Vitamin D3 carboxylic acid derivative and antigen and preparation method thereof

Abstract

The invention relates to the technical field of haptens, in particular to a vitamin D3 carboxylic acid derivative as well as an antigen and a preparation method thereof. The structural formula of the vitamin D3 carboxylic acid derivative is as follows: # imgabs0 #. The vitamin D3 carboxylic acid derivative has excellent antibody binding activity, and a vitamin D3 antigen formed by coupling has high activity and inhibition rate. And the vitamin D3 carboxylic acid derivative is low in synthesis cost, short in period and high in yield.

Description

Technical Field

[0001] The present invention relates to the technical field of haptens, and in particular to vitamin D3 carboxylic acid derivatives and antigens thereof and a preparation method thereof. Background Art

[0002] Vitamin D3, also known as cholecalciferol, is a type of vitamin D. 7-dehydrocholesterol generated by the dehydrogenation of cholesterol can form cholecalciferol when irradiated with ultraviolet light. Therefore, the vitamin D source of cholecalciferol is 7-dehydrocholesterol.

[0003] Vitamin D3 has the following physiological functions: 1. Improve the body's absorption of calcium and phosphorus, so that the levels of plasma calcium and plasma phosphorus reach saturation. 2. Promote growth and bone calcification, and promote healthy teeth; 3. Increase phosphorus absorption through the intestinal wall and increase phosphorus reabsorption through the renal tubules; 4. Maintain normal levels of citrate in the blood; 5. Prevent amino acid loss through the kidneys.

[0004] The existing method of using vitamin D3 to form hapten derivatives is mainly to optimize the 17th or 25th position of vitamin D3, but the above hapten derivatives all have the problems of long synthesis route, high cost and low conversion rate.

[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 vitamin D3 carboxylic acid derivative and an antigen and a preparation method thereof. The embodiment of the present invention provides a novel vitamin D3 carboxylic acid derivative, which has excellent antibody binding activity, and the vitamin D3 antigen formed by coupling has high activity and inhibition rate. The vitamin D3 carboxylic acid derivative has low synthesis cost, short cycle and high yield.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides a vitamin D3 carboxylic acid derivative, the structural formula of which is shown below:

[0009]

[0010] In a second aspect, the present invention provides a method for preparing the vitamin D3 carboxylic acid derivatives described in the aforementioned embodiment, which is synthesized according to the following synthesis route:

[0011]

[0012] Here, X represents a halogen.

[0013] In an optional embodiment, the operation of S1 includes: mixing pregnenolone acetate, a halide and an azo compound to perform a halogenation reaction;

[0014] Preferably, the mass ratio of the pregnenolone acetate, the halide and the azo compound is 1:0.2-0.5:0.005-0.015;

[0015] Preferably, the reaction conditions include: temperature of 80-120°C, time of 3-10 minutes;

[0016] Preferably, the halide comprises a bromide, preferably dibromohydantoin, and the azo compound comprises azobisisobutyronitrile.

[0017] In an optional embodiment, the operation of S2 includes: mixing compound II with an ammonium substance to react;

[0018] Preferably, the molar ratio of the compound II to the ammonium substance is 1:1.5-3.5;

[0019] Preferably, the ammonium substance includes tetrabutylammonium fluoride.

[0020] In an optional embodiment, the operation of S3 includes: mixing compound III with a base to perform a hydrolysis reaction;

[0021] Preferably, the mass ratio of the compound III to the base is 1:0.5-1; the base comprises a hydroxide, preferably sodium hydroxide.

[0022] In an optional embodiment, the operation of S4 includes: mixing compound IV with an antioxidant to perform a photochemical ring-opening reaction under ultraviolet light;

[0023] Preferably, the mass ratio of the compound IV to the antioxidant is 1:0.8-1.5;

[0024] Preferably, the antioxidant comprises BHT.

[0025] In an optional embodiment, the operation of S5 includes: mixing compound V with an antioxidant to perform a double bond isomerization reaction;

[0026] Preferably, the mass ratio of the compound V to the antioxidant is 1:0.9-1;

[0027] Preferably, the antioxidant comprises BHT.

[0028] In an optional embodiment, the operation of S6 includes: mixing compound VI and O-(carboxymethyl)hydroxylamine hemihydrochloride to perform an addition reaction;

[0029] Preferably, the mass ratio of the compound VI to the O-(carboxymethyl)hydroxylamine hemihydrochloride is 1:1.5-2.5;

[0030] Preferably, the reaction temperature is 40-60°C and the reaction time is 4-6 hours.

[0031] In a third aspect, the present invention provides a vitamin D3 antigen, the structural formula of which is shown below:

[0032] Wherein, A represents a substance to be coupled, and the substance to be coupled is selected from a carrier protein, a polymer carrier or a signal substance;

[0033] Preferably, the vitamin D3 antigen is selected from any one of the compounds represented by the following structural formulas:

[0034]

[0035] In an optional embodiment, the method comprises: activating the above-mentioned vitamin D3 carboxylic acid derivative and then coupling it with a coupling agent.

[0036] The present invention has the following beneficial effects: the present invention provides a new vitamin D3 hapten derivative, the new vitamin D3 carboxylic acid derivative has excellent antibody binding activity, and the vitamin D3 antigen formed by coupling has high activity and inhibition rate. The vitamin D3 carboxylic acid derivative has low synthesis cost, short cycle and high yield, and improves the existing problems of high production cost, long cycle and low yield of vitamin D3 related hapten or antigen. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] 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.

[0038] Figure 1 The liquid phase spectrum of the vitamin D3 carboxylic acid derivative provided in Example 1 of the present invention;

[0039] Figure 2 This is the mass spectrum of the vitamin D3 carboxylic acid derivative provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0040] 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.

[0041] The embodiment of the present invention provides a vitamin D3 carboxylic acid derivative, the structural formula of which is shown below:

[0042]

[0043] The present invention provides a method for preparing a vitamin D3 carboxylic acid derivative. The present invention adopts low-cost pregnenolone acetate as a basic raw material, and synthesizes a vitamin D3 (position 20) carboxylic acid derivative efficiently and at low cost through bromination and elimination, photocatalytic ring opening and double bond isomerization, and addition elimination. The specific synthesis route is as follows:

[0044]

[0045] Here, X represents a halogen, such as bromine, chlorine, fluorine, or iodine.

[0046] The specific process is as follows:

[0047] S1. Mixing pregnenolone acetate (compound I), a halide and an azo compound for halogenation reaction; wherein the mass ratio of pregnenolone acetate, the halide and the azo compound is 1:0.2-0.5:0.005-0.015, preferably 1:0.4:0.01; the reaction conditions include: a temperature of 80-120°C and a time of 3-10 minutes; the halide includes a bromide, preferably dibromohydantoin, and the azo compound includes azobisisobutyronitrile.

[0048] It should be noted that the above halide is dibromohydantoin which is only an example of the embodiment of the present invention, and other halides or bromides that can react in the prior art can also be used.

[0049] S2. Mixing compound II with an ammonium substance to react; wherein the molar ratio of compound II to the ammonium substance is 1:1.5-3.5, preferably 1:3; and the ammonium substance includes tetrabutylammonium fluoride.

[0050] It should be noted that the above-mentioned ammonium substance is tetrabutylammonium fluoride, which is only an example of the embodiment of the present invention. Ammonium substances that can react in the prior art, such as tetrabutylammonium chloride, may also be used.

[0051] S3, mixing compound III with a base to carry out a hydrolysis reaction; wherein the mass ratio of the compound III to the base is 1:0.5-1; the base comprises a hydroxide, preferably sodium hydroxide.

[0052] It should be noted that the above-mentioned base, sodium hydroxide, is only an example of the embodiment of the present invention, and alkaline substances that can react in the prior art, such as sodium alcoholate, potassium hydroxide, ammonia water, etc., can also be used.

[0053] S4. Mixing compound IV and an antioxidant to carry out a photochemical ring-opening reaction under ultraviolet light; wherein the mass ratio of compound IV to the antioxidant is 1:0.8-1.5; and the antioxidant includes BHT.

[0054] S5. Mixing compound V with an antioxidant to carry out double bond isomerization reaction; wherein the mass ratio of compound V to the antioxidant is 1:0.9-1; and the antioxidant includes BHT.

[0055] S6. Mix compound VI and O-(carboxymethyl)hydroxylamine hemihydrochloride to carry out addition reaction; wherein the mass ratio of compound VI to O-(carboxymethyl)hydroxylamine hemihydrochloride is 1:1.5-2.5; the reaction temperature is 40-60°C, and the reaction time is 4-6 hours.

[0056] The present invention also provides a vitamin D3 antigen, the structural formula of which is shown below:

[0057] Wherein, A represents the object to be coupled, and the object to be coupled is selected from a carrier protein, a polymer carrier or a signal substance.

[0058] Wherein, the carrier protein is selected from bovine serum albumin, human serum albumin, hemocyanin or ovalbumin.

[0059] 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.

[0060] 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.

[0061] When the substance to be coupled is a signal substance, the signal substance can also be indirectly coupled to the vitamin D3 carboxylic acid derivative through a carrier protein or a polymer carrier, that is, the carrier protein or the polymer carrier is coupled to the vitamin D3 carboxylic acid derivative through the coupling group of the vitamin D3 carboxylic acid derivative, and the signal substance is then coupled to the carrier protein or the polymer carrier.

[0062] Preferably, the vitamin D3 antigen is selected from any one of the compounds represented by the following structural formulas:

[0063]

[0064] The embodiment of the present invention also provides a method for preparing the above vitamin D3 antigen, comprising activating the vitamin D3 carboxylic acid derivative and then coupling it with a coupling agent.

[0065] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0066] Example 1

[0067] This embodiment provides a method for preparing a vitamin D3 antigen, comprising:

[0068] The synthesis was carried out according to the following synthesis route:

[0069]

[0070] , the specific operations are as follows:

[0071] S1: Brominated at position 7 of compound Ⅰ

[0072] Specific operation: In a 500mL eggplant-shaped bottle, dissolve 7.17g of pregnenolone acetate, 2.85g of dibromohydantoin and 82mg of azobisisobutyronitrile in 150mL of toluene and 150mL of cyclohexane, replace with argon and stir at 100 degrees Celsius for 3-5 minutes under argon protection, cool to room temperature and filter, rinse the filter cake with 10mL of cyclohexane, combine the filtrate and concentrate under reduced pressure to obtain the crude product of compound II, which can be directly used for the next step without further treatment.

[0073] S2: condensation reaction of compound II;

[0074] Specific operation: Dissolve compound II in 60 mL tetrahydrofuran in a 500 mL eggplant-shaped bottle, add 60 mL tetrabutylammonium fluoride (1M) tetrahydrofuran solution at 0 degrees Celsius, and then stir at room temperature overnight. Through plate chromatography (petroleum ether: ethyl acetate = 4:1), the product Rf = 0.3-0.4. Concentrate the tetrahydrofuran under reduced pressure, add 200 mL ethyl acetate and 200 mL water, collect the organic phase, dry and concentrate with anhydrous sodium sulfate, collect the components of petroleum ether: ethyl acetate = 20:1-10:1 by column chromatography, and spin dry to obtain 1.87 g of compound III, with a two-step yield of 26%.

[0075] S3: hydrolysis reaction of compound III;

[0076] Specific operation: 250mL eggplant-shaped bottle, dissolve 3g of compound III in a mixed solvent of 50mL tetrahydrofuran, 25mL water and 25mL methanol, cool to 0-5 degrees Celsius, add 1.35g of sodium hydroxide solid, and stir at room temperature overnight. Plate chromatography (petroleum ether: ethyl acetate = 4:1) shows that intermediate 3 is completely reacted, and the product Rf = 0.1-0.2. Methanol and tetrahydrofuran are concentrated under reduced pressure to precipitate a large amount of white solid, which is filtered under reduced pressure and the filter cake is collected and dried to obtain 2.6g of compound IV, with a yield of 98%.

[0077] S4: photocatalytic ring opening of compound IV;

[0078] Specific operation: 500mL watch glass, 2.6g compound IV and 2.5g antioxidant BHT were dissolved in 300mL tetrahydrofuran, and irradiated with ultraviolet light with a wavelength of 254nm for photochemical ring-opening reaction. After 3 hours, the remaining reaction liquid was collected, concentrated to dryness, and 100mL ethyl acetate was added. After stirring for 15 minutes, it was filtered under reduced pressure. The filtrate was collected and dried to obtain the crude intermediate V, which was further treated and directly transferred to the next step.

[0079] S5: Double bond isomerization of compound V;

[0080] Specific operation: In a 250 mL eggplant-shaped bottle, compound V of intermediate 5 and 0.5 g of antioxidant BHT were dissolved in 100 mL of anhydrous ethanol. After reflux for 8 hours under an argon atmosphere, the mixture was concentrated under reduced pressure and purified by column chromatography. The components of petroleum ether: ethyl acetate = 5:1 were collected and concentrated to obtain 0.5 g of compound VI.

[0081] S6: Formation of vitamin D3 (position 20) carboxylic acid derivative

[0082] Specific operation: 100 mg of compound VI, 208 mg of O-(carboxymethyl)hydroxylamine hemihydrochloride and 0.5 mL of diisopropylethylamine were dissolved in 10 mL of anhydrous methanol in a 50 mL eggplant-shaped bottle, heated to 60 degrees Celsius under an argon atmosphere, kept warm for 5 hours, concentrated the methanol under reduced pressure, added 20 mL of water to dissolve, adjusted the pH to 4-5 with 1 mol / L dilute hydrochloric acid, filtered under reduced pressure after a large amount of solid precipitated, and the filter cake was rinsed with 5 mL of water, collected and dried to obtain 100 mg of vitamin D3 (position 20) carboxylic acid derivative, compound VII, with a yield of 81%. MS: 386 (387-1).

[0083] S7: Synthesis of antigens VIII-Ⅰ and VIII-Ⅱ

[0084] Specific operation: Weigh 5 mg of vitamin D3 (position 20) carboxylic acid derivative (Compound VII), dissolve it in 200 ul dimethyl sulfoxide, then add 2 equivalents of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2 equivalents of N-hydroxysuccinimide, react for 2 hours, add to 3 mg / ml bovine serum albumin (BSA) or hemocyanin (KLH), react for 2 hours, dialyze to remove unreacted small molecules, and then concentrate to 3 mg / ml to obtain VIII-I and VIII-II.

[0085] Characterization

[0086] Compound VII was subjected to liquid phase analysis and mass spectrometry detection. The results are shown in Figure 1 and Figure 2 ,according to Figure 1 and Figure 2 It can be seen that the desired compound was synthesized.

[0087] Antigen activity assessment

[0088] 1) Antigen coating of 96-well plate: The synthetic antigen VIII-I of the present invention and the commercially available antigens (Hanzun VD-AG-001 and Shanghai Xinkai Pharmaceutical Technology VD-H0036) obtained above were diluted to 0.1ug / ml and 0.5ug / ml respectively with coating solution, and then 100ul was added to each well of the 96-well plate and coated at 4 degrees for 16 hours;

[0089] 2) After the above steps are completed, take out the coated plate and equilibrate it to room temperature, clean it twice with cleaning solution, then add 120ul of blocking solution to each well, block it at 37℃ for 1 hour, spin dry it, and place it in a drying room or electronic drying oven with a humidity of less than 30% to dry for 24 hours before use;

[0090] 3) Prepare antibody working solution: dilute VD antibody to 20ng / ml or 40ng / ml using diluent, mix well and set aside;

[0091] 4) Prepare enzyme-labeled secondary antibody working solution: dilute goat anti-mouse IgG-HRP into enzyme dilution solution at a 4K dilution ratio, mix well and set aside;

[0092] 5) Detection: Mix 50ul of the sample to be tested with 50ul of the antibody working solution, then place it in a 37°C constant temperature incubator for reaction for 30min, then wash it 5 times with the cleaning solution, then add 100ul of the secondary antibody enzyme working solution, and react in a 37°C constant temperature incubator for 30min; then wash it again 5 times with the cleaning solution, pat dry, add 100ul of the chemiluminescent substrate (luminescent A solution and luminescent B solution are mixed in a ratio of 1:1), react for 1min, and place it in a plate chemiluminescent immunoassay analyzer for reading.

[0093] The results are shown in Tables 1 and 2.

[0094] Table 1 Activity and linearity data of 40ng / ml VD antibody against different antigens

[0095]

[0096] Table 2 Activity and linearity data of 20ng / ml VD antibody against different antigens

[0097]

[0098] It can be seen from Tables 1 and 2 above that the vitamin D3 carboxylic acid derivatives provided in the embodiments of the present invention have antibody binding activity, and the formed vitamin D3 antigen has high activity and inhibition rate (C1 / C0).

[0099] 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 vitamin D3 carboxylic acid derivative, characterized in that Its structural formula is as follows:

2. A method for preparing the vitamin D3 carboxylic acid derivative according to claim 1, characterized in that: The synthesis was carried out according to the following synthesis route: Here, X represents a halogen.

3. The preparation method according to claim 2, characterized in that: The operation of S1 includes: mixing pregnenolone acetate, a halide and an azo compound to perform a halogenation reaction; Preferably, the mass ratio of the pregnenolone acetate, the halide and the azo compound is 1:0.2-0.5:0.005-0.015; Preferably, the reaction conditions include: temperature of 80-120°C, time of 3-10 minutes; Preferably, the halide comprises a bromide, preferably dibromohydantoin, and the azo compound comprises azobisisobutyronitrile.

4. The preparation method according to claim 2, characterized in that: The operation of S2 includes: mixing compound II with an ammonium substance to react; Preferably, the molar ratio of the compound II to the ammonium substance is 1:1.5-3.5; Preferably, the ammonium substance includes tetrabutylammonium fluoride.

5. The preparation method according to claim 2, characterized in that: The operation of S3 includes: mixing compound III with a base to perform a hydrolysis reaction; Preferably, the mass ratio of the compound III to the base is 1:0.5-1; the base comprises a hydroxide, preferably sodium hydroxide.

6. The preparation method according to claim 2, characterized in that: The operation of S4 includes: mixing compound IV with an antioxidant and performing a photochemical ring-opening reaction under ultraviolet light; Preferably, the mass ratio of the compound IV to the antioxidant is 1:0.8-1.5; Preferably, the antioxidant comprises BHT.

7. The preparation method according to claim 2, characterized in that: The operation of S5 includes: mixing compound V with an antioxidant to perform a double bond isomerization reaction; Preferably, the mass ratio of the compound V to the antioxidant is 1:0.9-1; Preferably, the antioxidant comprises BHT.

8. The preparation method according to claim 2, characterized in that: The operation of S6 comprises: mixing compound VI and O-(carboxymethyl)hydroxylamine hemihydrochloride to perform an addition reaction; Preferably, the mass ratio of the compound VI to the O-(carboxymethyl)hydroxylamine hemihydrochloride is 1:1.5-2.5; Preferably, the reaction temperature is 40-60°C and the reaction time is 4-6 hours.

9. A vitamin D3 antigen, characterized in that Its structural formula is as follows: Wherein, A represents a substance to be coupled, and the substance to be coupled is selected from a carrier protein, a polymer carrier or a signal substance; Preferably, the vitamin D3 antigen is selected from any one of the compounds represented by the following structural formulas:

10. A method for preparing the vitamin D3 antigen according to claim 9, characterized in that: include: The vitamin D3 carboxylic acid derivative according to claim 1 is activated and then coupled with a coupling agent.

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