Vitamin d3 carboxylic acid derivatives, antigens and methods of making same

Abstract

The present application relates to the technical field of hapten, in particular to vitamin D3 carboxylic acid derivative, antigen and preparation method thereof. The structural formula of vitamin D3 carboxylic acid derivative is shown as follows: the vitamin D3 carboxylic acid derivative has excellent antibody binding activity, the vitamin D3 antigen coupled has high activity and inhibition rate. And the synthesis cost of the vitamin D3 carboxylic acid derivative is low, the cycle is short, and the yield is high.

Description

Technical Field

[0001] This invention relates to the field of hapten technology, and more specifically, to vitamin D3 carboxylic acid derivatives, their antigens, and preparation methods. Background Technology

[0002] Vitamin D3, also known as cholecalciferol, is a type of vitamin D. Cholecalciferol is formed from 7-dehydrocholesterol produced by the dehydrogenation of cholesterol and is then exposed to ultraviolet light. Therefore, the vitamin D precursor of cholecalciferol is 7-dehydrocholesterol.

[0003] Vitamin D3 has the following physiological functions: 1. It enhances the body's absorption of calcium and phosphorus, ensuring that plasma calcium and phosphorus levels reach saturation. 2. It promotes growth and bone calcification, and promotes healthy teeth. 3. It increases phosphorus absorption through the intestinal wall and increases phosphorus reabsorption through the renal tubules. 4. It maintains normal levels of citrate in the blood. 5. It prevents the loss of amino acids through the kidneys.

[0004] Existing methods for forming hapten derivatives using vitamin D3 mainly involve optimizing positions 17 or 25 of vitamin D3. However, these hapten derivatives suffer from problems such as long synthetic routes, high costs, and low conversion rates.

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

[0006] The purpose of this invention is to provide a vitamin D3 carboxylic acid derivative, its antigen, and a preparation method thereof. This invention provides a novel vitamin D3 carboxylic acid derivative with excellent antibody-binding activity, and the vitamin D3 antigen formed by coupling with it exhibits high activity and inhibition rate. Furthermore, the synthesis of this vitamin D3 carboxylic acid derivative is low-cost, has a short cycle time, and high yield.

[0007] This invention is implemented as follows:

[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] Secondly, the present invention provides a method for preparing the vitamin D3 carboxylic acid derivative described in the foregoing embodiments, wherein the synthesis is carried out according to the following synthetic route:

[0011]

[0012] Where X represents halogen.

[0013] In an optional embodiment, the operation of S1 includes: mixing pregnenolone acetate, halide and azo compound to carry out 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: a temperature of 80-120°C and a time of 3-10 minutes;

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

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

[0018] Preferably, the molar ratio of 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 carry out a hydrolysis reaction;

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

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

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

[0024] Preferably, the antioxidant includes BHT.

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

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

[0027] Preferably, the antioxidant includes BHT.

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

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

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

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

[0032] Wherein, A represents the coupling agent, which is selected from carrier proteins, polymer carriers, or signaling agents;

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

[0034]

[0035] In an optional embodiment, the method includes: 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: It provides a novel vitamin D3 hapten derivative, which possesses excellent antibody-binding activity, and the resulting vitamin D3 antigen exhibits high activity and inhibition rate. Furthermore, the synthesis of this vitamin D3 carboxylic acid derivative is low-cost, has a short cycle time, and high yield, thus overcoming the problems of high cost, long cycle time, and low yield in the production of existing vitamin D3-related haptens or antigens. Attached Figure Description

[0037] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[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 a mass spectrum of the vitamin D3 carboxylic acid derivative provided in Example 1 of the present invention. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

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

[0042]

[0043] This invention provides a method for preparing a vitamin D3 carboxylic acid derivative. The method uses inexpensive pregnenolone acetate as a base 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 synthetic route is as follows:

[0044]

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

[0046] The specific process is as follows:

[0047] S1. Pregnenolone acetate (compound I), a halide, and an azo compound are mixed and subjected to a 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-mentioned halide, dibromohydantoin, is only an example of an embodiment of the present invention, and other halides or bromides that can react in the prior art can also be used.

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

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

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

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

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

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

[0055] S6. Compound VI and O-(carboxymethyl)hydroxylamine hemihydrochloride are mixed and subjected to an 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℃ and the reaction time is 4-6 hours.

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

[0057] Where A represents the coupling agent, which is selected from carrier proteins, polymer carriers, or signaling agents.

[0058] The carrier protein mentioned above is selected from bovine serum albumin, human serum albumin, hemocyanin, or ovalbumin.

[0059] The polymer carrier is selected from at least one of polysaccharides, polylysine, PEG (polyethylene glycol), polyethyleneimine, and dendritic polymers. Specifically, the polysaccharides include cross-linked sucrose and dextran; the dendritic polymers include polyethylene glycol and / or polypropyleneimine.

[0060] The signaling agent is selected from at least one of fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents, and nanoparticle-based 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 glucose-6-phosphate dehydrogenase; preferably, the radioactive isotope is selected from 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, and 47Sc. The chemiluminescent reagent is selected from at least one of 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, and 18F; preferably, the chemiluminescent reagent is selected from at least one of luminol and its derivatives, luciferin, luteolin and its derivatives, ruthenium bipyridine and its derivatives, acridinium ester and its derivatives, dioxane and its derivatives, rofenol and its derivatives, and peroxazone and its derivatives; preferably, the nanoparticle label 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, dispersed 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 signaling substance, the signaling substance can also be indirectly coupled to the vitamin D3 carboxylic acid derivative through a carrier protein or polymer carrier. That is, the carrier protein or polymer carrier is coupled to the vitamin D3 carboxylic acid derivative through the coupling group of the vitamin D3 carboxylic acid derivative, and the signaling substance is then coupled to the carrier protein or polymer carrier.

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

[0063]

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

[0065] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0066] Example 1

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

[0068] Perform the synthesis according to the following synthesis path:

[0069]

[0070] The specific steps are as follows:

[0071] S1: Brominated compound I at position 7

[0072] Specific procedures: In a 500mL flask, dissolve 7.17g of pregnenolone acetate, 2.85g of dibromohydantoin, and 82mg of azobisisobutyronitrile in 150mL of toluene and 150mL of cyclohexane. Purge the mixture with argon gas and stir at 100°C for 3-5 minutes under argon protection. After cooling to room temperature, filter the mixture. Wash the filter cake with 10mL of cyclohexane. Combine the filtrates and concentrate under reduced pressure to obtain the crude product of compound II. Proceed directly to the next step without further processing.

[0073] S2: Condensation reaction of compound II;

[0074] Specific procedures: In a 500 mL pear-shaped flask, compound II was dissolved in 60 mL of tetrahydrofuran. A 60 mL solution of tetrabutylammonium fluoride (1 M) in tetrahydrofuran was added dropwise at 0°C, followed by stirring overnight at room temperature. Plate chromatography (petroleum ether:ethyl acetate = 4:1) yielded a product Rf of 0.3–0.4. The tetrahydrofuran was concentrated under reduced pressure, and then 200 mL of ethyl acetate and 200 mL of water were added. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated. Column chromatography was used to collect fractions with petroleum ether:ethyl acetate ratios of 20:1–10:1, which were then evaporated to dryness to give 1.87 g of compound III, a two-step yield of 26%.

[0075] S3: Hydrolysis reaction of compound III;

[0076] Specific procedures: In a 250 mL flask, dissolve 3 g of compound III in a mixed solvent of 50 mL tetrahydrofuran, 25 mL water, and 25 mL methanol. Cool to 0-5°C, then add 1.35 g of solid sodium hydroxide and stir overnight at room temperature. Plate chromatography (petroleum ether: ethyl acetate = 4:1) showed that intermediate 3 reacted completely, with an Rf of 0.1-0.2 for the product. Concentrate the methanol and tetrahydrofuran under reduced pressure, precipitating a large amount of white solid. Filter under reduced pressure and collect the filter cake, drying to obtain 2.6 g of compound IV, with a yield of 98%.

[0077] S4: Photocatalytic ring-opening of compound IV;

[0078] Specific procedures: Dissolve 2.6g of compound IV and 2.5g of antioxidant BHT in 300mL of a 500mL petri dish. Perform a photochemical ring-opening reaction by irradiating with ultraviolet light at a wavelength of 254nm. After 3 hours, collect the remaining reaction solution, concentrate it to dryness, add 100mL of ethyl acetate, stir for 15 minutes, filter under reduced pressure, collect the filtrate, and evaporate to dryness to obtain the crude product of intermediate V. Proceed directly to the next step for further processing.

[0079] S5: Compound V double bond isomerization;

[0080] Specific procedures: In a 250mL flask, compound V of intermediate 5 and 0.5g of antioxidant BHT were dissolved in 100mL of anhydrous ethanol. The mixture was refluxed under an argon atmosphere for 8 hours, then concentrated under reduced pressure and purified by column chromatography. The fraction of petroleum ether:ethyl acetate = 5:1 was collected and concentrated to obtain 0.5g of compound VI.

[0081] S6: Forms a vitamin D3 (position 20) carboxylic acid derivative.

[0082] Specific procedures: In a 50 mL flask, 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. The mixture was heated to 60°C under an argon atmosphere and reacted for 5 hours. After concentrating the methanol under reduced pressure, 20 mL of water was added to dissolve the compound. The pH was adjusted to 4-5 with 1 mol / L dilute hydrochloric acid. A large amount of solid precipitated, and the mixture was filtered under reduced pressure. The filter cake was washed 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 procedure: Weigh 5 mg of vitamin D3 (position 20) carboxylic acid derivative (compound VII), dissolve it in 200 μL of dimethyl sulfoxide, then add 2 equivalents of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2 equivalents of N-hydroxysuccinimide. After reacting for 2 hours, add it to 3 mg / mL bovine serum albumin (BSA) or hemocyanin (KLH). After reacting for 2 hours, dialyze to remove unreacted small molecules, then concentrate to 3 mg / mL to obtain VIII-I and VIII-II.

[0085] Characterization

[0086] Compound VII was analyzed by liquid chromatography and mass spectrometry; the results are shown in [reference needed]. 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 plates: The synthetic antigen VIII-I obtained in the above embodiment of the present invention and commercially available antigens (Hanzun VD-AG-001 and Shanghai Xinkai Pharmaceutical Technology VD-H0036) were diluted to 0.1ug / ml and 0.5ug / ml respectively with coating solution, and then 100ul was added to each well of a 96-well plate and coated at 4 degrees for 16h.

[0089] 2) After the above steps are completed, remove the coated plate and bring it to room temperature. Clean it twice with cleaning solution, then add 120ul of sealing solution to each well and seal it at 37°C for 1 hour. After spin-drying, place it in a drying room or electronic drying oven with humidity less than 30% for 24 hours before use.

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

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

[0092] 5) Detection: Mix 50 μL of the sample to be tested with 50 μL of antibody working solution, and then incubate at 37°C for 30 min. After that, wash 5 times with washing solution, then add 100 μL of secondary antibody enzyme working solution and incubate at 37°C for 30 min. After that, wash 5 times with washing solution again, pat dry, add 100 μL of chemiluminescent substrate (chemiluminescence solution A and chemiluminescence solution B are mixed at a 1:1 ratio), react for 1 min, and then read the value in a plate chemiluminescence immunoassay analyzer.

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

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

[0095]

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

[0097]

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

[0099] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A vitamin D3 carboxylic acid derivative, characterized in that, Its structural formula is shown below: 。 2. A method for preparing the vitamin D3 carboxylic acid derivative according to claim 1, characterized in that, Perform the synthesis according to the following synthesis path: ， Where X represents halogen.

3. The preparation method according to claim 2, characterized in that, The operation of S1 includes: mixing pregnenolone acetate, halide and azo compound to carry out a halogenation reaction; The mass ratio of the pregnenolone acetate, the halide, and the azo compound is 1:0.2-0.5:0.005-0.015; The reaction conditions are: temperature 80-120℃, time 3-10 minutes; The halide is a bromide, and the azo compound is azobisisobutyronitrile.

4. The preparation method according to claim 3, characterized in that, The halide is dibromohydantoin.

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

6. The preparation method according to claim 2, characterized in that, The operation of S3 includes: mixing compound III with a base to carry out a hydrolysis reaction; The mass ratio of compound III to the base is 1:0.5-1; the base is a hydroxide.

7. The preparation method according to claim 6, characterized in that, The alkali is sodium hydroxide.

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

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

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

11. A vitamin D3 antigen, characterized in that, Its structural formula is shown below: In this context, A represents the coupling agent, which is selected from carrier proteins, polymer carriers, or signaling agents.

12. The vitamin D3 antigen according to claim 11, characterized in that, The vitamin D3 antigen is selected from any one of the compounds shown in the following structural formulas: and .

13. A method for preparing the vitamin D3 antigen according to claim 11, characterized in that, include: The vitamin D3 carboxylic acid derivative of claim 1 is activated and then coupled with a coupling agent.

Images