A compound RX4 and its role in anti-colon cancer drugs

Abstract

This invention discloses the role of the small molecule compound RX4 in the preparation of anti-colon cancer drugs. The structure of RX4 is shown in Figure (1). Our experimental results show that RX4 can inhibit the proliferation and colony formation of colon cancer cell lines RKO, HCT-116, and SW620 in a concentration-dependent manner, inhibit cell migration, and induce apoptosis, thereby achieving anti-tumor biological activity.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to compound RX4 and its application in the preparation of antitumor drugs. Background Technology

[0002] Colorectal cancer is a common malignant tumor of the digestive tract that occurs in the colon, and it is the third most common malignant tumor worldwide. In 2020, colorectal cancer accounted for 10% of all cancer cases globally and 9.4% of cancer deaths (second only to lung cancer). In 2020, colorectal cancer ranked second among all cancers in China, with 286,000 deaths that year, and the number is showing an increasing trend year by year. Colorectal cancer seriously endangers the safety of people in China and around the world.

[0003] Currently, the main treatments for colorectal cancer include surgical resection, radiotherapy, and chemotherapy. The five-year survival rate for early-stage colorectal cancer patients can reach about 90%, but for patients with advanced colorectal cancer, the five-year survival rate drops to 10%-30%. Colorectal cancer has a complex pathogenesis, is prone to recurrence and metastasis, and chemotherapy can lead to drug resistance and significant toxic side effects. Therefore, the development of novel anti-colorectal cancer drugs is of great significance for controlling the progression of colorectal cancer. Summary of the Invention

[0004] This invention provides a compound RX4, which has significant anti-colon cancer activity and can be used as a potential anti-colon cancer drug.

[0005] The present invention further provides the role of the compound RX4 in anti-colon cancer drugs.

[0006] The technical solution of the present invention is as follows:

[0007] A compound RX4 has the structure shown in formula (I):

[0008]

[0009] The structure of the small molecule compound RX4 of this invention is shown in the above formula, and its molecular formula is C. 15 H 10 O 12 P2. Through studying the pharmacological effects of the small molecule compound RX4, the inventors discovered that it has significant anti-colon cancer activity and can be considered a potential anti-colon cancer drug.

[0010] The present invention also provides the application of the compound RX4 in the preparation of an anticancer drug for the treatment and prevention of colon cancer.

[0011] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0012] This invention provides a novel compound RX4, which can significantly inhibit the proliferation and colony formation of colon cancer cells, inhibit migration and induce apoptosis, and can be used as a potential anti-colon cancer drug. Attached Figure Description

[0013] Figure 1 Synthetic routes of compounds RX4 and their analogues

[0014] Figure 2 This is a graph showing the results of RX4 inhibiting the proliferation of colon cancer cells in Example 2;

[0015] Figure 3 In Example 2, RX4 inhibited the formation of colon cancer cell clones;

[0016] Figure 4 In the scratch assay of Example 3, RX4 inhibited the migration of colon cancer cells;

[0017] Figure 5 Example 3: RX4 inhibits colon cancer cell migration in the Transwell assay;

[0018] Figure 6 Example 4: RX4-induced apoptosis in colon cancer cells during flow cytometry.

[0019] Figure 7 Example 4: Hoechst 33258 apoptosis assay, RX4-induced apoptosis in colon cancer cells;

[0020] Figure 8 The NMR spectrum of compound RX3;

[0021] Figure 9 This is the NMR spectrum of compound RX4. Detailed Implementation

[0022] Example 1

[0023] Synthesis method

[0024] Synthesis of α-rhein benzyl ester RX6

[0025] (1) Weigh out 800 mg (2.8 mmol) of rhein and 300 μL (2.8 mmol) of benzyl alcohol and add them to a 100 mL single-necked round-bottom flask. Add a stir bar and 40 mL of dichloromethane to dissolve. Cool in an ice bath for 10 min.

[0026] (2) Weigh 0.4 g of dicyclohexylcarbodiimide (DCC), 17.82 mmol and 0.02 g of 4-dimethylaminopyridine (DMAP) and add them to the reaction solution. After reacting for 1 h in an ice bath, move the mixture to room temperature and stir.

[0027] (3) TLC method to detect reaction progress: The developing solvent for TLC was PE:EA = 10:1. Until the reaction was completed.

[0028] (4) Post-reaction treatment: diatomaceous earth filtration, and dichloromethane is removed by vacuum concentration of the filtrate.

[0029] (5) Separation and purification: The product was separated and purified by column chromatography using silica gel-packed column. After rotary evaporation and drying, 450 mg of the product was obtained, with a yield of 43%. The product was an orange-yellow solid, designated RX6. The structural and characterization data are as follows:

[0030]

[0031] 1 H NMR (400 MHz, CDCl3) δ 12.06 (s, 1H), 11.99 (s, 1H), 8.47 (d, J =1.6 Hz, 1H), 7.99 (d, J = 1.7 Hz, 1H), 7.90 (dd, J = 7.5, 1.2 Hz, 1H), 7.75(t, J = 8.0 Hz, 1H), 7.54 – 7.38 (m, 5H), 7.36 (dd, J = 8.4, 1.1 Hz, 1H), 5.45 (s, 2H). 13 C NMR (400 MHz, CDCl3) δ 192.83, 180.95, 164.28, 162.84,162.42, 137.91, 137.82, 135.23, 133.94, 133.49, 128.80, 128.70, 128.56,125.49, 124.95, 120.45, 120.38, 118.34, 115.87, 67.82, 38.78, 23.05, 1.08.

[0032] b: Synthesis of benzyl rheate 4,5-phenolic hydroxyl-terminated dibenzyl phosphite RX1 and RX2

[0033] (1) Weigh out the raw material benzyl rhein (190 mg, 0.5 mmol), add it to a 100 mL single-necked round-bottom flask, add a stir bar and 10 mL of acetonitrile to dissolve it. Add 690 μL of carbon tetrachloride, 470 μL of N,N-diisopropylethylamine (DIPEA) and 50 mg of 4-dimethylaminopyridine (DMAP). Stir in an ice bath for 10 min.

[0034] (2) Weigh 320 μL of dibenzyl phosphite and slowly add it dropwise to the reaction system under ice bath conditions. Stir the reaction under ice bath conditions.

[0035] (3) TLC method to detect reaction progress: The developing solvent for TLC was PE:EA = 3:1. Until the reaction was completed.

[0036] (4) Post-reaction treatment: Add 5 mL of 0.5 mol / L potassium dihydrogen phosphate solution, extract three times with ethyl acetate, dry with anhydrous magnesium sulfate and concentrate by rotary evaporation.

[0037] (5) Separation and purification: The product was separated and purified by column chromatography using silica gel-packed column. After rotary evaporation and drying, 88 mg of monophosphate was obtained and labeled RX1, and 40 mg of diphosphate was obtained and labeled RX2. Both products were pale yellow solids. The structural and characterization data of RX1 and RX2 are as follows:

[0038]

[0039] RX1

[0040] 1 H NMR (400 MHz, CDCl3) δ 12.58 (s, 1H), 8.84 – 8.78 (m, 1H), 8.34 (t,J = 1.6 Hz, 1H), 7.84 (dd, J = 7.5, 1.2 Hz, 1H), 7.71 (dd, J = 8.4, 7.5 Hz,1H), 7.55 – 7.40 (m, 5H), 7.40 – 7.32 (m, 6H), 7.32 – 7.26 (m, 7H), 5.46 (s,2H), 5.38 – 5.23 (m, 4H). 13 C NMR (400 MHz, CDCl3) δ 187.14, 180.95, 163.68,162.74, 150.22, 136.85, 136.36, 135.67, 135.30, 135.21, 132.47, 128.82,128.79, 128.65, 128.60, 128.19, 125.68, 125.13, 119.54, 116.82, 70.68, 70.62,67.89, 33.86, 25.62, 24.95.

[0041]

[0042] RX2

[0043] 1 H NMR (400 MHz, CDCl3) δ 8.72 (d, J = 1.9 Hz, 1H), 8.34 (q, J = 1.6Hz, 1H), 8.11 (d, J = 7.6 Hz, 1H), 7.78 (dd, J = 8.2, 1.7 Hz, 1H), 7.69 (td,J = 8.0, 1.6 Hz, 1H), 7.54 – 7.40 (m, 6H), 7.33 – 7.17 (m, 24H), 5.46 (s,2H), 5.25 – 5.15 (m, 8H), 1.32 (d, J = 17.3 Hz, 3H), 0.11 (d, J = 1.7 Hz, 3H). 13 C NMR (400 MHz, CDCl3) δ 181.26, 179.95, 163.82, 135.43, 135.38,135.34, 135.28, 134.69, 134.52, 134.41, 128.66, 128.59, 128.56, 128.25,128.21, 125.10, 124.24, 70.61, 70.55, 70.53, 70.47, 67.82, 53.51.

[0044] c: Preparation of rhein 4,5-phenolic hydroxyl group attached to phosphite RX3

[0045] 160 mg of the monophosphate product RX was dissolved in 10 mL of anhydrous methanol. 20-50 mg of 5% Pd / C alumina loading and a stir bar were added, followed by the introduction of hydrogen gas. The hydrogen gas was bubbled through a balloon, and after three evacuations, the reaction mixture was stirred at room temperature for 60 min. The reaction mixture was then filtered through diatomaceous earth and washed with anhydrous potassium alcohol. After removing the solvent under reduced pressure, the product was dissolved in water and washed with dichloromethane (DCM) (2 x 5 mL). 12 mg of the debenzylated monophosphate product, designated RX3, was obtained by freeze-drying.

[0046] The structural and characterization data are as follows:

[0047]

[0048] RX3

[0049] 1H NMR (400 MHz, DMSO) δ 12.60 (s, 1H), 8.42 (s, 1H), 8.37 (s, 1H), 7.87 – 7.74 (m, 1H), 7.76 – 7.64 (m, 1H), 7.39 (d, J = 8.1 Hz, 1H). 13 C NMR(400 MHz, DMSO) δ 187.41, 181.61, 165.80, 161.92, 137.23, 136.97, 135.64,132.91, 128.36, 124.96, 123.07, 119.17, 117.36.

[0050] d: Preparation of rhein 4,5-phenolic hydroxyl group attached to bisphosphite RX4

[0051] 40 mg of the bisphosphonate product RX2 was dissolved in 10 mL of anhydrous methanol. A 5% Pd / C alumina loading (20-50 mg) and a stir bar were added, followed by the introduction of hydrogen gas. The hydrogen gas was bubbled through a balloon, and after three evacuations, the reaction mixture was stirred at room temperature for 60 min. The reaction mixture was then filtered through diatomaceous earth and washed with anhydrous potassium alcohol. After removing the solvent under reduced pressure, the product was dissolved in water and washed with dichloromethane (DCM) (2 x 5 mL). 10 mg of the debenzylated bisphosphonate product, designated RX4, was obtained by freeze-drying.

[0052] The structural and characterization data are as follows:

[0053]

[0054] RX4

[0055] 1 H NMR (400 MHz, DMSO) δ 8.41 (d, J = 1.7 Hz, 1H), 8.26 (d, J = 1.6Hz, 1H), 7.98 (d, J = 7.6 Hz, 1H), 7.85 (t, J = 7.9 Hz, 1H), 7.77 (d, J = 8.3Hz, 1H). 13 C NMR (400 MHz, DMSO) δ 182.11, 180.76, 165.88, 150.71, 135.80, 135.02, 134.77, 134.65, 128.85, 128.34, 122.89.

[0056] Example 2: RX4 significantly inhibited the proliferation and colony formation of colon cancer cells.

[0057] This embodiment utilizes the MTT assay to detect the inhibitory activity of RX4 on the proliferation of colon cancer cell lines. Three human colon cancer cell lines, RKO, HCT-116, and SW620, were selected for cell proliferation experiments. 10,000 cells / well were seeded into 96-well plates. After 24 hours of complete cell adhesion, different concentrations of RX4 (D, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, 100 μM) were added. After 72 hours of treatment, 25 μL / well of MTT solution (5 mg / mL) was added, and the plates were incubated at 37°C in a 5% CO2 incubator for another 4 hours. The supernatant was carefully aspirated from the wells, and 150 μL / well of DMSO solution was added to dissolve any crystals. The absorbance was measured at 490 nm using a microplate reader, and the half-maximal inhibitory concentration (IC50) was calculated using GraphPadPrism. The experimental results are as follows: Figure 2 As shown, RX4 can dose-dependently inhibit the proliferation of colon cancer cell lines after 72 hours of treatment.

[0058] Tumor cells possess the ability to proliferate indefinitely and form colonies. Through colony formation experiments, we found that RX4 significantly inhibited colony formation in colony cancer cell lines. In this example, cells were seeded at 2000 cells / well. After 24 hours of complete adhesion, the original culture medium was discarded and replaced with fresh medium. Drugs were added at concentration gradients of (0, 7.5, 15, 30 μM). After 48 hours of drug addition, the drugs were removed, and fresh culture medium was added. Once visible colonies formed, cells were fixed with 4% paraformaldehyde for 20 minutes, washed with PBS, and stained with crystal violet. The size and number of colonies in each well were compared. The results are as follows: Figure 3 As shown in the figure. The results indicate that RX4 can limit colon cancer cell colony formation in a concentration-dependent manner.

[0059] Example 3: RX4 significantly inhibits the migration of colon cancer cells.

[0060] The ability of RX4 to inhibit colon cancer cell migration was evaluated using a scratch assay. RKO cells were cultured in 6-well plates to 100% confluence, scratches were made using a sterile pipette tip, and the medium was replaced with 2% fresh medium. Different concentrations of RX4 (0, 7.5, 15, and 30 μM) were added simultaneously. The drug was withdrawn 24 h after addition, and images were taken under a 100× microscope at 0, 24, and 48 h. Results are shown below. Figure 4 The results showed that RX4 significantly and in a concentration-dependent manner inhibited the migration of colon cancer cells.

[0061] This embodiment further utilizes the Transwell assay to re-evaluate the effect of RX4 on colon cancer cell migration. 30,000 cells were seeded per well in each chamber. After 24 hours of adhesion, the upper chamber was replaced with serum-free medium, while the lower chamber contained 10% fresh culture medium. 24 hours after drug administration, the liquid in the upper chamber was aspirated, and the cells were fixed with 4% paraformaldehyde for 15 minutes, stained with crystal violet for 10 minutes, and then photographed under a 10× microscope. Figure 5 As shown, RX4 can significantly inhibit the migration of colon cancer cells.

[0062] Example 4: RX4 can significantly induce apoptosis in colon cancer cells.

[0063] The effect of RX4 on apoptosis in colon cancer cells was evaluated using flow cytometry. Colon cancer cells were evenly seeded into 6-well plates, and different concentrations of RX4 (0, 7.5, 15, and 30 μM) were added and incubated for 24 h. The effect of RX4 on apoptosis in colon cancer cells was then detected by flow cytometry using Annexin V-FITC / PI double staining. Results are as follows: Figure 6 As shown, RX4 can induce apoptosis in colon cancer cells in a dose-dependent manner.

[0064] In this embodiment, the Hoechst 33258 apoptosis staining kit was used to stain cell nuclei. The degree of nuclear deformation was determined by observing the fluorescence staining of the nuclei, thus reflecting the state of apoptosis. Apoptotic characteristics such as nuclear condensation and fragmentation were displayed as high fluorescence intensity or fragmented fluorescent dots, indicating that apoptosis had occurred. Colon cancer cells were evenly seeded into 6-well plates and treated with different concentrations of RX4 (7.5, 15, 30 μM) for 48 h. The cells were then fixed with 4% paraformaldehyde, stained with Hoechst 33258, and photographed under a fluorescence microscope. The results are as follows: Figure 7 As shown, RX4 can induce apoptosis in colon cancer cells in a concentration-dependent manner.

Claims

1. A compound RX4, characterized in that, The structure is shown in equation (I): 。 2. The use of compound RX4 as described in claim 1 in the preparation of an anticancer drug, characterized in that, The aforementioned anticancer drugs are used for the treatment and prevention of colon cancer.

3. The application of compound RX4 according to claim 2 in the preparation of anticancer drugs, characterized in that, The aforementioned anticancer drug is used to inhibit the proliferation and clonal formation of colon cancer cells, inhibit migration, and induce apoptosis.

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