Synthetic method and application of 19-O-propyl hydroxyl vardenafil and hapten

Abstract

The invention relates to a synthesis method and application of 19-O-propyl hydroxyl vardenafil and a hapten thereof, 2-propoxy cyanophenyl is used as an initial raw material, and 2-piperazin-1-yl ethanol is used for replacing N-ethyl piperazine for condensation, so that the reaction steps are simplified, and the water solubility and derivatization flexibility of the product are improved. The hapten prepared by introducing a carboxyl connecting arm to a 19-O-propyl hydroxyl vardenafil molecule has good immunogenicity, and the obtained antibody has high specificity. The established enzyme-linked immunosorbent assay method is high in detection sensitivity, IC15 is 8.85 ng / mL, IC50 is 157.8 ng / mL, the method can be used for rapidly and accurately detecting 19-O-propyl hydroxyl vardenafil illegally added in food and health care products, and public health can be guaranteed.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of food detection, and relates to an immunoassay technique, in particular to a synthesis method and application of 19-O-propylhydroxy vardenafil and a hapten. BACKGROUND

[0002] 19-O-propylhydroxy vardenafil is a new type of vardenafil structural analogue and belongs to a phosphodiesterase-5 (PDE-5) inhibitor derivative. The compound has similar pharmacological activity to vardenafil, can promote vascular smooth muscle relaxation by inhibiting PDE-5 enzyme activity, and thus enhance sexual function. Due to its significant physiological activity, 19-O-propylhydroxy vardenafil has been illegally added to food or health products in recent years to improve sexual function, which has brought potential risks to public health.

[0003] The existing synthesis methods of vardenafil and its structural analogues generally have problems such as many impurity by-products, complicated purification steps, high production cost and poor safety in industrial production. For example, in some synthesis routes, by-products are easily produced in the generation process of key intermediates, resulting in low purity of the final product, which needs to be purified for multiple times to meet the pharmaceutical quality standards; some synthesis methods need to use expensive catalysts (such as Pd / C), which not only increases the cost, but also has fire risk in the post-processing process, reducing the production safety; some synthesis steps need to be carried out under special reaction conditions, such as pressurized reaction equipment, which limits its application in industrial production. In addition, the traditional synthesis route of vardenafil is long, usually including 2-ethoxybenzonitrile as the starting material, and the target product is prepared through Pinner reaction, hydrazinolysis, cyclization, chlorosulfonation, amination and other multi-step reactions. The quality control of intermediates in each step is difficult, which directly affects the purity and yield of the final product.

[0004] Therefore, it is urgent to provide a synthesis method of 19-O-propylhydroxy vardenafil with short synthesis route, few reaction steps, few impurity by-products, high yield and suitable for industrialization, so as to overcome the deficiencies in the prior art, and provide a basis for the preparation of its hapten and the research of immunoassay method. SUMMARY

[0005] The present application is to overcome the deficiencies of the prior art, and to provide a synthesis method and application of 19-O-propylhydroxy vardenafil and a hapten with simple operation, high product yield and low production cost, low requirement for reaction conditions, and suitable for industrial production.

[0006] The technical problem of the present application is solved by adopting the following technical scheme: A method for synthesizing 19-O-propylhydroxyvardenafil includes the following steps: 2-propoxybenzonitrile is used as a starting material and undergoes an addition reaction to obtain 2-propoxybenzamide; then, it is reduced with N₂H₄•H₂O to obtain N-amino-2-propoxybenzamide; next, it undergoes a cyclization reaction with ethyl 3-(butyrylamino)-2-oxobutyrate to generate N-[1-[5-oxo-3-(2-propoxyphenyl)-4H-1,2,4-triazin-6-yl]ethyl]butyramide; finally, a second cyclization reaction is carried out under POCl₃ conditions to obtain 5-methyl-2-(2-propoxyphenyl)- 7-propyl-3H-imidazol[5,1-f][1,2,4]triazin-4-one was then sulfonated to give 3-(5-methyl-4-oxo-7-propyl-3H-imidazol[5,1-f][1,2,4]triazin-2-yl)-4-propoxybenzenesulfonyl chloride, which was then reacted with 2-piperazin-1-ylethanol to give the target product 2-[5-[4-(2-hydroxyethyl)piperazin-1-yl]sulfonyl-2-propoxyphenyl]-5-methyl-7-propyl-3H-imidazo[5,1-f][1,2,4]triazin-4-one, i.e., 19-O-propylhydroxyvardenafil, with the molecular formula C 24 H 34 N6O5S has a molecular weight of 504.22.

[0007] A method for preparing a 19-O-propylhydroxyvardenafil hapten and antigen, characterized in that: an active carboxyl group is introduced onto the synthesized 19-O-propylhydroxyvardenafil molecule; using the carboxyl group as a linker, a carboxyl arm chain is introduced into the 19-O-propylhydroxyvardenafil molecule via a succinic anhydride reaction to synthesize the 19-O-propylhydroxyvardenafil hapten; the resulting 19-O-propylhydroxyvardenafil hapten has the molecular formula C1. 28 H 38 N6O8S has a molecular weight of 618.25. The 19-O-propylhydroxyvardenafil hapten has a carboxyl group on the linker arm far from the molecular characteristic structure. Therefore, the 19-O-propylhydroxyvardenafil derivative can be coupled to the carrier protein BSA / OVA by the activated ester method to form the 19-O-propylhydroxyvardenafil antigen.

[0008] The synthesized 19-O-propylhydroxyvardenafil antigen was used in animal immunization experiments to obtain 19-O-propylhydroxyvardenafil antibody, which was detected by enzyme-linked immunosorbent assay (ELISA). The detection limit was IC50. 15 The concentration was 8.85 ng / mL, and the sensitivity was IC50. 50 It was 157.8 ng / mL.

[0009] The advantages and positive effects of this invention are: 1. This invention significantly reduces the formation of impurities such as vardenafil acid, vardenafil dimer, and 7-methylvardenafil by optimizing the synthetic route and reaction conditions. The product has high purity and high yield, reduces purification steps, and ensures drug quality.

[0010] 2. This invention uses mild and safe reaction conditions, without the need for high temperature and high pressure or precious metal catalysts, thus avoiding the safety hazards of traditional processes, reducing production risks and costs, and making it suitable for industrial production.

[0011] 3. The synthetic route of this invention using 2-propoxybenzonitrile as the starting material is simple and the reaction time is significantly shortened. The use of 2-piperazin-1-ylethanol instead of N-ethylpiperazine for condensation not only maintains the reactivity but also improves the water solubility of the product and the flexibility of subsequent derivatization.

[0012] 4. This invention establishes a complete method for impurity profiling and quality analysis during the synthesis process, which can achieve qualitative and quantitative control of 19-O-propylhydroxyvardenafil and its impurities, providing a scientific basis for drug quality standards.

[0013] 5. The hapten prepared by introducing a carboxyl linker in this invention exhibits good immunogenicity, the resulting antibody has high specificity, and the established enzyme-linked immunosorbent assay (ELISA) method has high sensitivity (IC50). 15 It was 8.85 ng / mL, IC50 50 (At 157.8 ng / mL), it can be used for illegal additive detection and safety monitoring. Attached Figure Description

[0014] Figure 1 This is a synthetic route diagram for 19-O-propylhydroxyvardenafil according to the method of the present invention; Figure 2 This is a schematic diagram of the synthetic route for the 19-O-propylhydroxyvardenafil hapten according to the method of the present invention. Figure 3 This is a structural diagram of the 19-O-propylhydroxyvardenafil hapten of the present invention. Figure 4 This is a synthetic route diagram of 19-O-propylhydroxyvardenafil antigen (VOA) according to the method of the present invention; Figure 5 This is a schematic diagram of the synthetic route for 19-O-propylhydroxyvardenafil antigen (BSA) according to the method of the present invention; Figure 6 This is the ELISA verification result of the 19-O-propylhydroxyvardenafil anti-antibody obtained by the method of the present invention; Figure 7 This is a schematic diagram of the test strip used in the method of the present invention; Figure 8 This is a schematic diagram of the detection results of the test strip of the method of the present invention. Detailed Implementation

[0015] The present invention will be further described in detail below through specific embodiments. The following embodiments are merely descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0016] Example 1 The detailed synthetic steps of 19-O-propylhydroxyvardenafil are as follows: like Figure 1 The first reactant 1 undergoes an addition reaction to obtain compound 2; compound 2 reacts with hydrazine hydrate to generate compound 3, which then undergoes a cyclization reaction with compound 4 to generate compound 5; compound 5 undergoes a cyclization reaction in the acid solvent POCl3 to generate intermediate 6; intermediate 6 reacts with chlorosulfonic acid to generate sulfonyl chloride intermediate 7; sulfonyl chloride intermediate 7 reacts with the second reactant to generate the target product 19-O-propylhydroxyvardenafil.

[0017] The first reactant is 2-propoxybenzonitrile; the second reactant is 2-piperazin-1-ylethanol, N-methylpiperazine, or water; compound 2 is 2-propoxybenzamide, compound 3 is N-amino-2-propoxybenzamide, compound 4 is ethyl 3-(butyrylamino)-2-oxobutyrate, compound 5 is N-[1-[5-oxo-3-(2-propoxyphenyl)-4H-1,2,4-triazin-6-yl]ethyl]butyramide, intermediate 6 is 5-methyl-2-(2-propoxyphenyl)-7-propyl-3H-imidazol[5,1-f][1,2,4]triazin-4-one, and intermediate 7 is 3-(5-methyl-4-oxo-7-propyl-3H-imidazol[5,1-f][1,2,4]triazin-2-yl)-4-propoxybenzenesulfonyl chloride.

[0018] The specific reaction steps are as follows: (1) 2-Propoxybenzonitrile (4.5 g, 27.92 mmol, 1 equivalent) was dissolved in toluene (50 mL), and AlCl3 (7 g, 52.50 mmol, 2.87 mL, 1.88 equivalent) and NH4Cl (3 g, 56.08 mmol, 2.01 equivalent) were added. The reaction mixture was stirred at 80 °C for 16 hours. The mixture was quenched by slow addition of H2O (150 mL). The resulting mixture was transferred to a separatory funnel, and the aqueous phase mixture was extracted with ethyl acetate (150 mL × 3). The combined organic layers were washed with brine (150 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 2-propoxybenzamide (2.5 g, 14.03 mmol, 50.25% yield) as a yellow solid, which was used directly for the next step without further purification.

[0019] (2) 2-Propoxybenzamide (2.1 g, 11.78 mmol, 1 equivalent) was dissolved in ethanol (30 mL), and N2H4•H2O (1 equivalent) was added. The reaction mixture was stirred at 20 °C for 1 hour. The reaction mixture was filtered and concentrated under reduced pressure to give crude N-amino-2-propoxybenzamide (2.1 g, 10.87 mmol, 92.23% yield) as a yellow solid, which was used directly for the next step without further purification.

[0020] (3) N-amino-2-propoxybenzamide (2.1 g, 10.87 mmol, 1 equivalent) and ethyl 3-(butyrylamino)-2-oxobutyrate 4 (2.34 g, 10.87 mmol, 1 equivalent) were dissolved in ethanol (30 mL). The reaction mixture was stirred at 80 °C for 4 hours. After filtration, the reaction mixture was concentrated under reduced pressure to obtain the residue. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate, from 1 / 0 to 5 / 1) to give N-[1-[5-oxo-3-(2-propoxyphenyl)-4H-1,2,4-triazin-6-yl]ethyl]butyramide (1.5 g, 4.36 mmol, 40.08% yield) as a colorless oil.

[0021] (4) N-[1-[5-oxo-3-(2-propoxyphenyl)-4H-1,2,4-triazin-6-yl]ethyl]butyramide (1.5 g, 4.36 mmol, 1 equivalent) was dissolved in DCE (dichloroethane) (30 mL), and POCl3 (667.81 mg, 4.36 mmol, 1 equivalent) was added. The reaction mixture was stirred at 80 °C for 4 hours under a nitrogen atmosphere. The mixture was quenched at 20 °C by slow addition of H2O (150 mL). The resulting mixture was transferred to a separatory funnel, and the aqueous phase mixture was extracted with ethyl acetate (150 mL × 3). The combined organic layers were washed with brine (150 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The resulting residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate, from 1 / 0 to 5 / 1) to give 5-methyl-2-(2-propoxyphenyl)-7-propyl-3H-imidazol[5,1-f][1,2,4]triazine-4-one (1 g, 3.06 mmol, 70.35% yield) as a white solid.

[0022] (5) 5-Methyl-2-(2-propoxyphenyl)-7-propyl-3H-imidazol[5,1-f][1,2,4]triazine-4-one (1 g, 3.06 mmol, 1 equivalent) was dissolved in chlorosulfonic acid (20 mL). The reaction mixture was stirred at 0 °C for 0.5 h. The mixture was quenched by slow addition of H2O (150 mL). The resulting mixture was transferred to a separatory funnel, and the aqueous phase mixture was extracted with ethyl acetate (150 mL × 3). The combined organic layers were washed with brine (150 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the residue. The resulting residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate, from 1 / 0 to 5 / 1) to give 3-(5-methyl-4-oxo-7-propyl-3H-imidazol[5,1-f][1,2,4]triazine-2-yl)-4-propoxybenzenesulfonyl chloride (1 g, 2.35 mmol, 76.82% yield) as a yellow solid.

[0023] (6) A solution of 3-(5-methyl-4-oxo-7-propyl-3H-imidazo[5,1-f][1,2,4]triazin-2-yl)-4-propoxybenzenesulfonyl chloride (0.6 g, 1.41 mmol, 1 equivalent) and 2-piperazin-1-ylethanol (0.2 g, 1.54 mmol, 1.09 equivalent) was dissolved in DMF (dimethylformamide) (10 mL), and K2CO3 (0.4 g, 2.89 mmol, 2.05 equivalent) was added. The reaction mixture was stirred at 25 °C for 1 hour. The mixture was quenched by slow addition of H2O (100 mL). The resulting mixture was transferred to a separatory funnel, and the aqueous layer was extracted with ethyl acetate (100 mL × 3). The combined organic layers were washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The resulting residue was subjected to pretreatment HPLC (column: Waters Xbrid GE BEH C). 18 The sample was purified to obtain 2-[5-[4-(2-hydroxyethyl)piperazin-1-yl]sulfonyl-2-propoxyphenyl]-5-methyl-7-propyl-3H-imidazo[5,1-f][1,2,4]triazin-4-one (0.23 g, 443.48 μmol, 31.41% yield), i.e., 19-O-propylhydroxyvardenafil, as a pale white solid. (250×50mm×10μm; mobile phase: [H2O(0.05% NH3H2O)-ACN]; gradient: 32%-62% B over 20.0 min).

[0024] Example 2 Synthesis of 19-O-propylhydroxyvardenafil hapten and antigen likeFigure 2 Weigh 40.00 mg (0.08 mmol) of 19-O-propylhydroxyvardenafil and dissolve it in 5 mL of anhydrous ethanol. While stirring slowly, add 12 mg (0.12 mmol) of succinic anhydride and react at room temperature for 24 hours in the dark to obtain a pale yellow liquid. The product is then concentrated by rotary evaporation to obtain a brown oily liquid, which is the 19-O-propylhydroxyvardenafil hapten, with the structural formula shown below. Figure 3 The structure of the derivatives was identified by nuclear mass spectrometry.

[0025] like Figure 4 , Figure 5 300 μl (0.015 mmol) of 19-O-propylhydroxyvardenafil hapten was dissolved in 1.5 mL of water. 200 μL each of 0.015 mmol NHS and 0.031 mmol EDC solution were added sequentially to achieve a molar ratio of 19-O-propylhydroxyvardenafil hapten:EDC:NHS of 1:2:1. After stirring at room temperature for 24 h, the mixture was slowly added to 5 mL of protein solution (25 mg BSA / OVA in 0.01 mol / L PBS solution), and stirred slowly for another 24 h to obtain 19-O-propylhydroxyvardenafil antigen. Dialysis with PBS solution at room temperature was performed for 3-4 days, with the dialysate changed twice daily.

[0026] Example 3 Preparation of 19-O-propylhydroxyvardenafil antibody Two New Zealand white rabbits were subcutaneously immunized six times at 2-week intervals with an immunogen (hapten-OVA or hapten-BSA) at 1 mg / mL concentration. Freund's complete and incomplete adjuvants were mixed with equal volumes of the immunogen for the initial injection and subsequent booster immunizations, respectively. After bleeding from the rabbits, the collected whole blood was coagulated and centrifuged at 4°C to separate the antiserum. The antiserum was purified using a Protein A-Sepharose 4 B affinity chromatography column.

[0027] Example 4 The specific steps for the enzyme-linked immunosorbent assay (ELISA) are as follows: (1) Coating of 96-well plate: Dilute the coating agent (19-O-propyl hydroxyvardenafil antigen) with coating solution, add 100 μL of diluted coating agent to each well (concentration set at 10 μg-20 μg / mL), and then incubate the coated 96-well plate in a 37℃ incubator for 3 hours.

[0028] After the reaction is complete, remove the coating solution (quickly turn upside down and shake dry), then wash three times with PBST (shake for two minutes to mix), and then pat dry (if there are air bubbles in the well after patting dry, turn upside down on filter paper and pat dry or puncture with a pipette tip).

[0029] (2) Blocking: Add 200 μL of blocking solution containing 0.5% skim milk powder to the plate, incubate at 37°C for 1 h, then remove the milk powder solution, wash three times with PBST and pat dry.

[0030] (3) Add antibody and standard solutions: Add 50 μL of PBS buffer to the control well, add 50 μL of standard solutions with different concentration gradients (0.1, 1, 10, 100, 500, 1000, 2000, 5000, 8000, 10000) to the sample well, add 50 μL of antibody dilution (4000 times) to each of the above wells, and add 100 μL of PBS buffer to the blank well.

[0031] The 96-well plate was incubated in a 37°C incubator for 1 h. The solution after the reaction was removed, and the plate was washed four times with PBST and then patted dry.

[0032] (4) Add enzyme-labeled secondary antibody: After dilution with PBS buffer, add 100 μL to each well (rabbit secondary antibody dilution: 10 μL added to 10 mL PBS, choose one).

[0033] The reaction was carried out in a constant temperature oven at 37℃ for 30 min. The solution after the reaction was removed, washed five times with PBST, and then patted dry.

[0034] (5) Color development: After mixing a certain volume of solution A and solution B, add 100 μL to each well (substrate A:substrate B = 11:0.38) and incubate for 15-20 min. (A: Dissolve 4.10 g sodium acetate, 1.58 g citric acid, and 1.25 g β-dextrin in 400 mL of ultrapure water. Dissolve 214.0 mg of hydrourea peroxide in 100 mL of pure water three times (dissolve once and wash off the undissolved part from the tube wall twice). Then add the solution to the previous 400 mL after each dissolution to ensure the total system is 500 mL. B: Dissolve 40.0 mg TMB in 4 mL of DMSO.) (6) Termination of reaction: Add 50 μL of sulfuric acid to each well to stop the colorimetric reaction. After termination, measure the od value within 30 min.

[0035] (7) Instrument detection: The absorbance value at 450 nm was measured using an ELISA reader (absorbance value (OD). The value of OD between 0.8 and 1.2 was selected as the antibody titer. The optimal antibody dilution factor and coating amount of the coating agent were selected based on the inhibition rate.

[0036] The ELISA validation results of the 19-O-propylhydroxyvardenafil anti-antibody are as follows: Figure 6 As shown.

[0037] Example 5 Preparation and detection of test strips (1) Assembly of test strips The Tijin rapid test strip is made up of an NC membrane (nitrocellulose membrane), a sample pad, an absorbent pad, and a PVC plastic base plate.

[0038] Using an XYZ 3D spray-scratch instrument, the coating antigen (19-O-propylhydroxyvardenafil antigen) was sprayed onto the NC membrane at a spray volume of 1 μL / cm as the test line (T line). Goat anti-rabbit IgG was sprayed onto the NC membrane using the same method and dosage as the control line (C line). The test line (T line) and control line (C line) were located in the middle of the NC membrane and spaced 6 mm apart from each other. After drying at 37°C for 12 h, the NC membrane was pasted onto the middle part of the backing plate, with the sample pad overlapping the T line end of the NC membrane by 1 mm. The absorbent pad was pasted onto the upper side of the cellulose membrane and overlapped with the cellulose membrane by 1 mm. The assembled test strips were cut into 3.5 mm wide strips using a chopper.

[0039] (2) Preparation of gold-labeled antibodies Take 1 mL of colloidal gold solution, add 0.2 mol / L K₂CO₃, adjust the pH of the solution to approximately 8.0, add 10 μg of the prepared 19-O-propylhydroxyvardenafil polyclonal antibody and incubate for 30 min, then add 10% wt BSA solution and incubate for another 30 min. Centrifuge at 10,000 rpm for 20 min at 4 °C, discard the supernatant, and resuspend in 200 μL of 0.2 mol / L pH 7.4 phosphate buffer (containing 0.5% v / v Tween-20, 0.5% wt LF, 5% wt sucrose, 0.3% wt polyvinylpyrrolidone (PVP), and 0.03% v / v procline-300), and store at 4 °C. The colloidal gold-mAb is obtained.

[0040] (3) Test strip detection After mixing the colloidal gold-mAb with the sample solution, the mixture was applied to the sample pad and analyzed chromatographically on an NC membrane. The presence of vardenafil-like substances in the sample affects the interaction between the colloidal gold-mAb, the 19-O-propylhydroxyvardenafil antigen, and the antibody on the test strip. In the absence of vardenafil-like substances, the colloidal gold-mAb heterotropically binds to the 19-O-propylhydroxyvardenafil antigen on the T line, forming a visible red band. Simultaneously, the colloidal gold-mAb also binds to the antibody on the C line, producing a red band, indicating a negative result. For samples containing vardenafil-like substances, the molecules of these substances bind to the colloidal gold-mAb, reducing the availability of interaction between the unbound colloidal gold-mAb and the 19-O-propylhydroxyvardenafil antigen on the T line. Therefore, the intensity of the band on the T line decreases, indicating a positive result. Regardless of the concentration of 19-O-propylhydroxyvardenafil and its analogues, the C line always displays a band as a quality control indicator. If there is no color on line C, the test strip is invalid. A diagram of the test strip and test results are shown below. Figure 7 , Figure 8 As shown.

[0041] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A 19-O-propylhydroxyvardenafil, characterized in that: Its molecular formula is C 24 H 34 N6O5S, with a molecular weight of 504.22, has the following structural formula: 。 2. A method for synthesizing 19-O-propylhydroxyvardenafil, characterized in that: This method involves using 2-propoxybenzonitrile as a starting material, undergoing an addition reaction to obtain 2-propoxybenzamide, followed by a reduction reaction with the starting material N2H4•H2O to obtain N-amino-2-propoxybenzamide; then, a cyclization reaction is carried out with ethyl 3-(butyrylamino)-2-oxobutyrate to generate N-[1-[5-oxo-3-(2-propoxyphenyl)-4H-1,2,4-triazin-6-yl]ethyl]butyramide, followed by a second cyclization reaction under POCl3 conditions to obtain 5-methyl-2-(2-propoxyphenyl)-7-propyl-3H- Imidazol[5,1-f][1,2,4]triazin-4-one was then sulfonated to give 3-(5-methyl-4-oxo-7-propyl-3H-imidazol[5,1-f][1,2,4]triazin-2-yl)-4-propoxybenzenesulfonyl chloride, which was then reacted with 2-piperazin-1-ylethanol to give the target product 2-[5-[4-(2-hydroxyethyl)piperazin-1-yl]sulfonyl-2-propoxyphenyl]-5-methyl-7-propyl-3H-imidazo[5,1-f][1,2,4]triazin-4-one, i.e., 19-O-propylhydroxyvardenafil.

3. A 19-O-propylhydroxyvardenafil hapten, characterized in that: Its structural formula is: 。 4. A method for preparing a 19-O-propylhydroxyvardenafil hapten and antigen, characterized in that: An active carboxyl group is introduced onto the 19-O-propylhydroxyvardenafil molecule as described in claim 1 or 2. Using this carboxyl group as a linker, a carboxyl arm chain is introduced into the 19-O-propylhydroxyvardenafil molecule via a succinic anhydride reaction to synthesize a 19-O-propylhydroxyvardenafil hapten. The resulting 19-O-propylhydroxyvardenafil hapten has the molecular formula C1. 28 H 38 N6O8S has a molecular weight of 618.

25. The 19-O-propylhydroxyvardenafil hapten has a carboxyl group on the linker arm far from the molecular characteristic structure. Therefore, the 19-O-propylhydroxyvardenafil derivative can be coupled to the carrier protein BSA / OVA by the activated ester method to form the 19-O-propylhydroxyvardenafil antigen.

5. An application of 19-O-propylhydroxyvardenafil, characterized in that: The synthesized 19-O-propylhydroxyvardenafil antigen was used in animal immunization experiments to obtain 19-O-propylhydroxyvardenafil antibody. This antibody was then applied to the detection of vardenafil in food, and the result was obtained by enzyme-linked immunosorbent assay (ELISA), with a detection limit of IC50. 15 The concentration was 8.85 ng / mL, and the sensitivity was IC50. 50 The concentration was 157.8 ng / mL.

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