Schiff base vanadium metal catechol complex as well as preparation method and application thereof

Abstract

The invention discloses a Schiff base vanadium metal catechol complex as well as a preparation method and application thereof. The structural formula of the Schiff base vanadium metal catechol complex is shown in the specification, and the Schiff base vanadium metal catechol complex is high in effective anti-cancer characteristic and very small in toxicity to normal cells. # imgabs0 #

Description

Technical Field

[0001] The invention relates to a Schiff base vanadium metal catechol complex and a preparation method and application thereof, belonging to the field of Schiff base vanadium metal catechol complexes. Background Art

[0002] There are three main treatment strategies used for cancer such as surgery, radiotherapy, and drug therapy. Cisplatin is the main chemotherapy drug used in cancer treatment all over the world. Through these, new inventions have emerged such as anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, antibody drugs, small interfering RNA (siRNA), immune checkpoint inhibitors such as anti-programmed (PD-1) cell death inhibitors, anti-cytotoxic T-lymphocyte-associated protein 4 (CLTA-4) antibodies are used in anti-cancer treatment. However, none of them has a satisfactory performance in ridding the world of cancer deaths because they have so many side effects. Therefore, there is a need to introduce a new compound that can effectively kill cancer cells with cytotoxicity. Summary of the invention

[0003] Purpose of the invention: The technical problem to be solved by the present invention is to provide a Schiff base vanadium metal catechol complex and its preparation method and application.

[0004] Technical solution: To solve the above technical problems, the present invention provides a Schiff base vanadium metal catechol complex or a pharmaceutically acceptable salt thereof. The structural formula of the Schiff base vanadium metal catechol complex is shown below:

[0005]

[0006] The present invention also provides a method for preparing the Schiff base vanadium metal catechol complex, comprising the following steps:

[0007] (1) adding a 2-hydroxy-1-naphthaldehyde solution to a methanol solution of N-(2-hydroxyethyl)-1,2-ethylamine; heating to reflux, filtering, washing the precipitate with cold ethanol and ether, and collecting a yellow solid compound 1;

[0008] (2) dissolving the yellow solid compound 1 in step (1) in methanol, adding vanadium sulfate hydrate solution, slowly adding sodium hydroxide solution, ice bathing, filtering the precipitate; washing with cold methanol, and drying to obtain a yellow solid compound 2;

[0009] (3) reacting 4-tert-butylcatechol, 1-adamantanol and trifluoroacetic acid under argon, filtering and washing until the filtrate is neutral, purifying and drying to obtain intermediate 3;

[0010] (4) Mixing the acetone solution containing the compound 2 described in step (2) with the intermediate 3 described in step (3), stirring under an argon atmosphere, filtering; drying; dissolving with acetone, adding n-hexane; filtering the dark purple microcrystalline precipitate in a vacuum filter to obtain the Schiff base vanadium metal catechol complex.

[0011] The present invention also provides the use of the Schiff base vanadium metal catechol complex or a pharmaceutically acceptable salt thereof in preparing a drug for treating and / or preventing cancer.

[0012] Wherein, the cancer includes pancreatic cancer.

[0013] The present invention also provides an anticancer drug that is less toxic to the human body and can selectively kill cancer cells, and observes the effect of this drug on pancreatic cancer, which is extremely lethal, so that it can subsequently produce good effects on other types of cancer cells.

[0014] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the Schiff base vanadium metal catechol complex of the present invention has high effective anti-cancer properties and very low toxicity to normal cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the NMR spectrum of the naphthalene dehydrogenation condensation product (intermediate 1) in CDCl3;

[0016] Figure 2 For intermediate 2 1 NMR spectrum in DMSO;

[0017] Figure 3 is [VO2(nap-Hshed)] in DMSO 51 V NMR;

[0018] Figure 4 for 1 1H NMR of the final compound in DMSO;

[0019] Figure 5 The final compound [VO(nap-Hshed)tbad] in CDCl3 51 V NMR spectrum;

[0020] Figure 6 The graph is the relationship between the average optical density value and time (hours) after treatment and removal of compounds in cells;

[0021] Figure 7 is the number of viable cells of MiaPaCa-2 cancer cell line v[VO(nap-Hshed)tbad] the dose concentration of the compound;

[0022] Figure 8 is the number of viable cells of hTERT-HPNE pancreatic normal cell line v the dose concentration of compound [VO(3-OMe)SALIMP(dtb)];

[0023] Fig. 9 is the surviving cell number v dose concentration of [VO(nap-Hshed)tbad] compound on hTERT-HPNE pancreatic normal cell line;

[0024] Fig.10 is the number of viable cells of MiaPaCa-2 cancer cell line v the dose concentration of compound [VO(3-OMe)SALIMP(dtb)];

[0025] Fig.11 MTT assay for [VO(nap-Hshed)tbad] component after treatment of MiaPaCa-2 cancer cells;

[0026] Fig.12 MTT assay of [VO(3-OMe)SALIMP(dtb)] components after treatment of MiaPaCa-2 cancer cells. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0028] Example 1 Synthesis of Schiff base vanadium metal catechol complex

[0029] Step 1:

[0030] Synthesis route:

[0031]

[0032] 20 mL of a methanol solution containing 2-hydroxy-1-naphthaldehyde (1.7 g, 10 mmol) was added to 15 mL of a methanol solution containing N-(2-hydroxyethyl)ethylenediamine (1.1 g, 10.5 mmol). The mixture was heated to reflux for 4 hours and then placed in a -20°C refrigerator overnight. The resulting yellow precipitate was filtered and rinsed with cold ethanol (20 mL) and ether (20 mL) to obtain 2.35 g (91%) of a yellow solid, Intermediate 1. 1H NMR (400 MHz, CDCl3), the data of the intermediate compound are as follows: δ8.80 (s, 1H), 7.87 (d, J = 8.4 Hz, 1H), 7.68 (d, J = 9.3 Hz, 1H), 7.61 (d, J = 7.9 Hz, 1H), 7.44 (ddd, J = 8.4, 7.0, 1.5 Hz, 1H), 7.23 (d, J = 7.7 Hz, 1H), 6.93 (d, J = 9.3 Hz, 1H), 3.75 (t, J = 5.8 Hz, 2H), 3.67 (t, J = 5.1 Hz, 2H), 3.03 (t, J = 5.8 Hz, 2H), 2.87 (t, J = 5.1 Hz, 2H). See the NMR spectrum for details. Figure 1 .

[0033] Step 2:

[0034] Synthesis route:

[0035]

[0036] In a 250 mL round bottom flask, add HPLC grade methanol (50 mL) and degas with argon for 20 minutes. Add intermediate 1 (1.3 g, 5.0 mol) to produce a yellow solution. Dissolve vanadium sulfate hydrate (0.91 g, 5.0 mmol, Sigma-Aldrich, CAS number no.123334-20-3) in degassed water (15 mL) to make the reaction mixture black. Stir the resulting mixture for 1 hour, then dissolve sodium hydroxide (0.40 g, 10 mmol) in water (8.0 mL) and slowly (over 5 minutes) add to the above mixed solution. Stir the reaction for 12 hours to produce a yellow precipitate. Place the reaction in an ice bath for 20 minutes, then filter the product and rinse with cold methanol (30 mL). Dry the solid under vacuum for 3 days to obtain 88% of a yellow solid (Intermediate 2). 1 The data of H NMR (d6-DMSO, 400 MHz) are as follows: δ9.76 (s, 1H), 8.39 (d, J = 8.6 Hz, 1H), 8.00 (d, J = 9.2 Hz, 1H), 7.89-7.82 (m, 1H), 7.59 (ddd, J = 8.5, 6.9, 1.5 Hz, 1H), 7.37 (t, J = 7.5 Hz, 1H), 7.05 (d, J = 9. 1Hz,1H),5.67(s,1H),4.83(t,J=5.3Hz,1H),4.32-4.23(m,1H),4.11(m,1H),3.83(q,J=5. 7Hz, 2H), 3.43-3.34 (m, 2H), 2.88 (dt, J=11.9, 5.1Hz, 1H), 2.68 (td, J=11.8, 4.8Hz, 1H). and δ51 The data of V NMR (d6-DMSO, 105MHz) is: -524ppm. See the NMR spectrum Figure 2 and Figure 3 .

[0037] Step 3:

[0038] Synthesis route:

[0039]

[0040] A 50 mL round bottom flask equipped with a stirring bar was flame dried and then purged with argon. 4-tert-butylcatechol (1.66 g, 10.0 mmol), 1-adamantanol (1.52 g, 10.0 mmol) and trifluoroacetic acid (15 mL) were added, and the reaction mixture was reacted under argon for 2 days, and a white precipitate was observed. The precipitate was filtered and washed with deionized water until the filtrate was neutral. The crude solid was purified with a flash column (n-hexane: ethyl acetate = 20: 1, v / v) and dried in vacuum for 2 days to obtain 2.34 g of intermediate 3 (78%). 1 The data of H NMR (400 MHz, CDCl3) are as follows: δ 6.84 (d, J = 2.3 Hz, 1H), 6.76 (d, J = 2.3 Hz, 1H), 5.44 (s, 1H, OH), 4.67 (s, 1H, OH), 2.13 (m, 6H), 2.09 (m, 3H), 1.79 (m, 6H), 1.27 (s, 9H).

[0041] Step 4:

[0042] Synthesis route:

[0043]

[0044] A 250 mL round-bottom Schlenk flask filled with ACS grade acetone (100 mL) was degassed with argon for 20 minutes. [VO2(nap-Hshed)] (Intermediate 2, 0.34 g, 1.0 mmol) was added to the degassed acetone, followed by the addition of Intermediate 3 (3-adamantyl-5-tert-butylcatechol, 0.30 g, 1.0 mmol). A dark purple solution was obtained after 1 hour and stirred under argon for 24 hours. The reaction mixture was vacuum filtered and then concentrated to dryness under reduced pressure. The purple residue was dissolved with a small amount of HPLC grade acetone and then n-hexane (100 mL) was added. The solution was stored at -20°C for 2-3 days. The dark purple microcrystalline precipitate was vacuum filtered, washed with cold hexane (2×20 mL), and dried under vacuum for 3 days to obtain 0.32 g (51%) of a purple solid ( Figure 4 and Figure 5 ).this 1The H NMR (400 MHz, DMSO) data of the final compound are shown below: 1 H NMR (400MHz, DMSO) δ9.58 (s, 1H), 8.33 (d, J = 8.6Hz, 1H), 7.95 (d, J = 9.1Hz, 1H), 7.82 (d, J = 7.9Hz, 1 H),7.57(t,J=7.8Hz,1H),7.34(t,J=7.5Hz,1H),7.00-6.82(m,1H),6.59-6.29(m,1H),6.22(s,1H ),4.89-4.62(m,2H),4.35(m,J=14.3Hz,1H),4.04(m,1H),3.76(s,1H),3.52(s,3H),2.81(m,1H), and δ 51 The data of V NMR (105 MHz, CDCl3) of the final compound [VO(nap-Hshed)tbad] was: 395.79 ppm.

[0045] Example 2 Cytotoxicity Experiment

[0046] The present invention uses normal pancreatic cell lines to conduct cytotoxicity experiments.

[0047] Among them, [VO(3-OMe)SALIMP(dtb)](Debbie C. Crans, Peter A. Lay, Heide A. Murakami, Aviva Levina, Kateryna Kostenkova, Andrew C. Bates, John F. Manganaro, Josef Grundy, Skyler A. Markham, Kameron L. Klugh. Vanadium Compounds and Methods of Making and Use The structural formula of Thereof,10975-043US1,2023.):

[0048]

[0049] The IC values ​​of vanadium complexes on normal pancreatic cell lines were determined by treating cells with DMSO solutions of compounds. 50 Value, the result is Figure 7 , 8 As shown, the IC of the complex [VO(nap-Hshed)tbad] against normal pancreatic cell lines50 The value is 0.858μM, and the IC value of [VO(3-OMe)SALIMP(dtb)] complex against normal pancreatic cell line is 50 The value is 0.829 μM.

[0050] The present invention uses a novel substituted Schiff base vanadium metal catechol complex, which has a good anti-cancer effect on pancreatic cancer cells. Due to the presence of aliphatic groups with good hydrophobicity in the ligand, it is easy to penetrate the lipid bilayer of the cell. Their hydrolysis products are non-toxic and easily excreted from the body, which makes the level of cytotoxic adverse reactions of these drugs in the body low.

[0051] Example 3 Determination of the anticancer properties of Schiff base vanadium metal catechol complexes

[0052] 1. One day before the composite treatment, MiaPaCa-2 pancreatic cancer cells (purchased from ATCC) were inoculated in 500 μL of DMEM medium (1×10 cells per well in a 24-well plate). 5 The cells were stored in an incubator at 37°C and 5% CO2.

[0053] 2. Prepare a stock solution of the vanadium complex in DMSO: weigh 1.244 mg of the [VO(nap-Hshed)tbad] substance prepared in Example 1 and immediately dissolve it in 20 ml of DMSO to prepare a 100 μM [VO(nap-Hshed)tbad] stock solution; weigh 1.057 mg [VO(3-OMe)SALIMP(dtb)] and immediately dissolve it in 20 ml of DMSO to prepare a 100 μM [VO(3-OMe)SALIMP(dtb)] stock solution.

[0054] 3. The old cell culture medium in each well was replaced with new culture medium. Except for one negative control well, each well was treated with a specified concentration of vanadium complex stock solution (freshly prepared [VO(nap-Hshed)tbad] stock solution or [VO(3-OMe)SALIMP(dtb)] stock solution was added to DMSO and cultured for 72 hours) and kept in an incubator.

[0055] Among them, for the [VO(nap-Hshed)tbad] compound, the final solution concentrations (final concentrations after treatment) of cell viability detection were 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, and 5 μM, respectively.

[0056] For [VO(3-OMe)SALIMP(dtb)] compounds, the final concentrations (final concentration after treatment) for cell viability assay were 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, and 4 μM, respectively.

[0057] 4. After 72 hours, remove the plate from the incubator, wash with PBS, and keep it in the incubator for 30 minutes. After adding 40 μL of trypsin, count the number of live cells under a microscope.

[0058] The results are shown in Table 1 and Figures 9-10 As shown:

[0059] Table 1

[0060]

[0061] according to Fig. 9 and Fig.10 The concentration at which half of the surviving cells in the negative control survived was determined as the IC 50 Therefore, the IC of [VO(nap-Hshed)tbad] 50 The IC value of [VO(3-OMe)SALIMP(dtb)] is 1.192 μM. 50 The value is 1.249 μM.

[0062] Example 4

[0063] The efficiency of these complexes was determined by MTT assay. 50 value:

[0064] 1. One day before compound treatment, MiaPaCa-2 pancreatic cancer cells (from ATCC) were inoculated in 100 mm plates (1×10 4 cells) and placed in an incubator.

[0065] 2. Remove the plate from the incubator and gently remove the culture medium. Then wash with PBS. Now, add 40 μL of trypsin and keep it in the incubator for 5 minutes. Count the cells after staining with trypan blue under a microscope. According to the observation, 4 mL of DMEM culture medium contains 6.3×10 6 cells. 4 As a standard for the experiment, mix 29.048 mL of culture medium with 952.48 μL of DMEM medium to form a cell stock solution. Now, add 200 μL of solution to each well that needs to be observed and keep it in the incubator.

[0066] 3. Treat cells with [VO(3-OMe)SALIMP(dtb)] compound

[0067] To prepare the stock solution of [VO(nap-Hshed)tbad] compound, 2.981 mg of [VO(nap-Hshed)tbad] compound was dissolved in 20 mL of DMSO; to prepare the stock solution of [VO(3-OMe)SALIMP(dtb)] compound, 2.654 mg of [VO(3-OMe)SALIMP(dtb)] compound was dissolved in 20 mL of DMSO. The stock solution of [VO(nap-Hshed)tbad] compound or [VO(3-OMe)SALIMP(dtb)] compound was added to the cell stock solution (1 μL was added to each well) for compound treatment and negative control. And stored in the incubator.

[0068] result:

[0069] 1. MTT 0 hours:

[0070] Add 10 μL of MTT reagent to the cell stock solution and store it in the incubator for 4 hours. Take it out from the incubator after 4 hours, take out 100 μL of culture medium, add 100 μL of DMSO solution to dissolve the formazan crystals, and store it in the incubator for 15 minutes. Take it out after 15 minutes and use a plate reader to take the UV absorbance value (Table 2). In the table below, 6-H refers to the [VO(nap-Hshed)tbad] complex; 13-S refers to the [VO(3-OMe)SALIMP(dtb)] complex.

[0071] Table 2

[0072]

[0073] 2. MTT test for 24 hours:

[0074] After 24 hours of compound treatment, the plate was taken out, 10 μL of MTT reagent was added, and it was kept in the incubator for 4 hours. After 4 hours, it was taken out from the incubator, 100 μL of culture medium was taken out, 100 μL of DMSO solution was added to dissolve the formazan crystals, and it was kept in the incubator for 15 minutes. After 15 minutes, it was taken out and the UV absorbance value was taken using a plate reader (Table 3).

[0075] Table 3

[0076]

[0077] 3. MTT assay for 48 hours:

[0078] After 48 hours of composite treatment, the plate was removed, 10 μL of MTT reagent was added, and the plate was kept in an incubator for 4 hours. After 4 hours, the plate was removed from the incubator, 100 μL of culture medium was removed, 100 μL of DMSO solution was added to dissolve the formazan crystals, and the plate was kept in an incubator for 15 minutes. After 15 minutes, the plate was removed and the UV absorbance value was taken using a plate reader (Table 4).

[0079] Table 4

[0080]

[0081]

[0082] 4. MTT 72 hours:

[0083] After 72 hours of composite treatment, the plate was taken out, 10 μL of MTT reagent was added, and the plate was kept in the incubator for 4 hours. After 4 hours, the plate was taken out from the incubator, 100 μL of culture medium was taken out, 100 μL of DMSO solution was added to dissolve the formazan crystals, and the plate was kept in the incubator for 15 minutes. After 15 minutes, the plate was taken out and the UV absorbance value was taken using a plate reader (Table 5).

[0084] Table 5

[0085]

[0086] The MTT test results of [VO(3-OMe)SALIMP(dtb)] and [VO(nap-Hshed)tbad] components after treatment of MiaPaCa-2 cancer cells are as follows Fig.11 and Fig.12 shown.

[0087] Therefore, the activity test of MTT method showed that both vanadium complexes [VO(nap-Hshed)tbad] and [VO(3-OMe)SALIMP(dtb)] had good antiproliferative properties. It showed good cytotoxicity against MiaPaCa-2 pancreatic cancer cell line.

[0088] MTT assay was used to detect the prolonged effect of vanadium complex on MiaPaCa-2 cell line. Figure 6 As shown, the cytotoxic effect of the vanadium complex on cancer cells lasts so long that the cancer cells cannot continue to proliferate after the compound is removed from the cells.

Claims

1. A Schiff base vanadium metal catechol complex or a pharmaceutically acceptable salt thereof, characterized in that: The structural formula of the Schiff base vanadium metal catechol complex is as follows:

2. A method for preparing the Schiff base vanadium metal catechol complex according to claim 1, characterized in that: The following steps are involved: (1) adding a 2-hydroxy-1-naphthaldehyde solution to an N-(2-hydroxyethyl)-1,2-ethylamine solution; Heat to reflux, filter and collect yellow solid compound 1; (2) dissolving the yellow solid compound 1 in step (1) in methanol, adding vanadium sulfate hydrate solution, slowly adding sodium hydroxide solution, ice bathing, filtering the precipitate; washing with cold methanol, and drying to obtain a yellow solid compound 2; (3) reacting 4-tert-butylcatechol, 1-adamantanol and trifluoroacetic acid under argon, filtering and washing until the filtrate is neutral, purifying and drying to obtain intermediate 3; (4) Mixing the acetone solution containing the compound 2 described in step (2) with the intermediate 3 described in step (3), stirring under an argon atmosphere, filtering; drying; dissolving with acetone, adding n-hexane; filtering the dark purple microcrystalline precipitate in a vacuum filter to obtain the Schiff base vanadium metal catechol complex.

3. Use of the Schiff base vanadium metal catechol complex or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug for treating cancer.

4. Use of the Schiff base vanadium metal catechol complex or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug for preventing cancer.

5. The use according to claim 3 or 4, characterized in that: The cancer includes pancreatic cancer.

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