Steroid derivative and preparation method thereof, and complete antigen derivative and preparation method thereof

Abstract

The invention relates to the technical field of haptens, in particular to a steroid derivative and a preparation method thereof, and a complete antigen derivative and a preparation method thereof. The steroid derivative comprises a steroid substance, a coupling group capable of carrying out a coupling reaction, a zwitterionic group and a linking group, the steroid substance and the zwitterionic group are bonded to the two sides of the linking group respectively, and the zwitterionic group is linked with the coupling group. According to the steroid derivative, isomeric impurities are reduced, so that the antigen homogeneity is better, the performance is more stable, meanwhile, the steroid derivative is good in hydrophilicity, precipitates are not prone to being generated in the subsequent coupling process, and the detection sensitivity and accuracy are improved.

Description

[0001] The present invention claims the priority of Chinese patent application No.: 202311477548.0, entitled: Steroid derivatives and methods for their preparation and complete antigen derivatives and methods for their preparation. The contents described in the description and claims of the priority document are fully introduced into the description of the present invention and are regarded as part of the original description of the present invention. Technical Field

[0002] The present invention relates to the technical field of haptens, in particular to steroid derivatives and preparation methods thereof and complete antigen derivatives and preparation methods thereof. Background Art

[0003] A hapten refers to an antigen that can bind to a corresponding antibody to produce an antigen-antibody reaction, but cannot stimulate the human or animal body to produce antibodies alone. It is only immunoreactive and not immunogenic. Haptens have an important influence on the body's functions. For example, hapten steroid hormones have a significant effect on the regulation of human metabolism and immune regulation. The steroid content in the human body reflects the health status, and the content of steroid hormones in the human body is usually determined by immunoassay. Immunoassay requires the development of antibodies against steroids, and steroids cannot induce an immune response to produce corresponding antibodies, so it is necessary to conjugate steroids with carrier proteins to form complete antigens. Furthermore, complete antigens containing steroids can also be coupled with signal substances to form labeled antigens, which are used to detect steroids as analytes in samples in competitive methods.

[0004] However, existing steroid derivatives on the market have poor hydrophilicity and are prone to precipitation when subsequently conjugated with carrier proteins, which in turn affects the performance of the complete antigen / labeled antigen.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The object of the present invention is to provide a steroid derivative and a preparation method thereof and a complete antigen derivative and a preparation method thereof. The steroid derivative provided in the embodiment of the present invention reduces isomeric impurities, makes the antigen homogeneity better, and has more stable performance. At the same time, the steroid derivative has good hydrophilicity and is not easy to produce precipitation in the subsequent coupling process, thereby improving the sensitivity and accuracy of detection.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides a steroid derivative, the structure of which includes a steroid substance and a zwitterionic structure, wherein the steroid substance and the zwitterionic structure are bonded via a bonding bond;

[0009] The zwitterionic structure includes a coupling group capable of undergoing coupling reaction, a zwitterionic group and a connecting group, the two sides of the connecting group are bonded to the steroid substance and the zwitterionic group respectively, and the zwitterionic group is linked to the coupling group.

[0010] In an optional embodiment, the steroid substance includes steroid substances, preferably cortisol, aldosterone, progesterone and testosterone.

[0011] In an optional embodiment, the cation in the zwitterionic group is a quaternary ammonium cation, and the anion is a sulfonate anion;

[0012] Preferably, the structural formula of the zwitterionic group is selected from any one of the groups shown in the following structural formulas:

[0013] Wherein, R1 represents a C1-C5 alkyl group, n represents 1-5, and A represents a C2-C6 carbocyclic ring;

[0014] Preferably, the structural formula of the zwitterionic group is selected from any one of the groups shown in the following structural formulas:

[0015]

[0016] Wherein, R1 represents a C1-C5 alkyl group, and n represents 1-5.

[0017] In an alternative embodiment, the linking group comprises a functional group of 2 to 50 atoms containing at least one of carbon, oxygen, sulfur, nitrogen and phosphorus;

[0018] Preferably, the linking group comprises any one of an amino acid residue, a nitro group, a nitroso group, a phosphate monoester bond, a phosphate diester bond, a phosphonate monoester bond, a phosphonate diester bond, an alkylamine, an amidine, a thioamide, an ether, a urea, a thiourea, a guanidine, an azo, a thioether, a carboxylate, a sulfonate, a phosphate, an amide, a thioester and an oxime group;

[0019] Preferably, the linking group is selected from any one of the groups represented by the following structural formulas:

[0020]

[0021] Where n represents 1-5.

[0022] In an alternative embodiment, the coupling group comprises a carboxylate;

[0023] Preferably, the coupling group is selected from any one of the groups described in the following structural formulas:

[0024] Here, n represents 1-5.

[0025] In an optional embodiment, the steroid derivative is selected from any one of the compounds represented by the following structural formulas:

[0026]

[0027] Wherein, R1 represents a C1-C5 alkyl group, n represents 1-5, and m represents 1-3;

[0028] Preferably, the steroid derivative is selected from any one of the compounds represented by the following structural formulas:

[0029]

[0030] In a second aspect, the present invention provides a method for preparing a steroid derivative as described in the aforementioned embodiment, comprising: reacting a steroid substance with a raw material containing a linking group to form an intermediate A;

[0031] Next, the intermediate A is reacted with a raw material containing a cation in a zwitterionic group to form an intermediate B;

[0032] Then, the intermediate B is reacted with a raw material containing a coupling group to form an intermediate C;

[0033] Then, the anion in the zwitterionic group is grafted onto the intermediate C.

[0034] In a third aspect, the present invention provides a complete antigen derivative, which comprises a substance to be coupled and the steroid derivative described in any one of the aforementioned embodiments; the substance to be coupled and the steroid derivative are coupled via the coupling group of the steroid derivative.

[0035] In an optional embodiment, the object to be coupled is selected from a carrier protein, a polymer carrier or a signal substance.

[0036] In a fourth aspect, the present invention provides a method for preparing the complete antigen derivative described in the aforementioned embodiment, comprising: activating the steroid derivative described in any one of the aforementioned embodiments, and then performing a coupling reaction with a substance to be coupled.

[0037] The present invention has the following beneficial effects: the present invention introduces zwitterionic groups as part of the connecting arm in the steroid derivatives to ensure a certain length of carbon chain, and at the same time, improves the hydrophilicity of the steroid derivatives, which is not easy to produce precipitation in the subsequent coupling process, thereby improving the sensitivity and accuracy of the detection. At the same time, structural impurities are eliminated, so that the antigen homogeneity is better and the performance is more stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0039] Figure 1 The linear difference and nonspecific adsorption difference result diagram of the complete antigen derivative provided in Detection Example 2 of the present invention;

[0040] Figure 2 A schematic diagram of a precipitation in which the coupling ratio of BSA as a carrier protein is 1:120 provided in Detection Example 1 of the present invention. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0042] In a first aspect, the present invention provides a steroid derivative, the structure of which includes a steroid substance and a zwitterionic structure, wherein the steroid substance and the zwitterionic structure are bonded via a bonding bond; wherein the zwitterionic structure includes a coupling group capable of undergoing a coupling reaction, a zwitterionic group and a connecting group, the two sides of the connecting group are bonded to the steroid substance and the zwitterionic group respectively, and the zwitterionic group is linked to the coupling group.

[0043] Specifically, the linking group may include about 2 to about 50 atoms or 4 to about 30 atoms (excluding hydrogen), and may include a chain of 2 to about 30 atoms, or 3 to about 20 atoms, each of which is independently selected from the group generally consisting of carbon, oxygen, sulfur, nitrogen and phosphorus. In some instances, part or all of the linking group may be a part of a molecule connected, such as, but not limited to, for example, an amino acid residue on a poly (amino acid). The number of heteroatoms in the linking group may be 0 to about 20, or 1 to about 15, or about 2 to about 10. The linking group may be aliphatic or aromatic. When heteroatoms are present, oxygen is generally present as an oxo or oxy group bonded to carbon, sulfur, nitrogen or phosphorus, and nitrogen is generally present as a nitro, nitroso or amino group generally bonded to carbon, oxygen, sulfur or phosphorus; sulfur is similar to oxygen; and phosphorus is generally bonded to carbon, sulfur, oxygen or nitrogen as a phosphonic acid mono- or diester and a phosphoric acid mono- or diester. Common functional groups that form a covalent bond between the linker and the molecule to be conjugated are alkylamines, amidines, thioamides, ethers, ureas, thioureas, guanidines, azos, thioethers and carboxylates, sulfonates and phosphates, amides and thioesters.

[0044] In most cases, the linking group further has a linking functional group (a functional group for reacting with a group), including non-oxycarbonyl groups of nitrogen and sulfur analogs, phosphate groups, amino groups, alkylating agents such as halo or tosylalkyl, oxy (hydroxyl or sulfur analogs, sulfhydryl) oxycarbonyl (e.g., aldehyde or ketone), or active olefins such as vinyl sulfone or α-, β-unsaturated esters, these functional groups can be connected to amine groups, carboxyl groups, active olefins, alkylating agents, such as bromoacetyl. When connecting amines and carboxylic acids or their nitrogen derivatives or phosphoric acid, amides, amidines and phosphoramides can be formed. When connecting thiols and activated olefins, thioethers are formed. When connecting thiols and alkylating agents, thioethers are formed. When connecting aldehydes and amines under reducing conditions, alkylamines are formed. When connecting ketones or aldehydes and hydroxylamines (including derivatives thereof, in which substituents replace the hydrogen of the hydroxyl group), oxime functional groups (=NO-) are formed. When connecting carboxylic acids or phosphoric acids and alcohols, esters are formed. When the linking functional group is, for example, a click chemistry group, including methyl tetrazine, trans-cyclooctene, azide, dibenzocyclooctyne, tetrazine, alkyne, cyclopropanecyclooctyne and cyclopropene, it can be linked to pairs of orthogonal click chemistry groups.

[0045] For example, in the embodiment of the present invention, the connecting group is selected from any one of the groups shown in the following structural formulas:

[0046]

[0047] Where n represents 1-5.

[0048] The cation in the zwitterionic group is a quaternary ammonium cation, and the anion is a sulfonate anion; the structural formula of the zwitterionic group is selected from any one of the groups shown in the following structural formulas:

[0049] Wherein, R1 represents a C1-C5 alkyl group, n represents 1-5, and A represents a C2-C6 carbocyclic ring; for example,

[0050] Wherein, R1 represents a C1-C5 alkyl group, and n represents 1-5.

[0051] The coupling group includes a carboxylate; specifically, the coupling group is selected from any one of the groups described in the following structural formulas:

[0052] Wherein, n represents 1-5. It should be noted that the dashed line in the group in the implementation of the present invention Indicates the connection site.

[0053] More specifically, the steroid derivative is selected from any one of the compounds represented by the following structural formulas:

[0054]

[0055] Among them, R1 represents a C1-C5 alkyl group, n represents 1-5, and m represents 1-3; for example, R1 represents a C1-C5 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, etc., n represents any value between 1-5 such as 1, 2, 3, 4 and 5, and m represents any value between 1-3 such as 1, 2 and 3.

[0056] Among them, steroid substances include cortisol, aldosterone, progesterone and testosterone. It should be noted that, although the steroid substances provided in the embodiment of the present invention are only steroid substances such as cortisol, aldosterone, progesterone and testosterone, other steroid substances may also be used, for example, estradiol and cholecalciferol.

[0057] Specifically, the steroid derivative provided in the embodiment of the present invention is selected from any one of the compounds represented by the following structural formulas:

[0058]

[0059] In a second aspect, the present invention provides a method for preparing a steroid derivative as described in the aforementioned embodiment, comprising: reacting a steroid substance with a raw material containing a linking group to form an intermediate A;

[0060] Next, the intermediate A is reacted with a raw material containing a cation in a zwitterionic group to form an intermediate B;

[0061] Then, the intermediate B is reacted with a raw material containing a coupling group to form an intermediate C;

[0062] Then, the anion in the zwitterionic group is grafted onto the intermediate C.

[0063] For example, the synthesis is carried out according to the following synthesis route:

[0064]

[0065] In a third aspect, the present invention provides a complete antigen derivative, which comprises a substance to be coupled and the steroid derivative described in any one of the aforementioned embodiments; the substance to be coupled and the steroid derivative are coupled via the coupling group of the steroid derivative.

[0066] Wherein, the substance to be coupled is selected from a carrier protein, a polymer carrier or a signal substance.

[0067] The carrier protein is selected from bovine serum albumin, human serum albumin, hemocyanin or ovalbumin.

[0068] The polymer carrier is selected from at least one of polysaccharide, polylysine, PEG (polyethylene glycol), polyethylene imine and dendrimer. Specifically, the polysaccharide includes cross-linked polysucrose and dextran; the dendrimer includes polyethylene glycol and / or polypropylene imine.

[0069] The signal substance is selected from at least one of fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents and nanoparticle markers; preferably, the fluorescent dye is selected from at least one of fluorescein dyes and their derivatives, rhodamine dyes and their derivatives, Cy series dyes and their derivatives, Alexa series dyes and their derivatives, and protein dyes and their derivatives; preferably, the enzyme is selected from any one of horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase and 6-phosphoglucose deoxygenase; preferably, the radioactive isotope is selected from 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc , 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu and 18F; preferably, the chemiluminescent agent is selected from at least one of luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetane and its derivatives, lophanite and its derivatives and peroxyoxalate and its derivatives; preferably, the nanoparticle marker is selected from any one of colloids, organic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles; preferably, the colloid is selected from at least one of colloidal metals, disperse dyes, dye-labeled microspheres and latex; preferably, the colloidal metal is selected from at least one of colloidal gold, colloidal silver and colloidal selenium.

[0070] When the substance to be coupled is a signal substance, the signal substance can also be indirectly coupled to the steroid derivative via a carrier protein or a polymer carrier, that is, the carrier protein or the polymer carrier is coupled to the steroid derivative via the coupling group of the steroid derivative, and the signal substance is further coupled to the carrier protein or the polymer carrier.

[0071] In a fourth aspect, the present invention provides a method for preparing the complete antigen derivative described in the aforementioned embodiment, comprising: activating the steroid derivative described in any one of the aforementioned embodiments, and then performing a coupling reaction with a substance to be coupled.

[0072] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0073] Example 1 Synthesis of testosterone derivatives containing type I quaternary ammonium ion linkers

[0074] An embodiment of the present invention provides a method for preparing a steroid derivative, comprising:

[0075] The synthesis was carried out according to the following synthesis route:

[0076]

[0077] , the specific steps are as follows:

[0078] The first step is to take a solution dissolved in methanol, add sodium acetate, O-(carboxymethyl)hydroxylamine hemihydrochloride and react at room temperature for 5 to 6 hours, feed in a molar ratio of 1:1.5 to 2.0:1.2 to 1.5 (in this embodiment, the molar ratio is 1:1.5:1.2), and the polar dichloromethane / methanol is 10:1 on a thin layer silica gel plate. After the reaction is completed, add 1N dilute hydrochloric acid to adjust the pH of the solution to 4.0, concentrate, extract with ethyl acetate, dry the organic phase with anhydrous sodium sulfate, concentrate, and vacuum dry to obtain a mixture of testosterone 3Z and 3E oxime carboxylic acid i (Ratio of Z / E=2:3), with a yield of about 93%.

[0079] The nuclear magnetic resonance hydrogen spectrum detection data of the obtained 3-position testosterone carboxyl derivative are as follows: 1 HNMR (DMSO_d6, 400MHz) δppm: 0.67and 0.68(s,3H,H-18), 0.77-1.02(m,4H), 1.03and1.06(s,3H,H-19), 1.17-1.53(m,7H), 1.72-1.87(m,4H), 2.16-2.28and2.82-2.94(m,4H), 3.41-3.45(t,1H), 4.44-4.47(m,3H), 5.67and6.31(d,1H), 12.70(s,1H). Compound i was analyzed and identified by chromatography / mass spectrometry (LC / MS): MS Calcd.: 361.2, MS Found: 360.2(M -1 ).

[0080] In the second step, the oxime carboxylic acid mixture i is taken into N,N-dimethylformamide, DCC and N-hydroxysuccinimide are added in a molar ratio of 1:1.2:1.2, and the reaction is carried out at room temperature for 5 hours. The polar dichloromethane / methanol is displayed on a thin layer silica gel plate at a ratio of 20:1. After the raw materials are almost completely reacted, the urea salt is removed by filtration, and the filtrate is added to a reaction bottle, 1.2 eq of 1-(ethoxycarbonylmethyl)piperazine is added, and the pH value of the reaction solution is adjusted to 8.0 by adding triethylamine. The reaction is continued at room temperature for 1 hour. The polar dichloromethane / methanol is displayed on a thin layer silica gel plate at a ratio of 20:1. The activated ester reacts completely, water is added, and the mixture is extracted with ethyl acetate. The organic phases are combined, dried over anhydrous sodium sulfate, and concentrated to obtain compound ii with a yield of about 82%.

[0081] The third step is to take compound ii, add methanol and 2eq of sodium hydroxide, react at 50°C for 1h, and use a thin layer silica gel plate with polar dichloromethane / methanol = 10:1. After the ethyl ester is completely hydrolyzed, add 1N hydrochloric acid to adjust the solution to pH = 7.0, concentrate to remove methanol, and then add 1N hydrochloric acid to adjust the solution to pH = 3.0 until no more white solid precipitates. Filter, wash the filter cake with a small amount of acetone, and dry in vacuo to obtain compound iii with a yield of about 77%.

[0082] The nuclear magnetic resonance hydrogen spectrum detection data of the obtained compound iii are as follows: 1 H NMR (DMSO_d6, 400MHz) δppm: 0.72and 0.73(s,3H,H-18),0.76-0.84(m,2H),1.03and1.05(s,3H,H-19),1.07-1.87(m,10H),2.08-2.27and2.82-2.90(m,5H),2.42-2.56(d,2H),2. 48-2.56(m,4H), 3.02(s,2H), 3.30-3.45(t,1H), 3.55-3.45(m,4H), 4. 32-4.36(d,2H), 4.49-4.53(t,1H), 5.69and6.40(d,1H), 12.70(s,1H). Compound iii was analyzed and identified by chromatography / mass spectrometry (LC / MS): MS Calcd.: 487.3, MS Found: 486.3 (M -1 ).

[0083] The fourth step is to dissolve compound iii in N,N-dimethylformamide, add HATU, 6-aminobutyric acid methyl ester hydrochloride and N,N-diisopropylethylamine in a molar ratio of 1:1.1:1.1:2.5, react at room temperature for 8 hours, and develop a thin layer silica gel plate with polar dichloromethane / methanol = 10:1. The raw material is almost completely converted. After post-treatment, ethyl acetate is added for extraction, the organic phases are combined, dried over anhydrous sodium sulfate, and concentrated to obtain compound iv with a yield of about 86%.

[0084] The fifth step is to dissolve compound iv in anhydrous 1,4-dioxane, add 1.5-2.0eq of 1,3-propane sultone, heat to 100°C and react for 2h, and apply polar dichloromethane / methanol = 10:1 on a thin layer silica gel plate. Ethyl acetate is added during cooling, and a large amount of white solid precipitates. The solid is filtered, and the filter cake is washed with a small amount of ethyl acetate and dried with an oil pump to obtain compound v with a yield of about 66%.

[0085] In the sixth step, compound v is dissolved in tetrahydrofuran and methanol, 2 eq of sodium hydroxide is added, and the raw material is hydrolyzed completely. Finally, the compound is separated and purified by preparative liquid chromatography to obtain a single Z-configuration testosterone hapten carboxyl derivative vi containing a zwitterionic group, with a yield of about 90%.

[0086] The nuclear magnetic resonance hydrogen spectrum detection data of the obtained compound vi are as follows: 1 H NMR (D2O, 400MHz) δppm: 0.78 (s, 3H), 0.81-0.90 (m, 2H), 1.03 (s, 3H,), 1.08-1.60 (m, 6H), 1.72-1.88 (m, 6H), 2.08-2.32 (m, 11H), 2.34-2.36 ( d,2H), 2.46-2.62(m,2H), 2.66-2.74(m,4H), 3.06(s,2H), 3.42-3.56( t,1H), 3.48(d,2H), 3.58-3.60(m,4H), 4.44-4.46(d,2H), 6.36(d,1H). Compound vi was analyzed and identified by chromatography / mass spectrometry (LC / MS): MS Calcd.: 694.3, MS Found: 695.4 (M +1 ).

[0087] Example 2 Synthesis of testosterone derivatives containing type II quaternary ammonium ion linkers

[0088] An embodiment of the present invention provides a method for preparing a steroid derivative, comprising:

[0089] The synthesis was carried out according to the following synthesis route:

[0090]

[0091] , the specific steps are as follows:

[0092] The first step is to dissolve testosterone in methanol, add sodium acetate and O-(carboxymethyl)hydroxylamine hemihydrochloride at room temperature for 5 to 6 hours, feed the materials in a molar ratio of 1:1.5:1.2, and display the polar dichloromethane / methanol on a thin layer silica gel plate at a ratio of 10:1. After the reaction is completed, add 1N dilute hydrochloric acid to adjust the solution to pH = 4.0, concentrate, extract with ethyl acetate, dry the organic phase with anhydrous sodium sulfate, concentrate, and vacuum dry to obtain a testosterone 3Z and 3E oxime carboxylic acid mixture T-001 (Ratio of Z / E = 2:3), with a yield of about 93%;

[0093] The second step is to dissolve the oxime carboxylic acid mixture T-001 in N,N-dimethylformamide, add DCC and N-hydroxysuccinimide in a molar ratio of 1:1.2:1.2, and react at room temperature for 5 hours. After the raw materials are almost completely reacted, the polar dichloromethane / methanol is 20:1 on a thin layer silica gel plate. After the reaction is almost complete, the urea salt is removed by filtration, and the filtrate is added to a reaction bottle, and 1.2 eq of N-(3-aminopropyl)-N-methylcarbamic acid tert-butyl ester is added. The pH value of the reaction solution is adjusted to 8.0 by adding triethylamine, and the reaction is continued at room temperature for 1 hour. The polar dichloromethane / methanol is 20:1 on a thin layer silica gel plate. The activated ester reacts completely, water is added, and the ethyl acetate is extracted. The organic phases are combined, dried over anhydrous sodium sulfate, and concentrated to obtain compound T-002 with a yield of about 86%;

[0094] Step 3: Take compound T-002, add dichloromethane and trifluoroacetic acid (volume ratio of about 3:1), react at room temperature for 2h, and apply thin layer silica gel plate with polar dichloromethane / methanol = 10:1. After Boc is completely hydrolyzed, vacuum concentrate to remove trifluoroacetic acid, add 0.1 mol / L sodium hydroxide to adjust the solution pH to 7.5, extract with ethyl acetate 2-3 times, combine the organic phases, dry over anhydrous sodium sulfate, and vacuum concentrate to obtain compound T-003 with a yield of about 85%;

[0095] Step 4: Compound T-003, methyl 4-bromobutyrate, potassium carbonate, dissolved in N,N-dimethylformamide, added in a molar ratio of 1:1.5:2.0, reacted at 80°C for 6h, thin layer silica gel plate display plate polar dichloromethane / methanol = 10:1, the raw material was almost completely converted, and after treatment, 1N hydrochloric acid was added to adjust the pH of the solution to about 2.0, and ethyl acetate was added for extraction, and the aqueous phase was retained and 1 mol / L sodium hydroxide was added to adjust the pH of the solution to about 7.0-8.0, and ethyl acetate was added for extraction twice, the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to obtain compound T-004, with a yield of about 77%;

[0096] Step 5: Add 1.5 eq of 1,3-propane sultone to compound T-004, heat to 140°C for reaction for 1-2 hours, and apply polar dichloromethane / methanol = 10:1 on a thin layer silica gel plate. Add ethyl acetate during cooling, and a large amount of white solid precipitates. Filter, wash the filter cake with a small amount of ethyl acetate, and dry with an oil pump to obtain compound T-005 with a yield of about 70%.

[0097] The sixth step is to dissolve the compound T-005 in tetrahydrofuran and methanol, add 2eq of sodium hydroxide, wait for the raw material to be completely hydrolyzed, and finally separate and purify by preparative liquid chromatography to obtain a single Z-configuration testosterone hapten carboxyl derivative containing a zwitterionic group T-006, with a yield of about 72%.

[0098] It should be noted that the compounds represented by the symbols T-001, T-002, T-003, T-004, T-005 and T-006 mentioned above are all as shown in the synthesis route.

[0099] Example 3 Preparation of complete testosterone antigen derivatives

[0100] This embodiment provides a method for preparing a complete antigen derivative, comprising:

[0101] The hapten derivative vi provided in Example 1 was mixed and activated at a ratio of vi:EDC:NHS=1:4:4 for 1 hour, and then added to a carrier protein solution with a concentration of 1 mg / ml to 6 mg / ml according to different coupling ratios, reacted for 1 hour, and then blocked with 1M glycine for 30 minutes, and then purified by dialysis to obtain the corresponding synthetic antigen; the specific coupling ratio and the selection of carrier protein are shown in Tables 1 to 3.

[0102] Example 4 Preparation of complete progesterone antigen derivatives

[0103] Take progesterone material and synthesize a progesterone derivative containing a quaternary ammonium ion linker according to the method and steps of Example 1. The synthesis route is as follows.

[0104]

[0105] The final product of the reaction was taken and the complete progesterone antigen derivative was prepared according to the method of Example 3.

[0106] Example 5 Preparation of complete cortisol antigen derivatives

[0107] Take the cortisol material and synthesize the cortisol derivative containing a quaternary ammonium ion linker according to the method and steps of Example 1. The synthesis route is as follows.

[0108]

[0109] The final product of the reaction was taken and the complete cortisol antigen derivative was prepared according to the method of Example 3.

[0110] Example 6 Preparation of complete aldosterone antigen derivatives

[0111] Take aldosterone material and synthesize an aldosterone derivative containing a quaternary ammonium ion linker according to the method and steps of Example 1. The synthesis route is as follows.

[0112]

[0113] The final product of the reaction was taken and the complete aldosterone antigen derivative was prepared according to the method of Example 3.

[0114] Comparative Example

[0115] The compound i and compound iii of Example 1 were used to prepare complete antigens according to the preparation method of complete antigen derivatives of Example 3. The specific coupling ratio and selection of carrier protein are shown in Tables 1 to 3.

[0116] Test Example 1

[0117] Observe whether precipitate is formed during the coupling process of Example 3 and the comparative example, and how much precipitate is formed. The results are shown in Tables 1 to 3.

[0118] Table 1 Complete antigen derivatives of BSA as carrier protein

[0119]

[0120] Table 2 Complete antigen derivatives of OVA as carrier protein

[0121]

[0122]

[0123] Table 3 Complete antigenic derivatives of KLH as carrier protein

[0124]

[0125] in, Figure 2 Indicates the precipitation of complete antigen derivatives formed by BSA as a carrier protein. Figure 2 From left to right, there are trace precipitation, obvious precipitation and large precipitation. According to Tables 1 to 3, the steroid derivatives provided by the embodiments of the present invention can significantly reduce the formation of precipitation, thereby improving the accuracy and sensitivity of the detection results.

[0126] Test Example 2

[0127] The complete antigen derivatives obtained by coupling Example 3 and the comparative example (coupling ratio 1:40 and coupling ratio 1:80) were paired with different antibodies to compete for binding to the testosterone quality control product, and the sample detection activity of the two complete antigens was evaluated;

[0128] The specific detection method is as follows:

[0129] 1) The streptavidin is chemically coupled to the magnetic microspheres, and then the conjugate is diluted to 0.1-0.3 mg / ml using a magnetic bead diluent, and the diluent is defined as the Ra component;

[0130] 2) labeling the testosterone antibody with biotin by chemical coupling, and then diluting it to 0.1-0.3 ug / ml using a labeling diluent, and defining the diluent as the Rb component;

[0131] 3) Several complete antigen derivatives to be evaluated are labeled with acridinium ester by chemical coupling, and then diluted to 0.02-0.08 ug / ml using a labeled diluent, which is defined as the Rc component;

[0132] 4) Take 25ul of the sample to be tested and mix with 50ul of Rb component for 5min, then add 50ul of Ra component and Rc component, react for 10min, after 4-5 washes, add acridinium ester excitation solution and use a luminometer to detect the luminescence value;

[0133] 5) Record and summarize the data to form the following attached data, see Figure 1 .

[0134] according to Figure 1 It can be seen that the linearity of the experimental group that is easy to precipitate during the coupling process will deteriorate, the inhibition rate is poor, especially the gradient of the high-value sample is not easy to separate, and the steroid derivative provided in the embodiment of the present invention can reduce specific adsorption and have a higher gradient in the high-value sample.

[0135] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A steroid derivative, characterized in that: Its structure includes a steroid substance and a zwitterionic structure, wherein the steroid substance and the zwitterionic structure are bonded via a bonding bond; The zwitterionic structure includes a coupling group capable of undergoing coupling reaction, a zwitterionic group and a connecting group, the two sides of the connecting group are bonded to the steroid substance and the zwitterionic group respectively, and the zwitterionic group is linked to the coupling group.

2. The steroid derivative according to claim 1, characterized in that The steroid substances include any one of cortisol, aldosterone, progesterone and testosterone.

3. The steroid derivative according to claim 1, characterized in that The cation in the zwitterionic group is a quaternary ammonium cation, and the anion is any one of a sulfonate anion, a carboxylate anion, a sulfate anion, a phosphate anion and an oxide anion; Preferably, the structural formula of the zwitterionic group is selected from any one of the groups shown in the following structural formulas: Wherein, R1 represents a C1-C5 alkyl group, n represents 1-5, and A represents a C2-C6 carbocyclic ring; Preferably, the structural formula of the zwitterionic group is selected from any one of the groups shown in the following structural formulas: Wherein, R1 represents a C1-C5 alkyl group, and n represents 1-5.

4. The steroid derivative according to claim 1, characterized in that The linking group includes a functional group of 2 to 50 atoms containing at least one of carbon, oxygen, sulfur, nitrogen and phosphorus; Preferably, the linking group comprises any one of an amino acid residue, a nitro group, a nitroso group, a phosphate monoester bond, a phosphate diester bond, a phosphonate monoester bond, a phosphonate diester bond, an alkylamine, an amidine, a thioamide, an ether, a urea, a thiourea, a guanidine, an azo, a thioether, a carboxylate, a sulfonate, a phosphate, an amide, a thioester and an oxime group; Preferably, the linking group is selected from any one of the groups represented by the following structural formulas: Where n represents 1-5.

5. The steroid derivative according to claim 1, characterized in that The coupling group includes a carboxylate; Preferably, the coupling group is selected from any one of the groups described in the following structural formulas: Here, n represents 1-5.

6. The steroid derivative according to any one of claims 1 to 5, characterized in that The steroid derivative is selected from any one of the compounds represented by the following structural formulas: Wherein, R1 represents a C1-C5 alkyl group, n represents 1-5, and m represents 1-3; Preferably, the steroid derivative is selected from any one of the compounds represented by the following structural formulas: 。 7. A method for preparing a steroid derivative according to claim 1, characterized in that: include: The steroid substance is reacted with a raw material containing a linking group to form an intermediate A; Next, the intermediate A is reacted with a raw material containing a cation in a zwitterionic group to form an intermediate B; Then, the intermediate B is reacted with a raw material containing a coupling group to form an intermediate C; Then, the anion in the zwitterionic group is grafted onto the intermediate C.

8. A complete antigen derivative, characterized in that: The method comprises a substance to be coupled and the steroid derivative according to any one of claims 1 to 6; the substance to be coupled and the steroid derivative are coupled via the coupling group of the steroid derivative.

9. The complete antigen derivative according to claim 8, characterized in that ,, the object to be coupled is selected from a carrier protein, a polymer carrier or a signal substance.

10. A method for preparing the complete antigen derivative according to claim 8, characterized in that: include: The steroid derivative according to any one of claims 1 to 6 is activated and then subjected to coupling reaction with the object to be coupled.

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