A preparation method of a chemiluminescent substrate

Abstract

The present invention belongs to the field of chemiluminescence immunoassay technology, and particularly relates to a preparation method of a chemiluminescent substrate. The preparation steps are as follows: Using compound 2 as a raw material, dissolving compound 2 in carbon tetrachloride, adding a catalyst and NBS for bromination reaction to obtain compound 3; adding compound 3 to triethyl phosphite, stirring at 20-30 °C for 10-24 h to obtain compound 4; mixing compound 4 with a mixture of acetone and an aqueous sodium hydroxide solution, stirring at 20-30 °C for 10-24 h to obtain the target compound. The experimental materials and experimental conditions adopted in the present invention are relatively simple, which improves the safety factor of the experiment, reduces the experimental cost, and can form a low-cost and safe chemiluminescent substrate production line.

Description

Technical Field

[0001] The present invention relates to the field of chemiluminescence immunoassay technology, and particularly to a preparation method of a chemiluminescent substrate. Background Art

[0002] The chemiluminescent substrate APS-5 is used on a fully automatic chemiluminescent immunoassay instrument and can emit a very strong light signal. Currently, it has been used in chemiluminescent immunoassay, with the highest detection sensitivity, stable detection results, and good repeatability, and has been popularized and used in medicine. However, due to its high price, its large-scale development and use are restricted. The main reason for the high price is that its synthesis process is complex and the synthesis conditions are difficult.

[0003] Patents US5772926A and CN1180349A respectively disclose a preparation method of a chemiluminescent substrate. In the intermediate steps, lithium diisopropylamide needs to be used at -78°C, and then a mixture of phosphorus oxychloride and pyridine is added dropwise. After reacting for a period of time, 3-hydroxypropionitrile is added dropwise to obtain a key intermediate, and then the target product is obtained by hydrolysis. The yield of this step is 30%. Patent CN104418887A discloses a preparation method of a compound that produces chemiluminescence in reaction with phosphatase. In this method, under the protection of N2, H and tetrahydrofuran are added, and then the temperature is lowered to -80°C, and the reaction is maintained at -80°C for 1 h, and then a tetrahydrofuran solution is added dropwise. Then the temperature is lowered to -20°C and a mixture is added, etc. Finally, the yield of the obtained intermediate is 52%. In the prior art, the reaction conditions for preparing chemiluminescent substrates are harsh, the operation is complex, and the yield is low, which is not suitable for batch production. Summary of the Invention

[0004] Aiming at the technical problems of harsh reaction conditions, complex operation, and low yield in the preparation technology of chemiluminescent substrates, the purpose of the present invention is to provide a preparation method of a chemiluminescent substrate.

[0005] To achieve the above purpose, the following main technical solutions are provided:

[0006] A preparation method of a chemiluminescent substrate, and the preparation technical route is as follows:

[0007]

[0008] The preparation method includes the following steps:

[0009] (1) Using compound 2 as a raw material, dissolving compound 2 in carbon tetrachloride, and then adding a catalyst and NBS for bromination reaction to obtain compound 3;

[0010] (2) Adding compound 3 to triethyl phosphite, and stirring at 20 - 30°C for 10 - 24 h to obtain compound 4;

[0011] (3) Mix compound 4 with a mixture of acetone and aqueous sodium hydroxide solution, and stir at 20 - 30 °C for 10 - 24 h to obtain the target compound 1.

[0012] Among them, the R group is C 1-6 haloalkyl, naphthyl, phenyl, substituted phenyl or heteroaryl, wherein the substituted phenyl contains 1 to 3 halogens, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, C(O)C 1-6 alkyl, CN or NO2 substituents.

[0013] In the above preparation method, the catalyst in step (1) is azobisisobutyronitrile.

[0014] In the above preparation method, preferably, the reaction temperature in step (2) is 22 - 28 °C, and the reaction time is 11 - 15 h; and / or the reaction temperature in step (3) is 22 - 28 °C, and the reaction time is 11 - 15 h.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] Compared with the technical solutions disclosed in the prior art, which require experimental conditions of maintaining a low temperature of -80 to -78 °C and multiple experimental steps, the reagents and reaction conditions used in the present invention are simpler, the experimental temperature adopted is easier to achieve, the steps are simpler, the operation is convenient, and the safety factor is higher. Detailed Embodiments

[0017] For the convenience of understanding, the present application will be described more comprehensively below, and preferred embodiments of the present application are given. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure content of the present invention more thorough and comprehensive.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0019] Examples

[0020] The chemical formula of the chemiluminescent substrate is:

[0021]

[0022]

[0023]

[0024] Each of the compounds 1-A to 1-F was prepared according to the following synthetic route.

[0025]

[0026] Example 1 Synthesis of Compound 1-A

[0027]

[0028] (1) Using compound 2 as the raw material, 3.65 g of compound 2 was dissolved in 50 ml of carbon tetrachloride, 1.32 g of azobisisobutyronitrile as the catalyst was added, and then 10 g of NBS was added for bromination reaction. After the reaction was complete, the reaction solution was added to a mixture of water and ethyl acetate (1:1, 100 ml), extracted and separated, the organic phase was collected, concentrated, and compound 3 was obtained;

[0029] (2) Compound 3 was added to 20 ml of triethyl phosphite (1 mol / L), stirred at 25 °C for 12 h, and the reaction was complete; the reaction solution was slowly added to ice water, stirred for 1 h, 50 ml of dichloromethane was added, extracted and separated, the organic phase was collected, dried, concentrated, and the residue was separated by column chromatography on a silica gel column to obtain compound 4;

[0030] (3) Compound 4 was mixed with a mixture of 170 ml of acetone and 12 ml of sodium hydroxide aqueous solution (2 mol / L), stirred at 25 °C for 12 h, filtered, and the filter cake was rinsed with a small amount of acetone to obtain compound 1-A. The weight of the final product was 4.48 g, the yield was 88%, and the purity was 98%.

[0031] Example 2 Synthesis of Compound 1-B

[0032]

[0033] (1) Using compound 2 as the raw material, 3.81 g of compound 2 was dissolved in 50 ml of carbon tetrachloride, 1.32 g of azobisisobutyronitrile as the catalyst was added, and then 10 g of NBS was added for bromination reaction; after the reaction was complete, the reaction solution was added to a mixture of water and ethyl acetate (1:1, 100 ml), extracted and separated, the organic phase was collected, concentrated, and compound 3 was obtained;

[0034] (2) Compound 3 was added to 20 ml of triethyl phosphite (1 mol / L), stirred at 30 °C for 10 h, and the reaction was complete; the reaction solution was slowly added to ice water, stirred for 1 h, 50 ml of dichloromethane was added, extracted and separated, the organic phase was collected, dried, concentrated, and the residue was separated by column chromatography on a silica gel column to obtain compound 4;

[0035] (3) The compound 4 was mixed with a mixture of 170 ml of acetone and 12 ml of aqueous sodium hydroxide solution (2 mol / L), stirred at 30 °C for 10 h, filtered, and the filter cake was rinsed with a small amount of acetone to obtain compound 1-B. The weight of the final product was 4.47 g, the yield was 85%, and the purity was 96%.

[0036] Example 3 Synthesis of Compound 1-C

[0037]

[0038] (1) Using compound 2 as the raw material, 3.67 g of compound 2 was dissolved in 50 ml of carbon tetrachloride, 1.32 g of azobisisobutyronitrile as the catalyst was added, and then 10 g of NBS was added for bromination reaction; after the reaction was complete, the reaction solution was added to a mixture of water and ethyl acetate (1:1, 100 ml), extracted and separated, the organic phase was collected, and after concentration, compound 3 was obtained;

[0039] (2) Compound 3 was added to 20 ml of triethyl phosphite (1 mol / L), stirred at 20 °C for 24 h, and the reaction was complete; the reaction solution was slowly added to ice water, stirred for 1 h, 50 ml of dichloromethane was added, extracted and separated, the organic phase was collected, dried, concentrated, and the residue was separated by column chromatography on a silica gel column to obtain compound 4;

[0040] (3) The compound 4 was mixed with a mixture of 170 ml of acetone and 12 ml of aqueous sodium hydroxide solution (2 mol / L), stirred at 20 °C for 24 h, filtered, and the filter cake was rinsed with a small amount of acetone to obtain compound 1-C. The weight of the final product was 4.39 g, the yield was 85%, and the purity was 95%.

[0041] Example 4 Synthesis of Compound 1-D

[0042]

[0043] (1) Using compound 2 as the raw material, 3.59 g of compound 2 was dissolved in 50 ml of carbon tetrachloride, 1.32 g of azobisisobutyronitrile as the catalyst was added, and then 10 g of NBS was added for bromination reaction; after the reaction was complete, the reaction solution was added to a mixture of water and ethyl acetate (1:1, 100 ml), extracted and separated, the organic phase was collected, and after concentration, compound 3 was obtained;

[0044] (2) Compound 3 was added to 20 ml of triethyl phosphite (1 mol / L), stirred at 22 °C for 15 h, and the reaction was complete; the reaction solution was slowly added to ice water, stirred for 1 h, 50 ml of dichloromethane was added, extracted and separated, the organic phase was collected, dried, concentrated, and the residue was separated by column chromatography on a silica gel column to obtain compound 4;

[0045] (3) Mix compound 4 with a mixture of 170 ml of acetone and 12 ml of aqueous sodium hydroxide solution (2 mol / L), stir at 22 °C for 15 h, filter, and wash the filter cake with a small amount of acetone to obtain compound 1-D. The weight of the final product is 4.33 g, the yield is 86%, and the purity is 96%.

[0046] Example 5 Synthesis of Compound 1-E

[0047]

[0048] (1) Using compound 2 as the raw material, dissolve 3.37 g of compound 2 in 50 ml of carbon tetrachloride, add 1.32 g of azobisisobutyronitrile as the catalyst, and then add 10 g of NBS for bromination reaction; after the reaction is complete, add the reaction solution to a mixture of water and ethyl acetate (1:1, 100 ml), extract and separate the layers, collect the organic phase, and concentrate to obtain compound 3;

[0049] (2) Add compound 3 to 20 ml of triethyl phosphite (1 mol / L), stir at 28 °C for 11 h until the reaction is complete; slowly add the reaction solution to ice water, stir for 1 h, add 50 ml of dichloromethane, extract and separate the layers, collect the organic phase, dry, concentrate, and subject the residue to column chromatography on a silica gel column to obtain compound 4;

[0050] (3) Mix compound 4 with a mixture of 170 ml of acetone and 12 ml of aqueous sodium hydroxide solution (2 mol / L), stir at 28 °C for 11 h, filter, and wash the filter cake with a small amount of acetone to obtain compound 1-E. The weight of the final product is 4.04 g, the yield is 85%, and the purity is 97%.

[0051] Example 6 Synthesis of Compound 1-F

[0052]

[0053] (1) Using compound 2 as the raw material, dissolve 3.61 g of compound 2 in 50 ml of carbon tetrachloride, add 1.32 g of azobisisobutyronitrile as the catalyst, and then add 10 g of NBS for bromination reaction; after the reaction is complete, add the reaction solution to a mixture of water and ethyl acetate (1:1, 100 ml), extract and separate the layers, collect the organic phase, and concentrate to obtain compound 3;

[0054] (2) Add compound 3 to 20 ml of triethyl phosphite (1 mol / L), stir at 25 °C for 12 h until the reaction is complete; slowly add the reaction solution to ice water, stir for 1 h, add 50 ml of dichloromethane, extract and separate the layers, collect the organic phase, dry, concentrate, and subject the residue to column chromatography on a silica gel column to obtain compound 4;

[0055] (3) The compound 4 was mixed with a mixture of 170 ml of acetone and 12 ml of aqueous sodium hydroxide solution (2 mol / L), stirred at 25 °C for 12 h, filtered, and the filter cake was rinsed with a small amount of acetone to obtain compound 1-F. The weight of the final product was 4.16 g, the yield was 86%, and the purity was 97%.

[0056] The above-described embodiments merely represent the implementation manners of the present application. The description is relatively specific and detailed, but it should not be construed as a limitation on the patent scope of the present application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all fall within the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims.

Claims

1. A preparation method of a chemiluminescent substrate, and the preparation technical route is as follows: It is characterized in that The preparation method comprises the following steps: (1) Using compound 2 as a raw material, dissolving compound 2 in carbon tetrachloride, and then adding a catalyst and NBS for bromination reaction to obtain compound 3; (2) Adding compound 3 into triethyl phosphite, and stirring at 20 - 30 °C for 10 - 24 h to obtain compound 4; (3) Mixing compound 4 with a mixture of acetone and an aqueous sodium hydroxide solution, and stirring at 20 - 30 °C for 10 - 24 h to obtain the target compound 1; wherein the R group is C 1-6 haloalkyl, naphthyl, phenyl, substituted phenyl or heteroaryl; Among them, the catalyst in the step (1) is azobisisobutyronitrile.

2. The preparation method of a chemiluminescent substrate according to claim 1, characterized in that, The substituted phenyl in the R group contains 1 to 3 halogens, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, C(O)C 1-6 alkyl, CN or NO2 substituents.

3. The preparation method of a chemiluminescent substrate according to claim 1, characterized in that, The reaction temperature in the step (2) is 22 - 28 °C, and the reaction time is 11 - 15 h; and / or the reaction temperature in the step (3) is 22 - 28 °C, and the reaction time is 11 - 15 h.

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