CN111518005B - Intermediate compound and preparation method and application thereof - Google Patents

Intermediate compound and preparation method and application thereof Download PDF

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CN111518005B
CN111518005B CN201910105699.0A CN201910105699A CN111518005B CN 111518005 B CN111518005 B CN 111518005B CN 201910105699 A CN201910105699 A CN 201910105699A CN 111518005 B CN111518005 B CN 111518005B
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CN111518005A (en
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章春奇
张向辉
赵卫国
刘宇卉
李临
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Kemei Boyang Diagnostic Technology Shanghai Co ltd
Chemclin Diagnostics Corp
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Chemclin Diagnostics Corp
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    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/32—Esters of carbamic acids having oxygen atoms of carbamate groups bound to carbon atoms of rings other than six-membered aromatic rings
    • C07C271/38—Esters of carbamic acids having oxygen atoms of carbamate groups bound to carbon atoms of rings other than six-membered aromatic rings with the nitrogen atom of at least one of the carbamate groups bound to a carbon atom of a six-membered aromatic ring
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    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/44—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members
    • C07D207/444—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5
    • C07D207/448—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. maleimide
    • C07D207/452—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. maleimide with hydrocarbon radicals, substituted by hetero atoms, directly attached to the ring nitrogen atom
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    • C07D217/00—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems
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    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
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    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
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    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
    • C07C2601/14—The ring being saturated
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2602/00—Systems containing two condensed rings
    • C07C2602/02—Systems containing two condensed rings the rings having only two atoms in common
    • C07C2602/14—All rings being cycloaliphatic
    • C07C2602/24—All rings being cycloaliphatic the ring system containing nine carbon atoms, e.g. perhydroindane
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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. The compounds of formula I can be used to determine the presence or amount of vitamin D analytes (including vitamin D2 and vitamin D3) and metabolites thereof in a sample containing the same.

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 which has two main forms: vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Vitamin D2 is derived from a nutritional supplementVitamin D3 is obtained from skin exposed to sunlight (ultraviolet radiation) and is mainly obtained from fish, liver oil, egg yolk, etc. in diets. Vitamin 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 kidney 2 D.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) D 2 And 25- (OH) D 3 . 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 C 1 -C 20 Alkyl radical, C 2 -C 20 Alkenyl and C 2 -C 20 Alkynyl, said alkyl, alkenyl and alkynyl being optionally selected from hydroxy, C 1 -C 10 Alkoxy radical, C 1 -C 10 One or more substituents of an ester group and an oxime group;
R 1 selected from hydrogen, hydroxy, C 1 -C 20 Alkyl radical, C 2 -C 20 Alkenyl radical, C 2 -C 20 Alkynyl, C 1 -C 20 Alkoxy radical, C 2 -C 20 Alkenyloxy and C 2 -C 20 An alkynyloxy group;
R 2 and R 3 Same or different, independently selected from hydrogen and C 1 -C 20 An alkyl group;
r' is at least R a Substituted C 6 -C 30 Aryl radical, C 5 -C 30 Heteroaryl or C 9 -C 30 A fused aromatic group,
R a is- (CH) 2 ) p X a Or- (CH) 2 ) p COX a Wherein X is a Is 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, X a Selected from N-maleimido, halogen, -NCO and-N 3 The halogen is preferably fluorine, chlorine, bromine or iodine.
In some embodiments of the foregoing, Z is selected from C 1 -C 10 Alkyl radical, C 2 -C 10 Alkenyl and C 2 -C 10 Alkynyl, said alkyl, alkenyl and alkynyl being optionally selected from hydroxy, C 1 -C 10 Alkoxy radical, C 1 -C 10 Ester groups and oxime groups.
In some embodiments of the foregoing, Z is selected from C 1 -C 10 Alkyl radical, C 2 -C 10 Alkenyl and C 2 -C 10 Alkynyl, said alkyl, alkenyl or alkynyl being optionally selected from hydroxy, C 1 -C 5 Alkoxy radical, C 1 -C 5 Ester group and oxime group.
In some embodiments of the foregoing, Z is C with a branch 4 -C 10 Alkyl, C with branched chain 4 -C 10 Alkenyl or C with a branch 4 -C 20 Alkynyl.
In some embodiments of the foregoing, the C with a branch is 4 -C 10 Alkyl, C with branched chain 4 -C 10 Alkenyl or C with a branch 4 -C 20 The 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, R 1 Selected from hydrogen, hydroxy, C 1 -C 10 Alkyl radical, C 2 -C 10 Alkenyl radical, C 2 -C 10 Alkynyl and C 1 -C 10 An alkoxy group; and/or, R 2 And R 3 Independently selected from hydrogen and C 1 -C 10 Alkyl, preferably selected from hydrogen and C 1 -C 5 An 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 foregoing, 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.
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 embodiments, the binding ligand for the small organic molecule is selected from the group consisting of antibodies to vitamin D and analogs thereof.
In some embodiments of the foregoing, R' is at least R a Substituted C 6 -C 20 Aryl radical, C 5 -C 20 Heteroaryl or C 9 -C 20 A fused aryl group.
In some embodiments of the foregoing, R' is at least R a Substituted C 6 -C 10 Aryl radical, C 5 -C 10 Heteroaryl or C 9 -C 10 Condensed aryl radicals, e.g. at least by R a Substituted phenyl, at least by R a Substituted pyridyl, at least by R a Substituted quinolyl radicals or substituted by at least R a A substituted isoquinolinyl group.
In some embodiments of the foregoing, the compound has a structure represented by formula A1 or formula A2,
Figure BDA0001966682920000041
in the formula A1, R 2 -R 5 The same or different, are independently selected from hydrogen and C 1 -C 20 Alkyl radical, C 2 -C 20 Alkenyl radical, C 2 -C 20 Alkynyl, C 1 -C 20 Alkoxy radical, C 6 -C 20 Aryl radical, C 5 -C 20 Heteroaryl group, C 9 -C 20 Condensed aryl, cyano, halogen, nitro, carboxyl, amino and C 1 -C 10 An alkyl-substituted amino group;
in the formula A2, R 2 -R 6 The same or different, are independently selected from hydrogen and C 1 -C 20 Alkyl radical, C 2 -C 20 Alkenyl radical, C 2 -C 20 Alkynyl, C 1 -C 20 Alkoxy radical, C 6 -C 20 Aryl radical, C 5 -C 20 Heteroaryl group, C 9 -C 20 Condensed aryl, cyano, halogen, nitro, carboxyl, amino and C 1 -C 10 Alkyl-substituted amino groups.
In some embodiments of the foregoing, in formula A1, R 2 -R 5 Independently selected from hydrogen, C 1 -C 10 Alkyl radical, C 1 -C 10 Alkenyl radical, C 1 -C 10 Alkynyl, C 1 -C 10 Alkoxy, cyano, halogen, nitro, amino and C 1 -C 5 An alkyl-substituted amino group; in the formula A2, R 2 -R 6 Independently selected from hydrogen, C 1 -C 10 Alkyl radical, C 1 -C 10 Alkenyl radical, C 1 -C 10 Alkynyl, C 1 -C 10 Alkoxy, cyano, halogen, nitro, amino and C 1 -C 5 Alkyl-substituted amino groups.
In some embodiments of the foregoing, in formula A1, R 2 -R 5 Independently selected from hydrogen, C 1 -C 5 Alkyl radical, C 1 -C 5 Alkenyl radical, C 1 -C 5 Alkynyl, C 1 -C 5 Alkoxy, cyano, halogen, nitro, amino and C 1 -C 5 Alkyl-substituted amino groups.
In some embodiments of the foregoing, in formula A2, R 2 -R 6 Independently selected from hydrogen, C 1 -C 5 Alkyl radical, C 1 -C 5 Alkenyl radical, C 1 -C 5 Alkynyl, C 1 -C 5 Alkoxy, cyano, halogen, nitro, amino and C 1 -C 5 Alkyl-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, R 1 、R 2 、R 3 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, R 1 、R 2 、R 3 The definition of (A) is the same as that above,
r is at least R 1 Substituted C 6 -C 30 Aryl radical, C 5 -C 30 Heteroaryl or C 9 -C 30 The aromatic group of the condensed aromatic group is,
R 1 is- (CH) 2 ) p X or- (CH) 2 ) p COX, wherein X is selected from the group consisting of a labeling moiety, 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, p is an integer from 0 to 10, such as an integer from 1 to 5, wherein said labeling moiety is derived from a labeling compound, and said biomacromolecule is derived from a labeling compoundIn part derived from biological macromolecules.
In some embodiments of the above technical solution, the small organic molecule is selected from biotin, fluorescein, rhodamine, a chemiluminescent molecule, dinitrophenol, acridinium ester, alkaline phosphatase and a labeled compound molecule, and the binding ligand of the small organic 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 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 embodiments, the binding ligand for the small organic molecule is selected from the group consisting of antibodies to vitamin D and analogs thereof.
In some embodiments of the foregoing, R is at least by R 1 Substituted C 6 -C 20 Aryl radical, C 5 -C 20 Heteroaryl or C 9 -C 20 Fused aryl, preferably R is at least R 1 Substituted C 6 -C 10 Aryl radical, C 5 -C 10 Heteroaryl or C 9 -C 10 Condensed aryl radicals, e.g. at least by R 1 Substituted phenyl, at least by R 1 Substituted pyridyl, at least by R 1 Substituted quinolyl radicals or substituted by at least R 1 Substituted 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, R 2 -R 5 Same or different, independently selected from hydrogen, C 1 -C 20 Alkyl radical, C 2 -C 20 Alkenyl radical, C 2 -C 20 Alkynyl, C 1 -C 20 Alkoxy radical, C 6 -C 20 Aryl radical, C 5 -C 20 Heteroaryl group, C 9 -C 20 Condensed aryl, cyano, halogen, nitro, carboxyl, amino and C 1 -C 10 An alkyl-substituted amino group;
in the formula III, R 2 -R 6 The same or different, are independently selected from hydrogen and C 1 -C 20 Alkyl radical, C 2 -C 20 Alkenyl radical, C 2 -C 20 Alkynyl, C 1 -C 20 Alkoxy radical, C 6 -C 20 Aryl radical, C 5 -C 20 Heteroaryl group, C 9 -C 20 Condensed aryl, cyano, halogen, nitro, carboxyl, amino and C 1 -C 10 Alkyl-substituted amino groups.
In some embodiments of the foregoing, formula II, R 2 -R 5 Independently selected from hydrogen, C 1 -C 10 Alkyl radical, C 1 -C 10 Alkenyl radical, C 1 -C 10 Alkynyl, C 1 -C 10 Alkoxy, cyano, halogen, nitro, amino and C 1 -C 5 An alkyl-substituted amino group; in the formula III, R 2 -R 6 Independently selected from hydrogen, C 1 -C 10 Alkyl radical, C 1 -C 10 Alkenyl radical, C 1 -C 10 Alkynyl, C 1 -C 10 Alkoxy, cyano, halogen, nitro, amino and C 1 -C 5 Alkyl-substituted amino groups.
In some embodiments of the foregoing, formula II, R 2 -R 5 Independently selected from hydrogen, C 1 -C 5 Alkyl radical, C 1 -C 5 Alkenyl radical, C 1 -C 5 Alkynyl, C 1 -C 5 Alkoxy, cyano, halogen, nitro, amino and C 1 -C 5 An alkyl-substituted amino group; in the formula III, R 2 -R 6 Independently selected from hydrogen, C 1 -C 5 Alkyl radical, C 1 -C 5 Alkenyl radical, C 1 -C 5 Alkynyl, C 1 -C 5 Alkoxy, cyano, halogen, nitro, amino and C 1 -C 5 Alkyl-substituted amino groups.
Detailed Description
In order that the invention may be readily understood, a more particular description of the invention will be rendered. 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 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) p Where p =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 C 1 -C 6 An alkyl ester.
The term "ester group" refers to a compound or fragment containing a carbon or heteroatom bonded to an oxygen atom that is 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-NH 2 . "alkylamino" includes the group-NH-thereof 2 Is bonded to at least one alkyl group. Examples of alkylamino include benzylamino, methylamino, ethylamino, phenethylamino and the like. "dialkylamino" includes wherein-NH 2 And a group in which the nitrogen atom of (a) 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 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 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) p Where p =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 a substituent, such 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, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an 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 C 1 -C 20 Alkyl radical, C 2 -C 20 Alkenyl and C 2 -C 20 Alkynyl, said alkyl, alkenyl and alkynyl being optionally selected from hydroxy, C 1 -C 10 Alkoxy radical, C 1 -C 10 One or more substituents of ester group and oxime group;
R 1 selected from hydrogen, hydroxy, C 1 -C 20 Alkyl radical, C 2 -C 20 Alkenyl radical, C 2 -C 20 Alkynyl, C 1 -C 20 Alkoxy radical, C 2 -C 20 Alkenyloxy and C 2 -C 20 An alkynyloxy group;
R 2 and R 3 Same or different, independently selected from hydrogen and C 1 -C 20 An alkyl group;
r' is at least R a Substituted C 6 -C 30 Aryl radical, C 5 -C 30 Heteroaryl or C 9 -C 30 A fused aromatic group,
R a is- (CH) 2 ) p X a Or- (CH) 2 ) p COX a Wherein X is a Is 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, X a Selected from N-maleimido, halogen, -NCO and-N 3 The halogen is preferably fluorine, chlorine, bromine or iodine.
In some embodiments of the foregoing, Z is selected from C 1 -C 10 Alkyl radical, C 2 -C 10 Alkenyl and C 2 -C 10 Alkynyl, said alkyl, alkenyl and alkynyl being optionally selected from hydroxy, C 1 -C 10 Alkoxy radical, C 1 -C 10 Ester groups and oxime groups.
In some embodiments of the foregoing, Z is selected from C 1 -C 10 Alkyl radical, C 2 -C 10 Alkenyl and C 2 -C 10 Alkynyl, said alkyl, alkenyl or alkynyl being optionally selected from hydroxy, C 1 -C 5 Alkoxy radical, C 1 -C 5 Ester groups and oxime groups.
In some embodiments of the foregoing, Z is C with a branch 4 -C 10 Alkyl, C with branched chain 4 -C 10 Alkenyl or C with a branch 4 -C 20 Alkynyl.
In some embodiments of the foregoing, the C with a branch is 4 -C 10 Alkyl, C with branched chain 4 -C 10 Alkenyl or C with a branch 4 -C 20 The terminal carbon atom of the alkynyl group is attached to a hydroxyl or protected hydroxyl group, preferably, Z is 4,4-dimethyl-4-hydroxybutyl.
In some embodiments of the foregoing, R 1 Selected from hydrogen, hydroxy, C 1 -C 10 Alkyl radical, C 2 -C 10 Alkenyl radical, C 2 -C 10 Alkynyl and C 1 -C 10 An alkoxy group; and/or, R 2 And R 3 Independently selected from hydrogen and C 1 -C 10 Alkyl, preferably selected from hydrogen and C 1 -C 5 An 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 R a Substituted C 6 -C 20 Aryl radical, C 5 -C 20 Heteroaryl or C 9 -C 20 A fused aryl group.
In the technical schemeIn some embodiments, R' is at least R a Substituted C 6 -C 10 Aryl radical, C 5 -C 10 Heteroaryl or C 9 -C 10 Condensed aryl radicals, e.g. at least by R a Substituted phenyl, at least by R a Substituted pyridyl, at least by R a Substituted quinolyl radicals or substituted by at least R a Substituted 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, R 2 -R 5 The same or different, are independently selected from hydrogen and C 1 -C 20 Alkyl radical, C 2 -C 20 Alkenyl radical, C 2 -C 20 Alkynyl, C 1 -C 20 Alkoxy radical, C 6 -C 20 Aryl radical, C 5 -C 20 Heteroaryl, C 9 -C 20 Condensed aryl, cyano, halogen, nitro, carboxyl, amino and C 1 -C 10 An alkyl-substituted amino group;
in the formula A2, R 2 -R 6 The same or different, are independently selected from hydrogen and C 1 -C 20 Alkyl radical, C 2 -C 20 Alkenyl radical, C 2 -C 20 Alkynyl, C 1 -C 20 Alkoxy radical, C 6 -C 20 Aryl radical, C 5 -C 20 Heteroaryl group, C 9 -C 20 Condensed aryl, cyano, halogen, nitro, carboxyl, amino and C 1 -C 10 Alkyl-substituted amino groups.
In some embodiments of the foregoing, in formula A1, R 2 -R 5 Independently selected from hydrogen, C 1 -C 10 Alkyl radical, C 1 -C 10 Alkenyl radical, C 1 -C 10 Alkynyl, C 1 -C 10 Alkoxy, cyano, halogen, nitro, amino and C 1 -C 5 An alkyl-substituted amino group; in the formula A2, R 2 -R 6 Independently selected from hydrogen, C 1 -C 10 Alkyl radical, C 1 -C 10 Alkenyl radical, C 1 -C 10 Alkynyl, C 1 -C 10 Alkoxy, cyano, halogen, nitro, amino and C 1 -C 5 Alkyl-substituted amino groups.
In some embodiments of the foregoing, in formula A1, R 2 -R 5 Independently selected from hydrogen, C 1 -C 5 Alkyl radical, C 1 -C 5 Alkenyl radical, C 1 -C 5 Alkynyl, C 1 -C 5 Alkoxy, cyano, halogen, nitro, amino and C 1 -C 5 Alkyl-substituted amino groups.
In some embodiments of the foregoing, in formula A2, R 2 -R 6 Independently selected from hydrogen, C 1 -C 5 Alkyl radical, C 1 -C 5 Alkenyl radical, C 1 -C 5 Alkynyl, C 1 -C 5 Alkoxy, cyano, halogen, nitro, amino and C 1 -C 5 Alkyl-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, R 1 、R 2 、R 3 And 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
of these, Z, R 1 、R 2 、R 3 The definition of (A) is the same as that above,
r is at least R 1 Substituted C 6 -C 30 Aryl radical, C 5 -C 30 Heteroaryl or C 9 -C 30 The aromatic group of the condensed aromatic group is,
R 1 is- (CH) 2 ) p X or- (CH) 2 ) p COX, 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 technical solution, the small organic molecule is selected from biotin, fluorescein, rhodamine, a chemiluminescent molecule, dinitrophenol, acridinium ester, alkaline phosphatase and a labeled compound molecule, and the binding ligand of the small organic 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 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, R 1 Is- (CH) 2 ) p X, 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, R 1 Is- (CH) 2 ) p COX, e.g., -CH 2 COX. 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 embodiments, the binding ligand for the small organic molecule is selected from the group consisting of antibodies to vitamin D and analogs thereof.
In some embodiments of the foregoing, R is at least R 1 Substituted C 6 -C 20 Aryl radical, C 5 -C 20 Heteroaryl or C 9 -C 20 Fused aryl, preferably R is at least R 1 Substituted C 6 -C 10 Aryl radical, C 5 -C 10 Heteroaryl or C 9 -C 10 Condensed aryl radicals, e.g. at least by R 1 Substituted phenyl, at least by R 1 Substituted pyridyl, at least by R 1 Substituted quinolyl radicals or substituted by at least R 1 Substituted 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, R 2 -R 5 The same or different, are independently selected from hydrogen and C 1 -C 20 Alkyl radical, C 2 -C 20 Alkenyl radical, C 2 -C 20 Alkynyl, C 1 -C 20 Alkoxy radical, C 6 -C 20 Aryl radical, C 5 -C 20 Heteroaryl, C 9 -C 20 Condensed aryl, cyano, halogen, nitro, carboxyl, amino and C 1 -C 10 An alkyl-substituted amino group;
in the formula III, R 2 -R 6 The same or different, are independently selected from hydrogen and C 1 -C 20 Alkyl radical, C 2 -C 20 Alkenyl radical, C 2 -C 20 Alkynyl, C 1 -C 20 Alkoxy radical, C 6 -C 20 Aryl radical, C 5 -C 20 Heteroaryl group, C 9 -C 20 Condensed aryl, cyano, halogen, nitro, carboxyl, amino and C 1 -C 10 Alkyl-substituted amino groups.
In some embodiments of the foregoing, formula II wherein R is 2 -R 5 Independently selected from hydrogen, C 1 -C 10 Alkyl radical, C 1 -C 10 Alkenyl radical, C 1 -C 10 Alkynyl, C 1 -C 10 Alkoxy, cyano, halogen, nitro, amino and C 1 -C 5 An alkyl-substituted amino group; in the formula III, R 2 -R 6 Independently selected from hydrogen, C 1 -C 10 Alkyl radical, C 1 -C 10 Alkenyl radical, C 1 -C 10 Alkynyl, C 1 -C 10 Alkoxy, cyano, halogen, nitro, amino and C 1 -C 5 Alkyl-substituted amino groups.
In some embodiments of the foregoing, formula II wherein R is 2 -R 5 Independently selected from hydrogen, C 1 -C 5 Alkyl, aryl, heteroaryl, and heteroaryl,C 1 -C 5 Alkenyl radical, C 1 -C 5 Alkynyl, C 1 -C 5 Alkoxy, cyano, halogen, nitro, amino and C 1 -C 5 An alkyl-substituted amino group; in the formula III, R 2 -R 6 Independently selected from hydrogen, C 1 -C 5 Alkyl radical, C 1 -C 5 Alkenyl radical, C 1 -C 5 Alkynyl, C 1 -C 5 Alkoxy, cyano, halogen, nitro, amino and C 1 -C 5 Alkyl-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) VD 3 50mg 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) VD 3 (62.4 umol), at a molar ratio of 1.2 (PMPI: 25- (OH) VD 3 ) 16.04mg of PMPI (p-maleimidophenyl isocyanate) (74.88 umol) 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 Na 2 SO 4 Drying, spin-drying, and purification by thin layer chromatography gave 15mg of compound 2 in 39% yield. 1 HNMR(300MHz,CDCl 3 ):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,H 3 ),5.06(dd,2H,CH 2 ),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 (5 kD) (2 umol) was weighed out and dissolved in 0.02M PBS (0.15M NaCl,25mM EDTA) pH7.2 buffer solution, 2.4mg (4 umol) of Compound 2 was added in a molar ratio of 2:1 and stirred at room temperature for 2h. Purifying by desalting column, and lyophilizing to obtain compound 3 lyophilized powder 12mg.
Figure BDA0001966682920000212
Synthesis example 3
Synthesis of chloromethyl aryl substituted vitamin D derivatives
Weighing compound 125- (OH) VD 3 50mg of the aqueous solution was dissolved in 2.5mL of anhydrous DMSO to prepare a 20mg/mL solution. A2 mL centrifuge tube was charged with 1.25mL of 25- (OH) VD 3 (62.4 umol) DMSO solution, according to the molar ratio of 3:1 added 31.37mg 4- (chloromethyl) phenyl isocyanate (187.2 umol), stirring at room temperature for 3h, to the reaction solution added saturated sodium chloride aqueous solution, using ethyl acetate extraction, anhydrous Na 2 SO 4 Drying, spin-drying, and purification by column chromatography gave 12.5mg of compound 4, 35% yield.
1 HNMR(300MHz,CDCl 3 ):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,CH 2 ),4.74–4.97(m,1H,H 3 ),5.03(dd,2H,CH 2 ),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 NH 2 PEGnBiotin (5 kD) (2 umol) was dissolved in 0.02M PBS (0.15M NaCl,25mM EDTA) pH7.2 buffer solution, 2.27mg (4 umol) of compound 4 was added in a molar ratio of 2:1, and stirred at 37 ℃ for 16 hours. Purifying by desalting column, and lyophilizing to obtain compound 5 lyophilized powder 14mg.
Figure BDA0001966682920000222
Synthesis example 5
Synthesis of chloromethyl heteroaryl substituted vitamin D derivatives
Weighing compound 125- (OH) VD 3 50mg, 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) VD 3 (62.4 umol) DMSO solution, 40.92mg of 8- (chloromethyl) -5-isocyanatoquinoline (187.2 umol) was added to 3:1 in terms of molar ratio, 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 Na 2 SO 4 Drying, spin-drying and purification by column chromatography gave 20mg of compound 6 in 52% yield.
1 HNMR(300MHz,CDCl 3 ):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,CH 2 ),4.70–4.94(m,1H,H 3 ),5.05(dd,2H,CH 2 ),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 NH 2 PEGnBiotin (5 kD) (2 umol) was dissolved in 0.02M PBS (0.15M NaCl,25mM EDTA) pH7.2 buffer solution, 2.48mg (4 umol) of Compound 6 was added in a molar ratio of 2:1 and stirred at 37 ℃ for 16 hours. Purifying by desalting column, and lyophilizing to obtain compound 7 lyophilized powder 13mg.
Figure BDA0001966682920000241
Synthesis example 7
Synthesis of succinate-substituted vitamin D-containing labels
2mg of compound 825-hydroxyvitamin D was weighed into a 2mL centrifuge tube 3 Hemisuccinate was dissolved in 1mL of anhydrous DMSO, and 1.9mg of EDAC and 2.3mg of NHS were added and stirred at room temperature for 1 hour.
Weighing 10mg of NH 2 PEGnBiotin (5 kD) (2 umol), dissolved in 0.02MPBS (0.15M NaCl,25mM EDTA) pH7.2 buffer solution, according to the molar ratio of 2:1 added activated 25-hydroxy vitamin D 3 Succinate, stirred at room temperature for 2h. 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 to 10mg/mL BSA (0.02M PBS, pH7.2, 25mM EDTA) buffer, 1M DTT was added to a final concentration of 10mM, vortexed, and allowed to stand at room temperature for 2 hours. Desalting with desalting column to remove excessive DTT. Then, compound 2 was added at a molar ratio of 10. Stir at room temperature for 2h and dialyze the protein into 0.02m pbs buffer, ph 7.2. Purification by dialysis yielded conjugate 10.
Figure BDA0001966682920000251
To a 2mL centrifuge tube was added 1mL of 10mg/mL BSA (0.02M PBS, pH7.2, 25mM EDTA) buffer, followed by 150uL of 20mg/mL of Compound 4 at a molar ratio of 10 (Compound 4. After stirring at room temperature for 24h, the protein was dialyzed into 0.02M PBS, pH7.2 buffer solution 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:
anti-25-OH VD 3 Antibody (Bioventix), biotinylated-25-OH VD 3 Compound 3 (self-produced), biotinylated-25-OH VD 3 Compound 5 (Secury), biotinylated-25-OH VD 3 Compound 7 (Secury), biotinylated-25-OH VD 3 Compound 9 (Secury), 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 VD 3 Preparation of microspheres of antibodies
In the first step, 10mg of carboxyl-functionalized microspheres were washed once in 2mL centrifuge tubes with 0.1M MES (pH 6.0) buffer, 10000rpm at 4 ℃ and 15min.
Secondly, 200uL 0.1M MES (pH 6.0) buffer solution is added for even ultrasonic dispersion, then, 8uL 1mg/mL anti-25-OH vitamin D sheepkin monoclonal antibody is added, then, 100uL 5mg/mL EDAC (0.1M MES) solution is added, and stirring is carried out for 4 hours at room temperature.
And step three, adding 50uL 200mg/mL BSA to block the carboxyl microspheres.
Third, the microspheres were washed three times by centrifugation with PBS buffer containing 0.5% Tween-20, and finally made to volume of 10mg/mL with PBS buffer.
Detection experiment
Experiment one:
the name of the experiment: different biotinylated aryl-substituted-25-OH VD 3 Detection of derivatives
Purpose of the experiment: screening for optimal biotinylated aryl substituted 25-OH VD 3 Derivatives of the same
Experiment design: the same reaction scheme, N-maleimide aryl substituted Bio-25-OH-VD was chosen 3 3 and chloromethyl aryl substituted Bio-25-OH-VD 3 5 Performance comparison
The experimental steps are as follows:
1. diluting the luminescent microspheres to 30 mu g/mL, biotinylating to-25-OH VD 3 The derivative 3,5 was diluted in a gradient of 5ng/mL, 0.5ng/mL, 0.05ng/mL, respectively.
2. A25-hydroxy vitamin D solution with the concentration of 1mg/mL is added into horse serum to prepare calibrators 1-6, and the calibrators are assigned with values of 0,4.07,8.15, 17.75, 33.13 and 65.12 respectively.
3. Adding a sample solution according to a reaction mode, and then sequentially adding the coated luminescent microspheres and the biotinylated 25-OH VD 3 Derivatives, 25uL each.
4. The first stage incubation was performed: incubate at 37 ℃ for 17min.
5. Add 175ul of universal solution.
6. Performing a second stage incubation: incubate at 37 ℃ for 15min.
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-VD 3 3 improving reagent signal amount and discrimination ratio
Bio-25-OH-VD 3 5 high.
The overall discrimination was best at a biotin reagent concentration of 5ng/mL.
And (4) experimental conclusion:
N-Maleimidoaryl substituted Bio-25-OH VD from calibrator signal quality and discrimination 3 3 reagent performance is superior to chloromethyl aryl substituted Bio-25-OH VD 3 5。
Experiment two:
the name of the experiment: different biotinylated aryl and heteroaryl substituted-25-OH VD 3 Detection of derivatives
Purpose of the experiment: heteroaryl substituted 25-OH VD 3 Derivatives and aryl substituted 25-OH VD 3 Comparison of the Properties of the markers of the derivatives
Experiment design: the same reaction scheme, choosing aryl substituted Bio-25-OH-VD 3 5 and aryl substituted Bio-25-OH-VD 3 7 Performance comparison
The experimental steps are as follows:
1. diluting the luminescent microspheres to 30 mu g/mL, biotinylating to-25-OH VD 3 The derivative 5,7 was diluted in a gradient of 5ng/mL, 0.5ng/mL, 0.05ng/mL, respectively.
2. A solution of 25-hydroxyvitamin D with the concentration of 1mg/mL is added into horse serum to prepare calibrators 1-6, and values are assigned, 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-OH VD 3 Derivatives, 25uL each.
4. A first stage incubation was performed: incubate at 37 ℃ for 17min.
5. Add 175ul of universal solution.
6. Performing a second stage incubation: incubate at 37 ℃ for 15min.
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 concentration 3 5 and Bio-25-OH-VD 3 7 lower end scores were closer, but Bio-25-OH-VD 3 5 overall reagent signal and discrimination was high. The overall discrimination was best at a biotin reagent concentration of 5ng/mL.
And (4) experimental conclusion:
aryl substituted Bio-25-OH-VD from calibrator signal and discrimination 3 5 reagent Performance superior to heteroaryl substituted Bio-25-OH-VD 3 7。
Experiment three:
the name of the experiment: different biotinylated 25-OH VD 3 Detection of derivatives
Purpose of the experiment: aryl substituted 25-OH VD 3 Derivatives and succinic acid substituted 25-OH VD 3 Comparison of the Properties of the markers of the derivatives
Experiment design: the same reaction scheme, choosing aryl substituted Bio-25-OH-VD 3 3 and succinic acid substituted Bio-25-OH-VD 3 9 Performance comparison
The experimental steps are as follows:
1. diluting the luminescent microspheres to 30 mu g/mL, biotinylating to-25-OH VD 3 Derivative 3,9 was diluted in 5ng/mL, 0.5ng/mL, 0.05ng/mL gradient, respectively.
2. A25-hydroxy vitamin D solution with the concentration of 1mg/mL is added into horse serum to prepare calibrators 1-6, and the calibrators are assigned with values of 0,4.07,8.15, 17.75, 33.13 and 65.12 respectively.
3. Adding a sample solution according to a reaction mode, and then sequentially adding the coated luminescent microspheres and the biotinylated 25-OH VD 3 Derivatives, 25uL each.
4. A first stage incubation was performed: incubate at 37 ℃ for 17min.
5. Add 175ul of universal solution.
6. Performing a second stage incubation: incubate at 37 ℃ for 15min.
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-VD 3 3 raising reagent signal amount and distinguishability ratio Bio-25-OH-VD 3 And 9 high.
The overall discrimination was best at a biotin reagent concentration of 5ng/mL.
The experimental conclusion is that:
from the calibrator signal quantity and discrimination, N-maleimidoaryl substituted Bio-25-OH VD 3 3 the performance of the reagent is superior to that of succinic acid-substituted Bio-25-OH VD 3 9。
Experiment four:
the name of the experiment: different biotinylated 25-OH VD 3 Detection of derivatives
Purpose of the experiment: exploration of different biotinylated 25-OH VD 3 Comparison 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 liquid for 5 times, adding not less than 400 μ L of washing liquid 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 liquid for 5 times, adding not less than 400 μ L of washing liquid 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: adding luminescent substrate liquid, standing and reacting for 5 minutes in a dark place at room temperature (20-27 ℃), and 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 VD 3 3 and Bio-25-OH VD 3 9 integral discrimination average ratio Bio-25-OH VD of detection sample 3 And 5, high.
Bio-25-OH VD 3 3 ratio Bio-25-OH VD 3 9 the integral discrimination of the detection sample is slightly higher, and the sensitivity of the detection of the low-end sample is higher.
The experimental conclusion is that:
from the discrimination of the test samples, N-maleimidoaryl substituted Bio-25-OH VD 3 3 the reagent has the best performance.
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 (2)

1. An intermediate compound selected from the group consisting of:
Figure 473293DEST_PATH_IMAGE001
Figure 796959DEST_PATH_IMAGE002
Figure 311117DEST_PATH_IMAGE003
Figure 503063DEST_PATH_IMAGE004
。
2. a process for the preparation of a compound according to claim 1, comprising the steps of: reacting a compound of formula B with R' NCO to produce an intermediate compound of formula A,
Figure 51856DEST_PATH_IMAGE005
wherein Z is
Figure 795690DEST_PATH_IMAGE006
，R 1 、R 2 、R 3 Are all hydrogen and are in the form of hydrogen,
in the formula A, R' is selected from
Figure 797144DEST_PATH_IMAGE007
、
Figure 792782DEST_PATH_IMAGE008
、
Figure 196082DEST_PATH_IMAGE009
3. A method for preparing a compound represented by formula 3, formula 5, formula 7, formula 10, or formula 11, comprising the steps of:
reacting the intermediate compound of claim 1 with a starting material selected from the group consisting of mercaptopolyethyleneglycol biotin, aminopolyethyleneglycol biotin, 8- (chloromethyl) -5-isocyanatoquinoline, mercaptobovine serum albumin, and amino bovine serum albumin, to produce a compound of formula 3, formula 5, formula 7, formula 10, or formula 11, wherein the molecular weight of the mercaptopolyethyleneglycol biotin and the aminopolyethyleneglycol biotin is 5000 daltons,
Figure 861549DEST_PATH_IMAGE010
Figure 350300DEST_PATH_IMAGE011
Figure 884049DEST_PATH_IMAGE012
。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997024127A1 (en) * 1995-12-29 1997-07-10 A And D Assay, Incorporated Labeled vitamin d compounds and the use thereof
WO2014121715A1 (en) * 2013-02-05 2014-08-14 北京九强生物技术股份有限公司 25-hydroxyl vitamin d detection kit and preparation method therefor
CN105636595A (en) * 2013-08-07 2016-06-01 欧根泰克诊断有限公司 25-oh vitamin d derivatives for determination of vitamin d metabolites

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WO1999067211A1 (en) * 1998-06-25 1999-12-29 Immundiagnostik Gesellschaft Für Produktion Und Vertrieb Von Labordiagnostika Mbh FUNCTIONAL VITAMIN D DERIVATIVES AND A METHOD FOR DETERMINING 25-HYDROXY-VITAMIN D AND 1α,25-DIHYDROXY-VITAMIN D
KR101838328B1 (en) * 2010-12-28 2018-03-13 퓨처 디아그노스틱스 비.브이. Release reagent for vitamin d
CN107652302B (en) * 2016-07-25 2020-09-15 深圳迈瑞生物医疗电子股份有限公司 Compounds, conjugates, kits and uses thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997024127A1 (en) * 1995-12-29 1997-07-10 A And D Assay, Incorporated Labeled vitamin d compounds and the use thereof
WO2014121715A1 (en) * 2013-02-05 2014-08-14 北京九强生物技术股份有限公司 25-hydroxyl vitamin d detection kit and preparation method therefor
CN105636595A (en) * 2013-08-07 2016-06-01 欧根泰克诊断有限公司 25-oh vitamin d derivatives for determination of vitamin d metabolites

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