Application of Quinadol B and structural analogue thereof in preparation of medicine for preventing and treating melanoma

Abstract

The invention discloses three novel Quinadline B structural analogues, and further discloses that Quinadline B and the structural analogues thereof have angiogenesis inhibition activity and have pharmaceutical application of preventing and treating melanoma.

Description

Technical Field

[0001] The invention belongs to the field of pharmacy and discloses a new use of Quinadoline B and its structural analogs in preparing drugs for preventing or treating pigment tumors. Background Art

[0002] Melanoma is an aggressive and metastatic tumor with an increasing incidence worldwide. Lung metastasis is considered the leading cause of death in melanoma patients and is one of the most difficult problems to solve in cancer treatment. Angiogenesis plays an important role in melanoma metastasis, which is coordinated by a series of cellular events, including endothelial cell proliferation, migration, invasion, and tube formation. Therefore, disrupting any aspect of tumor-associated angiogenesis may help prevent melanoma cells from metastasizing to other sites.

[0003] Quinadoline B is a small molecule alkaloid compound extracted from filamentous fungi. So far, it has been found to have biological activities such as inhibiting lipid droplet synthesis in mouse macrophages, cytotoxicity, and antiviral activities. At the same time, studies have predicted that Quinadoline B has a high gastrointestinal absorption rate, is not easy to pass through the blood-brain barrier, has a high ADMET evaluation potential, and has the potential for the development of drug lead compounds. The applicant previously extracted Quinadoline B and three novel structural analogs (compounds of formula I to formula III) from the fermentation of a marine microorganism, and for the first time found that these compounds have angiogenesis inhibitory effects. In-depth research has found that Quinadoline B can inhibit the number of new blood vessels sprouting from the chicken embryo chorioallantoic membrane (CAM), has the effect of inhibiting the lung metastasis of the B16F-10 cell line of C57BL / 6 mice, and can inhibit the invasion and lumen formation of human umbilical vein endothelial cells (HUVEC) in a dose-dependent manner, induce the destruction of the HUVEC cytoskeleton and cell migration, and the compound itself does not have HUVEC cytotoxicity. In addition, it was found that the potential anti-angiogenic effect of Quinadoline B involves the inhibition of ROCK / MYPT1 / MLC2 / coffin and FAK / src signaling pathways. The above studies show that Quinadoline B and its analogs have good potential for the development of anti-melanoma drug lead compounds. Summary of the invention

[0004]

[0005] One of the purposes of the present invention is to disclose three novel Quinadoline B structural analogs shown in Formulas 1 to 3;

[0006] The second purpose of the present invention is to disclose the new use of Quinadoline B and its analogs as drugs for preventing and treating melanoma.

[0007] The present invention confirms that Quinadoline B and its analogs have significant angiogenesis inhibitory effects through a series of in vitro and in vivo activity verification models, and deeply reveals the mechanism of action of Quinadoline B in inhibiting melanoma metastasis.

[0008] The content of the present invention is achieved through the following technical solutions:

[0009] The Tg (flila: EGFP) zebrafish model was used to screen compounds with anti-angiogenic activity (TG strain flila: EGFP zebrafish is a transgenic zebrafish embryo biological model with green fluorescent protein development characteristics, and the inhibitory effects of different compounds on zebrafish angiogenesis can be observed under a microscope). The experiment used zebrafish embryos 24 hours after fertilization, 0.1% DMSO was added to the blank control group, 10μM PTK787 was added to the positive control group, and 10μM drug was added to the experimental group. All groups of zebrafish embryos were treated for 48 hours and the angiogenesis status was observed under a confocal microscope. Four quinazoline indole alkaloid structures with significant angiogenesis inhibition were screened out (see Appendix). Figure 1 ), and selected Quinadolin B for further functional and mechanism verification:

[0010] 1) The zebrafish embryo model was treated with 2, 5, and 10 μM Quinadoline B for 48 h, 0.1% DMSO was used as a blank control, and 10 μM PTK 787 was used as a positive control. The results showed that the anti-angiogenic effect of Quinadoline B was dose-dependent (Appendix Figure 2 ).

[0011] 2) Quinadoline B inhibits angiogenesis in the CAM chicken embryo model. Embryos were exposed to different concentrations of QB. Neovascularization was observed and photographed 48 hours after treatment. n = 5. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 compared with the control group. The results showed that Quinadoline B could significantly inhibit angiogenesis in the CAM model (see Appendix Figure 3 ).

[0012] 3) B16F10 melanoma cells were inoculated into C57BL / 6J mice through the tail vein to establish an experimental lung metastasis model in mice; the direct effect of Quinadoline B on B16F10 was determined by MTT and wound healing assays; the vascular endothelial markers CD34 and VEGF in lung tissue were stained with vascular endothelial-specific antibodies to detect the expression of vascular endothelial growth factor. The results showed that mice treated with Quinadoline B significantly reduced the number of lung surface metastases and nodules of melanoma. At the same time, Quinadoline B had no direct effect on B16F10 cells, while the vascular endothelial growth factor markers in lung tissue after drug treatment were reduced (see Appendix). Figure 4 ).

[0013] 4) Quinadoline B was used to treat HUVEC migration and invasion assays. The results showed that the drug delayed the wound closure of HUVEC in a dose-dependent manner. Quinadoline B also significantly reduced the number of cell migration in the chamber in a dose-dependent manner. In the invasion assay, drug treatment significantly inhibited HUVEC invasion by reducing the number of invasive cells that passed through the Matrigel-coated permeable membrane barrier (see Appendix). Figure 5 ).

[0014] 5) HUVECs were seeded on Matrigel and treated with or without Quinadoline B at specified concentrations in ECM medium containing 2% FBS and 10 ng / mL VEGF. The capillary-like structures formed by HUVECs were then examined. The results showed that the drug had an effective anti-angiogenic effect in vitro (see Appendix). Figure 6 ).

[0015] 6) The time-dependent changes of actin filaments in the cytoskeleton of HUVEC were observed by confocal microscopy imaging. After 24h of QB treatment, stress fibers showed obvious breakage and fracture, and then the formation of actin stress fibers decreased after 48h. The results showed that Quinadoline B decomposed the actin filaments in the cytoskeleton (attached Figure 7 ).

[0016] 7) Quinadoline B was used to treat ROCK1 / ROCK2 kinases at different concentrations. The results showed that the drug inhibited ROCK kinase activity in a concentration-dependent manner. The expression of proteins in its downstream signaling pathway was also analyzed (see Appendix). Figure 8 ). BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Quinadolin B and its analogs inhibit angiogenesis in zebrafish embryos.

[0018] Figure 2 : Quinadolin B inhibits angiogenesis in zebrafish embryos in a dose-dependent manner. (A) Fluorescence images of zebrafish embryos treated with 2, 5, and 10 μM QB for 48 h, with 0.1% DMSO as blank control and 10 μM PTK 787 as positive control; (B) The anti-angiogenic effect of QB on zebrafish ISVs is dose-dependent. n=6, **P<0.001 compared with negative control. ISVs, intersegmental vessels.

[0019] Figure 3 : Quinadolin B inhibits neovascularization in the CAM model. Embryonic eggs were exposed to different concentrations of the drug. Neovascularization was observed and photographed 48 hours after treatment. n = 5. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 compared with the control group.

[0020] Figure 4 : Quinadolin B inhibits the formation of lung metastasis of B16F10 melanoma cells in mice. (A) Mice were inoculated with B16F10 melanoma cells via the tail vein. The mice were intraperitoneally injected with 10 mg / kg per day, and the control group was injected with the corresponding solvent DMSO for a total of 18 days. The figure shows the lung photos of C57BL / 6 mice after dissection. The number of metastatic nodules in each group was calculated, and the data are expressed as mean ± SD. (B) Effect of Quinadolin B on the viability of B16F10 melanoma cells. B16F10 melanoma cells were treated with different concentrations of the drug for 48 h and then evaluated by MTT assay. (C) Effect of Quinadolin B on the migration of B16F10 cells. B16F10 cells were treated with or without the drug for 24 h. (D) The body weight of mice was examined within 18 days of treatment with the solvent or Quinadolin B. (E) Immunohistochemical analysis of CD34 and VEGF protein expression in tumor tissues. Bar 200 μM. Data are expressed as mean ± SEM. Compared with the control group, **P<0.01.

[0021] Figure 5: Quinadolin B inhibits migration and invasion of HUVEC cells. (A) Inhibition of HUVEC migration by drugs in wound healing migration assay. HUVEC were treated with different concentrations of QB in ECM medium containing 2% FBS and 10 ng / mL VEGF. The dotted line indicates the area of ​​initial scratching. (B) Inhibitory effect of Quinadolin B on HUVEC migration. HUVEC treated with different concentrations of drugs were seeded in the upper chamber, and the lower chamber was filled with ECM medium containing 2% FBS and 10 ng / mL VEGF. The number of migrated cells was photographed and counted. (C) Inhibition of HUVEC invasion by Quinadolin B. HUVEC treated with different concentrations of drugs were seeded in the upper chamber transwell chamber pre-coated with Matrigel, and the lower chamber was filled with ECM medium containing 2% FBS and 10 ng / mL VEGF. The number of invading cells was photographed and counted. Data are expressed as mean ± SEM. Compared with the control group, *P<0.05, **P<0.01.

[0022] Figure 6 : Quinadolin B inhibits HUVEC tubule formation. The figure shows HUVEC treated with different concentrations of drugs in ECM medium containing 2% FBS and 10 ng / mL VEGF. HUVEC were seeded in 96-well plates coated with Matrigel and then treated with drugs for 6 h. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01 compared with the control group.

[0023] Figure 7 : Disassembly of actin stress fibers in HUVECs caused by Quinadolin B. HUVECs were treated with 10 μM of the drug for 24 and 48 h, respectively, and stained with rhodamine-labeled phalloidin to observe actin stress fibers.

[0024] Figure 8 :Quinadolin B effectively inhibits ROCK1 / 2 kinase activity and its downstream signaling pathway in HUVEC. (A) Quinadolin B effectively inhibits ROCK1 / 2 kinase activity. (B) HUVEC were treated with 10 μM drug for 15, 30, 45, and 60 min. The cells were then harvested and extracted for Western blot analysis. (A) Protein expression of the ROCK signaling pathway was determined by Western blotting. GAPDH was used as a loading control. The results are expressed as a percentage of the control group (mean ± SD; n = 3), **p < 0.01 compared with the control group.

[0025] Fig. 9:After exposure to VEGP (10 ng / mL) for 15, 30, 45, and 60 min, HUVEC were treated with 10 μM Quinadolin B for the indicated time. Whole cells were then extracted for Western blot analysis. The histogram shows the quantitative results of protein levels and was adjusted with the corresponding GAPDH protein level. Compared with the control group, each value is expressed as the multiple of the control mean ± SEM. *P < 0.05, **P < 0.01. Specific implementation plan:

[0026] According to the technical content disclosed in the present invention, those skilled in the art will have a better understanding of the essence of this patent, and the following implementation scheme is only for example.

[0027] Example 1

[0028] In this study, the CAM method was used to investigate the effects of Quinadolin B on angiogenesis and development in vivo. Fertilized chicken eggs were incubated with the air chamber facing upward for 24-48 hours. After embryonic development, the eggshell above the air chamber was uncovered, and Quinadolin B and DMSO were added to the CAM vascular plexus in the rubber ring. After 48 hours of observation, the branches and images of the CAM blood vessels were photographed and compared with the control group. The results showed that Quinadolin B could significantly inhibit CAM angiogenesis (see Appendix). Figure 3 ) and showed dose-dependency.

[0029] Example 2

[0030] B16F10 melanoma cells were inoculated into C57 / BL6J mice through the tail vein to establish an experimental lung metastasis model in mice. The mice were intraperitoneally injected with 10 mg / kg / d, and the control group was injected with the corresponding solvent DMSO for a total of 18 days. The mice were then dissected and the melanoma metastasis in the lungs was observed. Figure 4 A), The results showed that Quinadolin B could significantly inhibit the lung metastasis of mouse melanoma.

[0031] Example 3

[0032] To further verify the angiogenesis inhibitory activity of Quinadolin B, we conducted experiments on the migration, invasion and tubule formation of HUVEC human umbilical vein endothelial cells and observed the generation of HUVEC actin filaments under the influence of drugs under a microscope (see Appendix Figure 5 -Attached Figure 7 ). The molecular imprinting method was used to analyze the expression of downstream proteins (Appendix Figure 8 , 9 ), further elucidating the mechanism of Quinadolin B's inhibitory angiogenic activity.

[0033] Example 4

[0034] NMR data assignment and physicochemical properties of Quinadolin B structural analogs:

[0035] The compound of formula 1 is a white powdery solid with an optical rotation value of 44.0 (c 0.50, MeCN). IR(KBr)v max 3238, 2924, 1708, 1628, 1467, 1388, 1262, 1169, 773, 734, 694cm -1 The NMR data are shown in Table 1 below, and the mass spectrum data are HRESIMS m / z 400.1405[M+H] + (calcd for C 22 H 18 N5O3, 400.1410), UV(MeCN)λ max 205.6, 227.9, 301.4 nm. The planar structure of the compound was solved by the above data, and the stereo configuration was determined by comparing the ECD experimental data.

[0036] Table 1 Compounds of Formula 1 1 H NMR 13 C NMR HMBC 1 H- 1 H COSY data (DMSO-d6)

[0037]

[0038]

[0039] 1 H NMR (400 MHz), 13 C NMR (100MHz)

[0040] The compound of formula II is a white powdery solid with an optical rotation value of -56.0 (c 0.50, MeCN). IR(KBr)v max 3238, 2924, 1708, 1628, 1467, 1388, 1262, 1169, 773, 734, 694cm -1 , the NMR data are shown in Table 2 below, and the mass spectrum data is HRESIMS m / z 414.1558[M+H] + (calcd for C 23 H 20 N5O3, 414.1553), UV(MeCN)λ max206.7, 229.2, 303.0 nm. The planar structure of the compound was solved by the above data, and the stereo configuration was determined by comparing the ECD experimental data. ;

[0041] Table 2 Compounds of Formula II 1 H NMR 13 C NMR HMBC 1 H- 1 H COSY data (DMSO-d6)

[0042]

[0043]

[0044] 1 H NMR (400 MHz), 13 C NMR (100MHz)

[0045] The compound of formula III is a white powdery solid with an optical rotation value of -40.0 (c 0.50, MeCN). IR(KBr)v max 3229, 2929, 1701, 1628, 1478, 1387, 1171, 771, 731, 694cm -1 The NMR data are shown in Table 3 below, and the mass spectrum data is HRESIMS m / z 414.1562[M+H] + (calcd for C 23 H 20 N5O3, 414.1566), UV(MeCN)λ max 205.6, 227.9, 301.4 nm. The planar structure of the compound was solved by the above data, and the stereo configuration was determined by comparing the ECD experimental data.

[0046] Table 3 Compounds of Formula III 1 H NMR 13 C NMR HMBC 1 H- 1 H COSY data (DMSO-d6)

[0047]

[0048] 1 H NMR (400 MHz), 13 C NMR (100MHz)

[0049]

Claims

1. Compounds represented by Formula I to Formula III 2. Use of the compounds represented by Formula I to Formula III and Quinadoline B and pharmaceutically acceptable salts thereof in the preparation of drugs for preventing or treating melanoma.

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