Halogenated phenol butyl hapten compound as well as preparation method and application thereof
By introducing halogen substituents to phenol buty compounds, a hapten compound with terminal carboxyl groups is generated, which solves the problem of difficulty in introducing alkyl substituents on the N atoms of phenol buty compounds molecules in the prior art, and achieves simple synthesis and efficient detection of phenol buty hapten.
Patent Information
- Application Number
- CN202510176030.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-18
- Publication Date
- 2025-05-23
AI Technical Summary
The prior art is difficult to directly introduce alkyl substituents on the N atoms of the phenol buty compound molecules, resulting in complex and inefficient synthesis of phenol buty haptens.
By introducing a halogen substituent on the phenol buty compounds, a halogen-substituted phenol buty compounds are generated, and a specific preparation method is adopted, including reaction under the conditions of a solvent, a base and a phase transfer catalyst to produce a hapten compound with a terminal carboxy group.
The direct introduction of substituents on the N atom of the phenol buty compound is achieved, which simplifies the synthesis process of hapten, avoids the impurities introduced by multiple reactions, and improves the efficiency and accuracy of phenol buty detection.
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Figure CN120025275A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of drug detection, and in particular to a halogenated phenol hapten compound and a preparation method and application thereof. Background Art
[0002] For the detection of trace substances in the sample, immunoassay based on antibody-antigen reaction is the most common and efficient detection method in on-site testing. One of the key steps in immunoassay is the synthesis of haptens, which requires modification on the antigen molecule to introduce a group that can connect to the arm.
[0003] The introduction of the arm should maintain the chemical properties and structural information of the antigen molecule itself as much as possible. At the same time, the introduction steps should be simple and efficient. The antigen molecule connected with a special group is called a hapten molecule.
[0004] Phenolbuterol compounds are a powerful laxative and are one of the drugs that cannot be added to food. The molecular structure of phenolbuterol is simple, so the position of the arm introduction needs to be carefully selected to prepare phenolbuterol haptens. Due to the low activity of the N atom of phenolbuterol compounds, the current conventional methods cannot directly introduce alkyl substitution on the N atom of the phenolbuterol compound molecule.
[0005] Therefore, the present invention provides a one-step synthesis method of a hapten compound of phenolic acid, which can be further derived into a complete antigen and can be used to detect phenolic acid after antibody immunization. Summary of the invention
[0006] In order to solve the above problems in the prior art, the present invention provides a halogenated phenol hapten compound and a preparation method and use thereof.
[0007] In order to achieve the above object, the first aspect of the present invention provides a halogen-substituted phenolic hapten compound, the main feature of which is that the structural formula of the compound is as follows:
[0008]
[0009] Among them, Linker is selected from C 1-8 Alkyl, p-phenyl, m-phenyl, o-phenyl, 1,4-divinylphenyl, vinylphenyl, ethynylphenyl, 1,4-diethynylphenyl, biphenyl, 1,4-methylenephenyl, 9,10-anthryl; X is selected from fluorine, chlorine or bromine.
[0010] Preferably, the structural formula of the compound is as follows:
[0011]
[0012] The second aspect of the present invention provides a method for preparing the halogen-substituted phenolic hapten compound, the main feature of which is that the preparation method comprises: a halogenated carboxylic acid tert-butyl ester and a halogenated phenolic compound are kept at room temperature for 10 to 14 hours, then heated to 30 to 50°C and kept for 1 to 3 hours under the conditions of a solvent, a base and a phase transfer catalyst to react to generate the halogen-substituted phenolic hapten compound according to claim 1.
[0013] Preferably, the carboxylic acid in the tert-butyl halogenated carboxylate is selected from C 1-8 Alkyl carboxylic acids, p-phenyl carboxylic acids, m-phenyl carboxylic acids, o-phenyl carboxylic acids, 1,4-divinylphenyl carboxylic acids, vinylphenyl carboxylic acids, ethynylphenyl carboxylic acids, 1,4-diethynylphenyl carboxylic acids, biphenyl carboxylic acids, 1,4-methylenephenyl carboxylic acids, 9,10-anthryl carboxylic acids, and the halogenated phenolic acid compound is halogenated diacetylphenol.
[0014] Preferably, the solvent is N,N-dimethylformamide (DMF), the base is potassium tert-butoxide (t-BuOK), and the phase transfer catalyst is tetrabutylammonium fluoride (TBAF).
[0015] Preferably, at a temperature of -10 to -30°C, fluorobisacetyl butyl is dissolved in DMF, potassium tert-butoxide and tetrabutylammonium fluoride are added, and then a DMF solution of tert-butyl 5-bromovalerate is added dropwise, stirred and heated to room temperature, kept at room temperature for 10 to 14 hours, then heated to 30 to 50°C and kept for 1 to 3 hours; after cooling, a saturated ammonium chloride solution is added, the solvent is removed by rotary evaporation, ethyl acetate is added to dissolve, insoluble matter is removed by diatomaceous earth filtration, diatomaceous earth is washed with ether-ethyl acetate solution, the liquids are separated, extracted with dichloromethane, dried with anhydrous magnesium sulfate, the desiccant is removed by filtration, the solvent is removed by rotary evaporation, and the halogen-substituted phenol butyl hapten compound is purified by silica gel column chromatography to obtain the halogen-substituted phenol butyl hapten compound.
[0016] Preferably, the preparation method of the fluorobisacetylphenol is as follows: dissolving fluorobisacetylphenol in acetic anhydride, stirring and evaporating excess solvent.
[0017] The halogen-substituted phenolic hapten compound of the present invention and the preparation method thereof are halogen-substituted phenolic hapten compounds containing a terminal carboxyl group, which can be conveniently connected to proteins to prepare antigens through subsequent amidation, esterification and other reactions. It is one of the necessary technologies for rapid detection of phenolic hapten compounds and has application prospects in detection and the like.
[0018] The third aspect of the present invention provides a complete phenolic butylene antigen, the main feature of which is that it is obtained by substituting the halogen-substituted phenolic butylene hapten compound.
[0019] The fourth aspect of the present invention provides the use of the halogen-substituted phenolic acid complete antigen in the preparation of a product for detecting phenolic acid compounds.
[0020] Preferably, the product comprises immune antibodies, and the immune antibodies are prepared by using the halogen-substituted phenolic complete antigen.
[0021] The beneficial effects of the present invention are: successfully directly introducing a substituent on the N atom of the halogen-substituted phenolic compound molecule; and, no additional steps of protecting and removing the protecting groups on the phenolic compound are required, and the substituent is directly introduced on the phenolic compound, which can avoid the introduction of unexpected impurities in the multi-step reaction, affecting the synthesis of the complete antigen and the subsequent immunization steps. At the same time, the tert-butyl group retained on the carboxyl group can be removed in situ under mild conditions, without affecting the subsequent coupling steps, and no additional separation of carboxylic acid is required. Directly introducing the hydrocarbon chain reduces the influence of polar groups such as amide bonds on the molecule, thereby avoiding the influence on the subsequent immunization link. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 It is the high-resolution mass spectrum of fluorobisacetyl in Example 1.
[0023] Figure 2 is the fluorinated bisacetyl butyl in Example 1 1 H-NMR spectrum.
[0024] Figure 3 is the fluorinated bisacetyl butyl in Example 1 13 C-NMR spectrum.
[0025] Figure 4 It is the HPLC spectrum of fluorobisacetyl in Example 1.
[0026] Figure 5 This is the high-resolution mass spectrum of the halogen-substituted phenolic hapten compound A in Example 2.
[0027] Figure 6 is the halogen-substituted phenolic hapten compound A in Example 2 1 H-NMR spectrum.
[0028] Figure 7 is the halogen-substituted phenolic hapten compound A in Example 2 13 C-NMR spectrum.
[0029] Figure 8 A to Figure 8 D is a schematic diagram of the result determination of the colloidal gold reagent plate for rapid detection of phenols in Example 4.
[0030] Fig. 9This is a positive sample image for the result determination of the colloidal gold reagent plate for rapid detection of phenols in Example 4.
[0031] Fig.10 This is a negative sample image of the colloidal gold reagent plate result for rapid detection of phenols in Example 4. DETAILED DESCRIPTION
[0032] In order to more clearly describe the technical content of the present invention, further description is given below in conjunction with specific embodiments.
[0033] The tert-butyl protected small molecule halogen substituted phenolic hapten compound of the present invention is deprotected in situ and then coupled with the carrier protein bovine serum albumin (BSA) by a chemical method to form an immunogenic complex, and immune antibodies are obtained by animal immunization to prepare corresponding kit products.
[0034] Specifically, animal immunization is as follows: 6-8 week old Balb / c mice are selected, the coupled antigen is mixed with adjuvant, Freund's complete adjuvant and Freund's incomplete adjuvant are used, Freund's complete adjuvant is used for the first immunization, and Freund's incomplete adjuvant is used for the subsequent immunization. The antigen dose is 50-100 μg / mouse each time through subcutaneous and intraperitoneal injections, and the immunization procedure is as follows: after the first immunization, 2-3 booster immunizations are performed at intervals of 2-3 weeks, and spleen cells are taken for cell fusion 3-5 days after the last booster immunization.
[0035] Cell fusion is specifically as follows: Preparation of spleen cells: Aseptically obtain the spleen of the immunized mouse, grind and filter to obtain a spleen cell suspension. Preparation of myeloma cells: Select SP2 / 0 myeloma cells, culture to the logarithmic growth phase, and culture with RPMI1640 medium containing 10% calf serum. Splenocytes and myeloma cells are mixed in a certain ratio (5:1-10:1), polyethylene glycol (PEG) is added to promote cell fusion, and then cultured with HAT medium. Unfused spleen cells and myeloma cells die, and only fused hybridoma cells can grow.
[0036] Screening of positive hybridoma cells, preliminary screening: Use HAT culture medium to screen out hybridoma cells, and then use indirect ELISA method, etc., use small molecule antigens to coat the ELISA plate, and detect the hybridoma cell culture supernatant. The hybridoma cells that can specifically bind to the antigen are positive hybridoma cells.
[0037] Clonal culture: positive hybridoma cells are cloned and cultured using limiting dilution method or soft agar plate method to obtain cell clones formed by single cell proliferation, thus ensuring the uniqueness of secreted antibodies.
[0038] Large-scale preparation of monoclonal antibodies, in vivo culture: positive hybridoma cells are injected into the peritoneal cavity of syngeneic Balb / c mice, the mice produce ascites, which contains a large amount of monoclonal antibodies, and the ascites is collected to purify the antibodies; in vitro culture: large-scale cell culture is carried out using bioreactors, etc., the culture supernatant is collected, impurities are removed by centrifugation, filtration, etc., and then the monoclonal antibodies are purified by affinity chromatography, etc.
[0039] Identification of monoclonal antibodies and antibody titer determination: Use ELISA to determine the concentration and activity of the antibody.
[0040] Identification of antibody specificity: ELISA and colloidal gold methods are used to detect the binding of antibodies to small molecule antigens and other related antigens to verify specificity.
[0041] The production of colloidal gold reagent plate is specifically as follows: 1. Coating BSA-coupled antigen; 2. calcining colloidal gold; 3. labeling monoclonal antibody with colloidal gold to prepare colloidal gold-antibody complex, coating it on polyester film to prepare binding pad; 4. preparing sample buffer solution to prepare sample pad; combining the components, pasting, cutting, and assembling into colloidal gold detection reagent.
[0042] Example 1
[0043] The preparation method of fluorobisacetyl butyl provided by the present invention is specifically as follows:
[0044]
[0045] At room temperature, fluorophenol (CAS.No.867154-62-9, 335mg, 1mmol) was dissolved in 5mL acetic anhydride, stirred for 24 hours and then the excess solvent was evaporated. After cooling, saturated sodium bicarbonate solution was added, and the solvent was removed by rotary evaporation. After adding 150mL dichloromethane to dissolve, the insoluble matter was removed by diatomaceous earth filtration, and the diatomaceous earth was washed with 30mL dichloromethane solution, separated, extracted with dichloromethane 3 times, the organic phases were combined, and dried with anhydrous magnesium sulfate; the desiccant was removed by filtration, and the solvent was removed by rotary evaporation. Fluorobisacetyl butyl was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 4:1) to obtain white powder (350mg, 84%) of fluorobisacetyl butyl.
[0046] 1 H NMR(600MHz,dmso)δ10.87(s,1H),7.30(dd,J=8.5,2.7Hz,1H),7.26–7.16(m,4H),7.16–7.05(m,5H),6.98(dd,J=8.6,4.5Hz,1H),2.25(s,6H).
[0047] 13C NMR (151MHz, dmso) δ177.62,169.04,159.59,157.36,149.56,138.60,137.46,134.34,129.19,121.85,115.12,111.02,61.65,20.72.
[0048] (ESI / TOF)m / z:Calcd.for C24H18FNO5Na[M+Na]+442.1067; Found:442.1049.
[0049] The corresponding high-resolution mass spectra, hydrogen spectra, carbon spectra and high-performance liquid chromatography are shown in Figures 1 and 2. Figure 1 , 2 , 3, and 4.
[0050] Example 2
[0051] The preparation method of the fluorophenol hapten compound A provided by the present invention is specifically as follows:
[0052]
[0053] To the white powder (350 mg, 0.84 mmol) of fluorobisacetphenidine obtained in Example 1, 15 mL of the specified solvent was added and cooled to 0 ° C. A base (2 mmol) and a catalyst (0.1 mmol) were added, and then a solution (15 mL) of tert-butyl 5-bromopentanoate (351 mg, 1 mmol) in the specified solvent was added dropwise, stirred and slowly heated to a specified temperature T1, kept at T1 for a specified time t1, and then heated to a specified temperature T2 and kept heated for a specified time t2; after cooling, a saturated ammonium chloride solution was added, and the solvent was removed by rotary evaporation, 150 mL of ethyl acetate was added to dissolve, and the insoluble matter was removed by filtration through diatomaceous earth, and the diatomaceous earth was washed with 30 mL of ether-ethyl acetate 1:1 solution, and the liquid was separated, extracted 3 times with dichloromethane, and the organic phases were combined and dried with anhydrous magnesium sulfate; the desiccant was removed by filtration, and the solvent was removed by rotary evaporation. The tert-butyl protected fluorophenol butyl hapten compound A was obtained by purification by silica gel column chromatography (petroleum ether: ethyl acetate = 2:1) as a white solid, and the yield was shown in Table 1 below.
[0054] Table 1: Condition screening table
[0055]
[0056] 1H NMR (600MHz, Chloroform-d) δ7.28–7.24(m,2H),7.23–7.21(m,3H),7.04–7.01(m,4H),6.98(dd,J=8.0,2.6Hz,1H),6.86(dd,J=8. 5,4.1Hz,1H),3.76(t,J=7.3Hz,2H),2.27(d,J=8.4Hz,7H),2.02–1.96(m,1H),1.79–1.69(m,2H),1.68–1.57(m,4H),1.42(s,9H).
[0057] 13 C NMR(150MHz,Chloroform-d)δ176.10,171.91,168.67,159.42,157.82,149.47,138.02,133.57,1 28.82,121.09,114.46,113.71,108.85,79.74,61.14,39.63,34.26,27.52,26.10,21.76,20.56.
[0058] (ESI / TOF)m / z:Calcd.for C33H34NFO7Na[M+Na]+598.2217; Found:598.2215.
[0059] The corresponding high-resolution mass spectra, hydrogen spectra, and carbon spectra are shown as follows: Figure 5 , 6 , as shown in 7.
[0060] Example 3
[0061]
[0062] The hapten compound A (380 mg, 0.66 mmol) obtained in Example 2 was dissolved in DMF (5 mL), (DMAP) (5.6 mg, 0.05 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (382 mg, 2 mmol) were added, and then 100 mg of BSA in 0.1 M potassium phosphate buffer solution (15 mL) was added dropwise, and the reaction was carried out at 4 degrees Celsius for 72 hours. After the reaction, the above reaction solution was loaded into a 10 kD dialysis bag and dialyzed in PBS (pH 7.2) solution to remove excess hapten compounds. The solution was changed every 3 hours, at least 3 to 4 times, to ensure that the dialysis was complete and then freeze-dried to obtain the complete antigen.
[0063] Example 4
[0064] Prepare an aqueous solution of phenolphthalein diacetate fluoride at 1 μg / mL. The preparation of phenolphthalein diacetate fluoride can refer to Example 1. The complete antigen obtained in Example 3 is used for animal immunization to obtain immune antibodies, and a colloidal gold test strip is prepared. Take out the colloidal gold test strip, use a pipette to suck the corresponding solution, and add about 1 - 2 drops of the solution into the reagent sample application hole. After standing for 3 - 5 minutes, read the result.
[0065] As Figure 8 A to Figure 8 D shows, negative: a line appears at the test line (T line), and a line appears at the position of the control line (C line).
[0066] Positive: a line appears at the position of the control line (C line), and no line appears at the test line (T line).
[0067] Invalid: no line appears at the position of the control line (C line), and a line appears at the test line (T line); or, no line appears at the position of the control line (C line), and no line appears at the test line (T line).
[0068] As Fig. 9 shown, for the aqueous solution of phenolphthalein diacetate fluoride at 1 μg / mL, the result is positive.
[0069] As Fig.10 shown, for the aqueous solution, the result is negative.
[0070] In this specification, the present invention has been described with reference to its specific embodiments. However, it is obvious that various modifications and variations can still be made without departing from the spirit and scope of the present invention. Therefore, the specification and the drawings should be regarded as illustrative rather than restrictive.
Claims
1. A halogen-substituted phenolic hapten compound, characterized in that: The structural formula of the compound is as follows: Among them, Linker is selected from C 1-8 Alkyl, p-phenyl, m-phenyl, o-phenyl, 1,4-divinylphenyl, vinylphenyl, ethynylphenyl, 1,4-diethynylphenyl, biphenyl, 1,4-methylenephenyl, 9,10-anthryl; X is selected from fluorine, chlorine or bromine.
2. The halogen-substituted phenolic hapten compound according to claim 1, characterized in that: The structural formula of the compound is as follows:
3. A method for preparing the halogen-substituted phenolic hapten compound according to claim 1, characterized in that: The preparation method comprises: a halogenated carboxylic acid tert-butyl ester and a halogenated phenolic compound are kept at room temperature for 10 to 14 hours, and then heated to 30 to 50° C. and kept for 1 to 3 hours under the conditions of a solvent, a base and a phase transfer catalyst to react and generate the halogen-substituted phenolic hapten compound according to claim 1.
4. The method for preparing the halogen-substituted phenolic hapten compound according to claim 3, characterized in that: The carboxylic acid in the tert-butyl halogenated carboxylate is selected from C 1-8 Alkyl carboxylic acids, p-phenyl carboxylic acids, m-phenyl carboxylic acids, o-phenyl carboxylic acids, 1,4-divinylphenyl carboxylic acids, vinylphenyl carboxylic acids, ethynylphenyl carboxylic acids, 1,4-diethynylphenyl carboxylic acids, biphenyl carboxylic acids, 1,4-methylenephenyl carboxylic acids, 9,10-anthryl carboxylic acids, and the halogenated phenolic acid compound is halogenated diacetylphenol.
5. The preparation method according to claim 3, characterized in that: The solvent is DMF, the base is potassium tert-butoxide, and the phase transfer catalyst is tetrabutylammonium fluoride.
6. The preparation method according to claim 3, characterized in that: The preparation method is specifically as follows: at a temperature of -10 to -30°C, dissolving fluorobisacetyl butyl in DMF, adding potassium tert-butoxide and tetrabutylammonium fluoride, then dropwise adding a DMF solution of tert-butyl 5-bromovalerate, stirring and heating to room temperature, keeping at room temperature for 10 to 14 hours, then heating to 30 to 50°C and keeping for 1 to 3 hours; after cooling, adding a saturated ammonium chloride solution, rotary evaporation to remove the solvent, adding ethyl acetate to dissolve, filtering with diatomaceous earth to remove insoluble matter, washing the diatomaceous earth with an ether-ethyl acetate solution, separating the liquids, extracting with dichloromethane, drying with anhydrous magnesium sulfate, filtering to remove the desiccant, rotary evaporation to remove the solvent, and purifying with silica gel column chromatography to obtain the halogen-substituted phenol butyl hapten compound.
7. The preparation method according to claim 6, characterized in that: The preparation method of the fluorobisacetyl butyl is as follows: at room temperature, the fluorobisacetyl butyl is dissolved in acetic anhydride, stirred and excess solvent is evaporated.
8. A halogen-substituted phenolic complete antigen, characterized in that: The method is obtained by using the halogen-substituted phenolic hapten compound according to claim 1.
9. Use of the halogen-substituted phenolic acid complete antigen according to claim 8 in the preparation of a product for detecting phenolic acid compounds.
10. The use according to claim 9, characterized in that The product contains immune antibodies, and the immune antibodies are prepared by using the halogen-substituted phenolic complete antigen.