Magnolol triazole derivative as well as preparation method and application thereof

Abstract

The invention discloses a magnolol triazole derivative and a preparation method and application thereof, and belongs to the technical field of organic synthesis, the magnolol triazole derivative is a 1c or 1d derivative, the molecular formula of 1c is C38H32N6O4, and the molecular formula of 1d is C28H25N3O3. According to the magnolol triazole derivative and the preparation method and application thereof, stereotactic synthesis of the magnolol triazole derivative is achieved under a sodium ascorbate-Cu (I) catalytic system through a nitration-azidation-coupling three-step process, the total yield is guaranteed to be 80% or above, and meanwhile the yield of the magnolol triazole derivative is greatly improved. The three key technical bottlenecks of toxicity, selectivity and activity are successfully solved, and the method is suitable for industrial production of anti-cancer drug intermediates.

Description

Technical Field

[0001] The present invention relates to the technical field of organic synthesis, and in particular to a magnolol triazole derivative, a preparation method thereof and an application thereof. Background Art

[0002] As one of the diseases with the highest lethality rate globally, the treatment of cancer faces multiple technical bottlenecks. First, the problem of drug resistance is prominent. After targeted drug treatment, it is easy to induce gene mutations in tumor cells, resulting in a drug resistance rate of more than 50%. Secondly, traditional chemotherapy drugs have serious selectivity defects. Platinum drugs represented by cisplatin have a high killing rate for normal cells, and their LD 50 is only 5 - 10 mg / kg, and the therapeutic window is narrow. In addition, the problem of drug toxicity is significant. Chemotherapy drugs such as paclitaxel can cause serious side effects such as hair loss and bone marrow suppression, while the single - drug effective rate of targeted drugs is less than 20%. Phytochemicals have become an important direction for the development of anti - tumor drugs due to their multi - target and low - toxicity characteristics. Among them, magnolol, as a lignan active ingredient, can enhance the antioxidant stress ability by activating the Nrf2 pathway. However, it faces inherent defects such as low content (<0.1%) and fast metabolism in vivo (t 1 / 2 ≈2 h), making it difficult to meet the clinical medication requirements and severely restricting the application of the magnolol triazole system in the development of anti - cancer drugs. Summary of the Invention

[0003] The purpose of the present invention is to provide a magnolol triazole derivative, a preparation method thereof and an application thereof. Through a three - step process of nitration - azidation - coupling, the stereospecific synthesis of the magnolol triazole derivative is achieved under the catalysis of sodium ascorbate - Cu(I) system. While ensuring a total yield of more than 80%, the three key technical bottlenecks of toxicity, selectivity and activity are successfully solved, and it is suitable for the industrial production of anti - cancer drug intermediates.

[0004] To achieve the above purpose, the present invention provides a magnolol triazole derivative. The magnolol triazole derivative includes 1c and 1d. Among them, the molecular formula of 1c is C 38 H 32 N6O4, and its structural formula is:

[0005]

[0006] The molecular formula of 1d is C 28 H 25 N3O3, and its structural formula is:

[0007]

[0008] The present invention provides a preparation method of a magnolol triazole derivative, including the following steps:

[0009] S1, Nitration - Azidation Reaction: Add the substituted aniline to dilute hydrochloric acid. After cooling down, add the sodium nitrite solution. Stir in an ice bath and then transfer to room temperature. Add the sodium azide solution and react at room temperature. After monitoring the reaction completion by TLC, extract with ethyl acetate, combine the organic phases. Wash the combined organic phase A with saturated NaCl solution, dry over anhydrous Na2SO4, and rotary evaporate under reduced pressure to obtain intermediate 1a;

[0010] S2, Propiolic Acid Esterification Reaction: Add magnolol to anhydrous dichloromethane. After cooling down, add propiolic acid, let it stand, then add dicyclohexylcarbodiimide and 4 - dimethylaminopyridine, and transfer to room temperature for reaction. After monitoring the reaction completion by TLC, quench with water, extract with dichloromethane, combine the organic phases. Dry the combined organic phase B over anhydrous Na2SO4 and rotary evaporate under reduced pressure to obtain intermediate 1b;

[0011] S3, Cross - Coupling Reaction: Add intermediate 1a and intermediate 1b to an aqueous solution of tert - butanol, then add sodium ascorbate and anhydrous copper sulfate, and react at room temperature in the dark. After monitoring the reaction completion by TCL, extract with ethyl acetate, combine the organic phases. Wash the combined organic phase C with saturated NaCl solution, dry over anhydrous Na2SO4, and rotary evaporate under reduced pressure to obtain the crude product. The crude product is purified by silica gel column chromatography to obtain 1c and 1d.

[0012] Preferably, in S1, the mass fraction of HCl in the dilute hydrochloric acid is 10 - 12%;

[0013] The molar ratio of the substituted aniline, sodium nitrite solution, and sodium azide solution is 1:(1 - 1.4):(1 - 1.4);

[0014] The substituted aniline is one of 4 - methylaniline, 4 - methoxyaniline, and 4 - fluoroaniline.

[0015] Preferably, in S2, the molar ratio of magnolol, propiolic acid, dicyclohexylcarbodiimide, and 4 - dimethylaminopyridine is 1:(1 - 1.4):(1 - 1.4):(1 - 1.4).

[0016] Preferably, in S3, the molar ratio of intermediate 1a, intermediate 1b, sodium ascorbate, and anhydrous copper sulfate is 1:1:(0.08 - 0.12):(0.04 - 0.06);

[0017] The volume ratio of tert - butanol to water in the aqueous solution of tert - butanol is 1:(1 - 2).

[0018] Preferably, in S1, the temperature after cooling down is 0 - 4°C, the ice - bath stirring time is 30 - 60 min, and the reaction time at room temperature is 4 - 6 h.

[0019] Preferably, in S2, the temperature after cooling is 0-4°C, the standing time is 10-20 min, and the reaction time at room temperature is 4-6 h.

[0020] Preferably, in S3, the reaction time in the dark is 20-24 h.

[0021] The present invention provides a pharmaceutical composition comprising a combination of compound 1c and 1d with a pharmaceutically acceptable pharmaceutical carrier or excipient.

[0022] Preferably, the formulation of 1000 tablets containing 100 mg of active ingredient per tablet:

[0023] 100 g of compound 1c, 70 g of microcrystalline cellulose, 10 g of 5% povidone K30 ethanol solution, 10 g of croscarmellose sodium, 5 g of colloidal silicon dioxide, 3 g of magnesium stearate.

[0024] The present invention provides an application of magnolol triazole derivative. The above-mentioned magnolol triazole derivative is applied to the preparation of an anti-tumor drug, and the IC of the anti-tumor drug against breast cancer cell MDA-MB-231 50 ≤25 μM.

[0025] Therefore, the present invention adopts the above-mentioned magnolol triazole derivative, its preparation method and application, and has the following beneficial effects:

[0026] (1) Using substituted aniline as the starting material, through a three-step process of nitration - azidation - coupling, the stereospecific synthesis of magnolol triazole derivative is achieved under the catalysis of sodium ascorbate - Cu(I). The raw materials are simple and easy to obtain, the reaction conditions are mild, the production time is short, the production cost is low, and the single substitution rate reaches 82%, significantly improving the yield and stability of the active ingredient.

[0027] (2) By introducing the sodium ascorbate - Cu(I) catalytic system, spatial selectivity control is achieved, including the product 1c with a bidirectional substitution pattern and the unidirectional substitution pattern 1d. It has obvious inhibitory effects on three kinds of tumor cells 4T-1, MDA-MB-231 and Aml-12, and with the increase of time, the inhibitory effect is more obvious. Among them, the IC of 1c against MDA-MB-231 cells 50 is as low as 22.53 μM.

[0028] The following is a further detailed description of the technical solution of the present invention through the drawings and examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the reaction flow chart of Example 1 of the present invention;

[0030] Figure 2It is the inhibitory effect diagram of 1c on 4T-1 cells obtained in Example 1 of the present invention;

[0031] Figure 3 It is the inhibitory effect diagram of 1c on MDA-MB-231 cells obtained in Example 1 of the present invention;

[0032] Figure 4 It is the inhibitory effect diagram of 1c on Aml-12 cells obtained in Example 1 of the present invention. Detailed implementation manners

[0033] The technical solutions of the present invention will be further described below with reference to the drawings and examples.

[0034] Unless otherwise defined, the technical terms or scientific terms used in the present invention shall have the ordinary meanings understood by those of ordinary skill in the art to which the present invention belongs.

[0035] Example 1

[0036] As Figure 1 shown, a preparation method of magnolol triazole derivative includes the following steps:

[0037] S1. Nitration-azidation reaction: Add 10 mmol of 4-methylaniline to 10 mL of 10% dilute hydrochloric acid, cool down to 0 °C, dropwise add 5 mL of 12 mmol sodium nitrite solution first quickly and then slowly, stir in an ice bath for 30 min, then transfer to room temperature, dropwise add 6 mL of 12 mmol sodium azide solution, and react at room temperature for 4 h. After monitoring the reaction by TLC and it ends, extract three times with 120 mL of ethyl acetate, combine the organic phases, and then wash the combined organic phase A twice with 40 mL of saturated NaCl solution, dry over anhydrous Na2SO4, and rotary evaporate under reduced pressure to obtain intermediate 1a.

[0038] S2. Propiolic acid esterification reaction: Add 10 mmol of magnolol to 15 mL of anhydrous dichloromethane, cool down to 0 °C, add 12 mmol of propiolic acid, let it stand for 10 min, then dropwise add 12 mmol of dicyclohexylcarbodiimide and 12 mmol of 4-dimethylaminopyridine, and transfer to room temperature to react for 6 h. After monitoring the reaction by TLC and it ends, quench with water, extract three times with 60 mL of dichloromethane, combine the organic phases, dry the combined organic phase B over anhydrous Na2SO4, and rotary evaporate under reduced pressure to obtain intermediate 1b.

[0039] S3. Cross-coupling reaction: Add 10 mmol of intermediate 1a and 10 mmol of intermediate 1b to 16 mL of aqueous tert-butanol solution Then, 1 mmol of sodium ascorbate and 0.5 mmol of anhydrous copper sulfate were added, and the reaction was carried out in the dark at room temperature for 24 h. The reaction progress was monitored by TLC. After the reaction was completed, 90 mL of ethyl acetate was added for extraction three times, and the organic phases were combined. The combined organic phase C was washed twice with 40 mL of saturated NaCl solution, dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain 1c and 1d with a yield of 81.3%.

[0040] Among them, the molecular formula of 1c is C 38 H 32 N6O4, with a molecular weight of 626.2485. The biological name is N-[(4-methoxyphenoxy)-methyl]-5-amino-2-phenylpyridine-3-carboxamide, and the structural formula is:

[0041]

[0042] The molecular formula of 1d is C 28 H 25 N3O3, with a molecular weight of 451.1896. The biological name is 5-Hydroxy-N-[(4-trifluoromethylphenyl)-methyl]-2-(1-methylpyrrolidin-2-yl)pyridine-3-carboxamide, and the structural formula is:

[0043]

[0044] Example 2

[0045] As Figure 1 shown, a preparation method of magnolol triazole derivative comprises the following steps:

[0046] S1. Nitration-azidation reaction: 10 mmol of 4-methoxyaniline was added to 10 mL of 10% dilute hydrochloric acid, and the temperature was lowered to 4 °C. 5 mL of 11 mmol sodium nitrite solution was added dropwise first quickly and then slowly, and the mixture was stirred in an ice bath for 30 min. Then it was transferred to room temperature, and 6 mL of 11 mmol sodium azide solution was added dropwise, and the reaction was carried out at room temperature for 5 h. After the reaction was monitored by TLC and completed, it was extracted three times with 120 mL of ethyl acetate, and the organic phases were combined. The combined organic phase A was washed twice with 40 mL of saturated NaCl solution, dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain intermediate 1a.

[0047] S2, Propargylic acid esterification reaction: Add 10 mmol of magnolol to 15 mL of anhydrous dichloromethane, cool down to 0 °C, add 10 mmol of propargylic acid, let it stand for 10 min, then dropwise add 10 mmol of dicyclohexylcarbodiimide and 10 mmol of 4-dimethylaminopyridine, transfer to room temperature and react for 5 h. After monitoring the reaction by TLC until completion, quench with water, extract three times with 60 mL of dichloromethane, combine the organic phases, and the combined organic phase B is dried over anhydrous Na2SO4 and rotary evaporated under reduced pressure to obtain intermediate 1b.

[0048] S3, Cross-coupling reaction: Add 10 mmol of intermediate 1a and 10 mmol of intermediate 1b to 16 mL of an aqueous tert-butanol solution and then add 1.2 mmol of sodium ascorbate and 0.6 mmol of anhydrous copper sulfate, and react at room temperature in the dark for 20 h. Monitor the reaction process by TCL. After the reaction is completed, extract three times with 90 mL of ethyl acetate, combine the organic phases, wash the combined organic phase C twice with 40 mL of saturated NaCl solution, dry over anhydrous Na2SO4 and rotary evaporated under reduced pressure to obtain the crude product. The crude product is purified by silica gel column chromatography to obtain 1c and 1d, with a yield of 80.7%.

[0049] Example 3

[0050] As Figure 1 shown, a method for preparing a magnolol triazole derivative includes the following steps:

[0051] S1, Nitration-azidation reaction: Add 10 mmol of 4-fluoroaniline to 10 mL of 10% dilute hydrochloric acid, cool down to 0 °C, first quickly and then slowly dropwise add 5 mL of 14 mmol sodium nitrite solution, stir in an ice bath for 60 min, then transfer to room temperature, dropwise add 6 mL of 14 mmol sodium azide solution, and react at room temperature for 4 h. After monitoring the reaction by TLC until completion, extract three times with 120 mL of ethyl acetate, combine the organic phases, wash the combined organic phase A twice with 40 mL of saturated NaCl solution, dry over anhydrous Na2SO4 and rotary evaporated under reduced pressure to obtain intermediate 1a.

[0052] S2, Propargylic acid esterification reaction: Add 10 mmol of magnolol to 15 mL of anhydrous dichloromethane, cool down to 0 °C, add 14 mmol of propargylic acid, let it stand for 10 min, then dropwise add 14 mmol of dicyclohexylcarbodiimide and 14 mmol of 4-dimethylaminopyridine, transfer to room temperature and react for 4 h. After monitoring the reaction by TLC until completion, quench with water, extract three times with 60 mL of dichloromethane, combine the organic phases, and the combined organic phase B is dried over anhydrous Na2SO4 and rotary evaporated under reduced pressure to obtain intermediate 1b.

[0053] S3. Cross-coupling reaction: 10 mmol of intermediate 1a and 10 mmol of intermediate 1b were added to 14 mL of an aqueous tert-butanol solution . Then, 0.8 mmol of sodium ascorbate and 0.4 mmol of anhydrous copper sulfate were added, and the reaction was carried out in the dark at room temperature for 24 h. The reaction progress was monitored by TLC. After the reaction was completed, 90 mL of ethyl acetate was added for extraction three times, and the organic phases were combined. The combined organic phase C was washed twice with 40 mL of saturated NaCl solution, dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography to obtain 1c and 1d with a yield of 80.4%.

[0054] Four tumor cells, 4T-1 and MDA-MB-231, and normal Aml-12 liver cells were selected to study the antitumor effects of 1c and 1d prepared in Example 1. The preliminary preparations included: preparing the four tumor cells, 4T-1 and MDA-MB-231, and normal Aml-12 liver cells, selecting cells in the logarithmic growth phase with good growth status, washing, digesting, centrifuging, and resuspending. A small amount of the cell suspension was diluted 10-fold with PBS for cell counting, and the cell suspension concentration was adjusted to 5×10 4 cells / mL according to the results.

[0055] Test 1

[0056] The 4T-1 and MDA-MB-231 cells were seeded in 96-well plates at a density of 5×10 3 cells / well, and 100 μL of the cell suspension was added to each well. The plates were placed in a cell culture incubator at 37°C and 5% CO2 for static culture for 24 h to allow the cells to adhere. Two tumor cells were treated with 1c, 1d, magnolol, and paclitaxel at five concentration gradients for 24 h. The concentration gradients were set as 0, 6.25, 12.5, 25, and 50 μM. Among them, paclitaxel was used as a positive control drug, and a blank control well was set. The culture medium was carefully aspirated, and 10 μL of MTT solution was added to each well. The plates were continued to be incubated in the dark wrapped with tin foil in a cell culture incubator at 37°C and 5% CO2 for 4 h. Then, the supernatant was aspirated, and 150 μL of DMSO solution was added to each well. The 96-well plates were placed on a horizontal shaker and shaken at a low speed for 10 min to fully dissolve the formazan in the cells. Finally, the absorbance OD value was measured at 570 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and the survival rates of 4T-1 and MDA-MB-231 cells were expressed as a percentage of the blank control well, thereby obtaining the IC 50 50 of 1c, 1d, magnolol, and paclitaxel. The results are shown in Table 1. It can be seen from Table 1 that the inhibitory effects of compounds 1c and 1d on MDA-MB-231 and 4T-1 Aml-12 cells are better than those of magnolol and paclitaxel, and the IC 50The values are 22.53 and 27.43 μM, 25.44 and 29.42 μM respectively, indicating that compound 1c has significant inhibitory effects on both human and murine breast cancer cells, and is superior to the parent compound magnolol, compound 1d and paclitaxel.

[0057] Table 1 Detection results of the activities of different compounds against MDA-MB-231 and 4T-1 cells

[0058]

[0059] Test two

[0060] Using the same method, the inhibitory effects of 1c at concentrations of 0, 6.25, 12.5, 25, and 50 μM on the three types of cells were detected after treating 4T-1, MDA-MB-231, and Aml-12 cells for 24, 48, and 72 h, and the results are as Figures 2 - 4 shown.

[0061] From Figures 2 - 4 it can be seen that when the concentration of compound 1c is 6.25 μM, the viability of the three types of cells decreases. When the concentration is 12.5 μM, the inhibitory effect on the two types of tumor cells reaches about 40%. When the concentration is 25 μM, the inhibitory effect on the two types of tumor cells reaches about 50%. This indicates that the dosing concentration range of compound 1c from 6.25 to 25 μM is appropriate, and as the dosing concentration and time increase, the cell survival rate becomes lower and lower. Especially at the doses of 12.5, 25, and 50 μM of compound 1d, the survival rates of the three types of cells show a typical dose-dependent decrease. Compared with Aml-12 normal hepatocytes, the inhibitory effect of compound 1c on the two types of tumor cells is more obvious, indicating its lower toxicity. Research shows that compound 1c has anti-tumor effects and no toxic side effects, and can be used for the preparation of subsequent anti-tumor drugs and other research.

[0062] Therefore, the present invention adopts the above-mentioned magnolol triazole derivative, its preparation method and application, and realizes the stereospecific synthesis of the magnolol triazole derivative through a three-step process of nitration-azidation-coupling under the catalytic system of sodium ascorbate-Cu(I). While ensuring a total yield of more than 80%, it successfully solves the three key technical bottlenecks of toxicity, selectivity and activity, and is applicable to the industrial production of anti-cancer drug intermediates.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A honokiol triazole derivative, characterized in that, Magnolol triazole derivative is a derivative of 1c or 1d, wherein the molecular formula of 1c is C 38 H 32 N6O4, and its structural formula is: The molecular formula of 1d is C 28 H 25 N3O3, and its structural formula is:

2. The preparation method of a honokiol triazole derivative as described in claim 1, characterized in that, It includes the following steps: S1. Nitration - azidation reaction: Add the substituted aniline into dilute hydrochloric acid, cool down and then add sodium nitrite solution. After stirring in an ice bath and transferring to room temperature, add sodium azide solution and react at room temperature. After monitoring the end of the reaction by TLC, extract with ethyl acetate, combine the organic phases. Wash the combined organic phase A with saturated NaCl solution, dry over anhydrous Na2SO4, and rotary evaporate under reduced pressure to obtain intermediate 1a; S2. Propiolic acid esterification reaction: Add magnolol into anhydrous dichloromethane, cool down and then add propiolic acid, let it stand still, then add dicyclohexylcarbodiimide and 4 - dimethylaminopyridine, transfer to room temperature for reaction. After monitoring the end of the reaction by TLC, quench with water, extract with dichloromethane, combine the organic phases. Dry the combined organic phase B over anhydrous Na2SO4 and rotary evaporate under reduced pressure to obtain intermediate 1b; S3. Cross - coupling reaction: Add intermediate 1a and intermediate 1b into an aqueous solution of tert - butanol, then add sodium ascorbate and anhydrous copper sulfate, react in the dark at room temperature. After monitoring the end of the reaction by TCL, extract with ethyl acetate, combine the organic phases. Wash the combined organic phase C with saturated NaCl solution, dry over anhydrous Na2SO4 and rotary evaporate under reduced pressure to obtain the crude product. The crude product is purified by silica gel column chromatography to obtain 1c and 1d.

3. The preparation method according to claim 2, characterized in that, In S1, the mass fraction of HCl in the dilute hydrochloric acid is 10 - 12%; The molar ratio of the substituted aniline, sodium nitrite solution, and sodium azide solution is 1:(1 - 1.4):(1 - 1.4); The substituted aniline is one of 4 - methylaniline, 4 - methoxyaniline, and 4 - fluoroaniline.

4. The preparation method according to claim 2, wherein In S2, the molar ratio of magnolol, propiolic acid, dicyclohexylcarbodiimide, and 4 - dimethylaminopyridine is 1:(1 - 1.4):(1 - 1.4):(1 - 1.4); 5. The preparation method according to claim 2, characterized in that In S3, the molar ratio of intermediate 1a, intermediate 1b, sodium ascorbate, and anhydrous copper sulfate is 1:1:(0.08 - 0.12):(0.04 - 0.06); In the aqueous solution of tert - butanol, the volume ratio of tert - butanol to water is 1:(1 - 2).

6. The preparation method according to claim 2, characterized in that, In S1, the temperature after cooling is 0 - 4°C, the ice - bath stirring time is 30 - 60 min, and the reaction time at room temperature is 4 - 6 h.

7. The preparation method according to claim 2, wherein In S2, the temperature after cooling is 0 - 4°C, the standing time is 10 - 20 min, and the reaction time at room temperature is 4 - 6 h.

8. The preparation method according to claim 2, characterized in that, In S3, the reaction time in the dark is 20 - 24 h.

9. A pharmaceutical composition comprising the compounds 1c and 1d as claimed in claim 1 in combination with a pharmaceutically acceptable pharmaceutical carrier or excipient.

10. Use of a honokiol triazole derivative, characterized in that, Use the honokiol triazole derivative described in claim 1 in the preparation of an anti-tumor drug, and the anti-tumor drug has an IC 50 ≤ 25 μM against breast cancer cell MDA-MB-231.

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