Compounds, products and methods for determining the amount or presence of vitamin D

Abstract

The present invention relates to compounds of Formula I, which can be used to determine the presence or amount of vitamin D analytes (including vitamin D2 and vitamin D3) and their metabolites in samples containing the same. The present invention also relates to products, methods, and compositions for determining the presence or amount of vitamin D in a sample.

Description

[0001] This application is a divisional application of the Chinese patent application with the application date of February 1, 2019, application number "201910105697.1", and invention name "Compounds, products and methods for determining the content or presence of vitamin D". Technical Field

[0002] The present invention belongs to the field of biotechnology, and specifically relates to compounds, products and methods for detecting and determining the presence or amount of vitamin D analytes (including vitamin D2 and vitamin D3) and their metabolites in samples. Background Art

[0003] Vitamin D is a fat-soluble steroid prohormone found in two main forms: vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Vitamin D2 is obtained from nutritional supplements, while vitamin D3 is derived from skin exposed to sunlight (ultraviolet radiation) and from dietary sources such as fish, liver oil, and egg yolks. Vitamin D2 and D3 are metabolized in the liver to 25-OH vitamin D (25-(OH)D), which is then converted to 1,25-(OH)2D in the kidneys. 25-(OH)D is the major metabolite in the circulation, and therefore, the 25-(OH)D level can reflect the body's vitamin D level. Vitamin D in the blood exists in a protein-bound form. Blood vitamin D levels reported in clinical tests refer to the total 25-(OH)D, including 25-(OH)D2 and 25-(OH)D3. Accurate monitoring of total 25-OH vitamin D levels is crucial for clinical application.

[0004] Vitamin D plays an important role in maintaining bone mineral density. It and parathyroid hormone (PTH) play a key role in calcium balance. Vitamin D deficiency severely impairs the body's absorption of calcium and phosphorus, potentially leading to rickets, neonatal hypocalcemia, hypothyroidism, osteoporosis in the elderly, and other calcium metabolism disorders. Excessive vitamin D can lead to hypercalcemia and various aging-related diseases.

[0005] Vitamin D is present in almost all human tissues, and its role goes beyond simply maintaining calcium and phosphorus balance. Research indicates that vitamin D exerts its effects by binding to the vitamin D receptor (VDR). Recent epidemiological studies have linked vitamin D to a variety of diseases, including cancer, heart disease, hypertension, diabetes, autoimmune diseases, infectious diseases, and aging.

[0006] Currently, the main 25-OH vitamin D detection methods on the market include radioimmunoassay (RIA), liquid chromatography-tandem mass spectrometry (LC-MS), enzyme-linked immunosorbent assay (ELISA), and chemiluminescence assay (CLIA). Among them, chemiluminescence has become the development trend due to its high sensitivity, wide linear range, convenient operation, and pollution-free advantages. Summary of the Invention

[0007] The purpose of the present invention is to provide a composition, product and method for detecting the presence or amount of vitamin D analytes (including vitamin D2 and vitamin D3) and their metabolites in a sample containing the deficiencies in the prior art.

[0008] To achieve the above objectives, in a first aspect, the present invention provides a compound represented by Formula I.

[0009]

[0010] Wherein, Z is selected from C1-C 20 Alkyl, C2-C 20 Alkenyl and C2-C 20 Alkynyl, said alkyl, alkenyl and alkynyl groups are optionally selected from hydroxy, C1-C 10 Alkoxy, C1-C 10 The ester group and the oxime group are substituted with one or more substituents;

[0011] R1 is selected from hydrogen, hydroxy, C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C1-C 20 Alkoxy, C2-C 20 Alkenyloxy and C2-C 20 Alkynyloxy;

[0012] R2 and R3 are the same or different and are independently selected from hydrogen and C1-C 20 alkyl;

[0013] R is at least R 1 Substituted C6-C 30 Aryl, C5-C 30 Heteroaryl or C9-C 30 Fused aromatic groups,

[0014] R 1 -(CH2) p X or -(CH2) pCOX, wherein X is selected from a labeling moiety, a biomacromolecule moiety, an N-maleimide group connected to a labeling moiety or a biomacromolecule moiety, a member of a signal generating system, an organic small molecule, and a binding ligand or carrier of the organic small molecule, and p is an integer from 0 to 10, wherein the labeling moiety is derived from a labeled compound and the biomacromolecule moiety is derived from a biomacromolecule.

[0015] In some embodiments of the present invention, Z is selected from C1-C 10 Alkyl, C2-C 10 Alkenyl and C2-C 10 Alkynyl, said alkyl, alkenyl and alkynyl groups are optionally selected from hydroxy, C1-C 10 Alkoxy, C1-C 10 The ester group and the oxime group are substituted with one or more substituents.

[0016] In some of the above embodiments, Z is selected from C1-C 10 Alkyl, C2-C 10 Alkenyl and C2-C 10 Alkynyl, the alkyl, alkenyl or alkynyl group is optionally substituted with one or more substituents selected from hydroxyl, C1-C5 alkoxy, C1-C5 ester and oxime groups.

[0017] In some of the above embodiments, Z is a C4-C 10 Alkyl, branched C4-C 10 Alkenyl or branched C4-C 10 Alkynyl.

[0018] In some of the above embodiments, the C4-C 10 Alkyl, branched C4-C 10 Alkenyl or branched C4-C 10 The terminal carbon atom of the alkynyl group is linked to a hydroxy group or a protected hydroxy group.

[0019] In some of the above embodiments, Z is 4,4-dimethyl-4-hydroxybutyl, having the following structure:

[0020]

[0021] In some of the above embodiments, R1 is selected from hydrogen, hydroxy, C1-C 10 Alkyl, C2-C 10 Alkenyl, C2-C 10 Alkynyl and C1-C 10 Alkoxy.

[0022] In some of the above embodiments, R2 and R3 are independently selected from hydrogen and C1-C 10The alkyl group is preferably selected from hydrogen and C1-C5 alkyl.

[0023] In some of the above embodiments, the organic small molecule is selected from biotin, fluorescein, rhodamine, chemiluminescent molecules, dinitrophenol, acridinium ester, alkaline phosphatase and labeled compound molecules, and the binding ligand of the organic small molecule is selected from avidin, antibodies to fluorescein, antibodies to rhodamine, antibodies to chemiluminescent molecules and antibodies to dinitrophenol.

[0024] In some of the above embodiments, the member of the signal generating system is selected from fluorescent compounds, chemiluminescent compounds, sensitizers, enzymes and radioactive labels.

[0025] In some of the above embodiments, the member of the signal generating system comprises a particle.

[0026] In some of the above embodiments, the particles are selected from fluorescent particles, chemiluminescent particles, sensitizer particles and magnetic particles.

[0027] In some of the above embodiments, the biomacromolecule is selected from protein molecules, nucleic acid molecules, polysaccharide molecules and lipid molecules.

[0028] In some of the above embodiments, the binding ligand of the organic small molecule is selected from antibodies to vitamin D and its analogs.

[0029] In some of the above embodiments, p is 0, 1, 2, 3, 4 or 5.

[0030] In some of the above embodiments, R is at least R 1 Substituted C6-C 20 Aryl, C5-C 20 Heteroaryl or C9-C 20 Condensed aromatic group.

[0031] In some of the above embodiments, R is at least R 1 Substituted C6-C 10 Aryl, C5-C 10 Heteroaryl or C9-C 10 Fused aromatic groups, such as at least R 1 Substituted phenyl, at least R 1 Substituted pyridyl, at least R 1 Quinolinyl or at least R 1 Substituted isoquinolinyl.

[0032] In some of the above embodiments, the compound has a structure shown in Formula II or Formula III:

[0033]

[0034]

[0035] In Formula II, R 2 -R 5 the same or different, independently selected from hydrogen, C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C1-C 20 Alkoxy, C6-C 20 Aryl, C5-C 20 Heteroaryl, C9-C 20 Fused aromatic groups, cyano groups, halogen atoms, nitro groups, carboxyl groups, amino groups and C1-C 10 an alkyl-substituted amino group;

[0036] In formula III, R 2 -R 6 the same or different, independently selected from hydrogen, C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C1-C 20 Alkoxy, C6-C 20 Aryl, C5-C 20 Heteroaryl, C9-C 20 Fused aromatic groups, cyano groups, halogen atoms, nitro groups, carboxyl groups, amino groups and C1-C 10 Alkyl-substituted amino groups.

[0037] In some of the above embodiments, in Formula II, R 2 -R 5 independently selected from hydrogen, C1-C 10 Alkyl, C1-C 10 Alkenyl, C1-C 10 Alkynyl, C1-C 10 Alkoxy group, cyano group, halogen atom, nitro group, amino group and C1-C5 alkyl-substituted amino group.

[0038] In some of the above embodiments, in Formula III, R 2 -R 6 independently selected from hydrogen, C1-C 10 Alkyl, C1-C 10 Alkenyl, C1-C 10 Alkynyl, C1-C 10 Alkoxy group, cyano group, halogen atom, nitro group, amino group and C1-C5 alkyl-substituted amino group.

[0039] In some of the above embodiments, in Formula II, R 2 -R 5Independently selected from hydrogen, C1-C5 alkyl, C1-C5 alkenyl, C1-C5 alkynyl, C1-C5 alkoxy, cyano, halogen, nitro, amino and C1-C5 alkyl-substituted amino groups.

[0040] In some of the above embodiments, in Formula III, R 2 -R 6 Independently selected from hydrogen, C1-C5 alkyl, C1-C5 alkenyl, C1-C5 alkynyl, C1-C5 alkoxy, cyano, halogen, nitro, amino and C1-C5 alkyl-substituted amino groups.

[0041] In a second aspect, the present invention provides a product for determining the presence and / or amount of vitamin D in a sample, comprising the following components:

[0042] 1) the compound represented by the above formula I, and

[0043] 2) Specific binding member for vitamin D.

[0044] According to some of the above embodiments, the product is a composition or a kit.

[0045] According to some of the above embodiments, the sample is a biological sample or a non-biological sample.

[0046] According to some of the above embodiments, the specific binding member of vitamin D is an antibody against vitamin D and a ligand coupled to the antibody against vitamin D.

[0047] In a third aspect, the present invention provides the use of the above-mentioned product for determining the presence and / or amount of vitamin D in a sample in detecting the presence and / or amount of vitamin D in a sample.

[0048] In a fourth aspect, the present invention provides a method for determining the presence and / or amount of vitamin D in a sample, comprising the steps of:

[0049] 1) Providing the product provided in the second aspect of the present invention;

[0050] 2) forming a complex between the specific binding member for vitamin D and the compound of formula I;

[0051] 3) Measuring the amount of the complex, which is related to the presence and / or amount of vitamin D in the sample.

[0052] In a fifth aspect, the present invention provides a complex formed by the compound represented by formula I provided in the first aspect of the present invention and a specific binding member for vitamin D. DETAILED DESCRIPTION

[0053] To facilitate understanding of the present invention, the present invention will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0054] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of the range and any other specified or intervening values ​​in the specified range is encompassed within the present invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the present invention, subject to any express exclusions in the specified range. Where a specified range includes one or both limits, ranges excluding either or both of those included limits are also encompassed within the present invention.

[0055] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, preferred methods and materials are now described.

[0056] I. Terminology

[0057] The term "alkyl" refers to those alkyl groups having a specified number of carbon atoms, whether linear, branched, or cyclic. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, and the like.

[0058] The term "alkenyl" refers to a straight or branched hydrocarbon chain having the specified number of carbon atoms and at least one carbon-carbon double bond, which may occur at any position along the chain. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, dimethylpentenyl, and the like.

[0059] The term "alkynyl" refers to a straight or branched hydrocarbon with the specified number of carbon atoms containing at least one carbon-carbon triple bond. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, and the like.

[0060] "Aryl" includes groups having aromatic properties, including "conjugated" or polycyclic ring systems, which contain at least one aromatic ring and do not contain any heteroatoms in their ring structure. Examples include phenyl, benzyl, 1,2,3,4-tetrahydronaphthyl, and the like.

[0061] "Heteroaryl" refers to an aryl group as defined above, but having 1-4 heteroatoms in the ring structure, which may also be referred to as an "aromatic heterocycle" or "heteroaromatic compound". As used herein, the term "heteroaryl" refers to a stable 5-, 6-, or 7-membered monocyclic or 7-, 8-, 9-, 10-, 11-, or 12-membered bicyclic aromatic heterocycle consisting of carbon atoms and one or more heteroatoms, such as 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6 heteroatoms, or such as 1, 2, 3, 4, 5, or 6 heteroatoms, the heteroatoms being independently selected from nitrogen, oxygen, and sulfur. The nitrogen atom may be substituted or unsubstituted (i.e., N or NR, where R is hydrogen or another substituent as defined herein). The nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., N→O and S(O)). p , wherein p=1 or 2). However, it should be noted that the total number of sulfur and oxygen atoms in the aromatic heterocycle does not exceed 1.

[0062] Examples of heteroaryl groups include pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like.

[0063] The term "carboxyl" refers to -COOH or a C1-C6 alkyl ester thereof.

[0064] The term "ester group" refers to a compound or fragment containing a carbon atom or heteroatom bonded to an oxygen atom bonded to the carbon of a carbonyl group. The term "ester" includes alkoxycarbonyl groups such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, pentyloxycarbonyl, and the like.

[0065] The term "alkoxy" includes substituted and unsubstituted alkyl groups covalently bonded to an oxygen atom. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, isopropoxy, propoxy, butoxy, and pentoxy. The term "alkenyloxy" includes substituted and unsubstituted alkenyl groups covalently bonded to an oxygen atom. Examples of alkenyloxy groups include, but are not limited to, ethyleneoxy, propyleneoxy, butenyloxy, and pentenyloxy. The term "alkynyloxy" includes substituted and unsubstituted alkynyl groups covalently bonded to an oxygen atom. Examples of alkynyloxy groups include, but are not limited to, ethynyloxy, propynyloxy, butenyloxy, and pentenyloxy.

[0066] As used herein, "amine" or "amino" refers to an unsubstituted or substituted -NH2 group. "Alkylamino" includes groups in which the nitrogen atom of -NH2 is bonded to at least one alkyl group. Examples of alkylamino groups include benzylamino, methylamino, ethylamino, and phenethylamino. "Dialkylamino" includes groups in which the nitrogen atom of -NH2 is bonded to at least two alkyl groups. Examples of dialkylamino groups include, but are not limited to, dimethylamino and diethylamino. "Arylamino" and "diarylamino" include groups in which the nitrogen atom is bonded to at least one or two aryl groups, respectively. "Aminoaryl" and "aminoaryloxy" refer to aryl and aryloxy groups substituted with an amino group. "Alkylarylamino," "alkylaminoaryl," or "arylaminoalkyl" refers to an amino group bonded to at least one alkyl group and at least one aryl group. "Alkylaminoalkyl" refers to an alkyl, alkenyl, or alkynyl group bonded to a nitrogen atom, wherein the nitrogen atom is also bonded to an alkyl group. "Acylamino" includes groups in which the nitrogen atom is bonded to an acyl group. Examples of the acylamino group include, but are not limited to, an alkylcarbonylamino group, an arylcarbonylamino group, a carbamoyl group, and a ureido group.

[0067] "Aryl" includes groups having aromatic properties, including "conjugated" or polycyclic ring systems, which contain at least one aromatic ring and do not contain any heteroatoms in their ring structure. Examples include phenyl, benzyl, 1,2,3,4-tetrahydronaphthyl, and the like.

[0068] "Heteroaryl" refers to an aryl group as defined above, but having 1-4 heteroatoms in the ring structure, which may also be referred to as an "aromatic heterocycle" or "heteroaromatic compound". As used herein, the term "heteroaryl" refers to a stable 5-, 6-, or 7-membered monocyclic or 7-, 8-, 9-, 10-, 11-, or 12-membered bicyclic aromatic heterocycle consisting of carbon atoms and one or more heteroatoms, such as 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6 heteroatoms, or such as 1, 2, 3, 4, 5, or 6 heteroatoms, the heteroatoms being independently selected from nitrogen, oxygen, and sulfur. The nitrogen atom may be substituted or unsubstituted (i.e., N or NR, where R is hydrogen or another substituent as defined herein). The nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., N→O and S(O)). p , wherein p=1 or 2). However, it should be noted that the total number of sulfur and oxygen atoms in the aromatic heterocycle does not exceed 1.

[0069] Examples of heteroaryl groups include pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like.

[0070] In addition, the terms "aryl" and "heteroaryl" include polycyclic aryl and heteroaryl groups, for example, tricyclic, bicyclic, for example, naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzimidazole, benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline, naphthyridinyl, indole, benzofuran, purine, benzofuran, deazapurine, indolizine.

[0071] The cycloalkyl, heterocycloalkyl, aryl or heteroaryl ring can be substituted at one or more ring positions (e.g., a ring-forming carbon atom or a heteroatom such as a nitrogen atom) with a substituent as described above, for example, alkyl, alkenyl, alkynyl, halogen, hydroxy, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, arylalkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, arylalkyl, alkenylcarbonyl, alkoxycarbonyl, amino

[0014] The term "alkyl" refers to a group comprising a carbonyl, alkylthiocarbonyl, phosphate, phosphonate, phosphinate, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), amido (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonic acid, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Aryl and heteroaryl groups can also be fused or bridged with non-aromatic alicyclic or heterocyclic rings to form a polycyclic ring system (e.g., tetralin, methylenedioxyphenyl).

[0072] The term "oxime" refers to -C=N-OH.

[0073] II. Specific Implementation Plan

[0074] The present invention will be described in more detail below.

[0075] To achieve the above objectives, in a first aspect, the present invention provides a compound represented by formula I.

[0076]

[0077] Wherein, Z is selected from C1-C 20 Alkyl, C2-C 20 Alkenyl and C2-C 20 Alkynyl, said alkyl, alkenyl and alkynyl groups are optionally selected from hydroxy, C1-C 10 Alkoxy, C1-C 10 The ester group and the oxime group are substituted with one or more substituents;

[0078] R1 is selected from hydrogen, hydroxy, C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C20 Alkynyl, C1-C 20 Alkoxy, C2-C 20 Alkenyloxy and C2-C 20 Alkynyloxy;

[0079] R2 and R3 are the same or different and are independently selected from hydrogen and C1-C 20 alkyl;

[0080] R is at least R 1 Substituted C6-C 30 Aryl, C5-C 30 Heteroaryl or C9-C 30 Fused aromatic groups,

[0081] R 1 -(CH2) p X or -(CH2) p COX, wherein X is selected from a labeling moiety, a biomacromolecule moiety, an N-maleimide group connected to a labeling moiety or a biomacromolecule moiety, a member of a signal generating system, an organic small molecule, and a binding ligand or carrier of the organic small molecule, and p is an integer of 0-10, such as an integer of 1-5, wherein the labeling moiety is derived from a labeling compound and the biomacromolecule moiety is derived from a biomacromolecule.

[0082] In some embodiments of the present invention, Z is selected from C1-C 10 Alkyl, C2-C 10 Alkenyl and C2-C 10 Alkynyl, said alkyl, alkenyl and alkynyl groups are optionally selected from hydroxy, C1-C 10 Alkoxy, C1-C 10 The ester group and the oxime group are substituted with one or more substituents. For example, Z is a C1-C 10 Alkyl. For example, Z is C1-C 10 Alkoxy-substituted C1-C 10 For example, Z is a C2-C 10 For example, Z is C1-C 10 Alkoxy-substituted C2-C 10 Alkenyl.

[0083] In some of the above embodiments, Z is selected from C1-C 10 Alkyl, C2-C 10 Alkenyl and C2-C 10Alkynyl, wherein the alkyl, alkenyl or alkynyl group is optionally substituted with one or more substituents selected from hydroxy, C1-C5 alkoxy, C1-C5 ester and oxime. For example, Z is a C1-C5 alkyl substituted with hydroxy. For example, Z is a C1-C5 alkyl substituted with C1-C5 alkoxy. For example, Z is a C2-C5 alkenyl substituted with hydroxy. For example, Z is a C2-C5 alkenyl substituted with C1-C5 alkoxy.

[0084] In some of the above embodiments, Z is a C4-C 10 Alkyl, branched C4-C 10 Alkenyl or branched C4-C 10 For example, the side chain is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and tert-butyl. The number of the side chains can be one or more.

[0085] In some of the above embodiments, the C4-C 10 Alkyl, branched C4-C 10 Alkenyl or branched C4-C 10 The terminal carbon atom of the alkynyl group is connected to a hydroxyl group or a protected hydroxyl group. For example, the side chain is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and tert-butyl. The number of the side chains can be one or more.

[0086] In some of the above embodiments, Z is 4,4-dimethyl-4-hydroxybutyl, which has the following structure:

[0087]

[0088] In some of the above embodiments, R1 is selected from hydrogen, hydroxy, C1-C 10 Alkyl, C2-C 10 Alkenyl, C2-C 10 Alkynyl and C1-C 10 Alkoxy. Preferably, R1 can be hydrogen, hydroxyl, C1-C5 alkyl, C2-C5 alkenyl, C2-C5 alkynyl or C1-C5 alkoxy, etc. For example, R1 is hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.

[0089] In some of the above embodiments, R2 and R3 are independently selected from hydrogen and C1-C 10 Alkyl is preferably selected from hydrogen and C1-C5 alkyl. For example, R2 and R3 are both hydrogen. For example, R2 is C1-C5 alkyl and R3 is hydrogen. For example, R2 and R3 are both methyl. For example, R2 is hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl. For example, R3 is hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl.

[0090] In some of the above embodiments, R is at least R 1 Substituted C6-C 20 Aryl, C5-C 20 Heteroaryl or C9-C 20 Condensed aromatic group.

[0091] In some of the above embodiments, R is at least R 1 Substituted C6-C 10 Aryl, C5-C 10 Heteroaryl or C9-C 10 Condensed aromatic group.

[0092] In some of the above embodiments, R is at least R 1 substituted phenyl, pyridine, quinoline or isoquinoline.

[0093] In some other embodiments described above, X is a member of a signal generating system.

[0094] In some other embodiments described above, X is a small organic molecule.

[0095] In some other embodiments described above, X is a binding ligand or carrier of the organic small molecule.

[0096] In some of the above embodiments, the organic small molecule is selected from biotin, fluorescein, rhodamine, chemiluminescent molecules, dinitrophenol, acridinium ester, alkaline phosphatase and labeled compound molecules, and the binding ligand of the organic small molecule is selected from avidin, antibodies to fluorescein, antibodies to rhodamine, antibodies to chemiluminescent molecules and antibodies to dinitrophenol.

[0097] In some of the above embodiments, the member of the signal generating system is selected from fluorescent compounds, chemiluminescent compounds, sensitizers, enzymes and radioactive labels.

[0098] In some of the above embodiments, the member of the signal generating system comprises particles. Preferably, the particles are selected from fluorescent particles, chemiluminescent particles, sensitizer particles, and magnetic particles.

[0099] In some of the above embodiments, the binding ligand of the organic small molecule is selected from antibodies to vitamin D and its analogs.

[0100] In some of the above embodiments, the biomacromolecule is selected from protein molecules, nucleic acid molecules, polysaccharide molecules and lipid molecules.

[0101] In some other embodiments described above, R 1 -(CH2) pX, for example -(CH2)X. X can be a biomacromolecule moiety or an N-maleimide group attached to a biomacromolecule moiety. The biomacromolecule can be, for example, a biotin polyethylene glycol amino group (molecular weight can be greater than 1 Dalton, for example, between 2-100 Daltons, for example, between 2-80 Daltons) or bovine serum albumin (BSA).

[0102] In some other embodiments described above, R 1 -(CH2) p COX, for example -CH2COX. X can be a biomacromolecule moiety or an N-maleimide group attached to a biomacromolecule moiety. The biomacromolecule can be, for example, biotin-polyethylene glycol amino (molecular weight can be greater than 1 Dalton, for example, between 2 and 100 Daltons, for example, between 2 and 80 Daltons) or bovine serum albumin (BSA).

[0103] In some of the above embodiments, p is 0, 1, 2, 3, 4 or 5.

[0104] For example, at least R 1 Substituted phenyl, at least R 1 Substituted pyridyl, at least R 1 Quinolinyl or at least R 1 Substituted isoquinolinyl.

[0105] In some of the above embodiments, the compound has a structure shown in Formula II or Formula III:

[0106]

[0107] In Formula II, R 2 -R 5 the same or different, independently selected from hydrogen, C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C1-C 20 Alkoxy, C6-C 20 Aryl, C5-C 20 Heteroaryl, C9-C 20 Fused aromatic groups, cyano groups, halogen atoms, nitro groups, carboxyl groups, amino groups and C1-C 10 an alkyl-substituted amino group;

[0108] In formula III, R 2 -R 6 the same or different, independently selected from hydrogen, C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C1-C 20 Alkoxy, C6-C20 Aryl, C5-C 20 Heteroaryl, C9-C 20 Fused aromatic groups, cyano groups, halogen atoms, nitro groups, carboxyl groups, amino groups and C1-C 10 Alkyl-substituted amino groups.

[0109] In some of the above embodiments, in Formula II, R 2 -R 5 independently selected from hydrogen, C1-C 10 Alkyl, C1-C 10 Alkenyl, C1-C 10 Alkynyl, C1-C 10 Alkoxy group, cyano group, halogen atom, nitro group, amino group and C1-C5 alkyl-substituted amino group.

[0110] In some of the above embodiments, in Formula III, R 2 -R 6 independently selected from hydrogen, C1-C 10 Alkyl, C1-C 10 Alkenyl, C1-C 10 Alkynyl, C1-C 10 Alkoxy group, cyano group, halogen atom, nitro group, amino group and C1-C5 alkyl-substituted amino group.

[0111] In some of the above embodiments, in Formula II, R 2 -R 5 Independently selected from hydrogen, C1-C5 alkyl, C1-C5 alkenyl, C1-C5 alkynyl, C1-C5 alkoxy, cyano, halogen, nitro, amino and C1-C5 alkyl-substituted amino groups.

[0112] In some of the above embodiments, in Formula III, R 2 -R 6 Independently selected from hydrogen, C1-C5 alkyl, C1-C5 alkenyl, C1-C5 alkynyl, C1-C5 alkoxy, cyano, halogen, nitro, amino and C1-C5 alkyl-substituted amino groups.

[0113] In some of the above embodiments, in Formula II and / or Formula III, R 2 is selected from hydrogen, C1-C5 alkyl, C1-C5 alkenyl, C1-C5 alkynyl, C1-C5 alkoxy, cyano, halogen, nitro, amino and C1-C5 alkyl-substituted amino groups. 2 is hydrogen. For example, R 2 is hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.

[0114] In some of the above embodiments, in Formula II and / or Formula III, R 3is selected from hydrogen, C1-C5 alkyl, C1-C5 alkenyl, C1-C5 alkynyl, C1-C5 alkoxy, cyano, halogen, nitro, amino and C1-C5 alkyl-substituted amino groups. 3 is hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.

[0115] In some of the above embodiments, in Formula II and / or Formula III, R 4 is selected from hydrogen, C1-C5 alkyl, C1-C5 alkenyl, C1-C5 alkynyl, C1-C5 alkoxy, cyano, halogen, nitro, amino and C1-C5 alkyl-substituted amino groups. 4 is hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.

[0116] In some of the above embodiments, in Formula II and / or Formula III, R 5 is selected from hydrogen, C1-C5 alkyl, C1-C5 alkenyl, C1-C5 alkynyl, C1-C5 alkoxy, cyano, halogen, nitro, amino and C1-C5 alkyl-substituted amino groups. 5 is hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.

[0117] In some of the above embodiments, in Formula III, R 6 is selected from hydrogen, C1-C5 alkyl, C1-C5 alkenyl, C1-C5 alkynyl, C1-C5 alkoxy, cyano, halogen, nitro, amino and C1-C5 alkyl-substituted amino groups. 5 is hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.

[0118] In some of the above embodiments,

[0119] Specific examples of the compounds represented by formula I of the present invention include, but are not limited to, the following compounds:

[0120]

[0121] In a second aspect, the present invention provides a product for determining the presence and / or amount of vitamin D in a sample, comprising the following components:

[0122] 1) a compound according to the above formula I, and

[0123] 2) Specific binding member for vitamin D.

[0124] According to some of the above embodiments, the product is a composition or a kit.

[0125] According to some of the above embodiments, the sample is a biological sample or a non-biological sample.

[0126] According to some of the above embodiments, the specific binding member of vitamin D is an antibody against vitamin D and a ligand coupled to the antibody against vitamin D.

[0127] In a third aspect, the present invention provides the use of the above-mentioned product for determining the presence and / or amount of vitamin D in a sample in detecting the presence and / or amount of vitamin D in a sample.

[0128] In a fourth aspect, the present invention provides a method for determining the presence and / or amount of vitamin D in a sample, comprising the steps of:

[0129] 1) providing the compound represented by the above formula I;

[0130] 2) forming a complex between the specific binding member for vitamin D and the compound of formula I;

[0131] 3) Measuring the amount of the complex, wherein the amount of the conjugate is related to the presence and / or amount of vitamin D in the sample.

[0132] In a fifth aspect, the present invention provides a complex formed by the compound represented by the above-mentioned formula I and a specific binding member for vitamin D.

[0133] III. Examples

[0134] To make the present invention easier to understand, the present invention will be further described in detail below with reference to the following examples. These examples are merely illustrative and are not intended to limit the scope of application of the present invention. The raw materials or components used in the present invention can be obtained by commercial routes or conventional methods unless otherwise specified.

[0135] Reagents and instruments:

[0136] Synthesis example 1

[0137] Synthesis of Vitamin D Derivatives Containing N-Maleimidoaryl Substitutents

[0138] 50 mg of compound 12, 5-(OH)VD3, was weighed and dissolved in 2.5 mL of anhydrous DMSO to prepare a 20 mg / mL solution. In a 4 mL centrifuge tube, 1.25 mL of 2, 5-(OH)VD3 (62.4 μmol) was added. Then, 16.04 mg of PMPI (para-maleimidophenyl isocyanate) (74.88 μmol) was added at a molar ratio of 1.2:1 (PMPI:2, 5-(OH)VD3). The mixture was stirred at room temperature for 3 hours. Saturated aqueous sodium chloride was added to the reaction solution, which was extracted with ethyl acetate, dried over anhydrous Na2SO4, and spin-dried. The mixture was purified by thin-layer chromatography to yield 15 mg of compound 2 in a 39% yield. 1 HNMR(300MHz, CDCl3):0.56(s,3H,Me),0.90(d,3H,Me,J=5.8Hz),0.79–2.46(several m,19H),1.25(s,6H,Me),2.62(dd,1H,J=13.5,3.2Hz),2.75(d,1H,J=11.2Hz),3.35(dd,2H,J=11.5 ,6.1Hz),3.65(s,1H,OH),3.67(t,2H,J=5.7Hz),4.85–5.01(m,1H,H3),5.06(dd,2H,CH2),6.04and 6.22(2d,2H,CH,J=11.2Hz), 6.95(d,1H,CH), 7.00(d,1H,CH), 7.25–7.62(m,4H,ArH), 9.0(s,1H,NH).

[0139]

[0140] Synthesis example 2

[0141] Synthesis of Vitamin D Labeling Compounds Containing N-Maleimidoaryl Substitutents

[0142] 10 mg of SHPEGnBiotin (5kD) (2 μmol) was weighed and dissolved in 0.02 M PBS (0.15 M NaCl, 25 mM EDTA) pH 7.2 buffer. 2.4 mg (4 μmol) of compound 2 was added at a molar ratio of 2:1 and stirred at room temperature for 2 h. The mixture was purified by a desalting column and lyophilized to obtain 12 mg of lyophilized compound 3.

[0143]

[0144] Synthesis example 3

[0145] Synthesis of vitamin D derivatives containing chloromethyl aryl groups

[0146] 50 mg of compound 12,5-(OH)VD3, was weighed and dissolved in 2.5 mL of anhydrous DMSO to prepare a 20 mg / mL solution. In a 2 mL centrifuge tube, 1.25 mL of 2,5-(OH)VD3 (62.4 μmol) of DMSO solution was added. 31.37 mg of 4-(chloromethyl)phenyl isocyanate (187.2 μmol) was added at a molar ratio of 3:1. Stir at room temperature for 3 h. Saturated aqueous sodium chloride solution was added to the reaction solution, which was extracted with ethyl acetate, dried over anhydrous Na2SO4, and spin-dried. Purification by column chromatography afforded 12.5 mg of compound 4 in a 35% yield.

[0147] 1 HNMR(300MHz, CDCl3):0.54(s,3H,Me),0.88(d,3H,Me,J=5.8Hz),0.76–2.51(several m,19H),1.30(s,6H,Me),2.55(dd,1H,J=12.6,3.3Hz),2.64(d,1H,J=10.9Hz),3.23(dd,2H,J=10.5,5.8Hz) ,3.43(s,1H,OH),3.54(t,2H,J=4.6Hz),4.65(s,2H,CH2),4.74–4.97(m,1H,H3),5.03(dd,2H,CH2),5.95and 6.11(2d,2H,CH,J=11.2Hz), 7.41–7.68(m,4H,ArH), 8.5(s,1H,NH).

[0148]

[0149] Synthesis example 4

[0150] Synthesis of Vitamin D Labeling Compounds Containing Chloromethyl Aryl Substitution

[0151] 10 mg of NH2PEGnBiotin (5kD) (2 μmol) was weighed and dissolved in 0.02 M PBS (0.15 M NaCl, 25 mM EDTA) pH 7.2 buffer. 2.27 mg (4 μmol) of compound 4 was added at a molar ratio of 2:1 and stirred at 37°C for 16 h. The product was purified by a desalting column and lyophilized to obtain 14 mg of lyophilized compound 5.

[0152]

[0153] Synthesis example 5

[0154] Synthesis of vitamin D derivatives containing chloromethyl heteroaryl substitution

[0155] 50 mg of compound 125-(OH)VD3 was weighed and dissolved in 2.5 mL of anhydrous DMSO to prepare a 20 mg / mL solution. To a 4 mL centrifuge tube, 1.25 mL of 25-(OH)VD3 (62.4 μmol) of DMSO solution was added. 40.92 mg of 8-(chloromethyl)-5-isocyanatoquinoline (187.2 μmol) was added at a molar ratio of 3:1. Stir at room temperature for 3 h. Saturated aqueous sodium chloride solution was added to the reaction solution, which was extracted with ethyl acetate, dried over anhydrous Na2SO4, and spin-dried. Purification by column chromatography afforded 20 mg of compound 6 in a 52% yield.

[0156] 1 HNMR(300MHz, CDCl3):0.57(s,3H,Me),0.89(d,3H,Me,J=6.3Hz),0.73–2.49(several m,19H),1.33(s,6H,Me),2.57(dd,1H,J=12.4,3.6Hz),2.67(d,1H,J=11.3Hz),3.25(dd,2H,J=10.1,6.2Hz) ,3.46(s,1H,OH),3.58(t,2H,J=4.7Hz),4.64(s,2H,CH2),4.70–4.94(m,1H,H3),5.05(dd,2H,CH2),5.97and 6.15(2d,2H,CH,J=10.3Hz), 7.62–9.01(m,5H,ArH), 8.7(s,1H,NH).

[0157]

[0158] Synthesis example 6

[0159] Synthesis of Vitamin D Labeling Compounds Containing Chloromethyl Heteroaryl Substitutions

[0160] 10 mg of NH2PEGnBiotin (5kD) (2 μmol) was weighed and dissolved in 0.02 M PBS (0.15 M NaCl, 25 mM EDTA) pH 7.2 buffer. 2.48 mg (4 μmol) of compound 6 was added at a molar ratio of 2:1 and stirred at 37°C for 16 h. The product was purified by a desalting column and lyophilized to obtain 13 mg of lyophilized compound 7.

[0161]

[0162] Synthesis Example 7

[0163] Synthesis of succinate-substituted vitamin D markers

[0164] Take a 2 mL centrifuge tube, weigh 2 mg of compound 8 (25-hydroxyvitamin D3 hemisuccinate), dissolve it in 1 mL of anhydrous DMSO, add 1.9 mg of EDAC and 2.3 mg of NHS, and stir at room temperature for 1 h.

[0165] 10 mg of NH2PEGnBiotin (5 kD) (2 μmol) was weighed and dissolved in 0.02 M PBS (0.15 M NaCl, 25 mM EDTA) pH 7.2 buffer. Activated 25-hydroxyvitamin D3 succinate was added at a molar ratio of 2:1 and stirred at room temperature for 2 h. Compound 9 was obtained after purification via a desalting column.

[0166]

[0167] Synthesis example 8

[0168] Synthesis of BSA- and aryl-substituted vitamin D conjugates

[0169] To a 2 mL centrifuge tube, add 1 mL of 10 mg / mL BSA (0.02 M PBS, pH 7.2, 25 mM EDTA) buffer, add 1 M DTT to a final concentration of 10 mM, vortex to mix, and let stand at room temperature for 2 hours. Desalt the solution using a desalting column to remove excess DTT. Then, add 150 μL of 20 mg / mL compound 2 at a molar ratio of 10:1 (compound 2:BSA). Stir at room temperature for 2 hours, and dialyze the protein into 0.02 M PBS, pH 7.2. Dialysis purification yields conjugate 10.

[0170]

[0171] To a 2 mL centrifuge tube, add 1 mL of 10 mg / mL BSA (0.02 M PBS, pH 7.2, 25 mM EDTA) buffer, followed by 150 μL of 20 mg / mL compound 4 at a molar ratio of 10:1 (compound 4:BSA). Stir at room temperature for 24 h, then dialyze the protein into 0.02 M PBS, pH 7.2 buffer, and dialysis purification to obtain conjugate 11.

[0172]

[0173] Experiment Name: Development of a kit for quantitative detection of 25-hydroxyvitamin D by chemiluminescence

[0174] Experimental purpose: To develop a kit for the quantitative detection of 25-hydroxyvitamin D

[0175] Experimental design: Detection using competitive chemiluminescence

[0176] Reagents and instruments:

[0177] Anti-25-OH VD3 antibody (Bioventix), biotinylated-25-OH VD3 compound 3 (self-produced), biotinylated-25-OH VD3 compound 5 (self-produced), biotinylated-25-OH VD3 compound 7 (self-produced), biotinylated-25-OH VD3 compound 9 (self-produced), carboxyl microspheres (JSR), phosphate buffer (0.02 M PBS, pH 7.2), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDAC (Thermo Fisher), Tween-20, 0.1 M MES buffer (pH 6.0). LiCA HT (Shanghai Boyang Biotechnology Co., Ltd.), Hitachi high-speed refrigerated centrifuge.

[0178] Experimental steps:

[0179] Preparation of microspheres coated with anti-25-OH VD3 antibody

[0180] In the first step, 10 mg of carboxyl-functionalized microspheres were placed in a 2 mL centrifuge tube and washed once with 0.1 M MES (pH 6.0) buffer solution by centrifugation at 10,000 rpm at 4°C for 15 min.

[0181] In the second step, 200 μL of 0.1 M MES (pH 6.0) buffer was added and ultrasonically dispersed uniformly, and then 8 μL of 1 mg / mL anti-25-OH vitamin D sheep monoclonal antibody was added, followed by 100 μL of 5 mg / mL EDAC (0.1 M MES) solution, and stirred at room temperature for 4 h.

[0182] The third step was to add 50uL 200mg / mL BSA to block the carboxyl microspheres.

[0183] In the third step, the microspheres were washed three times by centrifugation with PBS buffer solution containing 0.5% Tween-20, and finally the volume was adjusted to 10 mg / mL with PBS buffer solution.

[0184] Detection experiment

[0185] Experiment 1:

[0186] Experimental Name: Detection of Different Biotinylated Aryl-Substituted-25-OH VD3 Derivatives

[0187] Experimental purpose: To screen the best biotinylated aryl-substituted 25-OH VD3 derivatives Experimental design: With the same reaction mode, the performance of Bio-25-OH-VD33 substituted with N-maleimide aryl and Bio-25-OH-VD35 substituted with chloromethyl aryl were compared.

[0188] Experimental steps:

[0189] 1. Dilute the luminescent microspheres to 30 μg / mL, and the biotinylated-25-OH VD3 derivatives 3 and 5 to 5 ng / mL, 0.5 ng / mL, and 0.05 ng / mL, respectively.

[0190] 2. Add horse serum to a 1 mg / mL 25-hydroxyvitamin D solution to prepare calibrators 1 to 6, and assign values ​​to them. The measured values ​​are 0, 4.07, 8.15, 17.75, 33.13, and 65.12, respectively.

[0191] 3. According to the reaction mode, add the sample solution, and then add the coated luminescent microspheres and biotinylated 25-OHVD3 derivatives in sequence, 25uL each.

[0192] 4. Carry out the first stage of incubation: incubate at 37°C for 17 minutes.

[0193] 5. Add 175ul universal solution.

[0194] 6. Perform the second stage of incubation: incubate at 37°C for 15 min.

[0195] 7. Read the numbers.

[0196] The results are shown in Table 1.

[0197] Table 1

[0198]

[0199] Calibrator discrimination: The results are shown in Table 2.

[0200] Table 2

[0201]

[0202]

[0203] Data Analysis:

[0204] From the perspective of signal quantity and calibrant discrimination, Bio-25-OH-VD33 improves the reagent signal quantity and discrimination more than Bio-25-OH-VD35.

[0205] The overall discrimination was best when the biotin reagent concentration was 5 ng / mL.

[0206] Experimental conclusion:

[0207] From the perspective of calibrant signal and discrimination, the performance of Bio-25-OHVD33 reagent substituted with N-maleimidoaryl group is better than that of Bio-25-OH VD35 reagent substituted with chloromethylaryl group.

[0208] Experiment 2:

[0209] Experimental Name: Detection of Different Biotinylated Aryl- and Heteroaryl-Substituted-25-OH VD3 Derivatives Experimental Purpose: Comparison of the performance of heteroaryl-substituted 25-OH VD3 derivatives and aryl-substituted 25-OH VD3 derivatives as labels Experimental Design: Using the same reaction mode, the performance of aryl-substituted Bio-25-OH-VD35 and aryl-substituted Bio-25-OH-VD37 were compared.

[0210] Experimental steps:

[0211] 1. Dilute the luminescent microspheres to 30 μg / mL, and the biotinylated-25-OH VD3 derivatives 5 and 7 to 5 ng / mL, 0.5 ng / mL, and 0.05 ng / mL, respectively.

[0212] 2. Add horse serum to a 1 mg / mL 25-hydroxyvitamin D solution to prepare calibrators 1 to 6, and assign values ​​to them. The measured values ​​are 0, 4.07, 8.15, 17.75, 33.13, and 65.12, respectively.

[0213] 3. According to the reaction mode, add the sample solution, and then add the coated luminescent microspheres and biotinylated 25-OHVD3 derivatives in sequence, 25uL each.

[0214] 4. Carry out the first stage of incubation: incubate at 37°C for 17 minutes.

[0215] 5. Add 175ul universal solution.

[0216] 6. Perform the second stage of incubation: incubate at 37°C for 15 min.

[0217] 7. Read the numbers.

[0218] The results are shown in Table 3.

[0219] Table 3

[0220]

[0221] Calibrator discrimination: The results are shown in Table 4.

[0222] Table 4

[0223] Cal1 / Cal2 1.58 1.78 1.19 1.55 1.67 1.15 Cal2 / Cal3 1.38 1.37 1.20 1.34 1.30 1.14 Cal3 / Cal4 1.60 1.33 1.10 1.52 1.25 1.07 Cal4 / Cal5 1.32 1.18 1.09 1.26 1.13 1.06 Cal5 / Cal6 1.99 1.14 0.95 1.71 1.10 0.96 Cal1 / Cal6 9.12 4.36 1.61 6.82 3.36 1.43

[0224] Data Analysis:

[0225] In terms of signal intensity and calibrator discrimination, at the same biotin reagent concentration, Bio-25-OH-VD35 and Bio-25-OH-VD37 had similar low-end discrimination, but Bio-25-OH-VD35 had higher overall reagent signal and discrimination. Overall discrimination was optimal at a biotin reagent concentration of 5 ng / mL.

[0226] Experimental conclusion:

[0227] From the perspective of calibrant signal and discrimination, the performance of the aryl-substituted Bio-25-OH-VD35 reagent is better than that of the heteroaryl-substituted Bio-25-OH-VD37.

[0228] Experiment 3:

[0229] Experiment Name: Detection of Different Biotinylated 25-OH VD3 Derivatives Experimental Purpose: Comparison of the performance of aryl-substituted 25-OH VD3 derivatives and succinate-substituted 25-OH VD3 derivatives Experimental Design: Using the same reaction mode, the performance of aryl-substituted Bio-25-OH-VD33 and succinate-substituted Bio-25-OH-VD39 were compared.

[0230] Experimental steps:

[0231] 1. Dilute the luminescent microspheres to 30 μg / mL, and the biotinylated-25-OH VD3 derivatives 3 and 9 to 5 ng / mL, 0.5 ng / mL, and 0.05 ng / mL, respectively.

[0232] 2. Add horse serum to a 1 mg / mL 25-hydroxyvitamin D solution to prepare calibrators 1 to 6, and assign values ​​to them. The measured values ​​are 0, 4.07, 8.15, 17.75, 33.13, and 65.12, respectively.

[0233] 3. According to the reaction mode, add the sample solution, and then add the coated luminescent microspheres and biotinylated 25-OHVD3 derivatives in sequence, 25uL each.

[0234] 4. Carry out the first stage of incubation: incubate at 37°C for 17 minutes.

[0235] 5. Add 175ul universal solution.

[0236] 6. Perform the second stage of incubation: incubate at 37°C for 15 min.

[0237] 7. Read the numbers.

[0238] The results are shown in Table 5.

[0239] Table 5

[0240]

[0241] Calibrator discrimination: The results are shown in Table 6.

[0242] Table 6

[0243]

[0244]

[0245] Data Analysis:

[0246] From the perspective of signal quantity and calibrant discrimination, Bio-25-OH-VD33 improves the reagent signal quantity and discrimination more than Bio-25-OH-VD39.

[0247] The overall discrimination was best when the biotin reagent concentration was 5 ng / mL.

[0248] Experimental conclusion:

[0249] From the perspective of calibrant signal and discrimination, the performance of the N-maleimidoaryl-substituted Bio-25-OHVD33 reagent is better than that of the succinate-substituted Bio-25-OH VD39.

[0250] Experiment 4:

[0251] Experiment Name: Detection of Different Biotinylated 25-OH VD3 Derivatives

[0252] Experimental purpose: To explore the performance comparison of different biotinylated 25-OH VD3 derivatives in plate-based chemiluminescence

[0253] Experimental design: Same reaction mode, plate-based chemiluminescence detection

[0254] Experimental steps:

[0255] 1. Avidin 2ug / ml coating, 100ul / well, 4℃ overnight

[0256] 2. Washing: Wash the microplate 5 times with diluted washing solution. Add no less than 400 μL of washing solution to each well, soak for 10 seconds each time, and finally pat dry on clean absorbent paper.

[0257] 3. Add 3, 5, 9, and 100 μl of Biotin-25-OH VD3 compound diluted in CB (1 / 10000) per well, respectively.

[0258] 4. Washing: Wash the microplate 5 times with diluted washing solution. Add no less than 400 μL of washing solution to each well, soak for 10 seconds each time, and finally pat dry on clean absorbent paper.

[0259] 5. Add sample: add 50μL of sample to each well. Add antibody: add 100μL of antibody to each well.

[0260] 6. Incubation: Mix by oscillating on a microplate oscillator for 5 seconds, seal the reaction plate with a sealing film, and incubate at 37°C for 2 hours.

[0261] 7. Washing: Wash the microplate 5 times with diluted washing solution. Add no less than 400 μL of washing solution to each well, soak for 10 seconds each time, and finally pat dry on clean absorbent paper.

[0262] 8. Add enzyme marker: Except for the blank control well, add 100 μL of enzyme marker to each well.

[0263] 9. Incubation: Mix by oscillating on a microplate oscillator for 5 seconds, seal the reaction plate with a sealing film, and incubate at 37°C for 1 hour.

[0264] 10. Washing: Wash the microplate 5 times with diluted washing solution. Add no less than 400 μL of washing solution to each well, soak for 10 seconds each time, and finally pat dry on clean absorbent paper.

[0265] 11. Add substrate solution: Add 100 μL of freshly prepared chemiluminescent substrate working solution to each well. It is recommended to use an 8-channel pipette and oscillate with a microvibrator for 5 seconds.

[0266] 12. Detection: After adding the luminescent substrate solution, incubate the plate in the dark at room temperature (20-27°C) for 5 minutes. Immediately measure the luminescence value (RLU) of each well on a microplate luminometer (0.1-1.0 sec / well).

[0267] Test results: See Table 7 and Table 8.

[0268] Table 7

[0269] <![CDATA[Bio-25-OHVD33]]> <![CDATA[Bio-25-OHVD35]]> <![CDATA[Bio-25-OHVD39]]> S0 5970 1958 2655 S1 4325 1855 2409 S2 3640 1640 1999 S3 2057 1527 1702 S4 1854 1477 1364 S5 1536 1395 1520 S6 1230 1287 1273 S7 705 1063 922

[0270] Table 8

[0271] S0 / S1 1.38 1.06 1.10 S1 / S2 1.19 1.13 1.21 S2 / S3 1.77 1.07 1.18 S3 / S4 1.11 1.03 1.25 S4 / S5 1.21 1.06 0.90 S5 / S6 1.25 1.08 1.19 S6 / S7 1.74 1.21 1.38

[0272] Data Analysis:

[0273] The overall discrimination of the samples tested by Bio-25 OH VD33 and Bio-25-OH VD39 was higher than that of Bio-25-OH VD35.

[0274] Bio-25-OH VD33 has a slightly higher overall discrimination among samples than Bio-25-OH VD39, and has a higher sensitivity in detecting low-end samples.

[0275] Experimental conclusion:

[0276] In terms of the discrimination of the detected samples, the N-maleimidoaryl-substituted Bio-25-OH VD33 reagent performed best.

[0277] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation of the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein. On the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. A compound of formula II or formula III, in, Z is selected from C4-C 10 Alkyl, the branched C4-C 10 The terminal carbon atom of the alkyl group has a hydroxyl group attached to it; R1 is selected from hydrogen, C1-C5 alkyl; R2 and R3 are the same or different and are independently selected from hydrogen and C1-C5 alkyl; R 1 -(CH2) p X or -(CH2) p COX, wherein X is selected from a biomacromolecule moiety, an N-maleimide group connected to a biomacromolecule moiety, a member of a signal-generating system, an organic small molecule, and a binding ligand of the organic small molecule, and p is an integer from 0 to 10, wherein the biomacromolecule moiety is derived from a biomacromolecule, and the biomacromolecule is selected from a protein molecule, a nucleic acid molecule, a polysaccharide molecule, and a lipid molecule; the member of the signal-generating system is selected from a fluorescent compound, a sensitizer, an enzyme, and a radioactive label; the organic small molecule is selected from biotin, fluorescein, rhodamine, a chemiluminescent molecule, dinitrophenol, an acridinium ester, and alkaline phosphatase; and the binding ligand of the organic small molecule is selected from avidin, an antibody to fluorescein, an antibody to rhodamine, an antibody to a chemiluminescent molecule, and an antibody to dinitrophenol; In Formula II, R 2 -R 5 the same or different, independently selected from hydrogen, C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C1-C 20 Alkoxy, cyano, halogen, nitro, amino and C1-C 10 an alkyl-substituted amino group; In formula III, R 2 -R 6 the same or different, independently selected from hydrogen, C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C1-C 20 Alkoxy, cyano, halogen, nitro, amino and C1-C 10 Alkyl-substituted amino groups.

2. The compound according to claim 1, characterized in that C4-C with branched chains 10 The branched chain in the alkyl group is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.

3. The compound according to claim 1, characterized in that Z is 4,4-dimethyl-4-hydroxybutyl.

4. The compound according to any one of claims 1 to 3, characterized in that R1 is selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.

5. The compound according to any one of claims 1 to 3, characterized in that R2 is selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl; R3 is selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.

6. The compound according to any one of claims 1 to 3, characterized in that The members of the signal generating system include particles.

7. The compound according to claim 6, characterized in that The particles are selected from fluorescent particles, chemiluminescent particles, sensitizer particles and magnetic particles.

8. The compound according to any one of claims 1 to 3, characterized in that p is 0, 1, 2, 3, 4 or 5.

9. The compound according to any one of claims 1 to 3, characterized in that In Formula II, R 2 -R 5 independently selected from hydrogen, C1-C 10 Alkyl, C1-C 10 Alkenyl, C1-C 10 Alkynyl, C1-C 10 Alkoxy, cyano, halogen, nitro, amino and C1-C5 alkyl substituted amino; in formula III, R 2 -R 6 independently selected from hydrogen, C1-C 10 Alkyl, C1-C 10 Alkenyl, C1-C 10 Alkynyl, C1-C 10 Alkoxy group, cyano group, halogen atom, nitro group, amino group and C1-C5 alkyl-substituted amino group.

10. The compound according to any one of claims 1 to 3, characterized in that In Formula II, R 2 -R 5 are independently selected from hydrogen, C1-C5 alkyl, C1-C5 alkenyl, C1-C5 alkynyl, C1-C5 alkoxy, cyano, halogen, nitro, amino and C1-C5 alkyl-substituted amino groups; in Formula III, R 2 -R 6 Independently selected from hydrogen, C1-C5 alkyl, C1-C5 alkenyl, C1-C5 alkynyl, C1-C5 alkoxy, cyano, halogen, nitro, amino and C1-C5 alkyl-substituted amino groups.

11. A product for determining the presence and / or amount of vitamin D in a sample, comprising the following components: 1) A compound according to any one of claims 1 to 10, and 2) Specific binding member for vitamin D.

12. The product according to claim 11, characterized in that The sample is a biological sample or a non-biological sample.

13. The product according to claim 11 or 12, characterized in that The specific binding member of vitamin D is an antibody against vitamin D and a ligand coupled to the antibody against vitamin D.

14. The product according to claim 11 or 12, characterized in that The product is a composition or a kit.

15. Use of a product according to any one of claims 11 to 14 for detecting the presence and / or amount of vitamin D in a non-biological sample.

16. A method for determining the presence and / or amount of vitamin D in a non-biological sample, comprising the steps of: 1) Providing a product according to any one of claims 11 to 14; 2) allowing the specific binding member for vitamin D in the product to form a complex with the compound; 3) Measuring the amount of the complex, which is related to the presence and / or amount of vitamin D in the sample.

17. A complex formed by a compound according to any one of claims 1 to 10 and a specific binding member for vitamin D.

18. The composite according to claim 17, characterized in that The specific binding member of vitamin D is an antibody against vitamin D and a ligand coupled to the antibody against vitamin D.

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