Aripiprazole artificial hapten, artificial antigen, preparation method and application thereof

Abstract

The present invention discloses an artificial hapten of aripiprazole, an artificial antigen, and a preparation method and application thereof. The molecular structural formula of the artificial hapten of aripiprazole is shown in Formula I, and the molecular structural formula of the artificial antigen of aripiprazole is shown in Formula III. The present invention also discloses the application of the artificial hapten of aripiprazole in the preparation of anti-aripiprazole antibodies. The artificial hapten of aripiprazole of the present invention retains the characteristic structure of aripiprazole to the greatest extent, and has an active group that can be coupled with a carrier protein, and can be used as an antigenic determinant; the artificial antigen of aripiprazole obtained by further preparation can be immunized to obtain anti-aripiprazole antibodies with high affinity, high sensitivity and strong specificity, and the polyclonal antibodies obtained by immunizing New Zealand white rabbits can be used for rapid and accurate immunodetection and immunoanalysis of aripiprazole.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical engineering, and specifically relates to an artificial hapten and an artificial antigen of aripiprazole and a preparation method and application thereof. Background Art

[0002] Aripiprazole, also known as Aripiprazole in English, is a second-generation antipsychotic drug that has a bidirectional regulatory effect on the nervous system and is a stabilizer of DA transmitters. It is used to treat various types of schizophrenia. Foreign clinical trials have shown that this product has a significant effect on both positive and negative symptoms of schizophrenia, and can also improve associated emotional symptoms and reduce the relapse rate of schizophrenia.

[0003] The more common adverse drug reactions reported (>10%) include: anxiety, headache, insomnia, dizziness, drowsiness, nausea and vomiting, urinary incontinence, and stiff neck; the occasional (1%-10%) include: akathisia, constipation, rash, cough, and nasal congestion; the uncommon (<1%) include: tremor, fever, bone pain, myalgia, and blurred vision.

[0004] At present, the commonly used detection and analysis methods for aripiprazole are mainly: gas chromatography (GC), gas-mass spectrometry (GC-MS), high performance liquid chromatography (HPLC), liquid-mass spectrometry (LC-MS), etc. These instrumental analysis methods have extremely high sensitivity and precision, but require expensive instruments, equipment and specially trained technicians, and are not suitable for screening and testing of batch samples and on-site instant testing. The immunoassay method developed in recent years has the advantages of simple operation, high efficiency, sensitivity, and suitability for large-scale testing. It has been widely used in the detection of various contraband, etc. In order to establish an immunoassay method for aripiprazole, it is necessary to obtain aripiprazole complete antigen with antigenic activity. Summary of the invention

[0005] The present invention provides an artificial hapten of aripiprazole, which is prepared by chemical modification using a metabolite of aripiprazole as a precursor, retains the characteristic structure of aripiprazole to the greatest extent, has an active group that can be coupled with a carrier protein, and can serve as an antigenic determinant.

[0006] In order to achieve the above technical objectives, the present invention is implemented through the following technical solutions:

[0007] The first object of the present invention is to provide an artificial hapten of aripiprazole, the molecular structure of which is shown in Formula I:

[0008]

[0009] The second object of the present invention is to provide a method for preparing the artificial hapten of aripiprazole, the method comprising the following steps:

[0010] (1) Acylation reaction of the hydroxy aripiprazole represented by formula II with triethylamine and di-tert-butyl dicarbonate to obtain a compound represented by formula A;

[0011] (2) the compound represented by formula A is subjected to substitution reaction with ethyl bromoacetate and sodium hydride to obtain the compound represented by formula B;

[0012] (3) The compound represented by formula B is deprotected in trifluoroacetic acid to obtain a compound represented by formula C;

[0013] (4) The compound represented by formula C is subjected to a hydrolysis reaction with an alkaline solution, and the pH value is adjusted to acidic to obtain the aripiprazole hapten represented by formula I.

[0014] The reaction formula of the method is as follows:

[0015]

[0016] The hydroxy aripiprazole shown in formula II is a metabolite of aripiprazole and the two have the same characteristic functional groups, so the hydroxy aripiprazole can be used to synthesize the artificial hapten of aripiprazole.

[0017] In the step (1), the molar ratio of hydroxy aripiprazole, triethylamine and di-tert-butyl dicarbonate is preferably 1:2-2.5:1.05-1.2;

[0018] In the step (2), the molar ratio of the compound represented by formula A, ethyl bromoacetate and sodium hydride is preferably 1:2-2.2:1.5-2;

[0019] The reaction temperature of step (2) is preferably heated to reflux reaction

[0020] The alkaline solution in step (4) is preferably a NaOH aqueous solution.

[0021] Further, the method is preferably carried out according to the following steps:

[0022] (1) Dissolve the hydroxy aripiprazole represented by formula II in tetrahydrofuran, place in an ice water bath at 0°C, add triethylamine, then add dropwise a tetrahydrofuran solution of di-tert-butyl dicarbonate, and react at room temperature with stirring for 4 to 5 hours; after the reaction is completed, evaporate the reaction solution under reduced pressure to dryness to obtain the compound represented by formula A;

[0023] The molar ratio of hydroxyaripiprazole, triethylamine and di-tert-butyl dicarbonate is 1:2-2.5:1.05-1.2;

[0024] The total volume amount of tetrahydrofuran is 10 to 30 mL / mmol based on the amount of hydroxy aripiprazole;

[0025] (2) The compound represented by formula A is dissolved in N,N-dimethylformamide, and ethyl bromoacetate and sodium hydride are added, and the mixture is stirred under reflux at 65° C. in an oil bath for 18 to 20 hours. After the reaction is completed, the reaction solution is evaporated to dryness under reduced pressure, extracted with water-dichloromethane, and the organic phase is taken, dried, and concentrated to obtain the compound represented by formula B;

[0026] The molar ratio of the compound represented by formula A, ethyl bromoacetate and sodium hydride is 1:2-2.2:1.5-2;

[0027] The volume amount of the N,N-dimethylformamide is 10 to 30 mL / mmol based on the amount of the compound represented by formula A;

[0028] (3) dissolving the compound represented by formula B in trifluoroacetic acid and dichloromethane, stirring and reacting at room temperature for 5 to 6 hours. After the reaction is completed, evaporating the reaction solution under reduced pressure to dryness to obtain the compound represented by formula C;

[0029] The trifluoroacetic acid is used in excess, and generally the volumetric amount of trifluoroacetic acid is 10 to 20 mL / g based on the mass of the compound represented by formula B;

[0030] The volume amount of the dichlorohexane is 10-20 mL / g based on the mass of the compound represented by formula B;

[0031] (4) dissolving the compound represented by formula C in methanol, adding tetrahydrofuran and 1N sodium hydroxide aqueous solution, stirring and reacting at room temperature for 3 to 5 hours, adjusting the pH to 3 after the reaction, extracting with water-ethyl acetate, taking the organic phase, drying and concentrating to obtain a solid product, and then purifying by thin layer chromatography to obtain the aripiprazole hapten represented by formula I;

[0032] The volume amount of the 1N sodium hydroxide aqueous solution is generally 35 to 50 mL / g based on the mass of the compound represented by formula C;

[0033] The volume amount of methanol is generally 15 to 30 mL / g based on the mass of the compound represented by formula C;

[0034] The volume amount of tetrahydrofuran is generally 10 to 25 mL / g based on the mass of the compound represented by formula C;

[0035] Generally, 1N hydrochloric acid is used to adjust the solution pH to 3;

[0036] The chromatographic solution of the thin layer chromatography is a mixed solution of 95 vol% ethanol, 1,4-dioxane, dichloromethane and ammonia water in a volume ratio of 8:1:10:1.

[0037] Through the above method, a linker is introduced to the carboxyl group of aripiprazole hapten I. The introduction of the linker at this modification site can retain the characteristic structure of aripiprazole to a large extent and does not occupy the active site of aripiprazole. The corresponding artificial antigen and antibody obtained have higher specificity.

[0038] The present invention also provides an artificial aripiprazole antigen, whose molecular structure is shown in Formula III:

[0039]

[0040] In formula (III), BGG is bovine gamma globulin.

[0041] The present invention also provides a method for preparing the artificial aripiprazole antigen III, comprising: combining the artificial aripiprazole hapten shown in formula I with bovine immunoglobulin gamma by an active ester method to obtain the artificial aripiprazole antigen shown in formula III.

[0042] Furthermore, when the active ester method is used to prepare the aripiprazole artificial antigen represented by formula III, the method comprises the following steps:

[0043] (a) mixing the artificial aripiprazole hapten of formula I, N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide at a molar ratio of 1:1-2:1-2, stirring at 20-30° C. for 18-20 hours, and centrifuging to obtain the supernatant after the reaction is completed;

[0044] Preferably, N,N-dimethylformamide solvent is added during the reaction; the volume amount of the N,N-dimethylformamide is generally 20-40 mL / mmol based on the amount of the aripiprazole artificial hapten substance shown in Formula I.

[0045] (b) adding the supernatant dropwise to the bovine immunoglobulin solution, mixing well, and then standing the resulting mixture at 3-5° C. overnight (usually for 15-24 hours), dialyzing, and centrifuging to obtain the supernatant to obtain the aripiprazole artificial antigen represented by formula III.

[0046] Furthermore, the bovine immunoglobulin G solution of the present invention is prepared by dissolving bovine immunoglobulin G in 0.01 M PBS buffer solution with a pH of 7.2-7.4.

[0047] In the step (b), the concentration of the bovine immunoglobulin G solution is 5 mg / mL, and the volume ratio of the supernatant to the bovine immunoglobulin G solution is 1:5.

[0048] The present invention selects bovine gamma globulin (BGG) as white or milky white powder, which is tasteless or has a slightly specific aroma and taste and is easily soluble in water. Its aqueous solution will produce casein precipitation when it meets acid. As a macromolecular carrier, it has the following advantages: 1. bovine gamma globulin can bind more artificial haptens of aripiprazole and has good immunogenicity; 2. from the experiment, the binding of bovine gamma globulin and artificial haptens of aripiprazole is more stable; 3. the artificial antigen of aripiprazole formed by the binding of bovine gamma globulin and artificial haptens of aripiprazole has better specificity of anti-aripiprazole antibodies obtained by animal immunization.

[0049] The present invention also provides the use of the artificial aripiprazole antigen in preparing anti-aripiprazole antibodies.

[0050] The present invention also provides an anti-aripiprazole antibody, which is a globulin obtained by immunizing an animal with the artificial aripiprazole antigen and can generate a specific immune reaction with aripiprazole.

[0051] The present invention also provides use of the anti-aripiprazole antibody in detecting aripiprazole.

[0052] The present invention can also provide a reagent for detecting aripiprazole, wherein the reagent comprises the anti-aripiprazole antibody.

[0053] The present invention also provides a kit for detecting aripiprazole, wherein the kit comprises the anti-aripiprazole antibody.

[0054] The experiment found that when the artificial aripiprazole antigen represented by Formula III was immunized into New Zealand white rabbits, the titer of the immune serum obtained was 1:70000. The results showed that the artificial aripiprazole antigen of the present invention can be used to obtain anti-aripiprazole antibodies with high affinity, high sensitivity and strong specificity through immunization, and the anti-aripiprazole antibodies can be used for immunodetection and analysis of aripiprazole.

[0055] Compared with the prior art, the present invention has the following beneficial effects:

[0056] The artificial aripiprazole hapten of the present invention retains the characteristic structure of aripiprazole to the greatest extent, and has an active group that can be coupled with a carrier protein, and can be used as an antigenic determinant cluster; the artificial aripiprazole antigen prepared further can be used to immunize to obtain anti-aripiprazole antibodies with high affinity, high sensitivity and strong specificity, and the titer of the immune serum obtained by immunizing New Zealand white rabbits can meet the needs, and can be used for rapid and accurate immunodetection and immunoanalysis of aripiprazole. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 is the reaction formula of the aripiprazole artificial antigen of the present invention.

[0058] Among them, (BOC) 2O represents di-tert-butyl dicarbonate, THF represents tetrahydrofuran, TEA represents triethylamine, DMF represents N,N-dimethylformamide, TFA represents trifluoroacetic acid, DCM represents dichloromethane, NHS represents N-hydroxysuccinimide, DCC represents cyclohexylcarbodiimide, BGG-NH 2 Represents bovine immunoglobulin G (there are many free amino groups on BGG, but only one is used here), the same below.

[0059] Figure 2 This is a liquid chromatogram of the artificial aripiprazole hapten represented by formula I prepared in Example 1.

[0060] Here, mAU stands for milliabsorptivity unit, and min stands for minute.

[0061] Figure 3 This is the ESI-MS analysis spectrum of the aripiprazole hapten represented by formula I prepared in Example 1.

[0062] In which, Intens. represents the signal intensity; m / z represents the charge-to-mass ratio.

[0063] Figure 4 These are the IRS spectra of bovine gamma globulin (BGG), aripiprazole artificial hapten (I), and aripiprazole artificial antigen (III).

[0064] Figure 5 This is a flow chart for the preparation of artificial aripiprazole antigen IV in comparative example 1.

[0065] Among them, BSA represents bovine serum albumin (there are many free amino groups on BSA, only one is used here to represent it), the same below.

[0066] Figure 6 This is a flow chart for the preparation of artificial aripiprazole antigen V in comparative example 2.

[0067] Figure 7 This is a flow chart for the preparation of artificial aripiprazole antigen VI in comparative example 3.

[0068] Figure 8 This is a flow chart for the preparation of artificial aripiprazole antigen VIII in comparative example 4.

[0069] Fig. 9 This is a flow chart for the preparation of artificial aripiprazole antigen IX in comparative example 5.

[0070] Fig.10 This is a flow chart for the preparation of artificial aripiprazole antigen X in comparative example 6.

[0071] Fig.11 This is a flow chart for the preparation of artificial aripiprazole antigen XI in comparative example 7. DETAILED DESCRIPTION

[0072] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods, but the protection scope of the present invention is not limited thereto.

[0073] Example 1

[0074] This embodiment provides a method for preparing an artificial aripiprazole antigen (reaction formula as shown in Figure 1 ), including the following steps:

[0075] (1) Preparation of artificial hapten (Formula I):

[0076] ① Weigh 200 mg (0.432 mmol) of hydroxy-aripiprazole into a 25 mL single-mouth round-bottom flask, add 5 mL of tetrahydrofuran to dissolve, place in a 0°C ice-water bath and stir thoroughly, add 120 μL (0.864 mmol) of triethylamine, then add dropwise 109 μL (0.475 mmol) of di-tert-butyl dicarbonate in 2 mL of tetrahydrofuran solution, stir at room temperature for 4 hours. End the reaction, evaporate the reaction solution under reduced pressure to dryness to obtain 262 mg of a light yellow oily substance A, and directly proceed to the next step without purification.

[0077] ② Place 262 mg (0.432 mmol) of the yellowish oily substance A in the previous step (calculated according to the theoretical value) in a 25 mL single-mouth round-bottom flask, add 5 mL N, N-dimethylformamide to dissolve, then add 96 μL (0.862 mmol) of ethyl bromoacetate and 31 mg (0.775 mmol) of sodium hydride, place in a 65°C oil bath, reflux and stir to react for 18 hours. TLC detection shows that the reaction is basically complete, and the chromatographic liquid is ethyl acetate: petroleum ether = 1:10 (v / v), and the product R f =0.8. Stop the reaction, evaporate the reaction solution under reduced pressure, add 20 mL of water to the reaction residue, extract with 20 mL×3 of dichloromethane, combine the organic phases, wash the organic phases with 20 mL of purified water and 20 mL of saturated brine, take the organic phase, dry it with anhydrous magnesium sulfate for 15 minutes, filter and evaporate under reduced pressure to obtain a yellow oil B, with a yield of 243 mg, which is impure and directly enters the next step of reaction.

[0078] ③ Place 243 mg of the yellow oil B obtained in the previous step in a 25 mL single-mouth round-bottom flask, add 3 mL of trifluoroacetic acid and 3 mL of dichloromethane to dissolve, and stir at room temperature for 5 hours. End the reaction, evaporate the solvent under reduced pressure to obtain a yellow oil C, with a yield of 215 mg.

[0079] ④ Place 215 mg of the yellow oil C in a 50 mL single-mouth round-bottom flask, add 4.4 mL of methanol, 3.3 mL of tetrahydrofuran and 8.6 mL of 1N NaOH aqueous solution, add a stirrer, and stir at room temperature for 4 hours. TLC detected that the raw material had reacted completely, and the chromatographic solution was 95% ethanol: 1,4-dioxane: dichloromethane: ammonia water = 8:1:10:1 (v / v), and the product R f =0.4. Stop the reaction, adjust the solution pH to 3 with 1N hydrochloric acid, a large amount of precipitation is generated, extract with 30mL×3 ethyl acetate, combine the organic phases, wash the organic phases with 30mL purified water and 30mL saturated brine respectively, collect the organic phases, add anhydrous magnesium sulfate to dry for 15 minutes, filter, and evaporate to dryness under reduced pressure to obtain 212mg of light yellow solid product;

[0080] ⑤ The light yellow solid (212 mg) was separated by thin layer chromatography (the solvent and eluent were anhydrous ethanol, the chromatographic liquid was 95% ethanol: 1,4-dioxane: dichloromethane: ammonia water = 8: 1: 10: 1, Rf = 0.4) to obtain 148 mg (0.284 mmol) of a light yellow solid, i.e., the artificial hapten of aripiprazole (Formula I).

[0081] The liquid chromatogram of the prepared artificial hapten of aripiprazole (Formula I) is as follows: Figure 2 As shown, the ESI-MS analysis spectrum is as follows Figure 3 As shown, from Figure 2 It can be seen that the purity of the purified artificial hapten I of aripiprazole reaches more than 99%. Figure 3 It can be seen that the mass-to-charge ratio (m / z) of the molecular ion peak of the aripiprazole artificial hapten obtained in this example is 522, which is consistent with its theoretical molecular weight. It can be determined that the final compound obtained in step ⑤ is the aripiprazole artificial hapten (Formula I) designed by the present invention.

[0082] (2) Preparation of Aripiprazole Artificial Antigen III:

[0083] ⑥ Place 148 mg (0.284 mmol) of aripiprazole artificial hapten I in a 25 mL round-bottom flask, add 7.4 mL of N,N-dimethylformamide (DMF), 47 mg (0.413 mmol) of N-hydroxysuccinimide (NHS) and 85 mg (0.413 mmol) of cyclohexylcarbodiimide (DCC), and stir at 20°C for 18 hours. TLC detection shows that the reaction is basically complete, and the chromatographic solution is 95% ethanol: 1,4-dioxane: dichloromethane: ammonia water = 8:1:10:1 (v / v), and the product R f = 0.1-0.2. Stop the reaction, centrifuge the reaction solution at 8000RPM and 4°C for 5 minutes, and take the supernatant for later use.

[0084] ⑦ Weigh 14.5 g (0.0405 mol) of disodium hydrogen phosphate dodecahydrate, 43.875 g (0.75 mol) of sodium chloride, and 1.495 g (0.00958 mol) of sodium dihydrogen phosphate dihydrate, dissolve them in double distilled water and make up to 5.0 L to obtain 0.01 M PBS buffer solution with pH = 7.2-7.4.

[0085] ⑧ Weigh 0.185 g of bovine immunoglobulin G and dissolve it in 37 mL of the PBS buffer in step ⑦ to obtain a bovine immunoglobulin G solution with a concentration of 5 mg / mL.

[0086] ⑨ At room temperature, add the supernatant of step ⑥ dropwise to the rapidly stirred bovine immunoglobulin solution of ⑧ and continue stirring for 0.5 hours. The volume ratio of the supernatant to the bovine immunoglobulin solution is 1:5. The obtained mixture is stored at 4°C for 18 hours to obtain an artificial antigen mixture.

[0087] ⑩ The artificial antigen mixture was transferred into a dialysis bag and dialyzed 7 times with the PBS buffer in step ⑦. After the dialysis, the mixture was centrifuged at 8000RPM and 4°C for 5 minutes. The supernatant was taken to obtain the artificial antigen: aripiprazole-bovine gamma globulin conjugate (such as formula III). The IRS spectra of bovine gamma globulin (BGG), aripiprazole artificial hapten (I), and aripiprazole artificial antigen (III) are shown in Figure 4 .

[0088] Figure 4 In the figure, the three curves are the infrared absorption spectra of BGG, aripiprazole artificial hapten I, and aripiprazole artificial antigen III. It can be seen from the figure that BGG and aripiprazole artificial antigen III have similar absorption curves. This is the characteristic peak produced by the amino acids in BGG, indicating that aripiprazole artificial antigen III does contain BGG, and aripiprazole artificial hapten I and aripiprazole artificial antigen III have a similar absorption curve at 1200cm -1 The results show that the artificial antigen III of aripiprazole has similar absorption, indicating that the artificial hapten I of aripiprazole exists in the artificial antigen III of aripiprazole. This proves that the artificial antigen III of aripiprazole is successfully coupled.

[0089] Comparative Example 1

[0090] Prepare the artificial aripiprazole antigen (Formula IV), the reaction formula is as follows Figure 5 As shown, the following steps are included:

[0091] (1) Preparation of artificial hapten I of aripiprazole:

[0092] ①-⑤ are the same as in Example 1.

[0093] (2) Preparation of artificial antigen IV of aripiprazole:

[0094] ⑥-⑩ are similar to Example 1, except that bovine serum albumin (BSA) is replaced with bovine serum albumin for coupling with artificial hapten I, and finally aripiprazole artificial antigen IV is obtained.

[0095] Comparative Example 2

[0096] Prepare the artificial aripiprazole antigen (Formula V), the reaction formula is as follows Figure 6 As shown, the following steps are included:

[0097] (1) Preparation of artificial hapten I of aripiprazole:

[0098] ①-⑤ are the same as in Example 1.

[0099] (2) Preparation of artificial aripiprazole antigen V:

[0100] ⑥ Place 100 mg (0.192 mmol) of aripiprazole artificial hapten I in a 50 mL round-bottom flask, add 5 mL of N,N-dimethylformamide (DMF), then add 27 μL (0.192 mmol) of triethylamine and 25 μL (0.192 mmol) of isobutyl chloroformate, and stir at 30°C for 19 hours. TLC detection shows that the reaction is basically complete, and the chromatographic solution is 95% ethanol: 1,4-dioxane: dichloromethane: ammonia water = 8:1:10:1 (v / v), and the product R f = 0.1-0.2. After the reaction is completed, the reaction solution is centrifuged at 8000RPM and 4°C for 5 minutes, the precipitate is discarded, and the supernatant is taken.

[0101] ⑦ Weigh 14.5 g (0.0405 mol) of disodium hydrogen phosphate dodecahydrate, 43.875 g (0.75 mol) of sodium chloride, and 1.495 g (0.00958 mol) of sodium dihydrogen phosphate dihydrate, dissolve them in deionized water to make up to 5.0 L to obtain 0.01 M PBS buffer solution with pH = 7.2-7.4.

[0102] ⑧ Weigh 0.100 g of bovine immunoglobulin G and dissolve it in 20 mL of the PBS buffer prepared in step ⑦ to obtain a 5 mg / mL bovine immunoglobulin G solution.

[0103] ⑨ Under rapid stirring, slowly add the supernatant of step ⑥ to the bovine immunoglobulin G solution of step ⑧, the volume ratio of the supernatant to the bovine immunoglobulin G solution is 1:5, and the obtained mixture is stored at 4°C for 18 hours to obtain an artificial antigen mixture.

[0104] ⑩ Transfer the artificial antigen mixture into a dialysis bag and dialyze it 7 times with the PBS buffer in step ⑦. After the dialysis, centrifuge it at 8000RPM and 4°C for 5 minutes, and take the supernatant to obtain the aripiprazole artificial antigen V.

[0105] Comparative Example 3

[0106] Prepare the artificial aripiprazole antigen (Formula VI), the reaction formula is as follows Figure 7 , including the following steps:

[0107] (1) Preparation of artificial hapten I of aripiprazole:

[0108] ①-⑤ are the same as in Example 1.

[0109] (2) Preparation of artificial aripiprazole antigen VI:

[0110] ⑥-⑩ are similar to comparative example 2; except that bovine immunoglobulin G is replaced by bovine serum albumin for coupling with artificial hapten I, and finally aripiprazole artificial antigen VI is obtained.

[0111] Comparative Example 4

[0112] Prepare the artificial aripiprazole antigen (Formula VIII), the reaction formula is as follows Figure 8 , including the following steps:

[0113] (1) Preparation of artificial hapten VII of aripiprazole:

[0114] ① Weigh 200 mg (0.432 mmol) of hydroxy-aripiprazole into a 25 mL single-mouth round-bottom flask, add 5 mL of tetrahydrofuran to dissolve, place in a 0°C ice-water bath and stir, add 120 μL (0.864 mmol) of triethylamine, then add dropwise 109 μL (0.475 mmol) of di-tert-butyl dicarbonate in 2 mL of tetrahydrofuran solution, stir at room temperature for 4 hours. End the reaction, evaporate the reaction solution under reduced pressure to dryness to obtain 257 mg of a light yellow oily substance A, and directly proceed to the next step without purification.

[0115] ②Put 257mg (0.432mmol) (calculated according to theoretical value) of the pale yellow oil in the previous step into a 50mL single-mouth round-bottom flask, add 20mL of dichloromethane to dissolve, then add 173mg (1.73mmol) of succinic anhydride, 180μL (1.30mmol) of triethylamine and 20mg of DMAP, and stir in a 41℃ oil bath for 18 hours. The reaction was basically complete by TLC detection, and the chromatographic solution was 95% ethanol: 1,4-dioxane: dichloromethane: ammonia water = 8:1:10:1 (v / v), and the product R f =0.4,. The reaction was stopped, the reaction solution was cooled to room temperature, and the obtained brown oil was evaporated to dryness under reduced pressure.

[0116] ③ The brown oil was separated by thin layer chromatography (the solvent and eluent were anhydrous ethanol, the chromatographic liquid was 95% ethanol: 1,4-dioxane: dichloromethane: ammonia water = 8: 1: 10: 1 (v / v), Rf = 0.4) to obtain 34 mg (0.352 mmol) of light yellow solid D2.

[0117] ④ Place 34 mg (0.352 mmol) of the pale yellow solid D2 in a 25 mL single-mouth round-bottom flask, add 3 mL of trifluoroacetic acid and 3 mL of dichloromethane to dissolve, and stir at room temperature for 5 hours. End the reaction, evaporate the solvent under reduced pressure to obtain a yellow oil, which is separated by thin layer chromatography (the solvent and eluent are anhydrous ethanol, the chromatographic liquid is 95% ethanol: 1,4-dioxane: dichloromethane: ammonia water = 8: 1: 10: 1 (v / v), Rf = 0.3) to obtain another yellow oil 108 mg (0.192 mmol), i.e., artificial hapten VII of aripiprazole.

[0118] (2) Preparation of artificial aripiprazole antigen VIII:

[0119] ⑤ Place 108 mg (0.192 mmol) of aripiprazole artificial hapten VII in a 25 mL single-necked round-bottom flask, add 5.4 mL of N,N-dimethylformamide (DMF) to dissolve, then add 44 mg (0.384 mmol) of N-hydroxysuccinimide (NHS) and 79 mg (0.384 mmol) of cyclohexylcarbodiimide (DCC), stir and react at 20°C for 18 hours. After the reaction is completed, centrifuge the reaction solution at 8000 RPM and 4°C for 5 minutes, take the supernatant and set aside.

[0120] ⑥ Weigh 14.5 g (0.0405 mol) of disodium hydrogen phosphate dodecahydrate, 43.875 g (0.75 mol) of sodium chloride, and 1.495 g (0.00958 mol) of sodium dihydrogen phosphate dihydrate, dissolve them in deionized water to make up to 5.0 L to obtain 0.01 M PBS buffer solution with pH = 7.2-7.4.

[0121] ⑦ Weigh 0.135 g of bovine immunoglobulin G and dissolve it in 27 mL of the PBS buffer prepared in step ⑥ to obtain a bovine immunoglobulin G solution with a concentration of 5 mg / mL.

[0122] ⑧ At room temperature, the supernatant of step ⑤ was slowly added dropwise to the rapidly stirred bovine immunoglobulin G solution of step ⑦, the volume ratio of the supernatant to the bovine immunoglobulin G solution being 1:5, and the obtained mixture was allowed to react at 4°C for 18 hours to obtain an artificial antigen mixture.

[0123] ⑨ Transfer the artificial antigen mixture into a dialysis bag and dialyze it 7 times with the PBS in step ⑥. After the dialysis, centrifuge it at 8000RPM and 4°C for 5 minutes, and take the supernatant to obtain aripiprazole artificial antigen VIII.

[0124] Comparative Example 5

[0125] Preparation of artificial aripiprazole antigen IX, the reaction formula is as follows Fig. 9 As shown, the following steps are included:

[0126] (1) Preparation of artificial hapten VII of aripiprazole:

[0127] ①-④ are the same as Comparative Example 4.

[0128] (2) Preparation of Aripiprazole Artificial Antigen IX:

[0129] ⑤-⑨ are similar to comparative example 4, except that bovine immunoglobulin G is replaced by bovine serum albumin for coupling with artificial hapten VII to obtain aripiprazole artificial antigen IX.

[0130] Comparative Example 6

[0131] Preparation of artificial aripiprazole antigen X, the reaction formula is as follows Fig.10 As shown, the following steps are included:

[0132] (1) Preparation of artificial hapten VII of aripiprazole:

[0133] ①-④ are the same as Comparative Example 4.

[0134] (2) Preparation of artificial aripiprazole antigen X:

[0135] ⑤ Place 100 mg (0.177 mmol) of aripiprazole artificial hapten VII in a 25 mL round-bottom flask, add 5 mL of N,N-dimethylformamide (DMF), then add 25 μL (0.177 mmol) of triethylamine and 23 μL (0.177 mmol) of isobutyl chloroformate, stir at 30 ° C for 19 hours, after the reaction is completed, centrifuge at 8000 RPM, 4 ° C for 5 minutes, and take the supernatant.

[0136] ⑥ Weigh 14.5 g (0.0405 mol) of disodium hydrogen phosphate dodecahydrate, 43.875 g (0.75 mol) of sodium chloride, and 1.495 g (0.00958 mol) of sodium dihydrogen phosphate dihydrate, dissolve them in deionized water to make up to 5.0 L to obtain 0.01 M PBS buffer solution with pH = 7.4.

[0137] ⑦ Weigh 0.125 g of bovine immunoglobulin G and dissolve it in 25 mL of the PBS buffer prepared in step ⑥ to obtain a bovine immunoglobulin G solution with a concentration of 5 mg / mL.

[0138] ⑧ At room temperature, the supernatant of step ⑤ is slowly added dropwise to the rapidly stirred bovine immunoglobulin G solution of step ⑦, the volume ratio of the supernatant to the bovine immunoglobulin G solution is 1:5, and the obtained mixture is stored at 4°C for 18 hours to obtain an artificial antigen mixture.

[0139] ⑨ Transfer the artificial antigen mixture into a dialysis bag and dialyze it 7 times with the PBS in step ⑥. After the dialysis, centrifuge it at 8000RPM and 4°C for 5 minutes, and take the supernatant to obtain the aripiprazole artificial antigen X.

[0140] Comparative Example 7

[0141] This invention provides a method for preparing an artificial aripiprazole antigen XI (reaction process as shown in Fig.11 ), including the following steps:

[0142] (1) Preparation of artificial hapten VII of aripiprazole:

[0143] ①-④ are the same as Comparative Example 4.

[0144] (2) Preparation of artificial aripiprazole antigen Ⅺ:

[0145] ⑤-⑨ are similar to comparative example 6, except that bovine immunoglobulin G is replaced by bovine serum albumin for coupling with artificial hapten VII to obtain artificial aripiprazole antigen XI.

[0146] Performance determination of artificial antigen of aripiprazole

[0147] (1) Identification of artificial antigens of aripiprazole:

[0148] Molar absorption coefficient ε: PBS buffer was used to prepare artificial hapten solutions of aripiprazole with concentrations of 0 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, and 40 μg / mL. The maximum absorption wavelength of aripiprazole hapten was 290 nm as shown in the UV scanning diagram. The absorbance was measured at 290 nm, and parallel samples were made for each concentration. The calculation formula for the molar absorption coefficient (i.e., molar absorption coefficient) is: ε = absorbance / molar concentration.

[0149] Determination of protein concentration of conjugate: Prepare 1 mL of bovine immunoglobulin solution with concentrations of 0 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, and 120 μg / mL with PBS buffer, add 3 mL of Coomassie Brilliant Blue staining solution, mix immediately, warm in a 30°C water bath for 5 minutes, make parallel samples for each concentration, measure the absorbance at 655 nm, and draw a curve of the relationship between protein concentration and absorbance. Dilute the artificial antigen solution (prepared with PBS buffer) in a certain proportion, measure the absorbance of the artificial antigen at 655 nm, and read the corresponding protein concentration value of the artificial antigen solution from the curve.

[0150] Conjugation ratio determination: Prepare 100 μg / mL bovine immunoglobulin PBS solution, dilute the conjugate (i.e., aripiprazole artificial antigen) to 100 μg / mL with PBS, and measure the absorbance value A at 285 nm. 1 , using PBS as blank to measure the absorbance value A 2 , then the coupling ratio γ is: γ=[(A1 -A 2 ) / ε] / (100×10 -3 / 150000).

[0151] Where ε is the molar absorption coefficient (L / mol), 150000 is the molecular weight of bovine gamma globulin, and 100×10 -3 is the concentration of bovine immunoglobulin (μg / mL).

[0152] When bovine serum albumin is used as a carrier, the coupling ratio is calculated as follows: γ = [(A 1 -A 2 ) / ε] / (100×10 -3 / 66430); wherein 66430 is the molecular weight of bovine serum albumin.

[0153] The coupling ratios of various aripiprazole artificial antigens and the molar absorption coefficients of the corresponding haptens are shown in Table 1.

[0154] Table 1 Coupling ratios of various aripiprazole artificial antigens and corresponding molar absorption coefficients of haptens

[0155] serial number Artificial Antigen Coupling ratio Conjugate protein concentration Molar Absorption Coefficient Example 1 Ⅲ 17 3.241mg / mL 5224.46 Comparative Example 1 Ⅳ 15 3.536mg / mL 5224.46 Comparative Example 2 Ⅴ 15 2.832mg / mL 5224.46 Comparative Example 3 Ⅵ 13 3.115mg / mL 5224.46 Comparative Example 4 Ⅷ 11 2.592mg / mL 5118.45 Comparative Example 5 Ⅸ 19 3.141mg / mL 5118.45 Comparative Example 6 Ⅹ 9 2.317mg / mL 5118.45 Comparative Example 7 Ⅺ 10 2.622mg / mL 5118.45

[0156] As can be seen from Table 1, the structure of the artificial hapten, the activation method of the artificial hapten and the structure of the carrier protein all have an impact on the binding ratio when the artificial hapten is coupled to the carrier protein.

[0157] (2) Animal immunization

[0158] The eight prepared artificial aripiprazole antigens were used to immunize New Zealand white rabbits, and the titers of the immune sera obtained were tested by ELISA. The test results are shown in Table 2.

[0159] Table 2 The titer test results of each immune serum

[0160] serial number Aripiprazole artificial antigen Immune serum titer Example 1 Ⅲ 1:70000 Comparative Example 1 Ⅳ 1:56000 Comparative Example 2 Ⅴ 1:50000 Comparative Example 3 Ⅵ 1:30000 Comparative Example 4 Ⅷ 1:28000 Comparative Example 5 Ⅸ 1:25000 Comparative Example 6 Ⅹ 1:8000 Comparative Example 7 Ⅺ 1:10000

[0161] As can be seen from Table 2, compared with Example 1, the titers of the immune sera obtained by immunizing animals with artificial aripiprazole antigens in each comparative example cannot meet the requirements and cannot be used in immunoassays. However, the titer of the immune sera obtained by immunizing animals with artificial aripiprazole antigen III is 1:70,000, which can be fully used in immunoassays, providing a more convenient, rapid and accurate way for the detection of aripiprazole.

Claims

1. An artificial hapten of aripiprazole, characterized in that: Its molecular structure is shown in (Ⅰ): I。 2. The method for preparing the artificial hapten of aripiprazole according to claim 1, characterized in that: The method comprises the following steps: (1) Acylation reaction of hydroxy aripiprazole represented by formula II with triethylamine and di-tert-butyl dicarbonate to prepare a compound represented by formula A; (2) The compound represented by formula A is subjected to substitution reaction with ethyl bromoacetate and sodium hydride to obtain the compound represented by formula B; (3) The compound represented by formula B is deprotected in trifluoroacetic acid to obtain the compound represented by formula C; (4) hydrolyzing the compound represented by formula C with an alkaline solution, and then adjusting the pH to acidic to obtain the aripiprazole hapten represented by formula I; The reaction formula of the method is as follows: 。 3. The method according to claim 2, characterized in that In the step (1), the molar ratio of hydroxyaripiprazole, triethylamine and di-tert-butyl dicarbonate is 1:2-2.5:1.05-1.2; In the step (2), the molar ratio of the compound represented by formula A, ethyl bromoacetate and sodium hydride is 1:2-2.2:1.5-2; The reaction temperature of step (2) is 60-70°C; The alkaline solution in step (4) is a NaOH aqueous solution.

4. A method for preparing an artificial aripiprazole antigen having a molecular structural formula as shown in III, characterized in that: The method comprises: combining the artificial aripiprazole hapten of formula I of claim 1 with bovine immunoglobulin gamma by an active ester method to obtain the artificial aripiprazole antigen of formula III; Ⅲ； In formula III, BGG is bovine gamma globulin.

5. The method for preparing the artificial aripiprazole antigen according to claim 4, characterized in that: The method comprises the following steps: (a) mixing the artificial aripiprazole hapten of formula I, N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide at a molar ratio of 1:1-2:1-2, stirring at 20-30° C. for 18-20 hours, and centrifuging to obtain the supernatant after the reaction is completed; (b) adding the supernatant dropwise to the bovine immunoglobulin solution, mixing well, and then standing the resulting mixture at 3-5° C. overnight, dialyzing, and centrifuging to obtain the supernatant to obtain the aripiprazole artificial antigen represented by formula III.

6. The method for preparing the artificial aripiprazole antigen according to claim 5, characterized in that: In the step (b), the concentration of the bovine immunoglobulin solution is 5 mg / mL; The volume ratio of the supernatant to the bovine immunoglobulin solution was 1:

5.

7. Use of the aripiprazole hapten according to claim 1 or the aripiprazole artificial antigen prepared by the method according to claim 4 in preparing anti-aripiprazole antibodies.

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