Glutaminase micromolecular fluorescent probe and preparation method and application thereof

A glutaminase, fluorescent probe technology, applied in fluorescence/phosphorescence, chemical instruments and methods, analytical materials, etc., can solve the problems of false positives and false positives in experimental data, and achieve a simple test system with little solvent influence. Effect

Active Publication Date: 2019-12-20
CHINA PHARM UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But with H 2 o 2 Inhibitors that react will produce false positive results, such as using this method to determine the inhibitory activity of ebselen on GLS1, IC 50 About 9nM, so ebselen is considered to be a potent GLS1 inhibitor
However, studies have shown that ebselen does not inhibit glutaminase, but reacts with the H generated in the reaction 2 o 2 A reaction occurred, causing a false positive in the experimental data

Method used

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  • Glutaminase micromolecular fluorescent probe and preparation method and application thereof
  • Glutaminase micromolecular fluorescent probe and preparation method and application thereof
  • Glutaminase micromolecular fluorescent probe and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Preparation of fluorescent probe Probe 1

[0060]

[0061] 5-(But-3-yn-1-yl)-1,3,4-thiadiazol-2-amine (4)

[0062] Add thiosemicarbazide (1.39 g, 0.015 mol) to the phosphorus oxychloride solution of compound 3 (1.5 g, 0.015 mol), and the reaction mixture was refluxed for 4 hours at 80 ° C. After cooling to room temperature, the reaction mixture was added into 30ml of ice water, adjust the pH to 9 with sodium hydroxide, extract the aqueous layer with ethyl acetate (15ml*3), dry over anhydrous sodium sulfate, filter and concentrate to obtain 1.88g of tan powder, yield 80.2%. 1 H NMR (300MHz, DMSO-d 6 ): δ6.99(s, 2H), 2.95(t, J=7.0Hz, 2H), 2.83(d, J=2.4Hz, 1H), 2.50(dd, J=7.1, 4.6Hz, 2H).HRMS (ESI): m / z, calcd for C 6 h 7 N 3 S[M+H] + , 154.0433; found: 154.0434.

[0063] N-(5-(but-3-yn-1-yl)-1,3,4-thiadiazol-2-yl)-2-(pyridin-2-yl)acetamide (5)

[0064] To compound 4 (3g, 0.02mol) in N,N-dimethylformamide solution, add 2-pyridine acetate hydrochloride (3.74g, 0....

Embodiment 2

[0078] Preparation of fluorescent probe Probe 2

[0079] Scheme 2. Synthesis of probe 2 a

[0080]

[0081] a Reagents and conditions: (a) N-boc-ethylenediamine, trimethylamine, DMF, DCM, rt, 1h, 76%; (b) trifluoroacetic acid, DCM, rt, 10h, 81%; (c) 8, HATU, DIPEA, DMF, 35°C, 1h, 52%.(2-((7-nitrobenzo[1,2,5]oxadiazol-4-yl)amino)ethyl)carbamate tert-butyl ester (12)

[0082] To a solution of compound 11 (0.5 g, 2.51 mmol) in N,N-dimethylformamide was added mono-boc ethylenediamine (0.4 g, 2.51 mmol) and trimethylamine (0.28 g, 2.77 mmol) dissolved in dichloromethane ), the reaction mixture was stirred at room temperature for 1 h, the mixture was added to water (30 ml), the aqueous layer was extracted with ethyl acetate (10 ml * 3), dried over anhydrous sodium sulfate, filtered and concentrated to obtain a solid 0.62 g, yield 76%. 1 H NMR (400MHz, CDCl 3 ): δ8.49(d, J=8.6Hz, 1H), 7.69(s, 1H), 6.18(d, J=8.6Hz, 1H), 5.12(s, 1H), 3.63(s, 4H), 1.48 (s, 9H).HRMS(ESI): m / z, c...

Embodiment 3

[0088] Preparation of fluorescent probe Probe3

[0089] Scheme 3. Synthesis of probe 3 a

[0090]

[0091] N 1 -(2-(3-(3′,6′-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-6-yl)thioureido)ethyl )-N 5 -(5-(4-(5-(2-phenylacetylamino)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazol-2-yl ) glutaramide (probe 3)

[0092] Please refer to Example 1 for the synthesis method. Yellow solid, yield 36.2%. mp141-143℃. 1 H NMR (300MHz, DMSO-d 6 ): δ12.82(s, 1H), 10.99(s, 1H), 10.00(s, 1H), 9.47(s, 1H), 8.53(d, 2H), 8.37(d, 1H), 8.23(s, 1H), 8.02(d, 1H), 7.60(s, 1H), 7.44(d, 1H), 7.26(d, 1H), 5.34(d, 1H), 4.79-4.71(m, 2H), 3.24(d , 2H), 2.06(s, 3H)ppm; 13 CNMR (75MHz, DMSO-d 6 ): δ178.2, 172.0, 168.3, 164.4, 159.9, 158.5, 155.3, 154.5, 149.5, 145.8, 144.9, 144.5, 138.4, 137.9, 136.1, 133.7, 131.2, 125.4, 123.9, 18, 103.3, 116 57.7, 45.6, 37.67, 35.74, 34.99, 34.70, 28.7, 23.9ppm; HRMS (ESI): m / z, calcd for C 44 h 42 N 9 o 8 S 3 [M+H] + , 920.2313; found: 920.2319. ...

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Abstract

The invention discloses a glutaminase micromolecular fluorescent probe with a general formula (I) and a preparation method and application of the glutaminase micromolecular fluorescent probe. The structural module of the probe comprises a glutaminase binding group, a linker chain and a fluorescent reporter group. The glutaminase micromolecular fluorescent probe can be used for high-throughput screening of glutaminase inhibitors and guiding discovery and structure optimization of the glutaminase inhibitors. By using the probe with the structure as a tool molecule, the binding condition of a micromolecular compound and a target protein can be rapidly and accurately confirmed.

Description

technical field [0001] The invention relates to a fluorescent probe and its preparation method and use, in particular to a glutaminase small molecule fluorescent probe and its preparation method and use. Background technique [0002] The metabolism of cancer cells is significantly different from that of normal cells. In normal cells, glucose is mainly converted into pyruvate, which enters the TCA cycle of the mitochondria to generate energy. However, due to the Warburg effect, tumor cells convert most of the glucose into lactic acid and excrete the cells, which cannot be used in the TCA cycle. As compensation, tumor cells increase the level of glutaminase (GLS1), so that tumor cells can use glutamine as an energy source, and through the catalysis of GLS and glutamate dehydrogenase, enter TCA to generate energy. Therefore, targeted inhibition of GLS1 can selectively inhibit tumor growth without inhibiting the growth of normal cells, which is an effective strategy for the tr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D493/10C07D417/14C09K11/06G01N21/64
CPCC07D417/14C07D493/10C09K11/06C09K2211/1007C09K2211/1029C09K2211/1044C09K2211/1048C09K2211/1051C09K2211/1088G01N21/6445Y02P20/55
Inventor 李志裕卞金磊徐熙匡自建孟颖
Owner CHINA PHARM UNIV
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