Application of ionized ring-opened cucurbituril in improving light stability of colchicine

Abstract

The invention discloses an application of ionized ring-opened cucurbituril in improving the light stability of colchicine, and particularly relates to an inclusion compound prepared by carrying out inclusion reaction on colchicine and ionized ring-opened cucurbituril, so as to achieve the purpose of improving the light stability of colchicine. The invention aims to solve the technical problems that colchicine is poor in light stability and easy to degrade in storage and application. By forming a stable host-guest clathrate compound, the light stability of colchicine is remarkably improved, and activity reduction caused by illumination degradation of colchicine is effectively avoided; meanwhile, the clathrate compound can well retain the original anti-tumor biological activity of colchicine, and the clathrate compound prepared by the invention can be used as a stable active pharmaceutical ingredient, is used for preparing an anti-tumor pharmaceutical composition, and has an important application prospect in the field of pharmacy.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology, and specifically relates to a method for improving the photostability of colchicine. Background Technology

[0002] Colchicine is an important alkaloid widely used clinically due to its significant anti-inflammatory and anti-proliferative activities. For example, as a highly effective anti-gout drug, it works by reducing the levels of inflammatory factors such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). In the treatment of cardiovascular diseases, it protects the myocardium by reducing inflammatory responses. Most importantly, colchicine has a significant inhibitory effect on cell mitosis and can block the life cycle of cancer cells by inhibiting microtubule polymerization, demonstrating great potential for application in the field of anti-tumor therapy.

[0003] However, the molecular structure of colchicine makes it sensitive to light and exhibits poor photostability. It is prone to photodegradation during storage, transportation, and clinical use, leading to decreased drug content, reduced efficacy, and potentially the formation of unknown degradation products, posing safety risks. This inherent defect severely limits its dosage form development and wider clinical application. Therefore, improving the photostability of colchicine while maintaining its inherent biological activity is a pressing technical problem to be solved in this field.

[0004] To improve the stability, solubility, and other physicochemical properties of drug molecules, inclusion technology in supramolecular host-guest chemistry provides an effective strategy. This technology involves encapsulating the drug guest molecule within the cavity of the host molecule to form a stable inclusion complex, thereby providing physical shielding and protection for the guest molecule. Cucurbituril (CB[n]) is a class of high-performance rigid macrocyclic host molecules, and its binding ability to guests is usually much higher than that of traditional cyclodextrins. However, traditional cucurbituril (such as CB[5], CB[6], CB[7]) has extremely low solubility in water, which greatly limits its practical application in the biomedical field.

[0005] In recent years, open-ring cucurbitacins, as a novel class of cucurbitacin derivatives, have offered new possibilities for solving the aforementioned problems. Unlike traditional cucurbitacins, open-ring cucurbitacins possess a flexible C-shaped cavity with adjustable size, capable of accommodating a wider range of guest molecules with different structures. The aromatic ring sidewalls at both ends can also enhance binding with guests through π-π interactions. More importantly, by introducing ionizing groups (such as sulfonic acid groups) onto the aromatic ring, highly water-soluble ionized open-ring cucurbitacins can be obtained. These host molecules combine high water solubility, strong inclusion capacity, and good biocompatibility, making them ideal carriers for constructing novel drug delivery systems.

[0006] A search revealed several existing technologies related to colchicine. For example, Chinese patent CN1533765A discloses a colchicine microsphere lyophilized agent prepared via physical encapsulation, but it does not address the photostability issue at the molecular level through host-guest chemistry. Chinese patent CN111954524A and US patent US2024099999A1 disclose novel uses of colchicine in inhibiting tumor growth and metastasis, further confirming the importance of developing stable colchicine formulations, but they themselves do not provide technical solutions to address stability issues.

[0007] In conclusion, although the medicinal value of colchicine, especially its anti-tumor value, is widely recognized, its poor photostability remains a bottleneck restricting its development. Currently, there are no publicly available reports of using ionized open-ring cucurbituril as the main molecule to include colchicine, thereby enhancing its photostability at the molecular level while maintaining its anticancer activity. Summary of the Invention

[0008] The present invention aims to solve the technical problem that colchicine has poor photostability due to inherent defects in its molecular structure, and is easily degraded during storage and use, thus limiting its clinical application (especially in the field of anti-tumor). The present invention provides a method that can effectively protect colchicine, significantly improve its photostability, and at the same time not affect its biological activity.

[0009] This invention involves mixing colchicine with ionized ring-opening cucurbita to undergo an inclusion reaction to obtain an inclusion complex, thereby improving the photostability of colchicine. The inclusion complex is a stable supramolecular complex formed by non-covalent interactions, with ionized ring-opening cucurbita as the host molecule and colchicine as the guest molecule encapsulated within its cavity. The structural formula of the ionized ring-open cucurbituril is selected from the following: (I) or (II); In the formula, R = (CH2) n SO3A, where A = Na, H; n = 1 to 8.

[0010] The structural formula of colchicine is as follows:

[0011] Ionized ring-opening cucurbita and colchicine were mixed in a solvent at a molar ratio of 1:1 to 10 and stirred at 0℃-90℃ for 1-72 hours. The solid and liquid were separated, and the filtrate was dried to obtain the inclusion complex.

[0012] The drying method is freeze drying, vacuum concentration drying, or spray drying, and the solvent is selected from one or more of water, methanol, ethanol, petroleum ether, ethyl acetate, dimethyl sulfoxide, N,N-dimethylformamide, dichloromethane, tetrahydrofuran, and acetone.

[0013] Another object of the present invention relates to the use of the inclusion complex in the preparation of antitumor pharmaceutical compositions with enhanced photostability.

[0014] Compared with the prior art, the present invention has the following significant advantages: (1) Significantly improves the photostability of colchicine: This invention encapsulates colchicine molecules through the C-shaped flexible cavity of ionized ring-opening cucurbituril, forming a physical shield that effectively slows down the degradation rate of colchicine under natural light and ultraviolet light irradiation. Experiments have shown that the encapsulation complex formed can significantly improve the photostability of colchicine while retaining its original anticancer activity, thus solving a key bottleneck in its application. (2) The preparation method of the inclusion complex is simple and efficient and easy to industrialize: The preparation method of the present invention is simple to operate, the reaction conditions are mild (it can be carried out at room temperature and pressure), no harsh reaction environment or expensive catalyst is required, the whole process is safe and efficient, the product is easy to separate and purify, the yield is high, the quality is excellent, and it has the potential for large-scale industrial production. (3) Expanding the application prospects of colchicine: By solving the fundamental problem of poor photostability, this invention lays a solid foundation for the development of novel and stable colchicine drug formulations (such as injections, oral solutions, sustained-release formulations, etc.). This not only improves the quality controllability and storage period of the drug, but also has the potential to expand its clinical application value in the treatment of major diseases such as tumors, and has broad market prospects. Attached Figure Description

[0015] Figure 1 Comparative analysis of the proton NMR spectra of the main I-Col inclusion complex (R=(CH2)3SO3H): (a) colchicine; (b) main I-Col inclusion complex (R=(CH2)3SO3H); (c) open-ring cucurbituril I (R=(CH2)3SO3H); Figure 2 2D-ROESY spectrum of the main component I-Col inclusion complex (R=(CH2)3SO3H); Figure 3 Molecular docking diagram of the host I-Col inclusion complex (R=(CH2)3SO3Na); Figure 4X-ray diffraction patterns of the main component I-Col inclusion complex (R=(CH2)5SO3Na); (a) open-ring cucurbita I (R=(CH2)5SO3Na); (b) colchicine; (c) physical mixture of open-ring cucurbita I (R=(CH2)5SO3Na) and colchicine; (d) main component I-Col inclusion complex (R=(CH2)5SO3Na). Figure 5 Comparative infrared spectral analysis of the main component I-Col inclusion complex (R=(CH2)8SO3Na); (a) open-ring cucurbituril I (R=(CH2)8SO3Na); (b) colchicine; (c) physical mixture of open-ring cucurbituril (R=(CH2)8SO3Na) and colchicine; (d) main component I-Col inclusion complex (R=(CH2)8SO3Na); Figure 6 Comparative analysis of 1H NMR spectra of the main II-Col inclusion complex (R=(CH2)1SO3H): (a) colchicine; (b) main II-Col inclusion complex (R=(CH2)1SO3H); (c) ionized ring-opening cucurbituril II (R=(CH2)1SO3H); Figure 7 2D-ROESY spectrum of the main II-Col inclusion complex (R=(CH2)1SO3H); Figure 8 X-ray diffraction patterns of the main body II-Col inclusion complex (R=(CH2)3SO3H); (a) open-ring cucurbituril II (R=(CH2)3SO3H); (b) colchicine; (c) physical mixture of open-ring cucurbituril II (R=(CH2)3SO3H) and colchicine; (d) main body II-Col inclusion complex (R=(CH2)3SO3H). Figure 9 Molecular docking diagram of the main II-Col inclusion complex (R=(CH2)4SO3H); Figure 10 Comparative infrared spectral analysis of the main body II-Col inclusion complex (R=(CH2)7SO3Na); (a) open-ring cucurbituril II (R=(CH2)7SO3Na); (b) colchicine; (c) physical mixture of open-ring cucurbituril II (R=(CH2)7SO3Na) and colchicine; (d) main body II-Col inclusion complex (R=(CH2)7SO3Na); Figure 11Figure 1 shows a comparison of the colchicine retention rates of the inclusion complexes in Examples 1 or 2 under natural light irradiation; Figure 2 shows a comparison of the inclusion complexes with n=1; Figure 3 shows a comparison of the inclusion complexes with n=3; Figure 4 shows a comparison of the inclusion complexes with n=4; in the figures, a represents colchicine; b represents the main body I inclusion complex of Example 1; and c represents the main body II inclusion complex of Example 2. Figure 12 Figure 1 shows a comparison of the colchicine retention rates of the inclusion complexes in Examples 1 or 2 under natural light irradiation; Figure 2 shows a comparison of the inclusion complexes with n=5; Figure 3 shows a comparison of the inclusion complexes with n=7; Figure 4 shows a comparison of the inclusion complexes with n=8; In the figures, a represents colchicine; b represents the main body I inclusion complex of Example 1; and c represents the main body II inclusion complex of Example 2. Figure 13 Figure 1 shows a comparison of the colchicine retention rates of the inclusion complexes in Examples 1 or 2 under ultraviolet light irradiation; Figure 2 shows a comparison of the inclusion complexes with n=5; Figure 3 shows a comparison of the inclusion complexes with n=7; Figure 4 shows a comparison of the inclusion complexes with n=8; In the figures, a represents colchicine; b represents the main body I inclusion complex of Example 1; and c represents the main body II inclusion complex of Example 2. Figure 14 The diagram shows the comparative analysis of the anticancer activity of inclusion complexes; Figure (1) shows HeLa cells and Figure (2) shows HCT116 cells; in the figure, (a) is colchicine; (b) is the main body I-Col inclusion complex (R=(CH2)7SO3H); and (c) is the main body II-Col inclusion complex (R=(CH2)2SO3Na). Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0017] In the following examples, Subject I refers to the ionized open-ring cucurbita shown in structural formula I; Subject II refers to the ionized open-ring cucurbita shown in structural formula II; Guest Col refers to colchicine; Subject I-Col inclusion complex or Subject II-Col inclusion complex refers to the corresponding inclusion complex product.

[0018] Example 1: Preparation of the main component I-Col inclusion complex 1. Weigh 1.428 g (1 mmol) of host I (R=(CH2)1SO3H) and dissolve it in 15 mL of water. Separately, dissolve 2.394 g (6 mmol) of guest Col in 10 mL of methanol and add it dropwise to the host I solution. Stir the mixture vigorously at 80 °C for 48 hours. After filtration, concentrate the filtrate under reduced pressure and dry it to obtain host I-Col inclusion complex (R=(CH2)1SO3H) with a yield of 71.75%. 2. The preparation method of the main component I-Col inclusion complex (R=(CH2)2SO3Na) is the same as in step 1, with a product yield of 73.89%; 3. Weigh 1.453 g (1 mmol) of host I (R=(CH2)3SO3H) solid powder, add it to 15 mL of pure water, heat and stir at 30 °C for 30 minutes until completely dissolved, stop heating, and allow the solution to cool to room temperature (about 25 °C). Add 0.798 g (2 mmol) of guest Col powder to the cooled solution, and stir vigorously at 25 °C for 48 hours. Filter to remove unreacted guest Col and other insoluble substances, and freeze-dry the filtrate to obtain a white powdery host I-Col inclusion complex (R=(CH2)3SO3H), with a yield of 72.9%. The NMR characterization results are as follows: Figure 1 As shown, compared with the free guest Col, the proton signals (e.g., H-2, H-3, H-4, H-11) of the guest Col in the host I-Col inclusion complex exhibit significant chemical shift changes, confirming a strong interaction between host I and guest Col. Further analysis was performed using two-dimensional rotating frame nuclear Overhauser effect spectroscopy (2D-ROESY) (e.g., Figure 2 As shown in the figure, significant spatial correlation signals were observed between the H-1, H-2, H-3, H-4, and H-7 protons on the guest Col and the Ha, Hb, and Hh protons inside the cavity of the host I. This strongly confirms that the guest Col molecule has entered the cavity of the host I and formed a host-guest inclusion complex.

[0019] 1.541 g (1 mmol) of host I (R=(CH2)3SO3Na) solid powder was weighed and added to 15 mL of pure water. The mixture was heated and stirred at 50 °C for 45 minutes until completely dissolved. After cooling to room temperature, 1.995 g (5 mmol) of guest Col powder was added, and the mixture was stirred vigorously at 20 °C for 36 hours. Insoluble matter was removed by filtration. The filtrate was concentrated under reduced pressure and dried to obtain host I-Col inclusion complex (R=(CH2)3SO3Na), with a yield of 78.3%. The binding mode between host I (R=(CH2)3SO3Na) and guest Col was predicted using computer molecular docking simulation. The results are as follows: Figure 3The simulation results show that the host I and the guest Col tend to form a stable inclusion complex with a molar ratio of 1:1; the calculated binding energy is negative, indicating that the inclusion process is thermodynamically favorable and the two can spontaneously form a stable host-guest complex.

[0020] 4. The preparation method of the main component I-Col inclusion complex (R=(CH2)4O3Na) is the same as in step 1, and the product yield is 70.64%.

[0021] 5. The method is the same as step 3, except that main component I (R=(CH2)5SO3Na) is used, the molar ratio is 1:1, the reaction is carried out at 20℃ for 24 h, and then dried under reduced pressure, with a yield of 82.7%; X-ray diffraction (XRD) analysis of main component I-Col inclusion complex (R=(CH2)5SO3Na) ( Figure 4 The spectrum of the main component I-Col inclusion complex (R=(CH2)5SO3Na) shows amorphous diffuse peaks. Figure 4 d), while the sharp characteristic diffraction peaks of the raw material I and the guest Col disappeared, confirming the formation of a new amorphous substance.

[0022] 6. The preparation method of the main component I-Col inclusion complex (R=(CH2)6SO3Na) is the same as in step 1, and the product yield is 69.16%.

[0023] 7. The preparation method of the main component I-Col inclusion complex (R=(CH2)7SO3H) is the same as in step 1, and the product yield is 64.43%.

[0024] 8. The method is the same as step 3, except that main component I (R=(CH2)8SO3Na) is used, the molar ratio is 1:1, the reaction is carried out at 90℃ for 72 h, and then dried under reduced pressure. The yield is 88.9%. Fourier transform infrared spectroscopy (FT-IR) analysis of main component I-Col inclusion complex (R=(CH2)8SO3Na) is performed. Figure 5 The results show that the physical mixture ( Figure 5 c) is merely a simple superposition of the raw material spectra, while the inclusion complex ( Figure 5 In d), the characteristic absorption peaks of the raw materials shift, weaken in intensity, or are even masked, indicating that there are strong intermolecular interactions and that inclusion complexes are formed.

[0025] Example 2: Preparation of the main body II-Col inclusion complex 1. Weigh 1.341 g (1 mmol) of host II (R=(CH2)1SO3H) solid powder and add it to 15 mL of pure water. Heat and stir at 50 °C for 60 minutes until completely dissolved. Stop heating and let the solution cool to room temperature. Add 1.197 g (3 mmol) of guest Col to the cooled solution. Stir the mixture vigorously at 50 °C for 72 hours. After the reaction is complete, filter to remove insoluble matter. Freeze-dry the filtrate to obtain host II-Col inclusion complex (R=(CH2)1SO3H) with a yield of 66.2%. Nuclear magnetic resonance characterization results are as follows Figure 6 As shown, after the formation of the inclusion complex, the proton peak shape of the host II-Col inclusion complex (R=-(CH2)1SO3H) changed significantly, becoming broader and shifted, indicating that its chemical environment was altered due to the inclusion of the guest Col. (2D-ROESY spectrum) Figure 7 In the study, the H-7, H-8, and H-9 protons on the guest Col exhibited significant correlation signals with the Ha and Hb protons inside the cavity of the host II, further confirming the formation of the inclusion complex.

[0026] 2. The preparation method of the main body II-Col inclusion complex (R=(CH2)2SO3H) is the same as step 1 in Example 1, and the product yield is 61.22%.

[0027] 3. The method is the same as step 3 in Example 1, except that main component II (R=(CH2)3SO3H) is used, the molar ratio is 1:1, the reaction is carried out at 20℃ for 1 h, and the product is dried under reduced pressure, with a yield of 78.3%; XRD analysis of main component II-Col inclusion complex (R=(CH2)3SO3H) ( Figure 8 Similarly, it was shown that the inclusion complex ( Figure 8 d) The loss of the characteristic crystalline peaks of the raw material indicates the formation of an inclusion compound.

[0028] 4. The method is the same as step 3 in Example 1, except that host II (R=(CH2)4SO3H) is used, the molar ratio is 1:1, the reaction is carried out at 40℃ for 72 h, and the product is dried under reduced pressure, with a yield of 74.9%. Molecular docking simulation of host II-Col inclusion complex (R=(CH2)4SO3H) is also performed. Figure 9 It also supports a 1:1 stable enclosing mode.

[0029] 5. The preparation method of the main body II-Col inclusion complex (R=(CH2)5SO3H) is the same as step 1 in Example 1, and the product yield is 68.02%.

[0030] 6. The preparation method of the main body II-Col inclusion complex (R=(CH2)6SO3H) is the same as step 1 in Example 1, and the product yield is 71.07%.

[0031] 7. The method is the same as step 3 in Example 1, except that main component II (R=(CH2)7SO3Na) is used, the molar ratio is 1:1, the reaction is carried out at 40℃ for 48 h, and then freeze-dried, with a yield of 76.3%; FT-IR analysis of main component II-Col inclusion complex (R=(CH2)7SO3Na) ( Figure 10 The same conclusion as step 8 of Example 1 was obtained, confirming the formation of the inclusion compound.

[0032] 8. The preparation method of the main body II-Col inclusion complex (R=(CH2)8SO3H) is the same as step 1 in Example 1, and the product yield is 67.71%.

[0033] Example 3: Photostability Test of Inclusion Compounds from Examples 1 and 2 The free guest Col, the main I-Col inclusion complex (from Example 1), and the main II-Col inclusion complex (from Example 2) were respectively prepared into aqueous solutions with the highest ultraviolet absorption peak at 353 nm close to 1. They were then exposed to natural light or ultraviolet light under the same conditions, and the retention rate of colchicine in the solution was detected at regular intervals. The results of natural light illumination are shown in Figure 11 , 12 The figure shows that the inclusion effect significantly improved the stability of colchicine. Within 20 minutes of natural light exposure, there was little difference in the retention rate of the free Col group and the inclusion group. From 20 minutes to 80 minutes, the retention rate of the inclusion group was significantly higher than that of the free Col group. The results of ultraviolet light irradiation are shown in Figure 13 The results in the figure show that under strong ultraviolet light irradiation, Figure 13 (1) The inclusion effect of the host I-Col inclusion complex (R=(CH2)5SO3Na) was more significant. After 10 minutes of irradiation, the retention rate of the free guest Col was only 12.00%, while the retention rates of the host I-Col inclusion complex and the host II-Col inclusion complex were still as high as 80.40% and 72.14%, respectively. After 25 minutes of irradiation, the free guest Col was almost completely degraded (retention rate 9.17%), while the retention rates of the two inclusion complexes remained at 56.30% and 42.21%, respectively. The trends of the other three groups were also similar. Figure 13 (1) Consistency; the results of inclusion compounds with n=1-4 under ultraviolet light irradiation are consistent with the results of inclusion compounds with n=5-8. Figure 13 ); In summary, colchicine can significantly delay its photodegradation and improve its photostability by forming inclusion complexes with ionized ring-opening cucurbituril.

[0034] Example 10: Comparative test of antitumor activity of main body I-Col inclusion complex (R=(CH2)7SO3H) and main body II-Col inclusion complex (R=(CH2)2SO3Na).

[0035] (1) Weigh 1.584 g (1 mmol) of host II (R=(CH2)2SO3Na) and dissolve it in 15 mL of water. Separately, dissolve 2.793 g (7 mmol) of guest Col in 10 mL of tetrahydrofuran and add it dropwise to the host II solution. The mixture is stirred vigorously at 60 °C for 36 hours. After filtration, the filtrate is concentrated and dried under reduced pressure to obtain host II-Col inclusion complex (R=(CH2)2SO3Na) with a yield of 79.35%. (2) Using human cervical cancer cells (HeLa) and human colon cancer cells (HCT116) as models, the in vitro cytotoxicity of free guest Col, host I-Col inclusion complex (R=(CH2)7SO3H), and host II-Col inclusion complex (R=(CH2)2SO3Na) was tested respectively; The cytotoxicity of Col and its inclusion complexes was assessed using the MTT assay. Specifically, HeLa cells and HCT116 cells were cultured in T25 flasks until the cells reached 80% confluence, then passaged into 96-well plates and incubated for another 24 hours. Different concentrations of Col and its inclusion complexes were added to the wells, with final concentrations of 3.125, 6.25, 12.5, 25, 50, and 100 μM. After incubation for another 44 hours, 20 μL of MTT solution (5 mg / mL) was added to each well. After incubation for another 4 hours, the liquid from each well was aspirated and discarded, and 150 μL of DMSO solution was added. The absorbance was measured at 490 nm using a microplate reader.

[0036] The results are as follows Figure 14 As shown, cell viability decreased in a dose-dependent manner with increasing drug concentration. For HeLa cells and HCT116 cells, there was no significant difference in cell viability between the two inclusion complex groups and the free guest Col group. These results indicate that the inherent antitumor cytotoxicity of colchicine is well preserved after it is encapsulated to form inclusion complexes.

Claims

1. The application of an ionized ring-opening cucurbitaurea in improving the photostability of colchicine, characterized in that: Colchicine was subjected to an inclusion reaction with ionized ring-opening cucurbituril to prepare an inclusion complex, thereby improving the photostability of colchicine. The chemical structural formula of the ionized ring-opening cucurbita is shown below: or ; In the formula: R=(CH2) n SO3A, where A = Na, H; n = 1 to 8.

2. The method for improving the photostability of colchicine according to claim 1, characterized in that: Ionized ring-opening cucurbita and colchicine were mixed in a solvent at a molar ratio of 1:1 to 10 and stirred at 0℃-90℃ for 1-72 hours. The solid and liquid were separated, and the filtrate was dried to obtain the inclusion complex.

3. The application according to claim 1, characterized in that: Inclusion compounds are used in the preparation of photostable antitumor drugs.

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