the final target compound PDIC-NOC with a yield of 74.20%, which was a dark red solid.

[0056] Figure 2 This is the mass spectrum of PDIC-NOC obtained in this comparative example. Figure 3 This is the 1H NMR spectrum of PDIC-NOC obtained in this comparative example. Figure 4 This is a graph showing the UV absorption and fluorescence emission results of PDIC-NOC obtained in this comparative example. Figure 2-4 The characterization data are as follows:

[0057] 1H NMR (400 MHz, CF3COOD): δ 9.25 (d, J = 3.0 Hz, 4H), 8.36–8.06 (m,8H), 3.92 (d, J = 3.0 Hz, 12H);

[0058] ESI-MS: m / z calcd 768.47; found 384.1 [(M 2+ / 2)];

[0059] The maximum ultraviolet absorption wavelength is 520 nm, and the maximum fluorescence emission wavelength is 556 nm.

[0060] Comparative Example 2

[0061] This comparative example provides a method for preparing the comparative compound PDIC-NFC, which includes the following steps:

[0062] 200 mg (0.4 mmol) of 1,6,7,12-tetrachloro-3,4,9,10-perylenetetracarboxylic acid dianhydride, 0.6 mL (4.0 mmol) of 4-(dimethylamino)benzylamine, 10 mL of glacial acetic acid, and 8 mL of NMP were added sequentially to a three-necked flask, and the mixture was reacted at 120 °C for 24 h under N2 protection.

[0063] After the reaction was completed, the mixture was cooled to room temperature (24 °C), and 15 mL of 2 mol / L HCl was added and stirred for 20 min. Then, 250 mL of anhydrous ethanol was added while stirring, and the mixture was allowed to stand overnight. The mixture was then filtered and dried under vacuum to obtain crude hydrochloride. The crude hydrochloride was purified by column chromatography using CH2Cl2 / CH3OH (V / V = 8:1) as the eluent and dried under vacuum at 60 °C for 10 h to obtain the final target compound PDIC-NFC, with a yield of 73.91%.

[0064] Figure 5 This is the mass spectrum of PDIC-NFC obtained in this comparative example. Figure 6 This is the 1H NMR spectrum of the PDIC-NFC obtained in this comparative example. Figure 7 This is a diagram showing the ultraviolet absorption and fluorescence emission results of the PDIC-NFC obtained in this comparative example. Figure 5-7 The characterization data are as follows:

[0065] 1H NMR (400 MHz, CF3COOD): δ 9.20 (s, 4H), 8.06 (dd, J = 87.2, 8.2 Hz, 8H), 5.99 (s, 4H), 3.78 (s, 12H);

[0066] ESI-MS: m / z calcd 796.52; found 795.4755 [M 2+ ];

[0067] The maximum ultraviolet absorption wavelength is 522 nm, and the maximum fluorescence emission wavelength is 564 nm.

[0068] Experiment 1: A549 Cell Proliferation Inhibition Experiment

[0069] A549 cells were seeded in a culture system. After the cells reached a suitable state for detection, different concentrations of PDIC-NLC, the comparative compound PDIC-NFC, and the comparative compound PDIC-NOC were added, with a negative control group included. The cells were treated under the specified culture conditions for 72 h. After treatment, changes in cell viability were detected using cell viability assays, and the IC50 was calculated based on the concentration-response curve. 50 .

[0070] The results are as follows Figure 11 As shown, the results indicate that PDIC-NLC, the comparative compound PDIC-NFC, and the comparative compound PDIC-NOC have different IC50 values ​​in A549 cells under 72-h experimental conditions. 50 The effective concentrations were 11.07 μM, 18.13 μM, and 17.80 μM, respectively. This indicates that PDIC-NLC has a better inhibitory effect on A549 cell proliferation under these experimental conditions, and is superior to the aforementioned comparative compounds.

[0071] Experiment 2: Cloning Experiment

[0072] Cloning experiments were conducted on PDIC-NLC, the comparative compound PDIC-NFC, and the comparative compound PDIC-NOC under negative control, 0.01 μM, 0.1 μM, 1 μM, 10 μM, and 100 μM conditions. The experimental results are as follows: Figure 12 As shown in the figure, the colony formation of all three compound treatment groups decreased with increasing treatment concentration, with PDIC-NLC showing a more significant change at lower concentrations.

[0073] The above results are consistent with the trend of the A549 cell proliferation inhibition experiment and can be used as supplementary effect data.

[0074] Experiment 3: Experiment on Changes in Reactive Oxygen Species

[0075] Reactive oxygen species (ROS) were detected in the PDIC-NLC treatment group and the negative control group. Results are as follows: Figure 14 As shown in the figure, the relative fluorescence intensity of the treatment group was higher than that of the negative control group, with an average value of 1.000000 and an average value of 2.089342 for the treatment group. This result indicates that PDIC-NLC treatment is accompanied by changes in reactive oxygen species levels.

[0076] Experiment 4: Annexin V / 7-AAD Detection

[0077] The PDIC-NLC treatment group and the negative control group were analyzed by Annexin V / 7-AAD flow cytometry. Results are as follows: Figure 15 As shown in the figure, the proportions in quadrants Q1, Q2, Q3, and Q4 of the negative control group were 1.99, 0.82, 0.26, and 96.9, respectively; while the proportions in quadrants Q1, Q2, Q3, and Q4 of the PDIC-NLC treatment group were 51.6, 25.7, 0.62, and 22.1, respectively. These results indicate that PDIC-NLC treatment is accompanied by changes in cell population proportions, which can serve as supplementary data on the technical efficacy.

[0078] Experiment 5: Experiment on Changes in Related Protein Bands

[0079] Western blot analysis was used to detect changes in relevant protein bands in PDIC-NLC under treatment conditions of 0 μM, 10 μM, 20 μM, and 30 μM. The results are as follows: Figure 16 As shown in the figure, with increasing PDIC-NLC treatment concentration, the bands of Cleaved Caspase-1 and GSDMD-N showed an increasing trend, while the internal control ACTIN band remained relatively stable overall. This result indicates that PDIC-NLC treatment is accompanied by changes in related protein bands and can serve as supplementary data on the effectiveness of the technique.

[0080] Experiment 6: Cellular Uptake Time-Dependent Experiment

[0081] Time-dependent uptake of PDIC-NLC was detected. PDIC-NLC was added to the cell system at a concentration of 10 μM, and the changes in PE-A channel fluorescence intensity were detected at different time points. The fluorescence intensity of the PE-A channel was used as the detection index. The results are as follows: Figure 13 As shown in the figure, the average fluorescence intensities at 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, and 8 h were 8300.8, 543949.3667, 859021.7333, 1174844.733, 1194204.7, 1234438.433, and 1281762.467, respectively. These results indicate that the cellular uptake signal of PDIC-NLC generally increases over time.

[0082] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A tetrachloroperylene diimide compound PDIC-NLC, characterized in that, The structural formula is: 。 2. The tetrachloroperylene diimide compound PDIC-NLC according to claim 1, characterized in that, The tetrachloroperylene diimide compound PDIC-NLC is a compound with tetrachloroperylene diimide as the core skeleton and 4-(dimethylaminomethyl)phenyl substituents attached to both imide positions.

3. A method for preparing the tetrachloroperylene diimide compound PDIC-NLC as described in claim 1, characterized in that, The preparation method includes the following steps: S1. 1,6,7,12-tetrachloro-3,4,9,10-perylenetetracarboxylic acid dianhydride and 4-(dimethylaminomethyl)aniline are added to the reaction system and reacted under inert gas protection to obtain a reaction solution. S2. Add hydrochloric acid to the reaction solution obtained in S1 and stir to obtain a mixture; S3. Add anhydrous ethanol to the mixture obtained in S2 while stirring. After standing overnight, filter and dry under vacuum to obtain a crude product containing salt. Purify the crude product containing salt by column chromatography to obtain tetrachloroperylene diimide compound PDIC-NLC.

4. The method for preparing the tetrachloroperylene diimide compound PDIC-NLC according to claim 3, characterized in that, The reaction system described in S1 is NMP / glacial acetic acid, with a volume ratio of 4:5 between NMP and glacial acetic acid; the inert gas is N2; the reaction temperature is 120 °C, and the reaction time is 24 h.

5. The method for preparing a tetrachloroperylene diimide compound PDIC-NLC according to claim 4, characterized in that, The mass-to-volume ratio of 1,6,7,12-tetrachloro-3,4,9,10-perylenetetracarboxylic acid dianhydride, 4-(dimethylaminomethyl)aniline, and the reaction system is 200 mg: 0.6 mL: 18 mL.

6. The method for preparing the tetrachloroperylene diimide compound PDIC-NLC according to claim 3, characterized in that, The concentration of hydrochloric acid in S2 is 2 mol / L, the volume ratio of the reaction solution to the hydrochloric acid is 6:5, and the stirring time is 20 min.

7. The method for preparing the tetrachloroperylene diimide compound PDIC-NLC according to claim 3, characterized in that, The volume ratio of the mixture to anhydrous ethanol in S3 is 1:8; the vacuum drying temperature is 50-80 ℃ and the time is 1-12 h; the column chromatography purification uses CH2Cl2 / CH3OH as the eluent, and the volume ratio of CH2Cl2 to CH3OH is 8:

1.

8. The use of the tetrachloroperylene diimide compound PDIC-NLC as described in claim 1 in inhibiting tumor cell proliferation.

9. The application of the tetrachloroperylene diimide compound PDIC-NLC according to claim 8 in inhibiting tumor cell proliferation, characterized in that, The tumor cells are lung cancer cells.

10. The application of the tetrachloroperylene diimide compound PDIC-NLC according to claim 9 in inhibiting tumor cell proliferation, characterized in that, The lung cancer cells were A549 cells.

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