CN111518005A - Intermediate compound and preparation method and application thereof - Google Patents

Intermediate compound and preparation method and application thereof Download PDF

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CN111518005A
CN111518005A CN201910105699.0A CN201910105699A CN111518005A CN 111518005 A CN111518005 A CN 111518005A CN 201910105699 A CN201910105699 A CN 201910105699A CN 111518005 A CN111518005 A CN 111518005A
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CN111518005B (en
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章春奇
张向辉
赵卫国
刘宇卉
李临
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Beyond Diagnostics Shanghai Co ltd
Chemclin Diagnostics Corp
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Abstract

The invention discloses an intermediate compound shown as a formula A, and a preparation method and application thereof. The intermediate compound shown in the formula A can be used for synthesizing the compound shown in the formula I. Compounds of formula I are useful for determining vitamin D containing analytes (including vitamin D2 and vitamin D C)Vitamin D3) and metabolites thereof.

Description

Intermediate compound and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an intermediate compound and a preparation method and application thereof.
Background
Vitamin D is a lipid-soluble steroid prohormone that has two main forms: vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Vitamin D2 is obtained from a nutritional supplement, whereas vitamin D3 is derived from skin exposed to sunlight (ultraviolet radiation) and is obtained mainly from fish, liver oil, egg yolk, and other diets. Vitamins D2 and D3 are metabolized in the liver to 25-OH vitamin D (25- (OH) D) and then converted to 1, 25- (OH) in the kidney2D. 25- (OH) D is the major metabolite in the circulation, and thus, 25- (OH) D values may reflect vitamin D levels in the body. Vitamin D in blood exists in a protein-bound form. The clinical test report shows that the vitamin D in blood refers to the sum of 25- (OH) D, including 25- (OH) D2And 25- (OH) D3. Accurate monitoring of total 25-OH vitamin D levels is critical for clinical use.
Vitamin D plays an important role in maintaining bone mineral density, and it plays a critical role in calcium balance with parathyroid hormone (PTH). Vitamin D deficiency seriously affects the absorption of calcium and phosphorus in human bodies, and can cause rickets, hypocalcemia of newborns, hypothyroidism, osteoporosis of middle-aged and elderly people and other calcium metabolism abnormal diseases. Vitamin D overdose can lead to hypercalcemia and various aging-related diseases.
Vitamin D is present in almost all human tissues and its role is not merely to maintain the balance of calcium and phosphorus. Studies have shown that vitamin D acts by binding to the Vitamin D Receptor (VDR), and recent epidemiological studies have found that vitamin D is associated with a variety of diseases-cancer, heart disease, hypertension, diabetes, autoimmune diseases, infectious diseases and aging.
Currently, the commercially available 25-OH vitamin D detection methods mainly include Radioimmunoassay (RIA), liquid chromatography-tandem mass spectrometry (LC-MS), enzyme-linked immunosorbent assay (ELISA) and chemiluminescence assay (CLIA). Among them, the chemiluminescence method is developed based on its advantages of high sensitivity, wide linear range, convenient operation, no pollution, etc.
Disclosure of Invention
The invention aims to provide an intermediate compound and a preparation method of the intermediate compound aiming at the defects of the prior art. The intermediate compound can be used for synthesizing the compound shown in the formula I, and therefore, the invention also provides a preparation method of the compound shown in the formula I. The compounds of formula I are useful for detecting the presence or amount of vitamin D analytes (including vitamin D2 and vitamin D3) and metabolites thereof in a sample containing the same.
In order to achieve the above object, in a first aspect, the present invention provides an intermediate compound represented by formula a,
Figure BDA0001966682920000021
wherein Z is selected from C1-C20Alkyl radical, C2-C20Alkenyl and C2-C20Alkynyl, said alkyl, alkenyl and alkynyl being optionally selected from hydroxy, C1-C10Alkoxy radical, C1-C10One or more substituents of ester group and oxime group;
R1selected from hydrogen, hydroxy, C1-C20Alkyl radical, C2-C20Alkenyl radical, C2-C20Alkynyl, C1-C20Alkoxy radical, C2-C20Alkenyloxy and C2-C20An alkynyloxy group;
R2and R3Same or different, independently selected from hydrogen and C1-C20An alkyl group;
r' is at least RaSubstituted C6-C30Aryl radical, C5-C30Heteroaryl or C9-C30The aromatic group of the condensed aromatic group is,
Rais- (CH)2)pXaOr- (CH)2)pCOXaWherein X isaIs a reactive group capable of reacting with the labeling compound, the biomacromolecule, the member of the signal producing system, the small organic molecule, and the binding partner or carrier of said small organic molecule, p is an integer from 0 to 10, for example p is 0, 1,2,3,4 or 5.
In some embodiments of the foregoing, XaSelected from N-maleimido, halogen, -NCO and-N3The halogen is preferably fluorine, chlorine, bromine or iodine.
In some embodiments of the foregoing, Z is selected from C1-C10Alkyl radical, C2-C10Alkenyl and C2-C10Alkynyl, said alkyl, alkenyl and alkynyl being optionally selected from hydroxy, C1-C10Alkoxy radical, C1-C10Ester groups and oxime groups.
In some embodiments of the foregoing, Z is selected from C1-C10Alkyl radical, C2-C10Alkenyl and C2-C10Alkynyl, said alkyl, alkenyl or alkynyl being optionally selected from hydroxy, C1-C5Alkoxy radical, C1-C5Ester groups and oxime groups.
In some embodiments of the foregoing, Z is C with a branch4-C10Alkyl, C with branched chain4-C10Alkenyl or C with a branch4-C20Alkynyl.
In some embodiments of the foregoing, the C with a branch is4-C10Alkyl, C with branched chain4-C10Alkenyl or C with a branch4-C20Of alkynyl groupsThe terminal carbon atom has a hydroxyl or protected hydroxyl group attached, preferably Z is 4, 4-dimethyl-4-hydroxybutyl.
In some embodiments of the foregoing, R1Selected from hydrogen, hydroxy, C1-C10Alkyl radical, C2-C10Alkenyl radical, C2-C10Alkynyl and C1-C10An alkoxy group; and/or, R2And R3Independently selected from hydrogen and C1-C10Alkyl, preferably selected from hydrogen and C1-C5An alkyl group.
In some embodiments of the above embodiments, the small organic molecule is selected from the group consisting of biotin, fluorescein, rhodamine, a chemiluminescent molecule, dinitrophenol, acridinium ester, alkaline phosphatase, and a labeled compound molecule, and the binding partner of the small organic molecule is selected from the group consisting of avidin, an antibody to fluorescein, an antibody to rhodamine, an antibody to a chemiluminescent molecule, and an antibody to dinitrophenol.
In some embodiments of the above technical scheme, the member of the signal generating system is selected from the group consisting of a fluorescent compound, a chemiluminescent compound, a sensitizer, an enzyme, and a radioactive label.
In some embodiments of the above technical solution, the member of the signal producing system comprises a particle, preferably the particle is selected from the group consisting of a fluorescent particle, a chemiluminescent particle, a sensitizer particle and a magnetic particle.
In some embodiments of the above technical scheme, the biomacromolecule is selected from the group consisting of a protein molecule, a nucleic acid molecule, a polysaccharide molecule and a lipid molecule.
In some embodiments of the foregoing, the binding partner of the small organic molecule is selected from antibodies to vitamin D and analogs thereof.
In some embodiments of the foregoing, R' is at least RaSubstituted C6-C20Aryl radical, C5-C20Heteroaryl or C9-C20A fused aryl group.
In some embodiments of the foregoing, R' is at least RaSubstitutionC of (A)6-C10Aryl radical, C5-C10Heteroaryl or C9-C10Condensed aryl radicals, e.g. at least by RaSubstituted phenyl, at least by RaSubstituted pyridyl, at least by RaSubstituted quinolyl radicals or substituted by at least RaSubstituted isoquinolinyl groups.
In some embodiments of the foregoing, the compound has a structure represented by formula A1 or formula A2,
Figure BDA0001966682920000041
in the formula A1, R2-R5The same or different, are independently selected from hydrogen and C1-C20Alkyl radical, C2-C20Alkenyl radical, C2-C20Alkynyl, C1-C20Alkoxy radical, C6-C20Aryl radical, C5-C20Heteroaryl group, C9-C20Condensed aryl, cyano, halogen, nitro, carboxyl, amino and C1-C10An alkyl-substituted amino group;
in the formula A2, R2-R6The same or different, are independently selected from hydrogen and C1-C20Alkyl radical, C2-C20Alkenyl radical, C2-C20Alkynyl, C1-C20Alkoxy radical, C6-C20Aryl radical, C5-C20Heteroaryl group, C9-C20Condensed aryl, cyano, halogen, nitro, carboxyl, amino and C1-C10Alkyl-substituted amino groups.
In some embodiments of the foregoing, in formula A1, R2-R5Independently selected from hydrogen, C1-C10Alkyl radical, C1-C10Alkenyl radical, C1-C10Alkynyl, C1-C10Alkoxy, cyano, halogen, nitro, amino and C1-C5An alkyl-substituted amino group; in the formula A2, R2-R6Independently selected from hydrogen, C1-C10Alkyl radical, C1-C10Alkenyl radical, C1-C10Alkynyl, C1-C10Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups.
In some embodiments of the foregoing, in formula A1, R2-R5Independently selected from hydrogen, C1-C5Alkyl radical, C1-C5Alkenyl radical, C1-C5Alkynyl, C1-C5Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups.
In some embodiments of the foregoing, in formula A2, R2-R6Independently selected from hydrogen, C1-C5Alkyl radical, C1-C5Alkenyl radical, C1-C5Alkynyl, C1-C5Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups.
In a second aspect, the present invention provides a process for the preparation of an intermediate compound according to formula a above, comprising the steps of: reacting a compound of formula B with R' NCO to produce an intermediate compound of formula A,
Figure BDA0001966682920000051
wherein, Z, R1、R2、R3And R' is as defined above.
The third aspect of the present invention also provides a preparation method of the compound represented by the formula I, which comprises the following steps: reacting an intermediate compound of formula A according to the first aspect of the invention with a starting material selected from the group consisting of a label compound, a biomacromolecule, a member of a signal producing system, a small organic molecule, and a binding partner for said small organic molecule or a carrier to produce a compound of formula I,
Figure BDA0001966682920000052
wherein, Z, R1、R2、R3The definition of (A) is the same as that above,
r is at least R1Substituted C6-C30Aryl radical, C5-C30Heteroaryl or C9-C30The aromatic group of the condensed aromatic group is,
R1is- (CH)2)pX or- (CH)2)pCOX, wherein X is selected from a labeling moiety derived from a labeling compound, a biomacromolecule moiety, an N-maleimido group to which the labeling moiety or biomacromolecule moiety is attached, a member of a signal generating system, a small organic molecule, and a binding ligand or carrier for said small organic molecule, and p is an integer from 0 to 10, such as an integer from 1 to 5.
In some embodiments of the above embodiments, the small organic molecule is selected from the group consisting of biotin, fluorescein, rhodamine, a chemiluminescent molecule, dinitrophenol, acridinium ester, alkaline phosphatase, and a labeled compound molecule, and the binding partner of the small organic molecule is selected from the group consisting of avidin, an antibody to fluorescein, an antibody to rhodamine, an antibody to a chemiluminescent molecule, and an antibody to dinitrophenol.
In some embodiments of the above technical scheme, the member of the signal generating system is selected from the group consisting of a fluorescent compound, a chemiluminescent compound, a sensitizer, an enzyme, and a radioactive label.
In some embodiments of the above technical solution, the member of the signal producing system comprises a particle, preferably the particle is selected from the group consisting of a fluorescent particle, a chemiluminescent particle, a sensitizer particle and a magnetic particle.
In some embodiments of the above technical scheme, the biomacromolecule is selected from the group consisting of a protein molecule, a nucleic acid molecule, a polysaccharide molecule and a lipid molecule.
In some embodiments of the foregoing, the binding partner of the small organic molecule is selected from antibodies to vitamin D and analogs thereof.
In some embodiments of the foregoing, R isAt least by R1Substituted C6-C20Aryl radical, C5-C20Heteroaryl or C9-C20Fused aryl, preferably R is at least R1Substituted C6-C10Aryl radical, C5-C10Heteroaryl or C9-C10Condensed aryl radicals, e.g. at least by R1Substituted phenyl, at least by R1Substituted pyridyl, at least by R1Substituted quinolyl radicals or substituted by at least R1Substituted isoquinolinyl groups.
In some embodiments of the foregoing, the compound of formula I has a structure represented by formula II or formula III:
Figure BDA0001966682920000061
Figure BDA0001966682920000071
in the formula II, R2-R5The same or different, are independently selected from hydrogen and C1-C20Alkyl radical, C2-C20Alkenyl radical, C2-C20Alkynyl, C1-C20Alkoxy radical, C6-C20Aryl radical, C5-C20Heteroaryl group, C9-C20Condensed aryl, cyano, halogen, nitro, carboxyl, amino and C1-C10An alkyl-substituted amino group;
in the formula III, R2-R6The same or different, are independently selected from hydrogen and C1-C20Alkyl radical, C2-C20Alkenyl radical, C2-C20Alkynyl, C1-C20Alkoxy radical, C6-C20Aryl radical, C5-C20Heteroaryl group, C9-C20Condensed aryl, cyano, halogen, nitro, carboxyl, amino and C1-C10Alkyl-substituted amino groups.
In some embodiments of the foregoing, formula II wherein R is2-R5Independent of each otherSelected from hydrogen, C1-C10Alkyl radical, C1-C10Alkenyl radical, C1-C10Alkynyl, C1-C10Alkoxy, cyano, halogen, nitro, amino and C1-C5An alkyl-substituted amino group; in the formula III, R2-R6Independently selected from hydrogen, C1-C10Alkyl radical, C1-C10Alkenyl radical, C1-C10Alkynyl, C1-C10Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups.
In some embodiments of the foregoing, formula II wherein R is2-R5Independently selected from hydrogen, C1-C5Alkyl radical, C1-C5Alkenyl radical, C1-C5Alkynyl, C1-C5Alkoxy, cyano, halogen, nitro, amino and C1-C5An alkyl-substituted amino group; in the formula III, R2-R6Independently selected from hydrogen, C1-C5Alkyl radical, C1-C5Alkenyl radical, C1-C5Alkynyl, C1-C5Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups.
Detailed Description
In order that the invention may be readily understood, a detailed description of the invention is provided below. However, before the invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Where a range of values is provided, it is understood that each intervening value, to the extent that there is no stated or intervening value in that stated range, to the extent that there is no such intervening value, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where a specified range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. 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, the preferred methods and materials are now described.
I. Term(s) for
The term "alkyl" refers to those alkyl groups that are straight, branched, or cyclic, having the indicated number of carbon atoms. 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.
The term "alkenyl" refers to a straight or branched hydrocarbon chain having the indicated number of carbon atoms and at least one carbon-carbon double bond, which may occur at any point along the chain. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, dimethyl pentenyl, and the like.
The term "alkynyl" refers to a straight or branched chain hydrocarbon of the indicated 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.
"aryl" includes groups having aromatic character, including "conjugated" or polycyclic systems containing at least one aromatic ring and not containing any heteroatoms in the ring structure. Examples thereof include phenyl, benzyl, 1,2,3, 4-tetrahydronaphthyl and the like.
"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" is meant to include 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, e.g., 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6A heteroatom, or for example 1,2,3,4, 5 or 6 heteroatoms, 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 other substituent as defined herein). The nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., N → O and S (O))pWherein p is 1 or 2). It should be noted, however, that the total number of sulfur and oxygen atoms in the aromatic heterocycle does not exceed 1.
Examples of heteroaryl groups include pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like.
The term "carboxyl" means-COOH or its C1-C6An alkyl ester.
The term "ester group" refers to a compound or fragment containing a carbon or heteroatom bonded to an oxygen atom bonded to a carbon on a carbonyl group. The term "ester" includes alkoxycarbonyl groups such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, pentoxycarbonyl and the like.
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, pentoxy, and the like. 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, vinyloxy, propenyloxy, butenyloxy, pentenyloxy, and the like. The term "alkynyloxy" includes substituted and unsubstituted alkynyl groups covalently bonded to an oxygen atom. Examples of alkynyloxy include, but are not limited to, ethynyloxy, propynyloxy, butynyloxy, pentynyloxy, and the like.
As used herein, "amine" or "amino" refers to unsubstituted or substituted-NH2. "alkylamino" includes the group-NH-thereof2And a group of compounds in which the nitrogen atom of (a) is bonded to at least one alkyl group. Examples of alkylamino include benzylamino, methylamino, ethylamino, phenethylamino and the like. "dialkylamino" includes wherein-NH2And a group in which the nitrogen atom of (a) is bonded to at least two alkyl groups. Examples of dialkylamino groups includeBut are not limited to, dimethylamino and diethylamino. "arylamino" and "diarylamino" include groups in which a nitrogen atom is bonded to at least one or two aryl groups, respectively. "aminoaryl" and "aminoaryloxy" refer to amino-substituted aryl, aryloxy groups. "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 that is also bonded to an alkyl group. "amido" includes groups in which a nitrogen atom is bonded to an acyl group. Examples of amido include, but are not limited to, alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido.
"aryl" includes groups having aromatic character, including "conjugated" or polycyclic systems containing at least one aromatic ring and not containing any heteroatoms in the ring structure. Examples thereof include phenyl, benzyl, 1,2,3, 4-tetrahydronaphthyl and the like.
"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" is meant to include a stable 5, 6 or 7 membered monocyclic or 7, 8, 9, 10, 11 or 12 membered bicyclic aromatic heterocyclic ring 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, 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 other substituent as defined herein). The nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., N → O and S (O))pWherein p is 1 or 2). It should be noted, however, that the total number of sulfur and oxygen atoms in the aromatic heterocycle does not exceed 1.
Examples of heteroaryl groups include pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like.
Furthermore, the terms "aryl" and "heteroaryl" include polycyclic aryl and heteroaryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzimidazole, benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline, naphthyridinyl, indole, benzofuran, purine, benzofuran, deazapurine, indolizine.
The cycloalkyl, heterocycloalkyl, aryl or heteroaryl ring may be substituted at one or more ring positions (e.g., ring carbon atoms or heteroatoms, such as nitrogen atoms) with substituents as described above, e.g., alkyl, alkenyl, alkynyl, halogen, hydroxy, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, arylalkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, arylalkyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonate, phosphinate, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), amido (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), Amidino, imino, mercapto, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonic, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or aromatic or heteroaromatic fragment. The aryl and heteroaryl groups can also be fused or bridged to non-aromatic alicyclic or heterocyclic rings to form a polycyclic ring system (e.g., tetralin, methylenedioxyphenyl).
The term "oximino" refers to-C ═ N-OH.
Detailed description of the preferred embodiments
The present invention will be described in more detail below.
In order to achieve the above object, in a first aspect, the present invention provides an intermediate compound represented by formula a,
Figure BDA0001966682920000101
wherein Z is selected from C1-C20Alkyl radical, C2-C20Alkenyl and C2-C20Alkynyl, said alkyl, alkenyl and alkynyl being optionally selected from hydroxy, C1-C10Alkoxy radical, C1-C10One or more substituents of ester group and oxime group;
R1selected from hydrogen, hydroxy, C1-C20Alkyl radical, C2-C20Alkenyl radical, C2-C20Alkynyl, C1-C20Alkoxy radical, C2-C20Alkenyloxy and C2-C20An alkynyloxy group;
R2and R3Same or different, independently selected from hydrogen and C1-C20An alkyl group;
r' is at least RaSubstituted C6-C30Aryl radical, C5-C30Heteroaryl or C9-C30The aromatic group of the condensed aromatic group is,
Rais- (CH)2)pXaOr- (CH)2)pCOXaWherein X isaIs a reactive group capable of reacting with the labeling compound, the biomacromolecule, the member of the signal producing system, the small organic molecule, and the binding partner or carrier of said small organic molecule, p is an integer from 0 to 10, for example p is 0, 1,2,3,4 or 5.
In some embodiments of the foregoing, XaSelected from N-maleimido, halogen, -NCO and-N3The halogen is preferably fluorine, chlorine, bromine or iodine.
In some embodiments of the foregoing, Z is selected from C1-C10Alkyl radical, C2-C10Alkenyl and C2-C10Alkynyl, said alkyl, alkenyl and alkynyl being optionally selected from hydroxy, C1-C10Alkoxy radical, C1-C10Ester groups and oxime groups.
In some embodiments of the foregoing, Z is selected from C1-C10Alkyl radical、C2-C10Alkenyl and C2-C10Alkynyl, said alkyl, alkenyl or alkynyl being optionally selected from hydroxy, C1-C5Alkoxy radical, C1-C5Ester groups and oxime groups.
In some embodiments of the foregoing, Z is C with a branch4-C10Alkyl, C with branched chain4-C10Alkenyl or C with a branch4-C20Alkynyl.
In some embodiments of the foregoing, the C with a branch is4-C10Alkyl, C with branched chain4-C10Alkenyl or C with a branch4-C20The terminal carbon atom of the alkynyl group is attached to a hydroxyl group or a protected hydroxyl group, preferably, Z is 4, 4-dimethyl-4-hydroxybutyl.
In some embodiments of the foregoing, R1Selected from hydrogen, hydroxy, C1-C10Alkyl radical, C2-C10Alkenyl radical, C2-C10Alkynyl and C1-C10An alkoxy group; and/or, R2And R3Independently selected from hydrogen and C1-C10Alkyl, preferably selected from hydrogen and C1-C5An alkyl group.
In some embodiments of the above embodiments, the small organic molecule is selected from the group consisting of biotin, fluorescein, rhodamine, a chemiluminescent molecule, dinitrophenol, acridinium ester, alkaline phosphatase, and a labeled compound molecule, and the binding partner of the small organic molecule is selected from the group consisting of avidin, an antibody to fluorescein, an antibody to rhodamine, an antibody to a chemiluminescent molecule, and an antibody to dinitrophenol.
In some embodiments of the above technical scheme, the member of the signal generating system is selected from the group consisting of a fluorescent compound, a chemiluminescent compound, a sensitizer, an enzyme, and a radioactive label.
In some embodiments of the above technical solution, the member of the signal producing system comprises a particle, preferably the particle is selected from the group consisting of a fluorescent particle, a chemiluminescent particle, a sensitizer particle and a magnetic particle.
In some embodiments of the above technical scheme, the biomacromolecule is selected from the group consisting of a protein molecule, a nucleic acid molecule, a polysaccharide molecule and a lipid molecule.
In some embodiments of the foregoing, the binding partner of the small organic molecule is selected from antibodies to vitamin D and analogs thereof.
In some embodiments of the foregoing, R' is at least RaSubstituted C6-C20Aryl radical, C5-C20Heteroaryl or C9-C20A fused aryl group.
In some embodiments of the foregoing, R' is at least RaSubstituted C6-C10Aryl radical, C5-C10Heteroaryl or C9-C10Condensed aryl radicals, e.g. at least by RaSubstituted phenyl, at least by RaSubstituted pyridyl, at least by RaSubstituted quinolyl radicals or substituted by at least RaSubstituted isoquinolinyl groups.
In some embodiments of the foregoing, the compound has a structure represented by formula A1 or formula A2,
Figure BDA0001966682920000121
in the formula A1, R2-R5The same or different, are independently selected from hydrogen and C1-C20Alkyl radical, C2-C20Alkenyl radical, C2-C20Alkynyl, C1-C20Alkoxy radical, C6-C20Aryl radical, C5-C20Heteroaryl group, C9-C20Condensed aryl, cyano, halogen, nitro, carboxyl, amino and C1-C10An alkyl-substituted amino group;
in the formula A2, R2-R6The same or different, are independently selected from hydrogen and C1-C20Alkyl radical, C2-C20Alkenyl radical, C2-C20Alkynyl, C1-C20Alkoxy radical, C6-C20Aryl radical, C5-C20Heteroaryl group, C9-C20Condensed aryl, cyano, halogen, nitro, carboxyl, amino and C1-C10Alkyl-substituted amino groups.
In some embodiments of the foregoing, in formula A1, R2-R5Independently selected from hydrogen, C1-C10Alkyl radical, C1-C10Alkenyl radical, C1-C10Alkynyl, C1-C10Alkoxy, cyano, halogen, nitro, amino and C1-C5An alkyl-substituted amino group; in the formula A2, R2-R6Independently selected from hydrogen, C1-C10Alkyl radical, C1-C10Alkenyl radical, C1-C10Alkynyl, C1-C10Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups.
In some embodiments of the foregoing, in formula A1, R2-R5Independently selected from hydrogen, C1-C5Alkyl radical, C1-C5Alkenyl radical, C1-C5Alkynyl, C1-C5Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups.
In some embodiments of the foregoing, in formula A2, R2-R6Independently selected from hydrogen, C1-C5Alkyl radical, C1-C5Alkenyl radical, C1-C5Alkynyl, C1-C5Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups.
Examples of the intermediate compound of formula a according to the present invention include, but are not limited to, compounds selected from the group consisting of:
Figure BDA0001966682920000141
Figure BDA0001966682920000151
in a second aspect, the present invention provides a process for the preparation of an intermediate compound according to formula a above, comprising the steps of: reacting a compound of formula B with R' NCO to produce an intermediate compound of formula A,
Figure BDA0001966682920000152
wherein, Z, R1、R2、R3And R' is as defined above.
The third aspect of the present invention also provides a preparation method of the compound represented by the formula I, which comprises the following steps: reacting an intermediate compound of formula A according to the first aspect of the invention with a starting material selected from the group consisting of a label compound, a biomacromolecule, a member of a signal producing system, a small organic molecule, and a binding partner for said small organic molecule or a carrier to produce a compound of formula I,
Figure BDA0001966682920000161
wherein, Z, R1、R2、R3The definition of (A) is the same as that above,
r is at least R1Substituted C6-C30Aryl radical, C5-C30Heteroaryl or C9-C30The aromatic group of the condensed aromatic group is,
R1is- (CH)2)pX or- (CH)2)pCOX, wherein X is selected from a labeling moiety derived from a labeling compound, a biomacromolecule moiety, an N-maleimido group to which the labeling moiety or biomacromolecule moiety is attached, a member of a signal generating system, a small organic molecule, and a binding ligand or carrier for said small organic molecule, and p is an integer from 0 to 10, such as an integer from 1 to 5.
In some embodiments of the above embodiments, the small organic molecule is selected from the group consisting of biotin, fluorescein, rhodamine, a chemiluminescent molecule, dinitrophenol, acridinium ester, alkaline phosphatase, and a labeled compound molecule, and the binding partner of the small organic molecule is selected from the group consisting of avidin, an antibody to fluorescein, an antibody to rhodamine, an antibody to a chemiluminescent molecule, and an antibody to dinitrophenol.
In some embodiments of the above technical scheme, the member of the signal generating system is selected from the group consisting of a fluorescent compound, a chemiluminescent compound, a sensitizer, an enzyme, and a radioactive label.
In some embodiments of the above technical solution, the member of the signal producing system comprises a particle, preferably the particle is selected from the group consisting of a fluorescent particle, a chemiluminescent particle, a sensitizer particle and a magnetic particle.
In some embodiments of the above technical scheme, the biomacromolecule is selected from the group consisting of a protein molecule, a nucleic acid molecule, a polysaccharide molecule and a lipid molecule.
In some other embodiments of the foregoing, R1Is- (CH)2)pX, e.g. - (CH)2) And (4) X. X may be a biomacromolecule moiety or an N-maleimido group to which a biomacromolecule moiety is attached. The biomacromolecule is for example biotin polyethylene glycol amino (which may have a molecular weight of more than 1 dalton, for example between 2 and 100 dalton, for example between 2 and 80 dalton) or Bovine Serum Albumin (BSA).
In some other embodiments of the foregoing, R1Is- (CH)2)pCOX, e.g., -CH2COX. X may be a biomacromolecule moiety or an N-maleimido group to which a biomacromolecule moiety is attached. The biomacromolecule is for example biotin polyethylene glycol amino (which may have a molecular weight of more than 1 dalton, for example between 2 and 100 dalton, for example between 2 and 80 dalton) or Bovine Serum Albumin (BSA). In some embodiments of the foregoing, the binding partner of the small organic molecule is selected from antibodies to vitamin D and analogs thereof.
In some embodiments of the foregoing, R is at least one substituted phenyl groupR1Substituted C6-C20Aryl radical, C5-C20Heteroaryl or C9-C20Fused aryl, preferably R is at least R1Substituted C6-C10Aryl radical, C5-C10Heteroaryl or C9-C10Condensed aryl radicals, e.g. at least by R1Substituted phenyl, at least by R1Substituted pyridyl, at least by R1Substituted quinolyl radicals or substituted by at least R1Substituted isoquinolinyl groups.
In some embodiments of the foregoing, the compound of formula I has a structure represented by formula II or formula III:
Figure BDA0001966682920000171
in the formula II, R2-R5The same or different, are independently selected from hydrogen and C1-C20Alkyl radical, C2-C20Alkenyl radical, C2-C20Alkynyl, C1-C20Alkoxy radical, C6-C20Aryl radical, C5-C20Heteroaryl group, C9-C20Condensed aryl, cyano, halogen, nitro, carboxyl, amino and C1-C10An alkyl-substituted amino group;
in the formula III, R2-R6The same or different, are independently selected from hydrogen and C1-C20Alkyl radical, C2-C20Alkenyl radical, C2-C20Alkynyl, C1-C20Alkoxy radical, C6-C20Aryl radical, C5-C20Heteroaryl group, C9-C20Condensed aryl, cyano, halogen, nitro, carboxyl, amino and C1-C10Alkyl-substituted amino groups.
In some embodiments of the foregoing, formula II wherein R is2-R5Independently selected from hydrogen, C1-C10Alkyl radical, C1-C10Alkenyl radical, C1-C10Alkynyl, C1-C10Alkoxy, cyano, halogenA, nitro, amino and C1-C5An alkyl-substituted amino group; in the formula III, R2-R6Independently selected from hydrogen, C1-C10Alkyl radical, C1-C10Alkenyl radical, C1-C10Alkynyl, C1-C10Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups.
In some embodiments of the foregoing, formula II wherein R is2-R5Independently selected from hydrogen, C1-C5Alkyl radical, C1-C5Alkenyl radical, C1-C5Alkynyl, C1-C5Alkoxy, cyano, halogen, nitro, amino and C1-C5An alkyl-substituted amino group; in the formula III, R2-R6Independently selected from hydrogen, C1-C5Alkyl radical, C1-C5Alkenyl radical, C1-C5Alkynyl, C1-C5Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups.
In some embodiments of the foregoing, the compound of formula I is selected from at least one of the following compounds:
Figure BDA0001966682920000191
Figure BDA0001966682920000201
example III
In order that the present invention may be more readily understood, the following detailed description will proceed with reference being made to examples, which are intended to be illustrative only and are not intended to limit the scope of the invention. The starting materials or components used in the present invention may be commercially or conventionally prepared unless otherwise specified.
Reagents and instrumentation:
synthesis example 1
Synthesis of derivatives containing N-maleimidoaryl substituted vitamin D
Weighing 125- (OH) VD350mg of the aqueous solution was dissolved in 2.5mL of anhydrous DMSO to prepare a 20mg/mL solution. A4 mL centrifuge tube was charged with 1.25mL of 25- (OH) VD3(62.4umol) in a molar ratio of 1.2:1 (PMPI: 25- (OH) VD)3) 16.04mg of PMPI (p-maleimidophenyl isocyanate) (74.88umol) was added thereto, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was added with saturated aqueous sodium chloride solution, extracted with ethyl acetate, and extracted with anhydrous Na2SO4Drying, spin-drying, and purification by thin layer chromatography gave 15mg of compound 2 in 39% yield.1HNMR(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).
Figure BDA0001966682920000211
Synthesis example 2
Synthesis of N-maleimidoaryl substituted vitamin D containing labels
10mg of SHPEGnBiotin (5kD) (2umol) was weighed out and dissolved in 0.02M PBS (0.15M NaCl, 25mM EDTA) pH7.2 buffer solution, 2.4mg (4umol) of Compound 2 was added at a molar ratio of 2:1 and stirred at room temperature for 2 h. Purifying by desalting column, and lyophilizing to obtain compound 3 lyophilized powder 12 mg.
Figure BDA0001966682920000212
Synthesis example 3
Synthesis of chloromethyl aryl substituted vitamin D derivatives
Weighing 125- (OH) VD350mg of the aqueous solution was dissolved in 2.5mL of anhydrous DMSO to prepare a 20mg/mL solution. 2mL of centrifuge tube was added to 1.25mL of 25- (OH) VD3(62.4. mu. mol) of DMSO solution, 31.37mg of 4- (chloromethyl) phenyl isocyanate (187.2. mu. mol) was added in a molar ratio of 3:1, the mixture was stirred at room temperature for 3 hours, a saturated aqueous solution of sodium chloride was added to the reaction mixture, the mixture was extracted with ethyl acetate, and the mixture was extracted with anhydrous Na2SO4Drying, spin-drying, and purification by column chromatography gave 12.5mg of compound 4, 35% yield.
1HNMR(300MHz,CDCl3):0.54(s,3H,Me),0.88(d,3H,Me,J＝5.8Hz),0.76–2.51(severalm,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).
Figure BDA0001966682920000221
Synthesis example 4
Marker for synthesizing chloromethyl aryl substituted vitamin D
Weighing 10mg NH2PEGnBiotin (5kD) (2umol) was dissolved in 0.02M PBS (0.15M NaCl, 25mM EDTA) pH7.2 buffer, 2.27mg (4umol) of Compound 4 was added at a molar ratio of 2:1, and stirred at 37 ℃ for 16 h. Purifying by desalting column, and lyophilizing to obtain compound 5 lyophilized powder 14 mg.
Figure BDA0001966682920000222
Synthesis example 5
Synthesis of chloromethyl heteroaryl substituted vitamin D derivatives
Weighing 125- (OH) VD350mg of the aqueous solution was dissolved in 2.5mL of anhydrous DMSO to prepare a 20mg/mL solution. A4 mL centrifuge tube was charged with 1.25mL of 25- (OH) VD3(62.4. mu. mol) of DMSO solution, 40.92mg of 8- (chloromethyl) -5-isocyanatoquinoline (187.2. mu. mol) was added in a molar ratio of 3:1, the mixture was stirred at room temperature for 3 hours, a saturated aqueous solution of sodium chloride was added to the reaction mixture, the mixture was extracted with ethyl acetate, and the mixture was extracted with ethyl acetateAnhydrous Na2SO4Drying, spin-drying and purification by column chromatography gave 20mg of compound 6 in 52% yield.
1HNMR(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).
Figure BDA0001966682920000231
Synthesis example 6
Marker for synthesizing chloromethyl heteroaryl substituted vitamin D
Weighing 10mg NH2PEGnBiotin (5kD) (2umol) was dissolved in 0.02M PBS (0.15M NaCl, 25mM EDTA) pH7.2 buffer, 2.48mg (4umol) of Compound 6 was added at a molar ratio of 2:1, and stirred at 37 ℃ for 16 h. Purifying by desalting column, and lyophilizing to obtain compound 7 lyophilized powder 13 mg.
Figure BDA0001966682920000241
Synthesis example 7
Synthetic succinate-substituted vitamin D-containing markers
2mg of compound 825-hydroxyvitamin D was weighed into a 2mL centrifuge tube3Hemisuccinate was dissolved in 1mL of anhydrous DMSO, and 1.9mg of EDAC and 2.3mg of NHS were added thereto, followed by stirring at room temperature for 1 hour.
Weighing 10mg NH2PEGnBiotin (5kD) (2umol), dissolved in 0.02MPBS (0.15M NaCl, 25mM EDTA) pH7.2 buffer solution, activated 25-hydroxyvitamin D was added in a molar ratio of 2:13Succinate, stirred at room temperature for 2 h. Purifying by a desalting column to obtain a compound 9.
Figure BDA0001966682920000242
Synthesis example 8
Synthetic BSA conjugates with and aryl substituted vitamin D
A2 mL centrifuge tube was added with 1mL of 10mg/mL BSA (0.02M PBS, pH7.2, 25mM EDTA) buffer, 1M DTT was added to a final concentration of 10mM, vortexed, mixed, and allowed to stand at room temperature for 2 h. Desalting with desalting column to remove excessive DTT. Then, 150uL of compound 2 (20 mg/mL) was added at a molar ratio of 10:1 (Compound 2: BSA). Stir at room temperature for 2h and dialyze the protein into a buffer solution of 0.02MPBS, pH 7.2. Purification by dialysis yielded conjugate 10.
Figure BDA0001966682920000251
A2 mL centrifuge tube was charged with 1mL of 10mg/mL BSA (0.02M PBS, pH7.2, 25mM EDTA) buffer, followed by 150uL of 20mg/mL Compound 4 at a molar ratio of 10:1 (Compound 4: BSA). After stirring at room temperature for 24h, the protein was dialyzed into 0.02M PBS, pH7.2 buffer, and purified by dialysis to give conjugate 11.
Figure BDA0001966682920000252
The name of the experiment: development of kit for quantitative detection of 25-hydroxyvitamin D by chemiluminescence method
Purpose of the experiment: research and development of kit for quantitatively detecting 25-hydroxyvitamin D
Experiment design: detection by competitive chemiluminescence
Reagents and instrumentation:
resisting 25-OH VD3Antibody (Bioventix), biotinylated-25-OH VD3Compound 3 (self-produced), biotinylated-25-OH VD3Compound 5 (Secury), biotinylated-25-OH VD3Compound 7 (Secury), biotinylated-25-OH VD3Compound 9 (authigenic), carboxyl microspheres (JSR), phosphate buffer (0.02M PBS, pH 7.2), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDAC (thermo fisher), Tween-20, 0.1M MES buffer (pH 6.0). LiCA HT (Shanghai Boyang Biotechnology Co., Ltd.), Hitachi high-speed refrigerated centrifuge
The experimental steps are as follows:
coating for resisting 25-OH VD3Preparation of microspheres of antibodies
First, 10mg of carboxyl functionalized microspheres were taken in a 2mL centrifuge tube and washed once with 0.1M MES (pH 6.0) buffer at 4 ℃ at 10000rpm for 15 min.
Secondly, 200uL of 0.1M MES (pH 6.0) buffer solution is added for even ultrasonic dispersion, 8uL of 1mg/mL anti-25-OH vitamin D goat monoclonal antibody is added, then 100uL of 5mg/mL EDAC (0.1M MES) solution is added, and stirring is carried out at room temperature for 4 hours.
And thirdly, adding 50uL of 200mg/mL BSA blocked carboxyl microspheres.
And thirdly, centrifuging and washing the microspheres for three times by using PBS (phosphate buffer solution) containing 0.5% Tween-20, and finally fixing the volume to 10mg/mL by using the PBS.
Detection experiment
Experiment one:
the name of the experiment: different biotinylated aryl-substituted-25-OH VD3Detection of derivatives
Purpose of the experiment: screening for optimal biotinylated aryl substituted 25-OH VD3Derivatives of the same
Experiment design: the same reaction scheme, N-maleimide aryl substituted Bio-25-OH-VD was chosen33 and chloromethyl aryl substituted Bio-25-OH-VD35 Performance comparison
The experimental steps are as follows:
1. diluting the luminescent microspheres to 30 mu g/mL, biotinylating to-25-OH VD3Derivatives 3 and 5 were diluted in a gradient of 5ng/mL, 0.5ng/mL and 0.05ng/mL, respectively.
2. Adding horse serum into a 25-hydroxy vitamin D solution with the concentration of 1mg/mL to prepare calibrators 1-6, and assigning values to the calibrators, wherein the values are respectively 0, 4.07, 8.15, 17.75, 33.13 and 65.12.
3. Adding a sample solution according to a reaction mode, and then sequentially adding the coated luminescent microspheres and the biotinylated 25-OHV3Derivatives, 25uL each.
4. A first stage incubation was performed: incubate at 37 ℃ for 17 min.
5. Add 175ul of universal solution.
6. Performing a second stage incubation: incubate at 37 ℃ for 15 min.
7. And (6) reading.
Figure BDA0001966682920000271
And (3) division of the calibration product:
Cal1/Cal2 1.58 1.77 1.19 1.80 2.17 1.74
Cal2/Cal3 1.37 1.36 1.19 1.10 1.37 1.24
Cal3/Cal4 1.59 1.32 1.10 1.50 1.43 1.25
Cal4/Cal5 1.31 1.18 1.08 1.65 1.41 1.11
Cal5/Cal6 1.97 1.14 0.95 3.01 1.51 1.18
Cal1/Cal6 8.94 4.28 1.59 14.81 8.99 3.52
and (3) data analysis:
from the viewpoint of the distinction between the signal amount and the calibrator, Bio-25-OH-VD33 improving reagent signal amount and discrimination ratio
Bio-25-OH-VD35 high.
The overall discrimination was best at a biotin reagent concentration of 5 ng/mL.
And (4) experimental conclusion:
from the calibrator signal quantity and discrimination, N-maleimidoaryl substituted Bio-25-OH VD33 reagent performance is superior to that of chloromethyl aryl substituted Bio-25-OH VD35。
Experiment two:
the name of the experiment: different biotinylated aryl and heteroaryl substituted-25-OH VD3Detection of derivatives
Purpose of the experiment: heteroaryl substituted 25-OH VD3Derivatives and aryl substituted 25-OH VD3Comparison of the Properties of the markers of the derivatives
Experiment design: same reaction scheme, choosing aryl substituted Bio-25-OH-VD35 and aryl substituted Bio-25-OH-VD37 Performance comparison
The experimental steps are as follows:
1. diluting the luminescent microspheres to 30 mu g/mL, biotinylating to-25-OH VD3Derivatives 5, 7 were diluted in a gradient of 5ng/mL, 0.5ng/mL, 0.05ng/mL, respectively.
2. Adding horse serum into a 25-hydroxy vitamin D solution with the concentration of 1mg/mL to prepare calibrators 1-6, and assigning values to the calibrators, wherein the values are respectively 0, 4.07, 8.15, 17.75, 33.13 and 65.12.
3. Adding a sample solution according to a reaction mode, and then sequentially adding the coated luminescent microspheres and the biotinylated 25-OHV3Derivatives, 25uL each.
4. A first stage incubation was performed: incubate at 37 ℃ for 17 min.
5. Add 175ul of universal solution.
6. Performing a second stage incubation: incubate at 37 ℃ for 15 min.
7. And (6) reading.
Figure BDA0001966682920000281
And (3) division of the calibration product:
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
and (3) data analysis:
from the viewpoint of the discrimination between the signal amount and the calibrator, Bio-25-OH-VD is used at the same biotin reagent concentration35 and Bio-25-OH-VD37 lower end scores were closer, but Bio-25-OH-VD35 overall reagent signal and discrimination was high. The overall discrimination was best at a biotin reagent concentration of 5 ng/mL.
And (4) experimental conclusion:
aryl substituted Bio-25-OH-VD from calibrator signal and discrimination35 reagent Performance superior to heteroaryl substituted Bio-25-OH-VD37。
Experiment three:
the name of the experiment: different biotinylated 25-OH VD3Detection of derivatives
Purpose of the experiment: aryl substituted 25-OH VD3Derivatives and succinic acid substituted 25-OH VD3Comparison of the Properties of the markers of the derivatives
Experiment design: same reaction scheme, choosing aryl substituted Bio-25-OH-VD33 and succinic acid substituted Bio-25-OH-VD39 Performance comparison
The experimental steps are as follows:
1. diluting the luminescent microspheres to 30 mu g/mL, biotinylating to-25-OH VD3Derivatives 3 and 9 were diluted in a gradient of 5ng/mL, 0.5ng/mL and 0.05ng/mL, respectively.
2. Adding horse serum into a 25-hydroxy vitamin D solution with the concentration of 1mg/mL to prepare calibrators 1-6, and assigning values to the calibrators, wherein the values are respectively 0, 4.07, 8.15, 17.75, 33.13 and 65.12.
3. Adding a sample solution according to a reaction mode, and then sequentially adding the coated luminescent microspheres and the biotinylated 25-OHV3Derivatives, 25uL each.
4. A first stage incubation was performed: incubate at 37 ℃ for 17 min.
5. Add 175ul of universal solution.
6. Performing a second stage incubation: incubate at 37 ℃ for 15 min.
7. And (6) reading.
Figure BDA0001966682920000301
And (3) division of the calibration product:
Cal1/Cal2 1.80 2.17 1.75 1.76 2.03 1.56
Cal2/Cal3 1.10 1.37 1.25 1.10 1.31 1.17
Cal3/Cal4 1.50 1.44 1.25 1.46 1.34 1.16
Cal4/Cal5 1.66 1.41 1.11 1.57 1.29 1.07
Cal5/Cal6 3.05 1.52 1.19 2.40 1.32 1.11
Cal1/Cal6 15.12 9.23 3.61 10.64 6.09 2.50
and (3) data analysis:
from the viewpoint of the distinction between the signal amount and the calibrator, Bio-25-OH-VD33 raising reagent signal amount and distinguishability ratio Bio-25-OH-VD3And 9 high.
The overall discrimination was best at a biotin reagent concentration of 5 ng/mL.
And (4) experimental conclusion:
from the calibrator signal quantity and discrimination, N-maleimidoaryl substituted Bio-25-OH VD33 the performance of the reagent is superior to that of succinic acid-substituted Bio-25-OH VD39。
Experiment four:
the name of the experiment: different biotinylated 25-OH VD3Detection of derivatives
Purpose of the experiment: exploration of different biotinylated 25-OH VD3Comparison of the Performance of the derivatives on plate-type chemiluminescence
Experiment design: same reaction mode, detection by plate chemiluminescence
The experimental steps are as follows:
1. avidin 2ug/ml coated, 100 ul/well, 4 ℃ overnight
2. Washing the plate: washing the microporous plate with diluted washing solution for 5 times, adding no less than 400 μ L of washing solution into each hole, soaking for 10 s each time, and drying on clean absorbent paper.
3. CB diluted Biotin-25-OH VD3 compound (1/10000)3, 5, 9, 100 ul/well was added.
4. Washing the plate: washing the microporous plate with diluted washing solution for 5 times, adding no less than 400 μ L of washing solution into each hole, soaking for 10 s each time, and drying on clean absorbent paper.
5. Adding a sample: 50 μ L of sample was added to each well. Adding an antibody: add 100. mu.L of antibody per well
6. And (3) incubation: mix well for 5 seconds with a micro-shaker, seal the plate with a sealing membrane, incubate for 2 hours at 37 ℃.
7. Washing the plate: washing the microporous plate with diluted washing solution for 5 times, adding no less than 400 μ L of washing solution into each hole, soaking for 10 s each time, and drying on clean absorbent paper.
8. Adding an enzyme marker: in addition to the blank control wells, 100. mu.L of enzyme label was added to each well
9. And (3) incubation: mix well for 5 seconds with a micro-shaker, seal the plate with a sealing membrane, incubate for 1 hour at 37 ℃.
10. Washing the plate: washing the microporous plate with diluted washing solution for 5 times, adding no less than 400 μ L of washing solution into each hole, soaking for 10 s each time, and drying on clean absorbent paper.
11. Adding a substrate solution: add 100 μ L of the prepared chemiluminescent substrate working solution into each well, and use 8-channel pipette, mix for 5 seconds with shaking by micro-shaker.
12. And (3) detection: and (3) adding the luminescent substrate solution, standing for 5 minutes at room temperature (20-27 ℃) in a dark place, immediately and sequentially measuring the luminescent value (RLU) of each hole on a microplate luminescence analyzer for 0.1-1.0 second/hole.
And (3) detection results:
Figure BDA0001966682920000311
Figure BDA0001966682920000321
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
and (3) data analysis:
Bio-25OH VD33 and Bio-25-OH VD39 integral discrimination average ratio Bio-25-OH VD of detection sample35 high.
Bio-25-OH VD33 ratio Bio-25-OH VD39 the integral discrimination of the detection sample is slightly higher, and the sensitivity of the detection of the low-end sample is higher.
And (4) experimental conclusion:
from the discrimination of the test samples, N-maleimidoaryl substituted Bio-25-OH VD33 the best performance of the reagent.
It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.

Claims (30)

1. An intermediate compound, the structure of which is shown in formula A,
Figure RE-FDA0002093967630000011
wherein Z is selected from C1-C20Alkyl radical, C2-C20Alkenyl and C2-C20Alkynyl, said alkyl, alkenyl and alkynyl being optionally selected from hydroxy, C1-C10Alkoxy radical, C1-C10One or more substituents of ester group and oxime group;
R1selected from hydrogen, hydroxy, C1-C20Alkyl radical, C2-C20Alkenyl radical, C2-C20Alkynyl, C1-C20Alkoxy radical, C2-C20Alkenyloxy and C2-C20An alkynyloxy group;
R2and R3Are identical to each otherOr different, independently selected from hydrogen and C1-C20An alkyl group;
r' is at least RaSubstituted C6-C30Aryl radical, C5-C30Heteroaryl or C9-C30The aromatic group of the condensed aromatic group is,
Rais- (CH)2)pXaOr- (CH)2)pCOXaWherein X isaIs a reactive group capable of reacting with the labeling compound, the biomacromolecule, the member of the signal producing system, the small organic molecule, and the binding partner or carrier of said small organic molecule, p is an integer from 0 to 10, for example p is 0, 1,2,3,4 or 5.
2. The compound of claim 1, wherein X isaSelected from N-maleimido, halogen, -NCO and-N3The halogen is preferably fluorine, chlorine, bromine or iodine.
3. A compound according to claim 1 or 2, wherein Z is selected from C1-C10Alkyl radical, C2-C10Alkenyl and C2-C10Alkynyl, said alkyl, alkenyl and alkynyl being optionally selected from hydroxy, C1-C10Alkoxy radical, C1-C10Ester groups and oxime groups.
4. A compound according to any one of claims 1 to 3, wherein Z is selected from C1-C10Alkyl radical, C2-C10Alkenyl and C2-C10Alkynyl, said alkyl, alkenyl or alkynyl being optionally selected from hydroxy, C1-C5Alkoxy radical, C1-C5Ester groups and oxime groups.
5. A compound according to any one of claims 1 to 4, wherein Z is C with a branch4-C10Alkyl, C with branched chain4-C10Alkenyl or C with a branch4-C20Alkynyl.
6. The compound of any one of claims 1 to 5, wherein C is branched4-C10Alkyl, C with branched chain4-C10Alkenyl or C with a branch4-C20The terminal carbon atom of the alkynyl group is attached to a hydroxyl group or a protected hydroxyl group, preferably, Z is 4, 4-dimethyl-4-hydroxybutyl.
7. A compound according to any one of claims 1 to 6, wherein R is1Selected from hydrogen, hydroxy, C1-C10Alkyl radical, C2-C10Alkenyl radical, C2-C10Alkynyl and C1-C10An alkoxy group; and/or, R2And R3Independently selected from hydrogen and C1-C10Alkyl, preferably selected from hydrogen and C1-C5An alkyl group.
8. The compound of any one of claims 1-7, wherein the small organic molecule is selected from the group consisting of biotin, fluorescein, rhodamine, chemiluminescent molecules, dinitrophenol, acridinium esters, alkaline phosphatase, and labeled compound molecules, and wherein the binding partner of the small organic molecule is selected from the group consisting of avidin, an antibody to fluorescein, an antibody to rhodamine, an antibody to chemiluminescent molecules, and an antibody to dinitrophenol.
9. The compound of any one of claims 1-8, wherein the member of the signal producing system is selected from the group consisting of a fluorescent compound, a chemiluminescent compound, a sensitizer, an enzyme, and a radiolabel.
10. A compound according to any one of claims 1 to 9, wherein the member of the signal producing system comprises a particle, preferably the particle is selected from the group consisting of a fluorescent particle, a chemiluminescent particle, a sensitizer particle and a magnetic particle.
11. A compound according to any of claims 1 to 10 wherein the biomacromolecule is selected from the group consisting of a protein molecule, a nucleic acid molecule, a polysaccharide molecule and a lipid molecule.
12. A compound according to any one of claims 1 to 11 wherein the binding ligand for the small organic molecule is selected from antibodies to vitamin D and analogues thereof.
13. The compound of any one of claims 1-12, wherein R' is at least one of RaSubstituted C6-C20Aryl radical, C5-C20Heteroaryl or C9-C20A fused aryl group.
14. The compound of any one of claims 1-13, wherein R' is at least one of RaSubstituted C6-C10Aryl radical, C5-C10Heteroaryl or C9-C10Condensed aryl radicals, e.g. at least by RaSubstituted phenyl, at least by RaSubstituted pyridyl, at least by RaSubstituted quinolyl radicals or substituted by at least RaSubstituted isoquinolinyl groups.
15. The compound of any one of claims 1-14, wherein the compound has a structure represented by formula A1 or formula A2,
Figure RE-FDA0002093967630000031
in the formula A1, R2-R5The same or different, are independently selected from hydrogen and C1-C20Alkyl radical, C2-C20Alkenyl radical, C2-C20Alkynyl, C1-C20Alkoxy radical, C6-C20Aryl radical, C5-C20Heteroaryl group, C9-C20Condensed aryl, cyano, halogen, nitro, carboxyl, amino and C1-C10An alkyl-substituted amino group;
in the formula A2, R2-R6The same or different, are independently selected from hydrogen and C1-C20Alkyl radical, C2-C20Alkenyl radical, C2-C20Alkynyl, C1-C20Alkoxy radical, C6-C20Aryl radical, C5-C20Heteroaryl group, C9-C20Condensed aryl, cyano, halogen, nitro, carboxyl, amino and C1-C10Alkyl-substituted amino groups.
16. A compound of claim 15, wherein R in formula A12-R5Independently selected from hydrogen, C1-C10Alkyl radical, C1-C10Alkenyl radical, C1-C10Alkynyl, C1-C10Alkoxy, cyano, halogen, nitro, amino and C1-C5An alkyl-substituted amino group; in the formula A2, R2-R6Independently selected from hydrogen, C1-C10Alkyl radical, C1-C10Alkenyl radical, C1-C10Alkynyl, C1-C10Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups.
17. A compound according to claim 15 or 16, wherein R in formula A12-R5Independently selected from hydrogen, C1-C5Alkyl radical, C1-C5Alkenyl radical, C1-C5Alkynyl, C1-C5Alkoxy, cyano, halogen, nitro, amino and C1-C5An alkyl-substituted amino group; in the formula A2, R2-R6Independently selected from hydrogen, C1-C5Alkyl radical, C1-C5Alkenyl radical, C1-C5Alkynyl, C1-C5Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups.
18. A compound according to any one of claims 1 to 17, selected from the following compounds:
Figure RE-FDA0002093967630000041
Figure RE-FDA0002093967630000051
19. a process for the preparation of a compound according to any one of claims 1 to 18, comprising the steps of: reacting a compound of formula B with R' NCO to produce an intermediate compound of formula A,
Figure RE-FDA0002093967630000052
Figure RE-FDA0002093967630000061
wherein, Z, R1、R2、R3R' is as defined in claims 1-18.
20. A process for the preparation of a compound of formula I, comprising the steps of: reacting an intermediate compound of formula a according to any one of claims 1-18 with a starting material selected from the group consisting of a labeling compound, a biomacromolecule, a member of a signal producing system, a small organic molecule, and a binding ligand or carrier for the small organic molecule to produce a compound of formula I,
Figure RE-FDA0002093967630000062
wherein, Z, R1、R2、R3Is as defined in claims 1-18,
r is at least R1Substituted C6-C30Aryl radical, C5-C30Heteroaryl or C9-C30The aromatic group of the condensed aromatic group is,
R1is- (CH)2)pX or- (CH)2)pCOX, wherein X is selected from a labeling moiety derived from a labeling compound, a biomacromolecule moiety, an N-maleimido group to which the labeling moiety or biomacromolecule moiety is attached, a member of a signal generating system, a small organic molecule, and a binding ligand or carrier for said small organic molecule, and p is an integer from 0 to 10, such as an integer from 1 to 5.
21. The method according to claim 20, wherein the small organic molecule is selected from the group consisting of biotin, fluorescein, rhodamine, a chemiluminescent molecule, dinitrophenol, acridinium ester, alkaline phosphatase, and a labeling compound molecule, and the binding partner of the small organic molecule is selected from the group consisting of avidin, an antibody to fluorescein, an antibody to rhodamine, an antibody to a chemiluminescent molecule, and an antibody to dinitrophenol.
22. The method of claim 20 or 21, wherein the member of the signal producing system is selected from the group consisting of a fluorescent compound, a chemiluminescent compound, a sensitizer, an enzyme, and a radioactive label.
23. A compound according to any one of claims 20 to 22 wherein the member of the signal producing system comprises a particle, preferably the particle is selected from the group consisting of a fluorescent particle, a chemiluminescent particle, a sensitizer particle and a magnetic particle.
24. A compound according to any of claims 21 to 23 wherein the biomacromolecule is selected from the group consisting of a protein molecule, a nucleic acid molecule, a polysaccharide molecule and a lipid molecule.
25. A compound according to any one of claims 20 to 24 wherein the binding ligand for the small organic molecule is selected from antibodies to vitamin D and analogues thereof.
26. The compound of any one of claims 20-25, wherein R is at least represented by R1Substituted C6-C20Aryl radical, C5-C20Heteroaryl or C9-C20Fused aryl, preferably R is at least R1Substituted C6-C10Aryl radical, C5-C10Heteroaryl or C9-C10Condensed aryl radicals, e.g. at least by R1Substituted phenyl, at least by R1Substituted pyridyl, at least by R1Substituted quinolyl radicals or substituted by at least R1Substituted isoquinolinyl groups.
27. The compound of any one of claims 20-26, wherein the compound of formula I has a structure according to formula II or formula III:
Figure RE-FDA0002093967630000071
in the formula II, R2-R5The same or different, are independently selected from hydrogen and C1-C20Alkyl radical, C2-C20Alkenyl radical, C2-C20Alkynyl, C1-C20Alkoxy radical, C6-C20Aryl radical, C5-C20Heteroaryl group, C9-C20Condensed aryl, cyano, halogen, nitro, carboxyl, amino and C1-C10An alkyl-substituted amino group;
in the formula III, R2-R6The same or different, are independently selected from hydrogen and C1-C20Alkyl radical, C2-C20Alkenyl radical, C2-C20Alkynyl, C1-C20Alkoxy radical, C6-C20Aryl, heteroaryl, and heteroaryl,C5-C20Heteroaryl group, C9-C20Condensed aryl, cyano, halogen, nitro, carboxyl, amino and C1-C10Alkyl-substituted amino groups.
28. The method according to any one of claims 20 to 27, wherein R in formula II2-R5Independently selected from hydrogen, C1-C10Alkyl radical, C1-C10Alkenyl radical, C1-C10Alkynyl, C1-C10Alkoxy, cyano, halogen, nitro, amino and C1-C5An alkyl-substituted amino group; in the formula III, R2-R6Independently selected from hydrogen, C1-C10Alkyl radical, C1-C10Alkenyl radical, C1-C10Alkynyl, C1-C10Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups.
29. The method according to any one of claims 20 to 28, wherein in formula II, R is2-R5Independently selected from hydrogen, C1-C5Alkyl radical, C1-C5Alkenyl radical, C1-C5Alkynyl, C1-C5Alkoxy, cyano, halogen, nitro, amino and C1-C5An alkyl-substituted amino group; in the formula III, R2-R6Independently selected from hydrogen, C1-C5Alkyl radical, C1-C5Alkenyl radical, C1-C5Alkynyl, C1-C5Alkoxy, cyano, halogen, nitro, amino and C1-C5Alkyl-substituted amino groups.
30. A compound according to any one of claims 20 to 29, wherein the compound of formula I is selected from the following compounds:
Figure RE-FDA0002093967630000091
Figure RE-FDA0002093967630000101
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