CN113149930B - Cell sugar transport channel inhibitor - Google Patents

Abstract

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

Description

A cellular sugar transport channel inhibitor

technical field

The invention belongs to the technical field of biomedicine, and relates to a cell sugar transport channel inhibitor for inhibiting cell sugar nutrient absorption and targeting tumor Warburg effect, wherein the cell sugar transport channel is abbreviated as GLUT.

Background technique

At present, malignant tumors are a major disease that threatens human life and health. The best strategy to deal with tumors is to inhibit tumor growth without harming normal cells and tissues by targeting tumor-specific biological targets.

There are many methods for targeting anti-tumor, the basic principle is to use tumor biomarkers that exist specifically in tumor cells or tumor tissues, or have significant differences from normal cells or normal tissues, and selectively identify and inhibit these biomarkers. achieve the purpose of treating tumors. Studies have shown that one of the important physiological mechanisms for the continuous proliferation, spread and metastasis of malignant tumors is the continuous absorption and intake of the special nutrients it needs. These nutrients include sugar molecules, amino acids, vitamins, nucleosides, etc. German biochemist Otto Warberg found that healthy cells can efficiently use aerobic metabolism to produce enough energy from a limited source of carbohydrate nutrients, while most tumor cells still rely on large amounts of sugar even in the presence of sufficient oxygen. Absorption and sugar metabolism provide energy for itself, a phenomenon known as the Warburg effect. Warburg was therefore awarded the Nobel Prize in Physiology or Medicine in 1931 (Reference: Warburg, O. Science, 123: 309 (1956); "The Nobel Prize in Physiology or Medicine 1931". Nobelprize.org. <http: https://www.nobelprize.org/nobel_prizes/medicine/laureates/1931/>).

Based on the tumor Warburg effect, since tumor cells specifically and highly express the sugar transport channel GLUT compared with healthy cells, using GLUT as a tumor-specific marker is expected to achieve targeted therapy for tumors. As shown in formula (A), as a prior art, it was reported in literature that compound 1 as a GLUT inhibitor can prevent the sugar absorption and sugar accumulation of blood cancer cells HL-60 and U-937 (Salas M., et al., Am. J. Physiol. Cell Physiol., 305:C90 (2013)). Another literature reported that compound 2 could inhibit the glycolytic metabolism and tissue tumor cell growth of ovarian cancer (Yibao Ma, et al., Cancers, 11(1):33 (2019)). It has also been reported that compound 3 acts as a selective inhibitor of GLUT, but its effect in inhibiting tumor cells has not been verified (Siebeneicher H, et al. ChemMedChem. 2016, 11(20):2261-2271).

These GLUT inhibitors obtained by different drug screening methods provide a material basis for further verification of the clinical application of these lead compounds. However, the development of more validated GLUT-selective inhibitors is needed.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a cell sugar transport channel inhibitor that can effectively inhibit the cell sugar transport channel GLUT, and utilize the tumor Warburg effect, that is, the tumor cells overexpress GLUT compared with normal cells, and achieve the purpose of inhibiting the growth of the cell sugar transport channel. agent.

The second object of the present invention is to provide the use of a cell sugar transport channel inhibitor in the preparation of antitumor drugs or the use of a reagent for inhibiting the cell sugar transport channel GLUT.

The third object of the present invention is to provide a pharmaceutical composition for inhibiting tumor.

The fourth object of the present invention is to provide the use of a pharmaceutical composition for inhibiting tumors in the preparation of anti-tumor drugs.

The technical scheme of the present invention is summarized as follows:

A cell sugar transport channel inhibitor, the structure is shown in formula I:

in,

XR ₁ and XR ₂ are the same or different;

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

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

X is selected from O, S or NH;

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

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

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

Preferably, XR ₁ and XR ₂ are the same;

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

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

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 use of the above-mentioned cell sugar transport channel inhibitor in the preparation of antitumor drugs or the use of a reagent for inhibiting the cell sugar transport channel GLUT.

A pharmaceutical composition for inhibiting tumors, comprising the above-mentioned inhibitor of a cell sugar transport channel or a pharmaceutically acceptable salt or solvate thereof.

Use of the above-mentioned pharmaceutical composition for inhibiting tumor in the preparation of anti-tumor drugs.

The present invention has the following beneficial effects:

Experiments show that the cell sugar transport channel inhibitor of the present invention can target the cell sugar transport channel, and inhibit tumor cell proliferation by directly blocking the GLUT channel and the uptake of sugar nutrients by tumors.

Description of drawings

Figure 1 is a fluorescence absorption map of a cellular sugar transport channel inhibitor (I)-(A-1)-1;

Figure 2 is a fluorescence absorption map of a cellular sugar transport channel inhibitor (I)-(A-2)-1;

Figure 3 is a fluorescence absorption map of a cellular sugar transport channel inhibitor (I)-(A-3)-1;

Figure 4 shows the test results of human lung cancer A549 and breast cancer MDA-MB-468 cells expressing GLUT.

Detailed ways

The present invention will be described in further detail below through specific embodiments, which are intended to explain rather than limit the present invention.

A cell sugar transport channel inhibitor, hereinafter referred to as a 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-oxadiazole (II), its synthetic route is as follows:

4-Chloro-7-acetamidobenzo-2-oxa-1,3-oxadiazole (XIV, 678.3 mg, 3.0 mmol) was dissolved in 12 mL of acetic anhydride. With stirring at 0°C, 4 mL of concentrated nitric acid was slowly added dropwise to the mixture. And after stirring was continued at 0 degreeC for 3 hours, ice water was added. After stirring for 10 min, the mixture was filtered, and the filter cake was washed three times with cold water to obtain 753.3 mg of yellow solid (II) in a yield of 59%. ¹ HNMR (600MHz, DMSO-d6): δ11.51(s,1H), 8.19(s,1H), 2.19(s,3H).

The reaction formula is:

2. The synthesis of 4-chloro-6-nitro-7-aminobenzo-2-oxa-1,3-oxadiazole (III), its synthetic route is as follows:

II (256.6 _mg , 1.0 mmol) was added to a 50% wt. _H2SO4 aqueous solution (3 mL), and the mixture was stirred at 100°C for 2 hours. The mixture was cooled to room temperature, adjusted to pH 8 with saturated aqueous sodium bicarbonate solution, extracted with ethyl acetate (20 mL×2), and the organic phase was dried over anhydrous sodium sulfate. The solvent was removed with a rotary evaporator to obtain 191.1 mg of yellow solid (III) in a yield of 89%. ¹ HNMR (600MHz, DMSO-d6): δ9.83(s,1H), 9.35(s,1H), 8.14(s,1H).

The reaction formula is:

3. The synthesis of 4-chloro-6,7-diaminobenzo-2-oxa-1,3-oxadiazole (IV), its synthetic route is as follows:

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

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

The reaction formula is:

4. Synthesis of 4-tert-butyldimethylsiloxybenzoyl chloride (VII):

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

The obtained colorless liquid was dissolved in a mixed solvent of glacial acetic acid (50 mL), tetrahydrofuran (15 mL) and water (15 mL), and stirred at room temperature for 4 h. Ethyl acetate (60 mL) and water (60 mL) were added, 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 with a rotary evaporator, and the obtained residue was dried under high vacuum to obtain 3.7 g of 4-tert-butyldimethylsiloxybenzoic acid (VI) as a white solid with a yield of 68%.

VI (2.5 g, 10.0 mmol)), N,N-dimethylformamide (DMF) (2 drops) and anhydrous dichloromethane (DCM) (20 mL) were added under nitrogen protection. At 0 °C, oxalyl chloride (1.0 mL, 12.0 mmol) was added dropwise to the reaction solution, stirred at room temperature for 3 h, and the solvent was evaporated to dryness with a rotary evaporator. The obtained VII was used in the next step without further purification. reaction.

The reaction formula is:

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

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

The reaction formula is:

Example 2

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

1. Synthesis of 3-tert-butyldimethylsiloxybenzoyl chloride (X):

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

The obtained colorless liquid was dissolved in a mixed solvent of glacial acetic acid (50 mL), tetrahydrofuran (15 mL) and water (15 mL), and stirred at room temperature for 4 h. Ethyl acetate (60 mL) and water (60 mL) were added, 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 with a rotary evaporator, and the obtained residue was dried under high vacuum to obtain 3.7 g of 3-tert-butyldimethylsiloxybenzoic acid (IX) as a white solid with a yield of 87%.

IX (2.5 g, 10.0 mmol)), N,N-dimethylformamide (DMF) (2 drops) and anhydrous dichloromethane (DCM) (20 mL) were added under nitrogen protection. At 0 °C, oxalyl chloride (1.0 mL, 12.0 mmol) was added dropwise to the reaction solution, stirred at room temperature for 3 h, and the solvent was evaporated to dryness with a rotary evaporator to obtain 3-tert-butyldimethylsilicon. Oxybenzoyl chloride (X) was used in the next reaction without further purification.

The reaction formula is:

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

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

The reaction formula is:

Example 3

Synthesis of GLUT Inhibitor (I)-(A-3)-1:

1. Synthesis of 3,5-bis(tert-butyldimethylsiloxy)benzoyl chloride (XIII):

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

The obtained colorless liquid was dissolved in a mixed solvent of glacial acetic acid (50 mL), tetrahydrofuran (15 mL) and water (15 mL), and stirred at room temperature for 4 h. Ethyl acetate (60 mL) and water (60 mL) were added, 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 with a rotary evaporator, and the obtained residue was dried under high vacuum to obtain 4.0 g of 3,5-bis(tert-butyldimethylsiloxy)benzoic acid (XII) as a white solid in a yield of 52%.

Under nitrogen protection, 3,5-bis(tert-butyldimethylsiloxy)benzoic acid (XII, 3.8 g, 10.0 mmol)), N,N-dimethylformamide (DMF) (2 drops) were added ) and anhydrous dichloromethane (DCM) (20 mL). At 0 °C, oxalyl chloride (1.0 mL, 12.0 mmol) was added dropwise to the reaction solution, stirred at room temperature for 3 h, and the solvent was evaporated to dryness with a rotary evaporator to obtain 3-tert-butyldimethylsilicon. Oxybenzoyl chloride (XIII) was used in the next reaction without further purification.

The reaction formula is:

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

To a solution of 4-chloro-6,7-diaminobenzo-2-oxa-1,3-oxadiazole (IV, 184.6 mg, 0.6 mmol) in tetrahydrofuran (4 ml) was sequentially added 3-tert-butyldimethylmethane Siloxybenzoyl chloride (XIII, 918.4 mg, 2.4 mmol) in tetrahydrofuran (4 ml), potassium carbonate (207.3 mg, 1.5 mmol), the reaction solution was placed in a sealed tube and stirred at 80°C for 12 hours. The reaction solution was cooled to room temperature, filtered, and the filter residue was washed twice with DCM. The solvent of the reaction solution was removed by a rotary evaporator, and purified by silica gel column chromatography (petroleum ether:ethyl acetate=100:1) to obtain a pale yellow solid. The solid was dissolved in THF (4 mL), an appropriate amount of 1M tetra-n-butylammonium fluoride (TBAF) solution in tetrahydrofuran (THF) was added dropwise with stirring at room temperature, and after stirring for 0.5 h, dichloromethane (20 mL) was added, followed by water, Washed with saturated NaCl solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate:methanol=20:1) to give a pale yellow solid (I)-(A-3)-1 79.4 mg, yield 29%.

The reaction formula is:

Example 4

The synthesis of GLUT inhibitor (I)-(A-1)-2 was synthesized according to the method of Example 1, and the 4-chloro-7-acetamidobenzo-2-oxa-1,3-di The oxazole was replaced with 4-fluoro-7-acetamidobenzo-2-oxa-1,3-oxadiazole, and the others were the same as those in Example 1, and the GLUT inhibitor (I)-(A-1)-2 was prepared.

Example 5

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

Substitute 4-chloro-7-acetamidobenzo-2-oxa-1,3-oxadiazole of Example 1 with 4-fluoro-7-acetamidobenzo-2-oxa-1,3-diazole azole, other steps are the same as in Example 1, to prepare 4-fluoro-6,7-diaminobenzo-2-oxa-1,3-oxadiazole; then 4-fluoro-6,7-diaminobenzoxy -2-oxa-1,3-oxadiazole and 3-tert-butyldimethylsiloxybenzoyl chloride (X) prepared in Example 2 were reacted according to the steps of Example 2 to prepare a GLUT inhibitor ( I)-(A-2)-2.

Example 6

Synthesis of GLUT inhibitor (I)-(A-3)-2:

Substitute 4-chloro-7-acetamidobenzo-2-oxa-1,3-oxadiazole of Example 1 with 4-fluoro-7-acetamidobenzo-2-oxa-1,3-diazole azole, other steps are the same as in Example 1, to prepare 4-fluoro-6,7-diaminobenzo-2-oxa-1,3-oxadiazole; then 4-fluoro-6,7-diaminobenzoxy -2-oxa-1,3-oxadiazole and 3,5-bis(tert-butyldimethylsiloxy)benzoyl chloride (XIII) prepared in Example 3 were reacted according to the steps in Example 3 to prepare GLUT inhibitor (I)-(A-3)-2.

Example 7

The synthesis of GLUT inhibitor (I)-(A-1)-3 was synthesized according to the method of Example 1, and the 4-chloro-7-acetamidobenzo-2-oxa-1,3-di The azole was replaced with 4-benzenesulfonyl-7-acetamidobenzo-2-oxa-1,3-oxadiazole, and the others were the same as in Example 1, and GLUT inhibitor (I)-(A-1)-3 was prepared .

Example 8

Synthesis of GLUT inhibitor (I)-(A-2)-3:

The 4-chloro-7-acetamidobenzo-2-oxa-1,3-oxadiazole of Example 1 was replaced with 4-benzenesulfonyl-7-acetamidobenzo-2-oxa-1,3 -Diazole, other steps are the same as in Example 1, to prepare 4-benzenesulfonyl-6,7-diaminobenzo-2-oxa-1,3-oxadiazole; then 4-benzenesulfonyl-6, 7-diaminobenzo-2-oxa-1,3-oxadiazole and 3-tert-butyldimethylsiloxybenzoyl chloride (X) prepared in Example 2 were reacted according to the steps of Example 2, GLUT inhibitor (I)-(A-2)-3 was prepared.

Example 9

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

The 4-chloro-7-acetamidobenzo-2-oxa-1,3-oxaazole of Example 1 was replaced with 4-benzenesulfonyl--7-acetamidobenzo-2-oxa-1, 3-Diazole, other steps are 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-oxadiazole and 3,5-bis(tert-butyldimethylsiloxy)benzoyl chloride (XIII) prepared in Example 3, According to the steps of Example 3, the GLUT inhibitor (I)-(A-3)-3 was prepared.

Example 10

The synthesis of GLUT inhibitor (I)-(A-1)-4 was synthesized according to the method of Example 1, and the 4-chloro-7-acetamidobenzo-2-oxa-1,3-di The oxazole was replaced with 4-phenylsulfinyl-7-acetamidobenzo-2-oxa-1,3-oxadiazole, the others were the same as in Example 1, and the GLUT inhibitor (I)-(A-1)- 4.

Example 11

Synthesis of GLUT inhibitor (I)-(A-2)-4:

The 4-chloro-7-acetamidobenzo-2-oxa-1,3-oxadiazole of Example 1 was replaced with 4-benzenesulfinyl-7-acetamidobenzo-2-oxa-1, 3-Diazole, other steps are the same as in Example 1, to prepare 4-phenylsulfinyl-6,7-diaminobenzo-2-oxa-1,3-oxadiazole; then 4-phenylsulfinyl -6,7-Diaminobenzo-2-oxa-1,3-oxadiazole and 3-tert-butyldimethylsiloxybenzoyl chloride (X) prepared in Example 2, according to the method of Example 2 Step reaction, prepare GLUT inhibitor (I)-(A-2)-4.

Example 12

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

The 4-chloro-7-acetamidobenzo-2-oxa-1,3-oxadiazole of Example 1 was replaced with 4-benzenesulfinyl-7-acetamidobenzo-2-oxa-1, 3-Diazole, other steps are the same as in Example 1, to prepare 4-phenylsulfinyl-6,7-diaminobenzo-2-oxa-1,3-oxadiazole; then 4-phenylsulfinyl -6,7-Diaminobenzo-2-oxa-1,3-oxadiazole and 3,5-bis(tert-butyldimethylsiloxy)benzoyl chloride (XIII) prepared in Example 3, According to the steps of Example 3, the GLUT inhibitor (I)-(A-3)-4 was prepared.

Example 13

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

Synthesize following the method of Example 1, replacing the 4-chloro-7-acetamidobenzo-2-oxa-1,3-oxadiazole of Example 1 with 4-phenylmercapto-7-acetamidobenzo-2 -Oxa-1,3-oxadiazole, the others are the same as in Example 1, and GLUT inhibitor (I)-(A-1)-5 was prepared.

Example 14

Synthesis of GLUT inhibitor (I)-(A-2)-5:

The 4-chloro-7-acetamidobenzo-2-oxa-1,3-oxadiazole of Example 1 was replaced by 4-phenylmercapto-7-acetamidobenzo-2-oxa-1,3- oxadiazole, other steps are the same as in Example 1, to prepare 4-phenylmercapto-6,7-diaminobenzo-2-oxa-1,3-oxadiazole; then 4-phenylmercapto-6,7-diazole Aminobenzo-2-oxa-1,3-oxadiazole and 3-tert-butyldimethylsiloxybenzoyl chloride (X) prepared in Example 2 were reacted according to the steps of Example 2 to prepare GLUT Inhibitor (I)-(A-2)-5.

Example 15

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

The 4-chloro-7-acetamidobenzo-2-oxa-1,3-oxadiazole of Example 1 was replaced by 4-phenylmercapto-7-acetamidobenzo-2-oxa-1,3- oxadiazole, other steps are the same as in Example 1, to prepare 4-phenylmercapto-6,7-diaminobenzo-2-oxa-1,3-oxadiazole; then 4-phenylmercapto-6,7-diazole Aminobenzo-2-oxa-1,3-oxadiazole and 3,5-bis(tert-butyldimethylsiloxy)benzoyl chloride (XIII) prepared in Example 3, according to the procedure of Example 3 After the reaction, the GLUT inhibitor (I)-(A-3)-5 was prepared.

Example 16

The synthesis of GLUT inhibitor (I)-(A-1)-6 follows the method of Example 1, and the

4-Chloro-7-acetamidobenzo-2-oxa-1,3-oxadiazole was replaced by 4-dimethylaminosulfonyl-7-acetamidobenzo-2-oxa-1,3-diazole oxazole, and others were the same as in Example 1, and GLUT inhibitor (I)-(A-1)-6 was prepared.

Example 17

Synthesis of GLUT inhibitor (I)-(A-2)-6:

Substitute 4-chloro-7-acetamidobenzo-2-oxa-1,3-oxadiazole of Example 1 with 4-dimethylaminosulfonyl-7-acetamidobenzo-2-oxa- 1,3-Diazole, other steps are the same as in Example 1, to prepare 4-dimethylaminosulfonyl-6,7-diaminobenzo-2-oxa-1,3-oxadiazole; then 4- Dimethylaminosulfonyl-6,7-diaminobenzo-2-oxa-1,3-oxadiazole and 3-tert-butyldimethylsiloxybenzoyl chloride (X) prepared in Example 2 , and react according to the steps of Example 2 to prepare GLUT inhibitor (I)-(A-2)-6.

Example 18

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

Substitute 4-chloro-7-acetamidobenzo-2-oxa-1,3-oxadiazole of Example 1 with 4-dimethylaminosulfonyl-7-acetamidobenzo-2-oxa- 1,3-Diazole, other steps are the same as in Example 1, to prepare 4-dimethylaminosulfonyl-6,7-diaminobenzo-2-oxa-1,3-oxadiazole; then 4- Dimethylaminosulfonyl-6,7-diaminobenzo-2-oxa-1,3-oxadiazole and 3,5-bis(tert-butyldimethylsiloxy)benzene prepared in Example 3 Formyl chloride (XIII) was reacted according to the procedure of Example 3 to prepare GLUT inhibitor (I)-(A-3)-6.

Table 1 lists the structures of some compounds of general formula I

Table 1

Table 2 lists the nuclear magnetic resonance, mass spectrometry and fluorescence spectrum data of each compound in Table 1

Table 2

Test Example 1: Other Spectroscopic Tests of Representative Compounds of the Invention

1) Experimental apparatus: Thermo Scientific Varioskan LUX.

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

Test solvent: DMSO:PBS(pH=7.2)=1:9(V:V)

3) Experimental steps

(1) Each test compound was weighed.

(2) Dissolve the compound to 10 mM with DMSO, and vortex for 1 min to fully dissolve the compound.

(3) Dilute the compound to 500 uM with PBS-DMSO (1:1, volume ratio), and vortex to dissolve. Take 100ul of each compound and test solvent, add it to a black bottom transparent 96-well culture plate, and use a multi-function microplate reader to detect the absorption spectrum, excitation spectrum and emission spectrum.

4) Data processing: processed with OriginPro 8.5 to derive absorption spectrum, emission spectrum, and absorption-emission composite spectrum.

5) Results: as shown in Table 2. Excitation and emission spectra of representative GLUT inhibitors are shown in the accompanying drawings (Figures 1-3).

Test Example 2: Inhibitory effect of GLUT inhibitor on GLUT1

According to literature reports, human and mammalian erythrocytes mainly express the GLUT1 sugar transport channel protein for sugar absorption (M.Mueckler, C.Caruso, S.A.Baldwin, I.Blench, H.R.Morris, W.J.Allard, G.E.Lienhard, H.F.Lodish, Science, 1985 , 229, 941; (b) Montel-Hagen, S. Kinet, N. Manel, C. Mongellaz, R. Prohaska, J. L. Battini, J. Delaunay, M. Sitbon, N. Taylor, Cell, 2008, 132, 1039), In this experiment, human red blood cells and 2-deoxyglucose cell uptake test kits (Cosmo Bio Co. Ltd., Japan) were used to evaluate the GLUT inhibitory effect of the GLUT inhibitor molecules of the present invention. The specific experimental methods and results are as follows:

Human red blood cells were collected by conventional methods: 10 mL of cubital vein whole blood collected by citric acid anticoagulation was centrifuged at 1500 r/min for 5 minutes, the plasma and leukocytes on the surface of red blood cells were discarded, and the cells were washed with 10 mL of phosphate buffer (pH 7.4) 3 times (1500 r/min, 4 minutes), red blood cells were stored in phosphate buffered saline and stored in a refrigerator at 4 degrees until use. The above red blood cell buffer suspension was counted and added to 96-well plate with 5× ^{10 6} per well. Setting: ① In the blank control group, add 200 microliters of 2-deoxyglucose (purchased from Sigma) dissolved in PBS (pH 7.2) to each well. liter (final concentration of 1 mM); ② positive control group added 2-deoxyglucose and 10 μM cytochalasin B (purchased from Sigma) (total volume 200 μl) with a final concentration of 1 mM; 2-Deoxyglucose at a concentration of 1 mM and 100 [mu]M GLUT inhibitor compound of the invention: (200 [mu]l total volume).

The blank control group, the positive control group, and the test group were each set with 3 wells, and the experiment was incubated at 37°C for 1 hour. Cells were then washed 3 times (1500 r/min, 4 min) with PBS (pH=7.2) cooled to zero degrees, and then washed with 5 mL of Tris-HCl buffer (pH 8.0, purchased from Solarbio) containing 10 mM and 0.5% triton Lysis buffer of X-100 (purchased from Sigma) was performed at 80°C for 15 minutes. After lysis, the cells were placed at 4°C and centrifuged at 1500 r/min for 20 minutes. A 2-deoxyglucose uptake test was performed on the supernatant. The intracellular concentration of 2-deoxyglucose absorbed by erythrocytes through GLUT1 was obtained in the form of 2-deoxyglucose-6-phosphate using the 2-Deoxyglucose (2DG) Uptake measurement kit, purchased from Cosmo Bio Co. Ltd., Japan) for testing. The test results are as follows in Table 3 after conversion with the sugar absorption of the blank control group as 100%:

table 3:

The positive control compound, cytochalasin B, is a known highly active inhibitor of GLUT1 (Proc Natl AcadSci. 2016, 113(17):4711-4716). The results in Table 3 show that the compounds of the present invention show different degrees of inhibition on the 2-deoxyglucose absorption of GLUT.

Test Example 3: Effects of Compounds in Inhibiting Tumor Growth

(1) Cytotoxicity test:

Cytotoxicity experiments were performed using the MTS test method. Collect log-phase tumor cells, adjust the concentration of cell suspension, add 100 μL to each well, and plate the cells to adjust the density to 1000-10000 cells/well (the edge wells are filled with sterile PBS). Incubate at 5% CO ₂ at 37° C. until the cell monolayer covers the bottom of the well (96-well flat bottom plate), add a fixed concentration of GLUT inhibitor compound (50 μM), 100 μL per well, and set 5 replicate wells. Incubate for 96 hours at 37°C with 5% CO ₂ and observe under an inverted microscope. Add 100 μL of PMS (1 mg/mL, prepared in DPBS) to 2 mL MTS (2 mg/ml, prepared in DPBS) solution, and mix well to prepare MTS working solution. After centrifuging the above cell culture plate, discard the culture medium, rinse carefully with PBS for 2-3 times, before measuring the absorbance, add 100 μL of culture medium to each well of the 96-well plate, and then add 20 μL of MTS working solution. After 2 h incubation under %CO ₂ conditions, the OD value (optical density value) was detected at 490 nm.

Control group:

Under the same conditions as above, the tested GLUT inhibitor compound was not added, and finally the OD value of tumor cells was measured at 490 nm, which was used as a control for 100% survival of the cells.

The above experiments for each sample were repeated for 5 groups, the average OD value was taken, and the cell viability was calculated by comparing with the control group.

(2) Experimental results:

Table 4

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

Test Example 4: Test Experiment of Cancer Cells Highly Expressing Sugar Transport Channel Proteins

(1) Experimental steps of Western Blotting test:

1) Cultivate cells in a 100mm culture cell dish, and control the total amount of cells and cell density in each dish. After the cells were confluent, they were lysed with 600 μL of RIPA lysis solution (Biyuntian P0013B). Add 6x Loading Buffer (Leybold Technology D1010) to the cell lysate and incubate at 95°C in a water bath or metal bath for about 30min.

2), SDS-PAGE gel electrophoresis analysis (separating gel concentration is 10% or 12%), and the loading volume of each well is about 20 μL. After running the gel, prepare to transfer the membrane. The method is semi-dry rotation. The buffer used for membrane transfer is transfer buffer (as shown in Table 1 below), the membrane used is PVDF membrane (GE Company, soaked in methanol before use), and the filter paper is bio-rad brand (soaked with Transfer buffer before membrane transfer).

3), membrane transfer experiment: the order from bottom to top is: filter paper, PVDF membrane, glue, filter paper. After placing, use a test tube or glass rod to drive out the air bubbles in one direction. Generally, the cut membrane and filter paper are the same size as the entire separation gel. After that, close the lid on the instrument. The membrane transfer voltage was 20V, and the time was 35min-40min.

4) After transferring the membrane, take out the membrane, mark the front side (cut off a small triangle in the upper left corner), and then incubate with 5% nonfat milk powder (configured with TBST buffer) (milk powder brand: Solebao) at room temperature for 1h (20- 50mL range).

5) Wash the PVDF membrane with TBST buffer, then cut the membrane according to the position of the marker (the target protein and the internal reference are cut), and then incubate the primary antibody in a small box.

6) Incubate the primary antibody at 4°C overnight. The primary antibody was prepared with TBST + 0.5% nonfat dry milk (5 mL). The concentration of the primary antibody is determined according to the instructions of the antibody. Generally, the concentration used is the minimum concentration stated in the instructions, or 10 times higher than the minimum concentration in the instructions.

7), wash 3 times with TBST, the time is 5min, 10min, 10min respectively.

8) Incubate the secondary antibody at 4°C for 1h. The primary antibody was prepared with TBST + 0.5% nonfat dry milk (5 mL). The concentration of the secondary antibody is determined according to the specification of the antibody, and the lowest concentration in the specification is generally used.

9), wash 3 times with TBST, the time is 5min, 10min, 10min respectively.

10) Develop color with HRP enzyme-labeled chemiluminescence chromogenic solution (Millipore WBKLS0500), operate according to the instructions, and take pictures by exposure.

Table 5. 10×Transfer Buffer

Add ddH ₂ O to make up to 800 mL, and stir well. When using, take 80mL and dilute to 800mL, then add 20% methanol (200mL).

Table 6. 10×TBST Buffer

After adding ddH ₂ O to dissolve to about 900 mL, the pH was adjusted to 7.6, and the volume was adjusted to 1000 mL. Dilute to 1X when used and add 0.05% Tween 20.

(2) Experimental results:

The experimental results are shown in Figure 4. The experimental results show that the lung cancer and breast cancer used in the tumor suppressing effect of the GLUT inhibitor of the present invention both highly express the sugar transport channel protein compared with normal human cells.

Experiments show 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, the GLUT inhibitor compounds of the present invention listed above can target the sugar transport channel GLUT of cells and inhibit the absorption of sugar molecules. At the same time, tumor cells highly express GLUT sugar absorption channel protein due to the Warburg effect, and this type of GLUT inhibitor has an inhibitory effect on the growth of tumor cells.

Therefore, the cellular sugar transport channel inhibitor provided by the present invention can be used to prepare antitumor drugs.

As an anti-tumor pharmaceutical composition, including the above-mentioned GLUT inhibitor compounds (respectively (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), optionally one of them is the only active ingredient, and the rest are pharmaceutically acceptable A mixture of excipients, such as excipients or diluents.

As an anti-tumor pharmaceutical composition, including the above-mentioned GLUT inhibitor compounds (respectively (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 pharmaceutically acceptable salt of -(A-1)-6, (I)-(A-2)-6, (I)-(A-3)-6), is the only active ingredient.

As an anti-tumor pharmaceutical composition, including the above-mentioned GLUT inhibitor compounds (respectively (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) - Solvates of (A-1)-6, (I)-(A-2)-6, (I)-(A-3)-6),.

As an anti-tumor pharmaceutical composition, including the above-mentioned GLUT inhibitor compounds (respectively (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 rest are drug carriers.

As sugar transport channel protein GLUT inhibitory reagents, including the above-mentioned GLUT inhibitor compounds (respectively (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), optionally one of them is the only active ingredient, and the rest are pharmaceutically acceptable A mixture of excipients, such as excipients or diluents.

As sugar transport channel protein GLUT inhibitory reagents, including the above-mentioned GLUT inhibitor compounds (respectively (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), a pharmaceutically acceptable salt of which is the only active ingredient.

As sugar transport channel protein GLUT inhibitory reagents, including the above-mentioned GLUT inhibitor compounds (respectively (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), solvates.

As sugar transport channel protein GLUT inhibitory reagents, including the above-mentioned GLUT inhibitor compounds (respectively (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 rest are drug carriers.

Use of the above pharmaceutical composition in the preparation of antitumor drugs.

Use of the above compound in the preparation of a reagent for inhibiting cellular sugar transport channel protein GLUT.

At the same time, as a GLUT inhibitor, it can also provide a molecular tool for the study of molecular mechanisms related to cell nutrition metabolism, including tumors, by blocking the absorption of sugar molecules in cells.

The above content is a further detailed description of the present invention in conjunction with the specific embodiments, and it cannot be considered that the specific implementation of the present invention is limited to these descriptions. Below, some simple deductions or substitutions can also be made, all of which should be regarded as belonging to the protection scope of the present invention.

Claims (4)

1. a cell sugar transport channel inhibitor, it is characterized in that the structure is as shown in formula (I):

in, XR ₁ and XR ₂ are the same; XR ₁ represents mono- or di-substitution at different positions of the benzene ring; XR ₂ represents mono- or di-substitution at different positions of the benzene ring; 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. The use of a cell sugar transport channel inhibitor according to claim 1 in the preparation of antitumor drugs or the use of a reagent for inhibiting cell sugar transport channel GLUT. 3. A pharmaceutical composition for inhibiting tumor, characterized by comprising a cell sugar transport channel inhibitor according to claim 1 or a pharmaceutically acceptable salt or solvate thereof. 4. Use of the tumor-inhibiting pharmaceutical composition of claim 3 in the preparation of an anti-tumor drug.

Images