CN113149930B

CN113149930B - Cell sugar transport channel inhibitor

Info

Publication number: CN113149930B
Application number: CN202110411870.8A
Authority: CN
Inventors: 高清志; 刘胜男; 王欣瑜
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2021-04-16
Filing date: 2021-04-16
Publication date: 2022-09-02
Anticipated expiration: 2041-04-16
Also published as: CN113149930A

Abstract

The inventionDiscloses a cell sugar transport channel inhibitor, which has a structure shown in a formula (I):

Description

Cell sugar transport channel inhibitor

Technical Field

The invention belongs to the technical field of biological medicines, and relates to a cell sugar transport channel inhibitor for inhibiting sugar nutrient absorption of cells and targeting tumor Woberg effect, wherein a cell sugar transport channel is called GLUT for short.

Background

At present, malignant tumor is a major disease threatening human life health, and the best strategy for treating tumor is to inhibit tumor growth by targeting tumor-specific biological targets on the premise of not damaging normal cells and tissues.

The basic principle of the method is to utilize tumor biomarkers which exist specifically in tumor cells or tumor tissues or have obvious difference with normal cells or normal tissues, and achieve the purpose of treating tumors by selectively recognizing and inhibiting the biomarkers. Research has shown that malignant tumors can proliferate, spread and metastasize continuously, and one of the important physiological mechanisms is to continuously absorb and take up the special nutrients it needs. These nutrients include sugar molecules, amino acids, vitamins, nucleosides, and the like. Otto Woberg, a German biochemist, found that healthy cells can efficiently utilize aerobic metabolism to produce sufficient energy from a limited carbohydrate nutrient source, while most tumor cells rely on massive carbohydrate absorption and metabolism to provide energy to themselves even in the presence of sufficient oxygen, a phenomenon known as the Woberg effect. Woberg thus acquired The physiological or medical Nobel Prize in 1931 (ref: Warburg, O.science,123:309 (1956); "The Nobel Prize in Physiology or Medicine 1931". Nobel prize.org. < http:// www.nobelprize.org/Nobel _ prizes/medium/laurates/1931/>).

Based on the Woberg effect of the tumor, because tumor cells specifically and highly express the sugar transport channel GLUT compared with healthy cells, the GLUT is taken as a tumor specific marker, and the targeted therapy of the tumor is hopefully realized. As shown in formula (a), as a prior art, the literature reports that compound 1 as a GLUT inhibitor can prevent sugar absorption and sugar accumulation of leukemia cells HL-60 and U-937 (Salas m., et al., am.j. physiol. cell physiol.,305: C90 (2013)). There is also a report in the literature that compound 2 is able to inhibit glycolytic metabolism and tissue tumor cell growth in ovarian cancer (Yibao Ma, et al, Cancers,11(1):33 (2019)). Compound 3 has also been reported as a GLUT selective inhibitor, but its role in inhibiting tumor cells has not been demonstrated (Siebenenicher H, et al., ChemMedChem.2016,11(20): 2261-2271).

The GLUT inhibitors obtained by different drug screening methods provide a material basis for further verifying the clinical application of the lead compounds. There is a need for more validated inhibitors with GLUT selectivity.

Disclosure of Invention

The invention aims to provide a cell sugar transport channel inhibitor which can effectively inhibit a cell sugar transport channel GLUT and realizes the purpose of inhibiting the growth of a tumor by utilizing the Woberg effect of the tumor, namely the characteristic that the tumor cell excessively expresses the GLUT compared with a normal cell.

The second purpose of the invention is to provide the application of the cell sugar transport channel inhibitor in preparing anti-tumor drugs or in preparing reagents for inhibiting the cell sugar transport channel GLUT.

The third purpose of the invention is to provide a pharmaceutical composition for inhibiting tumors.

The fourth purpose of the invention is to provide the application of the pharmaceutical composition for inhibiting the tumor in preparing the anti-tumor drugs.

The technical scheme of the invention is summarized as follows:

a cell sugar transport channel inhibitor has a structure shown in formula I:

wherein,

XR ₁ and XR ₂ The same or different;

XR ₁ represents mono-or di-substitution at different positions of a benzene ring;

XR ₂ represents mono-or di-substitution at different positions of a benzene ring;

x is selected from O, S or NH;

R ₁ is a hydrogen atom, a C1-C7 linear alkyl group or a C3-C8 branched alkyl group;

R ₂ is hydrogen atom, C1-C7 straight chain alkyl or C3-C8 branched chain alkyl.

Y is selected from halogen, arylsulfonyl, arylsulfinyl, arylmercapto or aminosulfonyl.

Preferably, XR ₁ And XR ₂ The same;

x is O;

R ₁ is a hydrogen atom;

R ₂ is a hydrogen atom;

y is F, Cl, PhSO ₂ PhSO, PhS or SO ₂ N(Me) ₂ 。

The application of the cell sugar transport channel inhibitor in preparing an anti-tumor medicament or preparing a reagent for inhibiting a cell sugar transport channel GLUT.

A pharmaceutical composition for inhibiting tumor comprises the above-mentioned cell sugar transport channel inhibitor or its pharmaceutically acceptable salt or its solvate.

The application of the pharmaceutical composition for inhibiting the tumor in preparing an anti-tumor medicament.

The invention has the following beneficial effects:

experiments prove that the cell sugar transport channel inhibitor can target the cell sugar transport channel and inhibit the proliferation of tumor cells by directly blocking the GLUT channel and the uptake of sugar nutrients by tumors.

Drawings

FIG. 1 is a fluorescence absorbance spectrum of a cellular sugar transport channel inhibitor (I) - (A-1) -1;

FIG. 2 is a fluorescence absorption spectrum of a cellular sugar transport channel inhibitor (I) - (A-2) -1;

FIG. 3 is a fluorescence absorption spectrum of a cellular sugar transport channel inhibitor (I) - (A-3) -1;

FIG. 4 shows the results of GLUT expression in human lung carcinoma A549 and breast carcinoma MDA-MB-468 cells.

Detailed Description

The present invention will be described in further detail with reference to specific examples, which are provided by way of illustration and not by way of limitation.

A cell sugar transport channel inhibitor, GLUT inhibitor.

Example 1

Synthesis of GLUT inhibitor (I) - (A-1) -1:

1. the synthesis of 4-chloro-6-nitro-7-acetamidobenzo-2-oxa-1, 3-diazole (II) comprises the following synthetic route:

4-chloro-7-acetamidobenzo-2-oxa-1, 3-diazole (XIV, 678.3mg, 3.0mmol) was dissolved in 12mL of acetic anhydride. To the mixture was slowly added dropwise 4mL of concentrated nitric acid with stirring at 0 ℃. And after stirring at 0 ℃ for a further 3 hours, ice water was added. After stirring for 10min, filtration was carried out and the filter cake was washed 3 times with cold water to give 753.3mg of a yellow solid (II), 59% yield. ¹ H NMR(600MHz,DMSO-d6):δ11.51(s,1H),8.19(s,1H),2.19(s,3H).

The reaction formula is as follows:

2. the synthesis of 4-chloro-6-nitro-7-aminobenzo-2-oxa-1, 3-diazole (III) comprises the following steps:

II (256.6mg, 1.0mmol) was added to 50% wt. H ₂ SO ₄ Aqueous solution (3mL) and the mixture was stirred at 100 ℃ for 2 h. The mixture was cooled to room temperature and the pH adjusted to 8 with saturated aqueous sodium bicarbonateThen, the mixture was extracted with ethyl acetate (20 mL. times.2), and the organic phase was dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporator to give 191.1mg of (III) as a yellow solid in 89% yield. ¹ H NMR(600MHz,DMSO-d6):δ9.83(s,1H),9.35(s,1H),8.14(s,1H).

The reaction formula is as follows:

3. the synthesis of 4-chloro-6, 7-diaminobenzo-2-oxa-1, 3-diazole (IV) comprises the following steps:

to a solution of III (107.3mg, 0.5mmol) in THF (10ml) was added 10% Pd/C (20 mg). The mixture was stirred under hydrogen at 25 ℃ for 12 hours. The mixture was filtered through celite, washed 2 times with THF, the solvent removed by rotary evaporator and purified by silica gel column chromatography (petroleum ether: ethyl acetate 4: 1 to 2: 1) to give 64.6mg of (iv) as a red solid in 70% yield.

¹ H NMR(400MHz,DMSO-d ₆ ):δ7.29(s,1H),5.38(s,2H),5.16(s,2H)

The reaction formula is as follows:

4. synthesis of 4-t-butyldimethylsilyloxybenzoyl chloride (VII):

4-hydroxybenzoic acid (V, 3.0g, 21.7mmol), tert-butyldimethylsilyl chloride (TBSCl) (7.5g, 50.0mmol) and imidazole (3.4g, 50.0mmol) were dissolved in anhydrous N, N-Dimethylformamide (DMF) (30mL) and stirred at room temperature for 24 h. After completion of the reaction, acetone (150mL) was added to the reaction mixture, filtered, the filtrate was collected, concentrated under reduced pressure, and the resulting residue was dissolved in ethyl acetate (50mL), washed 3 times with saturated aqueous ammonium chloride solution, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give a colorless liquid.

The resulting colorless liquid was dissolved in a mixed solvent of glacial acetic acid (50mL), tetrahydrofuran (15mL) and water (15mL) and stirred at room temperature for 4 h. Ethyl acetate (60mL) and water (60mL) were added, the mixture was washed with saturated aqueous sodium bicarbonate solution, and the organic phase was collected and dried over anhydrous sodium sulfate. The solvent was evaporated to dryness using a rotary evaporator and the resulting residue was dried under high vacuum to give 3.7g of 4-tert-butyldimethylsilyloxybenzoic acid (VI) as a white solid in 68% yield.

VI (2.5g, 10.0mmol)), N, N-Dimethylformamide (DMF) (2 drops) and dry Dichloromethane (DCM) (20mL) were added under nitrogen. Oxalyl chloride (1.0m L, 12.0mmol) was added dropwise to the reaction at 0 ℃, stirred at room temperature for 3h, the solvent was evaporated to dryness using a rotary evaporator and the obtained vii was used in the next reaction without further purification.

The reaction formula is as follows:

5. synthesis of (I) - (A-1) -1

To a solution of IV (184.6mg, 0.6mmol) in tetrahydrofuran (4ml) were added a solution of VII (650.1mg, 2.4mmol) in tetrahydrofuran (4ml) and potassium carbonate (207.3mg, 1.5mmol) in this order, and the reaction mixture was stirred in a sealed tube at 80 ℃ for 12 hours. The reaction was cooled to room temperature and filtered, the residue was washed with DCM 2 times, the solvent was removed by rotary evaporator and purified by silica gel column chromatography (petroleum ether: ethyl acetate 40: 1) to give a pale yellow solid. The solid was dissolved in THF (4mL), an appropriate amount of 1M tetra-n-butylammonium fluoride (TBAF) in Tetrahydrofuran (THF) was added dropwise with stirring at room temperature, after stirring for 0.5h, dichloromethane (20mL) was added, followed by washing with water, a saturated NaCl solution, drying over anhydrous sodium sulfate, concentration under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate ═ 1: 1) to give (I) - (a-1) -153.4mg as a pale yellow solid in 21% yield.

The reaction formula is as follows:

example 2

Synthesis of GLUT inhibitor (I) - (a-2) -1:

1. synthesis of 3-t-butyldimethylsilyloxybenzoyl chloride (X):

3-hydroxybenzoic acid (VIII, 3.0g, 21.7mmol), tert-butyldimethylsilyl chloride (TBSCl) (7.5g, 50.0mmol) and imidazole (3.4g, 50.0mmol) were dissolved in anhydrous N, N-Dimethylformamide (DMF) (30mL) and stirred at room temperature for 24 h. After completion of the reaction, acetone (150mL) was added to the reaction mixture, filtered, the filtrate was collected, concentrated under reduced pressure, and the resulting residue was dissolved in ethyl acetate (50mL), washed 3 times with saturated aqueous ammonium chloride solution, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give a colorless liquid.

The resulting colorless liquid was dissolved in a mixed solvent of glacial acetic acid (50mL), tetrahydrofuran (15mL) and water (15mL), and stirred at room temperature for 4 h. Ethyl acetate (60mL) and water (60mL) were added, the mixture was washed with saturated aqueous sodium bicarbonate solution, and the organic phase was collected and dried over anhydrous sodium sulfate. The solvent was evaporated to dryness using a rotary evaporator and the resulting residue was dried under high vacuum to give 3.7g of 3-tert-butyldimethylsilyloxybenzoic acid (IX) as a white solid in 87% yield.

IX (2.5g, 10.0mmol)), N, N-Dimethylformamide (DMF) (2 drops) and dry Dichloromethane (DCM) (20mL) were added under nitrogen. Oxalyl chloride (1.0m L, 12.0mmol) was added dropwise to the reaction mixture at 0 deg.c, stirred at room temperature for 3 hours, and the solvent was evaporated to dryness using a rotary evaporator, and the resulting 3-t-butyldimethylsilyloxybenzoyl chloride (x) was used immediately in the next reaction without further purification.

The reaction formula is as follows:

2. synthesis of (I) - (A-2) -1

To a solution of IV (184.6mg, 0.6mmol) in tetrahydrofuran (4ml) were added a solution of X (650.1mg, 2.4mmol) in tetrahydrofuran (4ml) and potassium carbonate (207.3mg, 1.5mmol) in this order, and the reaction solution was stirred in a sealed tube at 80 ℃ for 12 hours. The reaction was cooled to room temperature and filtered, the residue was washed with DCM 2 times, the solvent was removed by rotary evaporator and purified by silica gel column chromatography (petroleum ether: ethyl acetate 40: 1) to give a pale yellow solid. The solid was dissolved in THF (4mL), an appropriate amount of 1M tetra-n-butylammonium fluoride (TBAF) in Tetrahydrofuran (THF) was added dropwise with stirring at room temperature, after stirring for 0.5h, dichloromethane (20mL) was added, followed by washing with water, a saturated NaCl solution, drying over anhydrous sodium sulfate, concentration under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate ═ 1: 1) to give (I) - (a-2) -153.4mg as a pale yellow solid in 30% yield.

The reaction formula is as follows:

example 3

Synthesis of GLUT inhibitors (I) - (a-3) -1:

1. synthesis of 3, 5-bis (tert-butyldimethylsilyloxy) benzoyl chloride (XIII):

3, 5-Dihydroxybenzoic acid (XI, 3.1g, 20.1mmol), tert-butyldimethylsilyl chloride (TBSCl) (10.6g, 70.4mmol) and imidazole (4.8g, 70.4mmol) were dissolved in anhydrous N, N-Dimethylformamide (DMF) (30mL) and stirred at room temperature for 24 h. After completion of the reaction, acetone (150mL) was added to the reaction mixture, filtered, the filtrate was collected, concentrated under reduced pressure, and the resulting residue was dissolved in ethyl acetate (50mL), washed 3 times with saturated aqueous ammonium chloride solution, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give a colorless liquid.

The resulting colorless liquid was dissolved in a mixed solvent of glacial acetic acid (50mL), tetrahydrofuran (15mL) and water (15mL), and stirred at room temperature for 4 h. Ethyl acetate (60mL) and water (60mL) were added, the mixture was washed with saturated aqueous sodium bicarbonate, and the organic phase was collected and dried over anhydrous sodium sulfate. The solvent was evaporated to dryness on a rotary evaporator and the resulting residue was dried under high vacuum to give 4.0g of 3, 5-bis (tert-butyldimethylsilyloxy) benzoic acid (XII) as a white solid in 52% yield.

Under nitrogen, 3, 5-bis (tert-butyldimethylsilyloxy) benzoic acid (XII, 3.8g, 10.0mmol), N, N-Dimethylformamide (DMF) (2 drops) and dry Dichloromethane (DCM) (20mL) were added. Oxalyl chloride (1.0m L, 12.0mmol) was added dropwise to the reaction mixture at 0 ℃ and stirred at room temperature for 3h, the solvent was evaporated to dryness using a rotary evaporator, and the resulting 3-t-butyldimethylsilyloxybenzoyl chloride (XIII) was used in the next reaction without further purification.

The reaction formula is as follows:

2. synthesis of (I) - (A-3) -1

To a solution of 4-chloro-6, 7-diaminobenzo-2-oxa-1, 3-diazole (IV, 184.6mg, 0.6mmol) in tetrahydrofuran (4ml) were added a solution of 3-t-butyldimethylsilyloxybenzoyl chloride (XIII, 918.4mg, 2.4mmol) in tetrahydrofuran (4ml) and potassium carbonate (207.3mg, 1.5mmol) in this order, and the reaction mixture was stirred in a sealed tube at 80 ℃ for 12 hours. The reaction was cooled to room temperature and filtered, the residue was washed with DCM 2 times, the solvent was removed by rotary evaporator and purified by silica gel column chromatography (petroleum ether: ethyl acetate 100: 1) to give a pale yellow solid. The solid was dissolved in THF (4mL), and after stirring at room temperature an appropriate amount of 1M tetra-n-butylammonium fluoride (TBAF) in Tetrahydrofuran (THF) was added dropwise, and after stirring for 0.5h, dichloromethane (20mL) was added, followed by washing with water, a saturated NaCl solution, drying over anhydrous sodium sulfate, concentration under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate: methanol ═ 20: 1) to give (I) - (a-3) -179.4 mg as a pale yellow solid, in 29% yield.

The reaction formula is as follows:

example 4

Synthesis of GLUT inhibitors (I) - (a-1) -2 the GLUT inhibitors (I) - (a-1) -2 were prepared following the procedure of example 1 substituting 4-chloro-7-acetamidobenzo-2-oxa-1, 3-diazole of example 1 with 4-fluoro-7-acetamidobenzo-2-oxa-1, 3-diazole, otherwise the same as example 1.

Example 5

Synthesis of GLUT inhibitor (I) - (a-2) -2:

4-fluoro-6, 7-diaminobenzo-2-oxa-1, 3-diazole was prepared by substituting 4-chloro-7-acetamidobenzo-2-oxa-1, 3-diazole from example 1 for 4-fluoro-7-acetamidobenzo-2-oxa-1, 3-diazole and carrying out the same procedures as in example 1; the GLUT inhibitor (I) - (A-2) -2 was prepared by reacting 4-fluoro-6, 7-diaminobenzo-2-oxa-1, 3-diazole with 3-tert-butyldimethylsilyloxybenzoyl chloride (X) prepared in example 2, as in example 2.

Example 6

Synthesis of GLUT inhibitors (I) - (a-3) -2:

4-fluoro-6, 7-diaminobenzo-2-oxa-1, 3-diazole was prepared by substituting 4-chloro-7-acetamidobenzo-2-oxa-1, 3-diazole from example 1 for 4-fluoro-7-acetamidobenzo-2-oxa-1, 3-diazole and carrying out the same procedures as in example 1; the reaction of 4-fluoro-6, 7-diaminobenzo-2-oxa-1, 3-diazole with 3, 5-bis (tert-butyldimethylsilyloxy) benzoyl chloride (XIII), prepared in example 3, was carried out according to the procedure of example 3 to prepare GLUT inhibitors (I) - (A-3) -2.

Example 7

Synthesis of GLUT inhibitors (I) - (a-1) -3 the GLUT inhibitors (I) - (a-1) -3 were prepared following the procedure of example 1 substituting 4-chloro-7-acetamidobenzo-2-oxa-1, 3-diazole of example 1 with 4-benzenesulfonyl-7-acetamidobenzo-2-oxa-1, 3-diazole and otherwise following example 1.

Example 8

Synthesis of GLUT inhibitors (I) - (a-2) -3:

4-chloro-7-acetamidobenzo-2-oxa-1, 3-diazole of example 1 was replaced with 4-benzenesulfonyl-7-acetamidobenzo-2-oxa-1, 3-diazole, and the other steps were the same as in example 1 to prepare 4-benzenesulfonyl-6, 7-diaminobenzo-2-oxa-1, 3-diazole; then, 4-benzenesulfonyl-6, 7-diaminobenzo-2-oxa-1, 3-diazole was reacted with 3-t-butyldimethylsilyloxybenzoyl chloride (X) prepared in example 2 by the procedure of example 2 to prepare GLUT inhibitors (I) - (A-2) -3.

Example 9

Synthesis of GLUT inhibitors (I) - (A-3) -3

4-chloro-7-acetamidobenzo-2-oxa-1, 3-diazole of example 1 is replaced with 4-benzenesulfonyl-7-acetamidobenzo-2-oxa-1, 3-diazole, and the other steps are the same as in example 1 to prepare 4-benzenesulfonyl-6, 7-diaminobenzo-2-oxa-1, 3-diazole; GLUT inhibitors (I) - (A-3) -3 were prepared by reacting 4-benzenesulfonyl-6, 7-diaminobenzo-2-oxa-1, 3-diazole with 3, 5-bis (tert-butyldimethylsilyloxy) benzoyl chloride (XIII), prepared in example 3, following the procedure of example 3.

Example 10

Synthesis of GLUT inhibitors (I) - (a-1) -4 the GLUT inhibitors (I) - (a-1) -4 were prepared following the procedure of example 1 substituting 4-chloro-7-acetamidobenzo-2-oxa-1, 3-diazole of example 1 with 4-benzenesulfinyl-7-acetamidobenzo-2-oxa-1, 3-diazole and otherwise following example 1.

Example 11

Synthesis of GLUT inhibitors (I) - (a-2) -4:

4-Chloro-7-Acylaminobenzo-2-oxa-1, 3-diazole from example 1 was replaced with 4-benzenesulfinyl-7-acetamidobenzo-2-oxa-1, 3-diazole, and the other steps were the same as in example 1 to prepare 4-benzenesulfinyl-6, 7-diaminobenzo-2-oxa-1, 3-diazole; GLUT inhibitor (I) - (A-2) -4 was prepared by reacting 4-benzenesulfinyl-6, 7-diaminobenzo-2-oxa-1, 3-diazole with 3-tert-butyldimethylsilyloxybenzoyl chloride (X) prepared in example 2, according to the procedure of example 2.

Example 12

Synthesis of GLUT inhibitors (I) - (A-3) -4

4-Chloro-7-Acylaminobenzo-2-oxa-1, 3-diazole from example 1 was replaced with 4-benzenesulfinyl-7-acetamidobenzo-2-oxa-1, 3-diazole, and the other steps were the same as in example 1 to prepare 4-benzenesulfinyl-6, 7-diaminobenzo-2-oxa-1, 3-diazole; the reaction of 4-benzenesulfinyl-6, 7-diaminobenzo-2-oxa-1, 3-diazole with 3, 5-bis (tert-butyldimethylsilyloxy) benzoyl chloride (XIII) prepared in example 3 was carried out according to the procedure of example 3 to prepare GLUT inhibitors (I) - (A-3) -4.

Example 13

Synthesis of GLUT inhibitors (I) - (A-1) -5

GLUT inhibitors (I) - (A-1) -5 were prepared following the procedure of example 1 substituting 4-chloro-7-acetamidobenzo-2-oxa-1, 3-diazole of example 1 with 4-benzenemercapto-7-acetamidobenzo-2-oxa-1, 3-diazole and otherwise following example 1.

Example 14

Synthesis of GLUT inhibitors (I) - (a-2) -5:

4-benzenesulfonyl-6, 7-diaminobenzo-2-oxa-1, 3-diazole was prepared by substituting 4-chlorobenzol-7-acetamidobenzo-2-oxa-1, 3-diazole obtained in example 1 with 4-benzenethiol-7-acetamidobenzo-2-oxa-1, 3-diazole and performing the same procedures as in example 1; the GLUT inhibitors (I) - (A-2) -5 were prepared by reacting 4-benzenemercapto-6, 7-diaminobenzo-2-oxa-1, 3-diazole with 3-tert-butyldimethylsilyloxybenzoyl chloride (X) prepared in example 2, as described in example 2.

Example 15

Synthesis of GLUT inhibitors (I) - (A-3) -5

4-benzenesulfonyl-6, 7-diaminobenzo-2-oxa-1, 3-diazole was prepared by substituting 4-chlorobenzol-7-acetamidobenzo-2-oxa-1, 3-diazole obtained in example 1 with 4-benzenethiol-7-acetamidobenzo-2-oxa-1, 3-diazole and performing the same procedures as in example 1; the reaction of 4-benzenemercapto-6, 7-diaminobenzo-2-oxa-1, 3-diazole with 3, 5-bis (tert-butyldimethylsilyloxy) benzoyl chloride (XIII) prepared in example 3 was carried out in accordance with the procedure of example 3 to prepare GLUT inhibitors (I) - (A-3) -5.

Example 16

Synthesis of GLUT inhibitors (I) - (A-1) -6 following the procedure of example 1, the synthesis of example 1

GLUT inhibitors (I) - (A-1) -6 were prepared as in example 1 except for 4-chloro-7-acetamidobenzo-2-oxa-1, 3-diazole which was replaced with 4-dimethylaminosulfonyl-7-acetamidobenzo-2-oxa-1, 3-diazole.

Example 17

Synthesis of GLUT inhibitors (I) - (a-2) -6:

4-chloro-7-acetamidobenzo-2-oxa-1, 3-diazole of example 1 was replaced with 4-dimethylaminosulfonyl-7-acetamidobenzo-2-oxa-1, 3-diazole, and the other steps were the same as in example 1 to prepare 4-dimethylaminosulfonyl-6, 7-diaminobenzo-2-oxa-1, 3-diazole; then, 4-dimethylaminosulfonyl-6, 7-diaminobenzo-2-oxa-1, 3-diazole was reacted with 3-tert-butyldimethylsilyloxybenzoyl chloride (X) prepared in example 2 by the procedure of example 2 to prepare GLUT inhibitors (I) - (A-2) -6.

Example 18

Synthesis of GLUT inhibitors (I) - (A-3) -6

4-chloro-7-acetamidobenzo-2-oxa-1, 3-diazole of example 1 was replaced with 4-dimethylaminosulfonyl-7-acetamidobenzo-2-oxa-1, 3-diazole, and the other steps were the same as in example 1 to prepare 4-dimethylaminosulfonyl-6, 7-diaminobenzo-2-oxa-1, 3-diazole; the GLUT inhibitors (I) - (A-3) -6 were prepared by reacting 4-dimethylaminosulfonyl-6, 7-diaminobenzo-2-oxa-1, 3-diazole with 3, 5-bis (tert-butyldimethylsilyloxy) benzoyl chloride (XIII), prepared in example 3, following the procedure of example 3.

Table 1 shows the structures of some of the compounds of the formula I

TABLE 1

Table 2 shows NMR, mass and fluorescence spectra data for each compound of Table 1

TABLE 2

Test example 1: other spectral measurements of representative compounds of the invention

1) An experimental instrument: thermo Scientific variosukan LUX.

2) Test compounds: GLUT inhibitor compounds prepared in the above examples.

Testing the solvent: DMSO, DMSO: PBS (pH 7.2) ═ 1:9(V: V)

3) Experimental procedure

(1) Each test compound was weighed.

(2) Dissolve compound to 10mM in DMSO, vortex for 1min, dissolve compound well.

(3) Compounds were diluted to 500uM with PBS-DMSO (1:1, volume ratio) and vortexed for dissolution. Taking 100ul of each compound and test solvent, adding into a black bottom-penetrating 96-hole culture plate, and detecting absorption spectrum, excitation spectrum and emission spectrum by using a multifunctional microplate reader.

4) Data processing: the absorbance, emission, and combined absorbance-emission spectra were derived by treatment with OriginPro 8.5.

5) As a result: as shown in table 2. Excitation and emission spectra of representative GLUT inhibitors are shown in the accompanying figures (fig. 1-3).

Test example 2: GLUT inhibitor inhibition of GLUT1

Human and mammalian erythrocytes were reported to predominantly express GLUT1 sugar transport channel protein for sugar uptake (m.mueckler, c.caroso, s.a.baldwin, i.Blench, h.r.morris, w.j.allard, g.e.lienhard, h.f.lodis, Science,1985,229,941; (b) Montel-Hagen, s.kinet, n.manel, c.montella z, r.prohaska, j.l.battini, j.delaunay, m.sitton, n.taylor, Cell,2008,132,1039) and the present experiment was evaluated for GLUT inhibition of the GLUT inhibitor molecules of the invention using human erythrocytes and a 2-deoxyglucose Cell uptake test kit (Cosmo Bio co.ltd., japan). The specific experimental methods and results are as follows:

collecting human red blood cells by a conventional method: citric acid anticoagulated collected elbow vein whole blood 10mL, centrifuged at 1500r/min for 5 minutes, the white blood cells on the surface of plasma and erythrocytes were discarded, and the cells were washed 3 times (1500r/min, 4 minutes) with 10mL of phosphate buffer (pH 7.4), and the erythrocytes were stored in a 4-degree refrigerator in phosphate buffer for use. The red blood cell buffer suspension was counted and 5X10 was added ⁶ Each well was added to a 96-well plate, and set: add 200. mu.l (1 mM final concentration) of 2-deoxyglucose (purchased from Sigma) in PBS (pH 7.2) per well in the blank control group; ② the positive control group was added with 2-deoxyglucose containing a final concentration of 1mM and 10. mu.M cytochalasin B (purchased from Sigma) (total volume 200. mu.l); testing group: separately, 2-deoxyglucose containing a final concentration of 1mM and 100. mu.M of the GLUT inhibitor compound of the invention: (total volume 200. mu.l).

The blank control group, the positive control group and the test group were each provided with 3 wells, and the experiment was incubated at 37 ℃ for 1 hour. The cells were then washed 3 times (1500r/min, 4 min) with PBS cooled to zero (pH 7.2) and lysed with 5ml lysis buffer containing 10mM Tris-HCl buffer (pH 8.0, available from Solarbio) and 0.5% triton X-100 (available from Sigma) at 80 ℃ for 15 min. After lysis, the cells were centrifuged at 1500 rpm for 20 minutes at 4 ℃. The supernatant was subjected to a 2-deoxyglucose uptake test. The intracellular concentration of 2-Deoxyglucose taken up by erythrocytes via GLUT1 was tested in the form of 2-Deoxyglucose-6-phosphate using a 2-Deoxyglucose cell Uptake kit (2-Deoxyglucose (2DG) Uptake measurement kit, available from Cosmo Bio Co. Ltd., Japan). The results are shown in Table 3 below, after conversion to 100% sugar uptake for the blank control group:

table 3:

the positive control compound, cytochalasin B, is a known highly active inhibitor of GLUT1 (Proc Natl Acad Sci.2016,113(17): 4711-. The results in table 3 show that the compounds of the invention show varying degrees of inhibition against 2-deoxyglucose uptake by GLUT.

Test example 3: application of compound in inhibiting tumor growth

(1) Cytotoxicity test:

the cytotoxicity test adopts MTS test method. Tumor cells in log phase are collected, cell suspension concentration is adjusted, 100 mu L of cell suspension is added into each well, the density of the cells to be detected is adjusted to 10000 cells/well by plating, and the marginal wells are filled with sterile PBS. At 5% CO ₂ Incubate at 37 ℃ until the cell monolayer is confluent at the bottom of the well (96-well flat bottom plate), add a fixed concentration of GLUT inhibitor compound (50 μ M) at 100 μ L per well, and set 5 wells in duplicate. At 5% CO ₂ Incubation was carried out at 37 ℃ for 96 hours, and observed under an inverted microscope. To a 2mL MTS (2mg/mL, prepared DPBS) solution, 100. mu.L of PMS (1mg/mL, prepared DPBS) was added, and mixed to prepare an MTS working solution. Centrifuging the above cell culture plate, discarding culture solution, carefully washing with PBS for 2-3 times, adding 100 μ L culture medium into each well of 96-well plate, adding 20 μ L MTS working solution, and performing 5% CO treatment at 37 deg.C ₂ After incubation for 2h under conditions, the OD (optical density) was measured at 490 nm.

Control group:

the OD of the tumor cells at 490nm was obtained as a control for 100% survival of the cells without addition of the GLUT inhibitor compound to be tested under the same conditions as described above.

The experiment for each sample was repeated for 5 groups, and the average OD value was taken to calculate the cell viability in comparison with the control group.

(2) The experimental results are as follows:

TABLE 4

As shown in Table 4, the inhibitors of the cellular sugar transport channel of the present invention have inhibitory effects on breast cancer cells and lung cancer cells with different intensities.

Test example 4: test experiment of cancer cell high expression sugar transport channel protein

(1) Western Blotting immunoblotting test Experimental procedure:

1) cells were cultured in 100mm cell culture dishes, and the total cell amount and cell density of each dish were controlled to be comparable. After the cells were confluent, they were lysed with 600. mu.L of RIPA lysate (Biyunyan P0013B). Adding 6x Loading Buffer (Laibao technology D1010) into cell lysate, and incubating in water bath or metal bath at 95 deg.C for about 30 min.

2) SDS-PAGE gel electrophoresis analysis (gel concentration of 10% or 12%), and the amount of the sample was about 20. mu.L per well. After the glue running is finished, a transfer film is prepared. The method is semi-dry turning. The buffer used for the Transfer membrane was a Transfer buffer (shown in Table 1 below), the membrane used was a PVDF membrane (GE, soaked with methanol before use), and the filter paper was bio-rad brand (soaked with a Transfer buffer before Transfer).

3) And a film transferring experiment: the sequence from bottom to top is: filter paper, PVDF membrane, glue, filter paper. After being placed, the air bubbles are expelled out along one direction by using a test tube or a glass rod, and the membrane and the filter paper which are generally cut have the same size as the whole separation gel. After that, the cover above the instrument was closed. The membrane-rotating voltage is 20V, and the time is 35-40 min.

4) After turning the membrane, take the membrane out, mark the front (cut a little triangle in the upper left corner), then with 5% skimmed milk powder (TBST buffer configuration) (milk powder brand: solebao) was incubated at room temperature for 1h (20-50mL, etc.).

5) Washing the PVDF membrane by using TBST buffer, cutting the membrane according to the marker position (cutting the target protein and the internal reference), and then respectively incubating primary antibodies in small boxes.

6) Primary antibody was incubated at 4 ℃ overnight. Primary antibody was prepared using TBST + 0.5% skim milk powder (5 mL). The concentration of the primary antibody is determined according to the specification of the antibody, and the concentration generally used is the lowest concentration in the specification or 10 times higher than the lowest concentration in the specification.

7) And washing with TBST for 3 times (5 min, 10 min).

8) The secondary antibody was incubated at 4 ℃ for 1 h. Primary antibody was prepared using TBST + 0.5% skim milk powder (5 mL). The concentration of the secondary antibody is determined according to the antibody specification, and the lowest concentration of the antibody specification is generally used.

9) And washing with TBST for 3 times (5 min, 10 min).

10) The resulting mixture was developed with a chemiluminescent developing solution labeled with HRP enzyme (Millipore WBKLS0500), and the resultant was photographed by exposure in accordance with the instructions.

TABLE 5.10 XTransfer Buffer

Add ddH ₂ And (4) metering the volume of O to 800mL, and fully stirring. When used, 80mL of the solution was diluted to 800mL and 20% methanol (200mL) was added.

TABLE 6.10 XTBST Buffer

Add ddH ₂ After dissolving O to about 900mL, the pH was adjusted to 7.6 and the volume was adjusted to 1000 mL. When used, dilute to 1 ×, add 0.05% Tween 20.

(2) The experimental results are as follows:

the results of the experiment are shown in FIG. 4. Experimental results show that compared with normal cells of a human body, lung cancer and breast cancer used in the GLUT inhibitor for playing a role in tumor inhibition both highly express sugar transport channel protein.

Experiments prove that (I) - (A-1) -1, (I) - (A-2) -1, (I) - (A-3) -1, (I) - (A-1) -2, (I) - (A-2) -2, (I) - (A-3) -2, (I) - (A-1) -3, (I) - (A-2) -3, (I) - (A-3) -3, (I) - (A-1) -4, (I) - (A-2) -4, (I) - (A-3) -4, (I) - (A-1) -5, (I) - (A-2) -5, (I) - (A-3) -5, (I) - (A-1) -6, (I) - (A-2) -6, (I) - (A-3) -6, and the above-listed GLUT inhibitor compounds of the invention are capable of inhibiting the uptake of sugar molecules by the cell's sugar transport channel GLUT. Meanwhile, the GLUT sugar absorption channel protein is highly expressed by the tumor cells due to the Woberg effect, and the GLUT inhibitor has an inhibiting effect on the growth of the tumor cells.

Therefore, the cell sugar transport channel inhibitor provided by the invention can be used for preparing antitumor drugs.

As a pharmaceutical composition for antitumor, the above GLUT inhibitor compounds (I) - (A-1) -1, (I) - (A-2) -1, (I) - (A-3) -1, (I) - (A-1) -2, (I) - (A-2) -2, (I) - (A-3) -2, (I) - (A-1) -3, (I) - (A-2) -3, (I) - (A-3) -3, (I) - (A-1) -4, (I) - (A-2) -4, (I) - (A-3) -4, (I) - (A-1) -5, and, (I) - (A-2) -5, (I) - (A-3) -5, (I) - (A-1) -6, (I) - (A-2) -6, (I) - (A-3) -6), optionally one of them being the only active ingredient, the rest being a mixture of pharmaceutically acceptable adjuvants, such as excipients or diluents.

As a pharmaceutical composition for antitumor, the above GLUT inhibitor compounds (I) - (A-1) -1, (I) - (A-2) -1, (I) - (A-3) -1, (I) - (A-1) -2, (I) - (A-2) -2, (I) - (A-3) -2, (I) - (A-1) -3, (I) - (A-2) -3, (I) - (A-3) -3, (I) - (A-1) -4, (I) - (A-2) -4, (I) - (A-3) -4, (I) - (A-1) -5, and, (I) A pharmaceutically acceptable salt of (A-2) -5, (I) - (A-3) -5, (I) - (A-1) -6, (I) - (A-2) -6, (I) - (A-3) -6) is the only active ingredient.

As a pharmaceutical composition for antitumor, the above GLUT inhibitor compounds (I) - (A-1) -1, (I) - (A-2) -1, (I) - (A-3) -1, (I) - (A-1) -2, (I) - (A-2) -2, (I) - (A-3) -2, (I) - (A-1) -3, (I) - (A-2) -3, (I) - (A-3) -3, (I) - (A-1) -4, (I) - (A-2) -4, (I) - (A-3) -4, (I) - (A-1) -5, and, (I) A solvate of (A-2) -5, (I) - (A-3) -5, (I) - (A-1) -6, (I) - (A-2) -6, or (I) - (A-3) -6).

As a pharmaceutical composition for antitumor, the above GLUT inhibitor compounds (I) - (A-1) -1, (I) - (A-2) -1, (I) - (A-3) -1, (I) - (A-1) -2, (I) - (A-2) -2, (I) - (A-3) -2, (I) - (A-1) -3, (I) - (A-2) -3, (I) - (A-3) -3, (I) - (A-1) -4, (I) - (A-2) -4, (I) - (A-3) -4, (I) - (A-1) -5, and, (I) - (A-2) -5, (I) - (A-3) -5, (I) - (A-1) -6, (I) - (A-2) -6, (I) - (A-3) -6) and the balance of drug carriers.

As sugar transport channel protein GLUT inhibitory agents, the above GLUT inhibitor compounds (I) - (A-1) -1, (I) - (A-2) -1, (I) - (A-3) -1, (I) - (A-1) -2, (I) - (A-2) -2, (I) - (A-3) -2, (I) - (A-1) -3, (I) - (A-2) -3, (I) - (A-3) -3, (I) - (A-1) -4, (I) - (A-2) -4, (I) - (A-3) -4, (I) - (A-1) -5, and, (I) - (A-2) -5, (I) - (A-3) -5, (I) - (A-1) -6, (I) - (A-2) -6, (I) - (A-3) -6), optionally one of them being the only active ingredient, the rest being a mixture of pharmaceutically acceptable adjuvants, such as excipients or diluents.

As sugar transport channel protein GLUT inhibitory agents, the above GLUT inhibitor compounds (I) - (A-1) -1, (I) - (A-2) -1, (I) - (A-3) -1, (I) - (A-1) -2, (I) - (A-2) -2, (I) - (A-3) -2, (I) - (A-1) -3, (I) - (A-2) -3, (I) - (A-3) -3, (I) - (A-1) -4, (I) - (A-2) -4, (I) - (A-3) -4, (I) - (A-1) -5, and, (I) A pharmaceutically acceptable salt of (A-2) -5, (I) - (A-3) -5, (I) - (A-1) -6, (I) - (A-2) -6, (I) - (A-3) -6) is the only active ingredient.

As sugar transport channel protein GLUT inhibitory agents, the above GLUT inhibitor compounds (I) - (A-1) -1, (I) - (A-2) -1, (I) - (A-3) -1, (I) - (A-1) -2, (I) - (A-2) -2, (I) - (A-3) -2, (I) - (A-1) -3, (I) - (A-2) -3, (I) - (A-3) -3, (I) - (A-1) -4, (I) - (A-2) -4, (I) - (A-3) -4, (I) - (A-1) -5, and, (I) A solvate of (A-2) -5, (I) - (A-3) -5, (I) - (A-1) -6, (I) - (A-2) -6, or (I) - (A-3) -6).

As sugar transport channel protein GLUT inhibitory agents, the above GLUT inhibitor compounds (I) - (A-1) -1, (I) - (A-2) -1, (I) - (A-3) -1, (I) - (A-1) -2, (I) - (A-2) -2, (I) - (A-3) -2, (I) - (A-1) -3, (I) - (A-2) -3, (I) - (A-3) -3, (I) - (A-1) -4, (I) - (A-2) -4, (I) - (A-3) -4, (I) - (A-1) -5, and, (I) - (A-2) -5, (I) - (A-3) -5, (I) - (A-1) -6, (I) - (A-2) -6, (I) - (A-3) -6) and the balance of drug carriers.

The application of the pharmaceutical composition in preparing antitumor drugs.

The application of the compound in preparing a cell sugar transport channel protein GLUT inhibiting reagent.

Meanwhile, the GLUT inhibitor can provide a molecular tool for researching molecular mechanisms related to cell nutrition metabolism including tumors by blocking sugar molecule absorption of cells.

The foregoing is a detailed description of the present invention with reference to specific embodiments, and it should not be considered that the present invention is limited to the specific embodiments, and it is understood that various changes and substitutions may be made by those skilled in the art without departing from the spirit and scope of the present invention.

Claims

1. An inhibitor of a cellular sugar transport channel, characterized by the structure as shown in formula (I):

wherein,

XR ₁ and XR ₂ The same;

x is O;

R ₁ is a hydrogen atom;

R ₂ is a hydrogen atom;

y is F, Cl, PhSO ₂ PhSO, PhS or SO ₂ N(Me) ₂ 。

2. Use of the inhibitor of a cellular sugar transport channel according to claim 1 for the preparation of an anti-tumor medicament or for the preparation of an agent inhibiting the cellular sugar transport channel GLUT.

3. A pharmaceutical composition for inhibiting tumor, comprising the inhibitor of the sugar transport channel of claim 1 or a pharmaceutically acceptable salt or solvate thereof.

4. Use of the pharmaceutical composition for inhibiting tumor according to claim 3 in the preparation of an antitumor medicament.