A piperidinyl derivative, its preparation and use

Abstract

This application discloses an isoluminol derivative, its preparation method, and its uses. The isoluminol derivative is shown in formula (I), and the definitions of each substituent in formula (I) are detailed in the specification. This isoluminol derivative exhibits significantly improved luminescence intensity, thus solving the problem of low detection sensitivity.

Description

Technical Field

[0001] This article relates to, but is not limited to, chemiluminescence technology, particularly an isoluminol derivative and its preparation method and uses. Background Technology

[0002] In 1977, Halman et al. applied chemiluminescence methods in their immunoassay research and established chemiluminescence immunoassay. Luminol, isoluminol (ABEI), and their derivatives were among the first chemiluminescent substances used in this method. After catalytic oxidation of the chemiluminescent substance, an excited-state intermediate is obtained. When the intermediate returns to its ground state, it emits photons, and the photon yield is detected. Luminol, chemically known as 3-aminophthalohydrazine, was first reported for its luminescent properties by Albrecht. Although the luminescent quantum yield of isoluminol is only 10% of that of luminol, modification of its amino group has increased the chemiluminescent yield by several times or even tens of times, and its luminescent stability is superior to that of luminol. Therefore, in the field of chemiluminescence immunoassay, isoluminol (ABEI) is widely used as a chemiluminescent marker in the clinical diagnosis of various biomarkers.

[0003] However, the luminescence intensity of existing isoluminol is not ideal, which can easily lead to low detection sensitivity. Summary of the Invention

[0004] The purpose of this application is to overcome the above-mentioned shortcomings and provide an isoluminol derivative and its preparation method to solve the problems of insufficient luminescence intensity and low detection sensitivity of existing isoluminol.

[0005] On the one hand, this application provides an isoluminol derivative, which is shown in formula (I):

[0006]

[0007] In formula (I), one of R1 and R3 is hydrogen, while the other, together with R2, forms an aromatic ring fused with the benzene ring to which they are attached, which is substituted with one or more substituents; the aromatic ring is selected from benzene, naphthalene, five-membered heteroaromatic rings and six-membered heteroaromatic rings; the substituent is selected from substituted phenyl and -N(R4)(R5), where R4 and R5 are each independently hydrogen, unsubstituted C1-C4 alkyl and amino-substituted C1-C4 alkyl, and the substituted benzene is a phenyl substituted with a carboxyl group or -N(R6)(R7), where R6 and R7 are each independently hydrogen, unsubstituted C1-C4 alkyl and amino-substituted C1-C4 alkyl.

[0008] In some embodiments of this application, in formula (I), one of R1 and R3 is hydrogen, while the other, together with R2, forms an aromatic ring fused with the benzene ring to which they are attached, which is substituted with one or more substituents; the aromatic ring is benzene; the substituent is -N(R4)(R5), where R4 and R5 are each independently hydrogen, an unsubstituted C1-C4 alkyl group, and an amino-substituted C1-C4 alkyl group.

[0009] In some embodiments of this application, R1 in formula (I) is hydrogen, and R3 together with R2 forms an aromatic ring substituted with one or more substituents and fused with the benzene ring to which they are attached; the aromatic ring is benzene; the substituents are -N(R4)(R5), where R4 and R5 are each independently hydrogen, an unsubstituted C1-C4 alkyl group, and an amino-substituted C1-C4 alkyl group.

[0010] In some embodiments of this application, R3 in formula (I) is hydrogen, and R1 and R2 together form an aromatic ring fused with the benzene ring to which they are attached, which is substituted with one or more substituents; the aromatic ring is benzene; the substituents are -N(R4)(R5), where R4 and R5 are each independently hydrogen, unsubstituted C1-C4 alkyl, and amino-substituted C1-C4 alkyl.

[0011] In some embodiments of this application, in formula (I), one of R1 and R3 is hydrogen, while the other, together with R2, forms an aromatic ring fused with the benzene ring to which they are attached, substituted with one or more substituents; the aromatic ring is benzene; the substituents are -N(R4)(R5), where R4 and R5 are each independently an unsubstituted C1-C4 alkyl and an amino-substituted C1-C4 alkyl.

[0012] In some embodiments of this application, R3 in formula (I) is hydrogen, and R1 and R2 together form an aromatic ring fused with the benzene ring to which they are attached, which is substituted with one or more substituents; the aromatic ring is benzene; the substituents are -N(R4)(R5), where R4 and R5 are each independently an unsubstituted C1-C4 alkyl and an amino-substituted C1-C4 alkyl.

[0013] In some embodiments of this application, R1 in formula (I) is hydrogen, and R3 together with R2 forms an aromatic ring substituted with one or more substituents and fused with the benzene ring to which they are attached; the aromatic ring is benzene; the substituents are -N(R4)(R5), where R4 and R5 are each independently an unsubstituted C1-C4 alkyl and an amino-substituted C1-C4 alkyl.

[0014] In some embodiments of this application, in formula (I), one of R1 and R3 is hydrogen, while the other, together with R2, forms an aromatic ring substituted with one or more substituents and fused with the benzene ring to which they are attached; the aromatic ring is benzene; the substituent is -N(R4)(R5), where one of R4 and R5 is methyl, ethyl, n-propyl or isopropyl, and the other is 3-aminopropyl or 4-aminobutyl.

[0015] In some embodiments of this application, R3 in formula (I) is hydrogen, and R1 and R2 together form an aromatic ring substituted with one or more substituents and fused with the benzene ring to which they are attached; the aromatic ring is benzene; the substituent is -N(R4)(R5), where one of R4 and R5 is methyl, ethyl, n-propyl or isopropyl, and the other is 3-aminopropyl or 4-aminobutyl.

[0016] In some embodiments of this application, R1 in formula (I) is hydrogen, and R3 together with R2 forms an aromatic ring substituted with one or more substituents and fused with the benzene ring to which they are attached; the aromatic ring is benzene; the substituent is -N(R4)(R5), where one of R4 and R5 is methyl, ethyl, n-propyl or isopropyl, and the other is 3-aminopropyl or 4-aminobutyl.

[0017] In some embodiments of this application, in formula (I), one of R1 and R3 is hydrogen, while the other, together with R2, forms an aromatic ring substituted with one or more substituents and fused with the benzene ring to which they are attached; the aromatic ring is benzene; the substituent is -N(R4)(R5), where one of R4 and R5 is ethyl and the other is 4-aminobutyl.

[0018] In some embodiments of this application, one of R1 and R3 in formula (I) is hydrogen, while the other, together with R2, forms a benzene with the 4-position substituted by -N(R4)(R5) and fused with the benzene ring to which they are attached; here, one of R4 and R5 is ethyl, and the other is 4-aminobutyl.

[0019] In one embodiment of this application, R3 in formula (I) is hydrogen, and R1 and R2 together form a phenyl group with -N(R4)(R5) substituted at the 4 position and fused with the benzene ring to which they are attached; here, one of R4 and R5 is ethyl and the other is 4-aminobutyl.

[0020] In one embodiment of this application, R1 in formula (I) is hydrogen, and R3 together with R2 forms a benzene with -N(R4)(R5) substituted at the 4 position and fused with the benzene ring to which they are attached; here, one of R4 and R5 is ethyl and the other is 4-aminobutyl.

[0021] In some embodiments of this application, R1 in formula (I) is hydrogen, and R3 together with R2 forms a five-membered heteroaromatic ring substituted with one or more substituents and fused with the benzene ring to which they are attached; the five-membered heteroaromatic ring is an imidazole or a thiazole; the substituent is a substituted phenyl group, wherein the substituted benzene is a phenyl group substituted with a carboxyl group or -N(R6)(R7), wherein R6 and R7 are each independently hydrogen, an unsubstituted C1-C4 alkyl group, and an amino-substituted C1-C4 alkyl group.

[0022] In some embodiments of this application, R1 in formula (I) is hydrogen, and R3 together with R2 forms a five-membered heteroaromatic ring substituted with one or more substituents and fused with the benzene ring to which they are attached; the five-membered heteroaromatic ring is a thiazole; the substituent is a substituted phenyl group, wherein the substituted benzene is a phenyl group substituted with a carboxyl group or -N(R6)(R7), wherein R6 and R7 are each independently an unsubstituted C1-C4 alkyl group and an amino-substituted C1-C4 alkyl group.

[0023] In some embodiments of this application, R1 in formula (I) is hydrogen, and R3 together with R2 forms a five-membered heteroaromatic ring substituted with one or more substituents and fused with the benzene ring to which they are attached; the five-membered heteroaromatic ring is a thiazole; the substituent is a 4-substituted phenyl, wherein the 4-substituted benzene is a 4-carboxyphenyl or a 4-N(R6)(R7)phenyl, wherein one of R6 and R7 is an unsubstituted C1-C4 alkyl and the other is an amino-substituted C1-C4 alkyl.

[0024] In some embodiments of this application, R1 in formula (I) is hydrogen, and R3 together with R2 forms a five-membered heteroaromatic ring substituted with one or more substituents and fused with the benzene ring to which they are attached; the five-membered heteroaromatic ring is a thiazole; the substituent is a 4-substituted phenyl, wherein the 4-substituted benzene is 4-carboxyphenyl or 4-N(R6)(R7)phenyl, wherein one of R6 and R7 is methyl, ethyl, n-propyl or isopropyl, and the other is 3-aminopropyl or 4-aminobutyl.

[0025] In some embodiments of this application, R1 in formula (I) is hydrogen, and R3 together with R2 forms a five-membered heteroaromatic ring substituted with one or more substituents and fused with the benzene ring to which they are attached; the five-membered heteroaromatic ring is a thiazole; the substituent is a 4-substituted phenyl, wherein the 4-substituted benzene is 4-carboxyphenyl or 4-N(R6)(R7)phenyl, wherein one of R6 and R7 is ethyl and the other is 4-aminobutyl.

[0026] In some embodiments of this application, formula (I) is one of the following compounds:

[0027]

[0028] Secondly, this application provides a method for preparing the above-mentioned isoluminol derivative, the method comprising the following steps:

[0029] The compound of formula (II) reacts with hydrazine to give the compound of formula (I);

[0030] In formula (II), the substituents R1, R3, and R3 are defined as the corresponding groups in formula (I).

[0031]

[0032] In the preparation method embodiments of this application, the reaction is carried out in an organic solvent, wherein the organic solvent is selected from C1-C4 alkanols, preferably methanol, ethanol or isopropanol, and more preferably anhydrous ethanol.

[0033] In the preparation method embodiments of this application, the reaction is carried out under heating conditions, preferably under reflux conditions.

[0034] In an embodiment of the preparation method of this application, the preparation method further includes a purification step after the reaction is completed: purification using hydrochloric acid and an alkali metal hydroxide (e.g., potassium hydroxide).

[0035] In the preparation method embodiment of this application, the purification step further includes recrystallization, for example, recrystallization using dimethylamide and water.

[0036] Thirdly, this application provides the application of the above-mentioned isoluminol derivatives in chemiluminescent immunoassay, wherein the isoluminol derivatives are used for chemiluminescent immunoassay of at least one of various hormones, tumor markers, infectious diseases, cardiovascular and myocardial markers in human serum.

[0037] Fourthly, this application provides a chemiluminescent substrate, which comprises the above-mentioned isoluminol derivative, and optionally further comprises a luminescence enhancer.

[0038] Fifthly, this application provides an immunodiagnostic reagent comprising the above-mentioned isoluminol derivative and a detection antigen or detection antibody; wherein the detection antigen or detection antibody is labeled with the isoluminol derivative.

[0039] In a sixth aspect, this application provides a chemiluminescence reagent kit, which includes the above-mentioned immunodiagnostic reagents and further includes a solid support; preferably, the solid support is microparticles; more preferably, the microparticles are magnetic nanospheres with a particle size of 0.1-5 μm; more preferably, the microparticles are linked to a capture antigen or a capture antibody.

[0040] In a seventh aspect, this application provides a chemiluminescence immunoassay system, wherein the chemiluminescence immunoassay system comprises the above-mentioned chemiluminescence reagent kit and a semi-automatic or fully automated immunoassay analyzer.

[0041] The isoluminol derivatives provided in this application improve the luminescence intensity and solve the problem of low detection sensitivity by modifying existing isoluminol.

[0042] Other features and advantages of this application will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the application. Other advantages of this application may be realized and obtained by means of the methods described in the description. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of the present invention will be described in detail below. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be arbitrarily combined with each other.

[0044] Explanation of terms in this invention embodiment:

[0045] NMR: Nuclear magnetic resonance, used to indicate the structural characterization of ABEI.

[0046] DMSO: Dimethyl sulfoxide, a commonly used solvent in the art.

[0047] DCC: N,N'-Dicyclohexylcarbodiimide.

[0048] NHS: N-hydroxysuccinimide.

[0049] Unless otherwise specified, the reagents and instruments used in the examples are conventionally selected in the art. Experimental methods not specifying particular conditions in the examples were performed under conventional conditions, such as those described in literature, books, or methods recommended by the manufacturer.

[0050] Example 1

[0051] Synthesis of ABEI-1

[0052]

[0053] step:

[0054] 1. Mix 261 g (mw 261, 1 mol) of 7-nitronaphthalene-1,2-dicarboxylic acid A1 with 400 mL of acetic anhydride, heat to reflux until completely dissolved, and then continue heating for 15 min. After the reaction is complete, remove the solvent by rotary evaporation, and recrystallize the residue in toluene to give 121 g of 7-nitro-o-naphthalenedicarboxylic anhydride A2 (yield: 50%).

[0055] 2. Take 121 g (mw 243, 0.498 mol) of 7-nitronaphthalene 1,2-dicarboxylic anhydride and dissolve it in 200 ml of 20% methylamine solution. Stir the mixture at 0 °C for 30 min. After the reaction is complete, adjust the pH to 2.0 with concentrated hydrochloric acid. A white precipitate will precipitate. Filter and collect the white precipitate, wash with water and dry. Add 200 ml of acetic anhydride to the obtained solid, dissolve completely, and heat under reflux for 15 min. After the reaction is complete, cool to room temperature, and the product will crystallize out. Filter to obtain crystals, wash with acetic acid and water successively, and dry to obtain 76.8 g of crystal N-methyl-7-nitronaphthalene 1,2-dicarboxyimide A3 (yield: 60%).

[0056] 3. Weigh 281g of SnCl2·2H2O (1.25mol) and dissolve it in 500ml of ultrapure water and 1.5L of concentrated hydrochloric acid. Then add 76.8g (mw 256, 0.3mol) of N-methyl-7-nitronaphthalene 1,2-dicarboximide to the solution and stir vigorously. The reaction solution gradually heats up and slowly becomes clear. Then let it stand overnight, and a light yellow solid precipitates. Collect the light yellow solid, wash it with a large amount of ultrapure water, and dry it to obtain 45.2g of 7-amino-N-methylnaphthalene 1,2-dicarboximide A4 (yield: 66%).

[0057] 4. Take 45.2 g of 7-amino-N-methylnaphthalene 1,2-dicarboximide (mw256, 0.2 mol) and 28 g of N-(4-bromobutyl)phthalimide (mw281, 0.1 mol), dissolve them in 300 mL of dimethylformamide, and heat under reflux for 24 h. After the reaction is complete, cool to room temperature, and a light yellow precipitate will precipitate. Filter and collect 27.7 g of N-methyl-7-(4-phthalimide-butyl)amino-naphthalene 1,2-dicarboximide A5 (yield: 68%). Recrystallize the product from an aqueous solution of acetic acid to obtain 22.5 g of analytically pure N-methyl-7-(4-phthalimide-butyl)amino-naphthalene dicarboximide.

[0058] 5. Take 0.158 mol of diethyl sulfate and 22.5 g of A5 (mw427, 0.0527 mol), and heat to 160 °C in an oil bath for 45 min under anhydrous conditions. After the reaction is complete, cool to room temperature and pour in 0.3 L of ice water, and a light yellow precipitate will precipitate. Filter and collect the precipitate, then dry it, and recrystallize the product in an aqueous solution of acetic acid to give 17.7 g of analytical grade A6 (yield: 73%).

[0059] 6. Dissolve 8 ml of 95% hydrazine in 300 ml of anhydrous ethanol, then add 17.7 g of A6 (mw 455, 0.039 mol), and heat to reflux for 3 h. Reduce to dryness by rotary evaporation to obtain an oily substance, and dry under high vacuum at 110 °C. Redissolve the obtained solid in dilute hydrochloric acid, filter to remove insoluble matter, adjust the pH of the filtrate to 9.0 with potassium hydroxide, precipitate the solid and collect it, recrystallize from 50% dimethylformamide-water to obtain 4.49 g of analytical grade ABEI-1 (yield: 35%).

[0060] 7. ABEI-1 structural characterization data: 1 H NMR (300MHz, dDMSO) δ11.99(s,2H),8.18(t,J=6.1Hz,1H),8.16(d,J=9.1Hz,1H),8.08(dd,J=9.2,2.5Hz,1H),7.89(d,J= 9.2, 2.5Hz, 1H), 3.78 (m, J = 7.0Hz, 2H), 2.69 (t, 2H), 1.49 (d, 2H), 1.52–1.46 (m, 2H), 1.40 (m, 2H), 1.12 (t, J = 6.6Hz, 3H).

[0061] 13 C NMR (300MHz, dDMSO) δ167.32, 158.41, 134.45, 127.45, 122.86, 117.11, 103.12, 53.13, 47.35, 41.78, 25.17, 12.96.

[0062] HRMS: C 18 H 22 The theoretical value of N4O2 is 326.1743, and the measured value is 326.1815.

[0063] Example 2

[0064] Synthesis of ABEI-2

[0065]

[0066] 1. Mix 261g (mw261, 1mol) of 6-nitronaphthalene-2,3-dicarboxylic acid B1 with 400ml of acetic anhydride, heat to reflux until completely dissolved, and then continue heating for 15min. After the reaction is complete, remove the solvent by rotary evaporation, and recrystallize the residue in toluene to give 121g of 6-nitronaphthalene-2,3-dicarboxylic anhydride B2 (yield: 50%).

[0067] 2. Take 121 g (mw 243, 0.498 mol) of 6-nitronaphthalene 2,3-dicarboxylic anhydride and dissolve it in 200 ml of 20% methylamine solution. Stir the mixture at 0 °C for 30 min. After the reaction is complete, adjust the pH to 2.0 with concentrated hydrochloric acid. A white precipitate will precipitate. Filter and collect the white precipitate, wash with water and dry. Add 200 ml of acetic anhydride to the obtained solid, dissolve completely, and heat under reflux for 15 min. After the reaction is complete, cool to room temperature, and the product will crystallize out. Filter to obtain crystals, wash with acetic acid and water successively, and dry to obtain 76.8 g of N-methyl-6-nitronaphthalene 2,3-dicarboxyimide B3 (yield: 60%).

[0068] 3. Weigh 281g of SnCl2·2H2O (1.25mol) and dissolve it in 500ml of ultrapure water and 1.5L of concentrated hydrochloric acid. Then add 76.8g (mw 256, 0.3mol) of N-methyl-6-nitronaphthalene 2,3-dicarboximide to the solution and stir vigorously. The reaction solution gradually heats up and slowly becomes clear. Then let it stand overnight, and a light yellow solid precipitates. Collect the light yellow solid, wash it with a large amount of ultrapure water, and dry it to obtain 45.2g of 6-amino-N-methylnaphthalene 2,3-dicarboximide B4 (yield: 66%).

[0069] 4. Take 45.2 g of 6-amino-N-methylnaphthalene 2,3-dicarboximide (mw256, 0.2 mol) and 28 g of N-(4-bromobutyl)phthalimide (mw281, 0.1 mol), dissolve them in 300 mL of dimethylformamide, and heat under reflux for 24 h. After the reaction is complete, cool to room temperature, and a light yellow precipitate will precipitate. Filter and collect 27.7 g of B5 (yield: 68%). Recrystallize the product from an aqueous solution of acetic acid to obtain 22.5 g of analytically pure B5.

[0070] 5. Take 0.158 mol of diethyl sulfate and 22.5 g of B5 (mw427, 0.0527 mol), and heat to 160 °C in an oil bath for 45 min under anhydrous conditions. After the reaction is complete, cool to room temperature and pour in 0.3 L of ice water, and a light yellow precipitate will precipitate. Filter and collect the precipitate, then dry it, and recrystallize the product in an aqueous solution of acetic acid to give 17.7 g of analytical grade B6 (yield: 73%).

[0071] 6. Dissolve 8 ml of 95% hydrazine in 300 ml of anhydrous ethanol, then add 17.7 g of B6 (mw 455, 0.039 mol), and heat to reflux for 3 h. Reduce to dryness by rotary evaporation to obtain an oily substance, and dry under high vacuum at 110 °C. Redissolve the obtained solid in dilute hydrochloric acid, filter to remove insoluble matter, adjust the pH of the filtrate to 9.0 with KOH, precipitate the solid and collect it, recrystallize from 50% dimethylformamide-water to obtain 4.49 g of analytical grade ABEI-2 (yield: 35%).

[0072] 7. ABEI-2 structural characterization data: 1 H NMR (300MHz, dDMSO) δ11.99 (s, 2H), 8.61 (t, J = 6.1Hz, 1H), 8.55 (d, J = 9.1Hz, 1H), 7.71 (dd, J = 9.2, 2.5Hz, 1H), 7.68 (d, J = 9.2, 2.5Hz, 1H), 7.10 (d, J=9.2, 2.5Hz, 1H), 3.78 (m, J=7.0Hz, 2H), 3.40 (q, J=7.0Hz, 2H), 2.69 (t, 2H),1.52(d,2H),1.50–1.46(m,2H),1.39(m,2H),1.12(t,J=6.6Hz,3H).

[0073] 13 C NMR (300MHz, dDMSO) δ167.32, 149.31, 136.75, 131.45, 121.86, 106.41, 53.13, 47.35, 41.78, 26.17, 12.96.

[0074] HRMS: C 18 H 22 The theoretical value of N4O2 is 326.1743, and the measured value is 326.3855.

[0075] Example 3

[0076] Synthesis of ABEI-3

[0077]

[0078] step:

[0079] 29.8 g (mw 149, 0.2 mol) of 1,4-formyl-N-ethylaniline was placed in a 1 L round-bottom flask, and 300 mL of [BMIM][BF4] (1-butyl-3-methylimidazolium tetrafluoroborate) was added to dissolve it. 98.8 g (mw 152, 0.65 mol) of 4-bromobutylamine and 18 g of K2CO3 (0.13 mol) were added. The mixture was reacted at 160-164 °C for 5 h under nitrogen protection, followed by purging with oxygen for another 3 h. After cooling to room temperature, 300 mL of ice water was added, and the mixture was allowed to stand at 2-8 °C for 24 h to precipitate. The precipitate was collected and dried to give 11 g of 4-formyl-N-ethyl-N-butylaminoaniline (yield: 25%).

[0080] 2. Take 6.49 g (mw 162, 40 mmol) of 4-aminophthalimide and 6.09 g (mw 76, 80 mmol) of ammonium thiocyanate in a 500 ml round-bottom flask, and resuspend in 100 ml of methanol; take 16.0 g of Br2 and dissolve in 50 ml of methanol to prepare a 100 mmol / 50 ml solution, and add Br2 (methanol) dropwise to the above solution under ice bath cooling, and continue to stir the reaction at 0 °C for 2 h; transfer to room temperature, continue to stir the reaction for 8 h, collect the precipitate, evaporate to dryness, and recrystallize dimethylformamide-benzene to give 2.19 g of yellow crystals, yield 25%;

[0081] 3. Take 2.19 g (mw 219, 10 mmol) of the above solid 1 in a 500 ml round-bottom flask, add 100 ml of ethanol to resuspend, and heat to 60 °C to dissolve; then add 100 ml of hydrazine, and reflux for 72 h; after the reaction is complete, evaporate to dryness, and recrystallize from ethanol to give 0.21 g of ASPH light yellow solid, yield 10%;

[0082] 4. Take 0.21 g ASPH (mw 209, 1 mmol) and 0.22 g p-formyl N-ethylbutylaminoaniline (mw 220, 1 mmol) and place them in a 250 ml round-bottom flask. Add 20 ml dimethylformamide, 20 ml 1.5 M sodium sulfite, and 0.63 g sodium dihydrogen phosphate in sequence. Stir the mixture at 80 °C for 1 h, cool to room temperature, and continue stirring for 3 h. Cool in an ice bath to obtain a solid. Collect the solid and recrystallize it from methanol to obtain 0.06 g of light yellow needle-like crystals of ABEI-3, with a yield of 15%.

[0083] 5. ABEI-3 structural characterization data: 1H NMR (300MHz, dDMSO) δ11.99(s,2H),9.1(t,J=6.1Hz,1H),8.58(d,J=9.1Hz,1H),7.71(dd,J=9.2,2.5Hz,2H),6.92(d,J=9.2,2.5Hz,2H ), 3.78(d,J=9.2,2.5Hz,2H), 3.40(q,J=7.0Hz,2H),2.69(m,2H),1.52(d,2H),1.50–1.47(m,2H),1.45(m,2H),1.12(t,J=6.9Hz,3H).

[0084] 13 C NMR (300MHz, dDMSO) δ167.32, 157.98, 149.61, 139.35, 121.45, 122.86, 53.13, 47.35, 41.78, 26.27, 12.96.

[0085] HRMS: C 21 H 23 The theoretical value of N5O2S is 409.1572, and the measured value is 409.1725.

[0086] Example 4

[0087] Synthesis of ABEI-4

[0088]

[0089] step:

[0090] 1. Take 6.49 g (mw 162, 40 mmol) of 4-aminophthalimide and 6.09 g (mw 76, 80 mmol) of ammonium thiocyanate in a 500 ml round-bottom flask, and resuspend in 100 ml of methanol; take 16.0 g of Br2 and dissolve in 50 ml of methanol to prepare a 100 mmol / 50 ml solution, and add Br2 (methanol) dropwise to the above solution under ice bath cooling, and continue to stir the reaction at 0 °C for 2 h; transfer to room temperature, continue to stir the reaction for 8 h, collect the precipitate, evaporate to dryness, and recrystallize dimethylformamide-benzene to give 2.19 g of yellow crystals, yield 25%;

[0091] 2. Take 2.19 g (mw 219, 10 mmol) of the above solid 1 in a 500 ml round-bottom flask, add 100 ml of ethanol to resuspend, and heat to 60 °C to dissolve; then add 100 ml of hydrazine, and reflux for 72 h; after the reaction is complete, evaporate to dryness, and recrystallize from ethanol to give 0.21 g of ASPH light yellow solid, yield 10%;

[0092] 3. Take 0.21 g ASPH (mw 209, 1 mmol) and 0.15 g formylbenzoic acid (mw 150, 1 mmol) and place them in a 250 ml round-bottom flask. Add 20 ml dimethylformamide, 20 ml 1.5 M sodium sulfite, and 0.63 g sodium dihydrogen phosphate in sequence. Stir the mixture at 80 °C for 1 h, cool to room temperature, and continue stirring for 3 h. Cool in an ice bath to obtain a solid. Collect the solid and recrystallize it from methanol to obtain 0.08 g of light yellow needle-like crystals of ABEI-4 (yield 20%).

[0093] 4. ABEI-4 structural characterization data: 1 H NMR (300MHz, dDMSO) δ12.71(s,1H),11.99(s,2H),9.1(t,J=6.1Hz,1H),8.58(d,J=9.1Hz,1H),8.10(dd,J=8.3,2.5Hz,2H),7.98(dd,J=9.2,2.5Hz,2H).

[0094] 13 C NMR (300MHz, dDMSO) δ167.32, 157.98, 149.61, 139.35, 129.45, 127.56.

[0095] HRMS: C 16 The theoretical value of H9N3O4S is 339.0314, and the measured value is 339.0825.

[0096] Test Example 1 Performance Verification

[0097] The following examples use the Maglumi2000plus fully automated chemiluminescence immunoassay analyzer manufactured by Shenzhen New Industries Biomedical Engineering Co., Ltd. for detection.

[0098] Maglumi2000 Chemiluminescence Analyzer, source: Shenzhen New Industries Biomedical Engineering Co., Ltd.

[0099] The anti-Müllerian hormone (AMH) antibody and AMH antigen were prepared by Shenzhen New Industries Biomedical Engineering Co., Ltd. according to standard experimental procedures.

[0100] The principle of the chemiluminescent immunoassay platform is as follows: The sample to be tested (serum) is added to the reaction vessel, followed by the addition of magnetic microspheres coated with AMH and ABEI-labeled AMH antibody solution. After incubating at 37°C for 15 minutes, an external magnetic field is applied to precipitate the reaction products. The supernatant is removed, and the mixture is washed with buffer solution. Then, chemiluminescent exciters (NaOH and H2O2) are added, and the relative light intensity emitted is detected.

[0101] 1. Validation of AMH-labeled antibody luminescence intensity

[0102] 1.1 Preparation of ABEI markers:

[0103] 1) ABEI, ABEI-1, ABEI-2, ABEI-3, and ABEI-4 were modified with succinic anhydride, activated by DCC and NHS, and then used for labeling;

[0104] 2) Accurately weigh 27.6 mg ABEI (Mw = 276.34, 1.0 equiv) and 20 mg (Mw = 100.07, 2 equiv) succinic anhydride into a 2 ml glass bottle, and add 0.5 ml pyridine;

[0105] 3) React in a 50℃ water bath at 200 rpm for 20 minutes with stirring (the solution will become clear after heating);

[0106] 4) After the reaction was completed, the pyridine solvent was dried by nitrogen, and 41.2 mg of 690 μL NHS solution (59.71 mg / mL @ dimethylformamide, 2 equivalents) and 23 mg of 690 μL DCC solution (33.33 mg / mL @ dimethylformamide, 2 equivalents) were added. The mixture was stirred overnight in a water bath at 25°C and 200 rpm to obtain a labeled activation solution of 20 mg / mL @ dimethylformamide.

[0107] 5) Following the standard ABEI activation procedure, ABEI-1, ABEI-2, ABEI-3, and ABEI-4 were modified with succinic anhydride and activated with DCC and NHS.

[0108] 1.2 AMH antibody-labeled ABEI modifier:

[0109] 1) Take 7 mg of AMH antibody (2 ml, 3.5 mg / ml) into a 14000 Da dialysis bag, place it in a beaker containing 4 L of 0.1 M carbonate buffer, dialyze for 2 h, take it out and measure the concentration with a spectrophotometer. The concentration is 3.4 mg / ml. Add 0.1 M carbonate buffer to dilute to 1 mg / ml, for a total of 6.8 ml.

[0110] 2) Take 1 ml of AMH antibody after dialysis into a 2 ml glass bottle, and repeat 5 times. The molar ratio of ABEI modifier to AMH antibody label is 40:1.

[0111] 3) ① 1 ml AMH antibody (1 mg / ml 0.1 M carbonate buffer) + 3.67 μL ABEI-HS (20 mg / ml dimethylformamide)

[0112] ② 1 ml AMH antibody (1 mg / ml @ 0.1 M carbonate buffer) + 4.33 μL ABEI-1-HS (20 mg / ml dimethylformamide)

[0113] ③ 1 ml AMH antibody (1 mg / ml @ 0.1 M carbonate buffer) + 4.33 μL ABEI-2-HS (20 mg / ml dimethylformamide)

[0114] ④ 1 ml AMH antibody (1 mg / ml @ 0.1 M carbonate buffer) + 5.44 μL ABEI-3-HS (20 mg / ml dimethylformamide)

[0115] ⑤ 1 ml of AMH antibody (1 mg / ml @ 0.1 M carbonate buffer) + 5.30 μL of ABEI-4-HS (20 mg / ml dimethylformamide);

[0116] 4) The reaction was carried out in a water bath at 25℃ with stirring at 150 rpm for 1 hour. ① and ⑥ became turbid, while ②, ③, ④, and ⑤ became clear.

[0117] 5) After labeling, transfer to a 14000Da dialysis bag, place in a beaker containing 5L of 0.01M PBS, and dialyze 3 times, 2 hours each time;

[0118] 6) Pass the dialyzed antigen through a 0.22 μm filter membrane;

[0119] 7) Take the 6 groups of labeled AMH antibodies and dilute them with 0.01M PBS at 1000, 2000, 4000, 8000, 16000, 30000 and 64000 times, and detect the luminescence intensity RLU on the luminescent immunoassay platform;

[0120] Dilution factor ABEI ABEI-1 ABEI-2 ABEI-3 ABEI-4 1000 times 469952 569912 529323 499977 539009 2000 times 246393 276343 256395 266309 276865 4000 times 137230 153670 147432 147780 147544 8000 times 68696 75696 68645 71697 69643 16000 times 35405 39421 38405 39411 38487 32000 times 18534 20533 19545 19539 19539 64000 times 9926 11915 10957 10028 11023

[0121] The results showed that by modifying existing isoluminol, the luminescence intensity of ABEI derivatives 1-4 was significantly improved compared to ABEI for antibodies with the same dilution factor, effectively solving the problem of low detection sensitivity.

[0122] While the embodiments disclosed in this application are as described above, the content is merely for the purpose of facilitating understanding of this application and is not intended to limit this application. Any person skilled in the art to which this application pertains may make any modifications and changes in the form and details of the implementation without departing from the spirit and scope disclosed in this application; however, the scope of protection of this application shall still be determined by the scope defined in the appended claims.

Claims

1. An isoluminol derivative, said isoluminol derivative as shown in formula (I): In formula (I), R1 is hydrogen, and R3, together with R2, forms a five-membered heteroaromatic ring substituted with one or more substituents and fused with the benzene ring to which they are attached; the five-membered heteroaromatic ring is an imidazole or a thiazole; the substituent is a substituted phenyl group, wherein the substituted phenyl group is a phenyl group substituted with a carboxyl group or -N(R6)(R7), wherein, R6 and R7 are each independently hydrogen, unsubstituted C1-C4 alkyl, and amino-substituted C1-C4 alkyl.

2. The isoluminol derivative according to claim 1, wherein, In formula (I), R1 is hydrogen, and R3, together with R2, forms a five-membered heteroaromatic ring fused with the benzene ring to which they are attached, which is substituted with one or more substituents; the five-membered heteroaromatic ring is a thiazole; the substituent is a 4-substituted phenyl, wherein the 4-substituted benzene is a 4-carboxyphenyl or a 4-N(R6)(R7)phenyl, wherein one of R6 and R7 is an unsubstituted C1-C4 alkyl and the other is an amino-substituted C1-C4 alkyl.

3. The isoluminol derivative according to claim 1 or 2, wherein, In formula (I), R1 is hydrogen, and R3, together with R2, forms a five-membered heteroaromatic ring substituted with one or more substituents and fused with the benzene ring to which they are attached; the five-membered heteroaromatic ring is a thiazole; the substituent is a 4-substituted phenyl, wherein the 4-substituted benzene is 4-carboxyphenyl or 4-N(R6)(R7)phenyl, wherein one of R6 and R7 is methyl, ethyl, n-propyl or isopropyl, and the other is 3-aminopropyl or 4-aminobutyl.

4. The isoluminol derivative according to claim 1, wherein, Formula (I) is one of the following compounds: 。 5. The method for preparing the isoluminol derivative according to claim 1, wherein the preparation method comprises the following steps: The compound of formula (II) reacts with hydrazine to give the compound of formula (I); In formula (II), the substituents R1, R3, and R3 are defined in the same way as the corresponding groups in formula (I). 。 6. A chemiluminescent substrate, said chemiluminescent substrate comprising any one of claims 1 to 4 isoluminol derivatives.

7. An immunodiagnostic reagent, said immunodiagnostic reagent comprising an isoluminol derivative as described in any one of claims 1 to 4, and a detection antigen or a detection antibody; wherein, The detection antigen or detection antibody is labeled with the isoluminol derivative.

8. A chemiluminescence reagent kit comprising the immunodiagnostic reagent of claim 7.

9. A chemiluminescent immunoassay system, wherein, The chemiluminescence immunoassay system comprises the chemiluminescence reagent kit and a semi-automatic or fully automated immunoassay analyzer as described in claim 8.

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