1,3,4-oxadiazole derivative and preparation method and application thereof

Abstract

The invention discloses a 1,3,4-oxadiazole derivative and a synthetic method thereof, and an application of the derivative serving as a fluorescent indicator to detection of intracellular calcium ions (Ca<2+>). The method comprises the following steps of: under the protection of inert gas, performing a wittig reaction on the 1,3,4-oxadiazole derivative and a calcium ion response group to obtain a crude product; and concentrating the crude product, and separating through a silica gel column to obtain a pure product. The 1,3,4-oxadiazole derivative has the advantages of high sensitivity and high selectivity on Ca<2+>, large Stocks displacement, proportional measurement and the like; the color of the indicator changes greatly before and after bonding with Ca<2+>, so that identification and judgment can be performed conveniently through naked eyes; and moreover, the indicator can be loaded into cells, so that an intracellular Ca<2+> signal and the concentration thereof can be detected conveniently under a laser confocal microscope.

Description

technical field

[0001] The invention relates to an oxazole compound, specifically an oxadiazole derivative and a preparation method thereof, and the application of the oxadiazole derivative as a fluorescent indicator in detecting intracellular calcium ions. Background technique

[0002] As a second messenger, calcium ions are widely involved in the regulation of various physiological activities of cells, such as muscle contraction, heart beating, secretion of endocrine and exocrine glands, synthesis and decomposition of glycogen, transport of electrolytes, various hormones, enzymes And the release of neurotransmitters, the synthesis of DNA, the regulation of cell metabolism and function, etc. In normal cells, free intracellular Ca 2+ The concentration is about 0.1μmol / L~1.0μmol / L, and the extracellular calcium ion concentration is 10,000 times higher than the intracellular calcium ion concentration, which is about 1.5mmol / L. The existence of a concentration gradient inside...

Examples

Embodiment 1

[0038] (1) Synthesis of 2-(4-ethoxy)-phenyl)-5-(4-methyl)-phenyl)-1,3,4-oxadiazole:

[0039]

[0040] 4-Methylbenzoic acid (1.81g, 13.3mmol), add 2mLSOCl 2 , After stirring and refluxing for 3 h, the reaction mixture was concentrated under reduced pressure to obtain a yellow solution, namely p-toluoyl chloride. The cooled yellow solution was slowly added dropwise to 10 mL of chloroform solution containing 4-ethoxyphenylenehydrazine (2.18 g, 12.1 mmol) and 1.1 mL of pyridine, and the mixed solution was stirred and refluxed for 3 h, then concentrated under reduced pressure , cooled to obtain a solid mixture. The solid mixture was washed with water until neutral, and then dried. The dry mixture was recrystallized by adding hot ethanol to obtain 3.24 g of white crystals. After drying the white crystals in vacuo, add 10mL POCl 3 After reflux for 7h, it was concentrated under reduced pressure and cooled to obtain a solid mixture. The solid mixture was washed with NaAC (50%, ...

Embodiment 2

[0051] The lithium salt of 1,3,4-oxadiazole derivatives with the following structure can be obtained by alkaline hydrolysis of the 1,3,4-oxadiazole derivatives produced in Example 1 by LiOH.

[0052]

Embodiment 3

[0054] Ca 2+ The response group is 5,5'-dialdehyde-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-ethyl tetraacetate, 2-(4-alkane Oxygen)-phenyl)-5-(4-bromomethyl)-phenyl)-1,3,4-oxadiazole reacts with a calcium ion responsive group in a molar ratio of 2:1, and the rest are the same as in the examples 1. Obtain 1,3,4-oxadiazole derivatives with the following structure.

[0055]

[0056] Yield 56%. Melting point: 60-62°C. The 1 H NMR (600MHz, CDCl 3 ) data are δ8.02(d, Ar-H, 4H), 7.80(d, Ar-H, 4H), 7.56(d, Ar-H, 4H), 7.38(d, Ar-H, 2H), 7.30( d, Ar-H, 2H), 7.04 (d, Ar-H, 4H, d, -CH=CH-, 2H), 6.90 (d, -CH=CH-, 2H), 6.85 (dd, Ar-H ,2H),4.60(s,-CH 2 -CH 2 -,4H),4.31(s,-CH 2 -,8H),4.10(q,-CH 2 -.12H),1.30(t,CH 3 -,18H).The 13 C NMR (600MHz, CDCl 3 ) data are δ170, 164, 159, 143, 138, 128, 121, 125, 115, 112, 69, 65, 61, 14.8 and 14.0. MALDI-TOF-MS: m / z: calcd for C 48 h 54 N 4 o 12 + :1168,found:1191.6904(M + +Na + ).Anal.Calcd for C 48 h 54 N 4 o 12 :C,67.79;H,5.86...

technical field

[0001] The invention relates to an oxazole compound, specifically an oxadiazole derivative and a preparation method thereof, and the application of the oxadiazole derivative as a fluorescent indicator in detecting intracellular calcium ions. Background technique

[0002] As a second messenger, calcium ions are widely involved in the regulation of various physiological activities of cells, such as muscle contraction, heart beating, secretion of endocrine and exocrine glands, synthesis and decomposition of glycogen, transport of electrolytes, various hormones, enzymes And the release of neurotransmitters, the synthesis of DNA, the regulation of cell metabolism and function, etc. In normal cells, free intracellular Ca 2+ The concentration is about 0.1μmol / L~1.0μmol / L, and the extracellular calcium ion concentration is 10,000 times higher than the intracellular calcium ion concentration, which is about 1.5mmol / L. The existence of a concentration gradient inside...

Examples

Embodiment 1

[0038] (1) Synthesis of 2-(4-ethoxy)-phenyl)-5-(4-methyl)-phenyl)-1,3,4-oxadiazole:

[0039]

[0040] 4-Methylbenzoic acid (1.81g, 13.3mmol), add 2mLSOCl 2 , After stirring and refluxing for 3 h, the reaction mixture was concentrated under reduced pressure to obtain a yellow solution, namely p-toluoyl chloride. The cooled yellow solution was slowly added dropwise to 10 mL of chloroform solution containing 4-ethoxyphenylenehydrazine (2.18 g, 12.1 mmol) and 1.1 mL of pyridine, and the mixed solution was stirred and refluxed for 3 h, then concentrated under reduced pressure , cooled to obtain a solid mixture. The solid mixture was washed with water until neutral, and then dried. The dry mixture was recrystallized by adding hot ethanol to obtain 3.24 g of white crystals. After drying the white crystals in vacuo, add 10mL POCl 3 After reflux for 7h, it was concentrated under reduced pressure and cooled to obtain a solid mixture. The solid mixture was washed with NaAC (50%, ...

Embodiment 2

[0051] The lithium salt of 1,3,4-oxadiazole derivatives with the following structure can be obtained by alkaline hydrolysis of the 1,3,4-oxadiazole derivatives produced in Example 1 by LiOH.

[0052]

Embodiment 3

[0054] Ca 2+ The response group is 5,5'-dialdehyde-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-ethyl tetraacetate, 2-(4-alkane Oxygen)-phenyl)-5-(4-bromomethyl)-phenyl)-1,3,4-oxadiazole reacts with a calcium ion responsive group in a molar ratio of 2:1, and the rest are the same as in the examples 1. Obtain 1,3,4-oxadiazole derivatives with the following structure.

[0055]

[0056] Yield 56%. Melting point: 60-62°C. The 1 H NMR (600MHz, CDCl 3 ) data are δ8.02(d, Ar-H, 4H), 7.80(d, Ar-H, 4H), 7.56(d, Ar-H, 4H), 7.38(d, Ar-H, 2H), 7.30( d, Ar-H, 2H), 7.04 (d, Ar-H, 4H, d, -CH=CH-, 2H), 6.90 (d, -CH=CH-, 2H), 6.85 (dd, Ar-H ,2H),4.60(s,-CH 2 -CH 2 -,4H),4.31(s,-CH 2 -,8H),4.10(q,-CH 2 -.12H),1.30(t,CH 3 -,18H).The 13 C NMR (600MHz, CDCl 3 ) data are δ170, 164, 159, 143, 138, 128, 121, 125, 115, 112, 69, 65, 61, 14.8 and 14.0. MALDI-TOF-MS: m / z: calcd for C 48 h 54 N 4 o 12 + :1168,found:1191.6904(M + +Na + ).Anal.Calcd for C 48 h 54 N 4 o 12 :C,67.79;H,5.86...

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