Preparation method and application of 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolite

Abstract

The invention belongs to the technical field of chemical pharmacy, and particularly relates to a preparation method and application of a 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolite. The preparation method provided by the invention comprises the following steps: under an alkaline condition, substituting hydrogen at a specific position on the structure of a compound as shown in a formula I by a group of a compound as shown in a formula II, then converting an acetoxy group on the structure of a substitution reaction product into a hydroxyl group, and performing Tempo oxidation to obtain a 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolite. Meanwhile, reaction parameters such as temperature, time and pH value in reaction steps such as substitution reaction are optimized, and a product is separated and purified by using liquid chromatography, so that the 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolite with high yield and purity can be prepared; the technical problem that in the prior art, a 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolite synthesis method is lacked is solved.

Description

Technical Field

[0001] The present application belongs to the field of chemical pharmaceutical technology, and in particular relates to a preparation method and application of a 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolite. Background Art

[0002] The API Cavosonstat N91115 is an innovative drug developed by Nivalis Therapeutics. Its Chinese name is 3-chloro-4-(6-hydroxy-quinolin-2-yl)-benzoic acid. Cavosonstat N91115 can be processed into oral tablets and oral capsules for the treatment of cystic fibrosis, asthma and other diseases. It has successfully obtained clinical trial approval from the FDA and completed Phase I and Phase II clinical trials in the United States.

[0003] Domestic Langyu Pharmaceutical Technology (Hangzhou) Co., Ltd. and Kangyuan Pharmaceutical Group Co., Ltd. introduced the raw material Cavosonstat N91115 (3-chloro-4-(6-hydroxy-quinolin-2-yl)-benzoic acid) mainly to develop asthma drugs in the form of oral capsule preparations; after taking Cavosonstat N91115, the metabolite (2R,3R,4R,5S,6R)-6-((2-(4-carboxy-2-chlorophenyl)quinolin-6-yl)oxy)-3,4,5-trihydroxytetrahydropyran-2-carboxylic acid compound will be produced under the action of various drug metabolizing enzymes in the human body; in order to investigate the biological and toxicological activities of the drug Cavosonstat N91115, 3-chloro-4-(6-hydroxy-quinolin-2-yl)-benzoic acid metabolites are needed as reference substances.

[0004] However, there is currently no literature reporting a synthesis method for 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolites. Therefore, it is necessary to develop a synthesis method for 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolites and provide a reference substance to meet the needs of investigating the quality of research drugs. Summary of the invention

[0005] In view of this, the present application provides a preparation method and application of 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolites, which are used to solve the technical problem of the lack of a synthesis method for 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolites in the prior art.

[0006] The first aspect of the present application provides a method for preparing a 3-chloro-4-(6-hydroxy-quinolin-2-yl)-benzoic acid metabolite, the preparation method comprising the steps of:

[0007] Step S1, adding the compound 3-chloro-4-(6-hydroxy-quinolin-2-yl)-benzoic acid represented by Formula I and a first alkaline substance into an organic solvent for heating reaction to obtain an alkaline heating reaction solution;

[0008] Step S2, adding the compound represented by formula II to an alkaline heated reaction solution cooled to room temperature to carry out a substitution reaction, thereby obtaining a substitution reaction solution containing the compound represented by formula III;

[0009] Step S3, adding a second alkaline substance to the substitution reaction solution containing the compound represented by formula III to carry out a hydrolysis reaction to obtain a compound represented by formula IV;

[0010] Step S4, adding tetramethylpiperidinyl oxide and a third alkaline substance to a solution of the compound represented by formula IV to carry out an oxidation reaction to obtain a 3-chloro-4-(6-hydroxy-quinolin-2-yl)-benzoic acid metabolite represented by formula V;

[0011] The compound shown in formula II is:

[0012] ;

[0013] The compound shown in formula III is:

[0014] ;

[0015] The compound shown in formula IV is:

[0016] .

[0017] Preferably, in step S1, the heating reaction temperature is 45°C to 65°C, and the time is 0.5h to 2h.

[0018] Preferably, in step S1, the first alkaline substance is selected from at least one of sodium hydroxide, potassium hydroxide, sodium tert-butoxide, and potassium tert-butoxide;

[0019] The organic solvent is selected from at least one of tetrahydrofuran, dimethylformamide and dimethyl sulfoxide.

[0020] Preferably, in step S2, the room temperature of the substitution reaction is 15° C. to 25° C., and the time is 1 h to 3 h.

[0021] Preferably, in step S3, the pH value of the hydrolysis reaction is 12-13, and the time is 8h-16h.

[0022] Preferably, in step S3, the second alkaline substance is selected from at least one of sodium hydroxide, lithium hydroxide, cesium carbonate, and diazabicyclo.

[0023] Preferably, in step S4, the temperature of the oxidation reaction is 5°C to 15°C, and the pH value is 6.5 to 8.0.

[0024] Preferably, in step S4, the third alkaline substance is selected from at least one of sodium bicarbonate, sodium hydroxide, calcium hypochlorite, and calcium hydroxide;

[0025] The solution of the compound represented by formula IV is an aqueous solution of the compound represented by formula IV.

[0026] Preferably, in step S1 and step S2, the molar ratio of the compound represented by formula I to the compound represented by formula II is 1:1-3.

[0027] Preferably, step S3 specifically includes:

[0028] Step S31, adding a second alkaline substance to the substitution reaction solution to perform hydrolysis, acidification with hydrochloric acid, and extraction with ethyl acetate in sequence to obtain a hydrolysis reaction solution containing the compound represented by formula IV;

[0029] Step S32, subjecting the hydrolysis reaction solution containing the compound represented by formula IV to separation and purification by liquid chromatography and freeze-drying in sequence to obtain the compound represented by formula IV.

[0030] Preferably, step S4 specifically includes:

[0031] Step S41, adding tetramethylpiperidinyl oxide and a third alkaline substance to a solution of the compound represented by formula IV to carry out an oxidation reaction, thereby obtaining an oxidation reaction solution containing the compound represented by formula V.

[0032] Step S42, subjecting the oxidation reaction solution containing the compound of formula V to liquid chromatography separation and purification, and freeze-drying in sequence to obtain the compound of formula IV, 3-chloro-4-(6-hydroxy-quinolin-2-yl)-benzoic acid metabolite.

[0033] The second aspect of the present application provides the use of a 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolite prepared by the preparation method described in the first aspect in drug detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1The mass spectrum of the 3-chloro-4-(6-hydroxy-quinolin-2-yl)-benzoic acid metabolite prepared by the preparation method provided in Example 1 of the present application;

[0036] Figure 2 The hydrogen nuclear magnetic resonance spectrum of the 3-chloro-4-(6-hydroxy-quinolin-2-yl)-benzoic acid metabolite prepared by the preparation method provided in Example 1 of the present application;

[0037] Figure 3 This is the carbon nuclear magnetic resonance spectrum of the 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolite prepared by the preparation method provided in Example 1 of the present application. DETAILED DESCRIPTION

[0038] The present application provides a preparation method and application of 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolites, which are used to solve the technical problem of the lack of a synthesis method for 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolites in the prior art.

[0039] The technical solution of the present application will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0040] In view of the current lack of a synthetic method for 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolites, it is difficult to meet the demand for drug detection reference substances for 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid; the present application provides a method for preparing 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolites, the preparation method comprising: firstly adding a compound 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid shown in formula I and a first alkaline substance to an organic solvent for heating reaction to obtain an alkaline heating reaction solution; after cooling the alkaline heated reaction solution to room temperature, adding the compound shown in formula II to carry out a substitution reaction to obtain a substitution reaction solution containing the compound shown in formula III; then adding a second alkaline substance to the substitution reaction solution to carry out a hydrolysis reaction to obtain a compound shown in formula IV; then adding tetramethylpiperidinyl oxide and a third alkaline substance to the solution of the compound shown in formula IV to carry out a Tempo oxidation reaction to obtain a compound shown in formula V, 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolite; the reaction process is schematically shown as follows:

[0041] .

[0042] In the preparation method of 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolites provided in the present application, firstly, a first alkaline substance and a compound 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid shown in formula I are heated to provide an alkaline environment, and then a compound shown in formula II is added at room temperature for a substitution reaction, so that hydrogen at a specific position on the structure of the compound shown in formula I is substituted, and the regional selectivity of the substitution reaction is good, and the compound group shown in formula II is connected to the compound shown in formula I to obtain a compound shown in formula III; then a second alkaline substance is used, and hydrochloric acid is used for acid adjustment and ethyl acetate is used for extraction, and the acetoxy group on the structure of the compound shown in formula III is converted into a hydroxyl group by a hydrolysis reaction to obtain a compound shown in formula IV; then a third alkaline substance is used to increase the pH of the reaction system, and Tempo oxidation is used to obtain 3-chloro-4- (6-Hydroxy-quinoline-2-yl)-benzoic acid metabolite, whose structure is shown in Formula V; it can be seen from the nuclear magnetic resonance hydrogen spectrum, nuclear magnetic resonance carbon spectrum, and mass spectrum that the preparation method provided in the present application successfully synthesizes the 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolite shown in Formula V; and the yield of the synthesized 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolite is relatively high, reaching more than 60%, and the purity is also more than 99.60%, which shows that the preparation method provided in the present application can efficiently provide the 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolite shown in Formula V, overcomes the defect of the current lack of a synthesis method for 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolites, and can meet the demand for drug detection reference substances for 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid.

[0043] As a preferred technical solution, in the preparation method of 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolites provided in the present application, in order to provide a better alkaline environment, the hydrogen at a specific position on the structure of the compound represented by formula I is effectively replaced; the present application first heats the compound represented by formula I and the first alkaline substance at a temperature of 45°C to 65°C for 0.5h to 2h, then lowers the temperature of the reaction system to room temperature (20°C±5°C) and then adds the compound represented by formula II to carry out a substitution reaction.

[0044] As a preferred technical solution, in the preparation method of 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolites provided in the present application, in order to effectively convert the acetoxy group in the structure of the compound represented by formula III obtained by the substitution reaction into a hydroxyl group; the present application controls the pH value of the reaction system to 12~13 after adding the second alkaline substance and reacts for 8h~16h, then adjusts the acid with hydrochloric acid and uses ethyl acetate for extraction.

[0045] As a preferred technical solution, in the method for preparing 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolites provided in the present application, in order to effectively Tempo-oxidize the compound represented by Formula IV, the present application controls the temperature of the oxidation reaction to be 5°C to 15°C and the pH value to be 6.5 to 8.0.

[0046] As a preferred technical solution, in the method for preparing 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolites provided by the present application, in order to promote the Tempo oxidation reaction, the present application controls the type of the third alkaline substance added in the oxidation reaction, and the third alkaline substance is selected from at least one of sodium bicarbonate, sodium hydroxide, calcium hypochlorite, and calcium hydroxide; calcium hypochlorite having both oxidizing and alkalinity is further preferred.

[0047] As a preferred technical solution, in the preparation method of 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolites provided in the present application, the compound represented by formula IV and the compound represented by formula V are also purified, and the purification process is to use liquid chromatography separation and purification and freeze-dry the eluate.

[0048] As a preferred technical solution, in the preparation method of 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolites provided in the present application, in order to fully access the compound group of formula II to the structure of the compound represented by formula I; the present application adds an excess of the compound represented by formula II during the substitution reaction, and the molar ratio of the compound represented by formula I to the compound represented by formula II is 1:1~3.

[0049] The following is a detailed description of a 3-chloro-4-(6-hydroxy-quinolin-2-yl)-benzoic acid metabolite provided by the present application in conjunction with an example.

[0050] Example 1

[0051] Example 1 of the present application provides a method for preparing a 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolite, the preparation method comprising a heating reaction step, a substitution reaction step, a hydrolysis reaction step, and an oxidation reaction step.

[0052] The heating reaction step comprises: adding 10.00 g of compound I (0.03 mol) and 250 mL of dimethyl sulfoxide into a 500 mL four-necked bottle under nitrogen protection, stirring until dissolved, adding 9.62 g of sodium hydroxide, heating to 55° C., and reacting for 1 hour to obtain an alkaline heating reaction solution;

[0053] The steps of the substitution reaction include: cooling to 20° C., adding 27.44 g of compound II (0.07 mol), reacting for 2 to 3 hours, and obtaining a substitution reaction solution containing a compound represented by formula III;

[0054] The hydrolysis reaction step comprises: adding 160 mL of water and 16.81 g of lithium hydroxide monohydrate to a substitution reaction solution containing a compound represented by formula III, adjusting the pH to 12-13, stirring the reaction for 12 h, adjusting the pH to 2.75 with hydrochloric acid after the reaction, a large amount of crystals precipitate, adding 200 mL of ethyl acetate, stirring until the crystals are dissolved, standing for phase separation, extracting the aqueous phase with 150 mL × 3 times of ethyl acetate, filtering the aqueous phase, and obtaining a hydrolysis reaction solution containing a compound represented by formula IV; then subjecting the hydrolysis reaction solution containing the compound represented by formula IV to liquid chromatography separation and purification, and freeze drying to obtain a compound represented by formula IV, with a mass of 10.63 g, a yield of 69%, and a purity of 99.65%; the diameter of the chromatographic column used in the liquid chromatography separation and purification process is 50 mm, the filler is Unisil8-120 C18, the mobile phase A is acetonitrile, the mobile phase B is 0.2% acetic acid aqueous solution, the elution method is gradient elution, and the gradient elution parameters are shown in Table 1.

[0055] The oxidation reaction comprises the following steps: adding 1.50 g of the compound shown in formula IV and 450 mL of water into a 500 mL four-necked bottle, stirring, adding 0.1 N sodium hydroxide solution to adjust the pH to 5.60, completely dissolving the solid, cooling to 10° C., slowly adding 25 mg of tetramethylpiperidinium oxide (Tempo) and 468 mg of calcium hypochlorite, controlling the temperature at 10° C. to react for 45 minutes, and adding calcium hypochlorite during the reaction process to control the pH to be between 6.5 and 8.0; after the reaction, the mother liquor is separated and purified by liquid chromatography, and freeze-dried to obtain the 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolite shown in formula V, with a mass of 1.05 g, a yield of 68%, and a purity of 99.66%; the diameter of the chromatographic column used in the liquid chromatography separation and purification process is 50 mm, and the filler is Unisil 8-120 C18, mobile phase A was acetonitrile, mobile phase B was 0.2% acetic acid aqueous solution for elution, the elution mode was gradient elution, and the gradient elution parameters are shown in Table 1.

[0056] Table 1: Gradient elution parameters during liquid chromatography separation and purification

[0057]

[0058] Example 2

[0059] Example 2 of the present application provides a method for preparing a 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolite, the preparation method comprising a heating reaction step, a substitution reaction step, a hydrolysis reaction step, and an oxidation reaction step.

[0060] The heating reaction step comprises: adding 10.00 g of compound I (0.03 mol) and 250 mL of dimethyl sulfoxide into a 500 mL four-necked bottle under nitrogen protection, stirring until dissolved, adding 9.62 g of potassium tert-butoxide, heating to 55° C., and reacting for 1 hour to obtain an alkaline heating reaction solution;

[0061] The steps of the substitution reaction include: cooling to 20° C., adding 13.72 g of compound II (0.07 mol), reacting for 2 to 3 hours, and obtaining a substitution reaction solution containing a compound represented by formula III;

[0062] The hydrolysis reaction step comprises: adding 160 mL of water and 16.81 g of sodium hydroxide to a substitution reaction solution containing a compound represented by formula III, adjusting the pH to 12-13, stirring the reaction for 12 h, adjusting the pH to 2.75 with hydrochloric acid after the reaction is completed, a large amount of crystals are precipitated, adding 200 mL of ethyl acetate, stirring until the crystals are dissolved, standing for phase separation, extracting the aqueous phase with 150 mL×3 times of ethyl acetate, filtering the aqueous phase, and obtaining a hydrolysis reaction solution containing a compound represented by formula IV; then subjecting the hydrolysis reaction solution containing the compound represented by formula IV to liquid chromatography separation and purification, and freeze drying to obtain a compound represented by formula IV, with a mass of 9.09 g, a yield of 59%, and a purity of 99.70%; the diameter of the chromatographic column used in the liquid chromatography separation and purification process is 50 mm, the filler is Unisil 8-120 C18, the mobile phase A is acetonitrile, the mobile phase B is 0.2% acetic acid aqueous solution, and the elution process is the same as that in Example 1.

[0063] The oxidation reaction step comprises: adding 1.50g of compound IV and 450mL of water into a 500mL four-necked bottle, stirring, adding 0.1N sodium hydroxide solution to adjust the pH to 4.95, completely dissolving the solid, cooling to 10°C, slowly adding 25mg of tetramethylpiperidinium oxide Tempo and 468mg of calcium hypochlorite, controlling the temperature at 10°C for reaction, and adding calcium hypochlorite during the reaction process to control the pH between 6.5 and 8.0; after the reaction is completed, the mother liquor is separated and purified by liquid chromatography, and freeze-dried to obtain a 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolite shown in formula V, with a mass of 0.89g, a yield of 58%, and a purity of 99.63%; the diameter of the chromatographic column used in the liquid chromatography separation and purification process is 50mm, the filler is Unisil 8-120 C18, the mobile phase A is acetonitrile, and the mobile phase B is 0.2% acetic acid aqueous solution for elution, and the elution process is the same as that in Example 1.

[0064] Example 3

[0065] Example 3 of the present application provides a method for preparing a 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolite, the preparation method comprising a heating reaction step, a substitution reaction step, a hydrolysis reaction step, and an oxidation reaction step.

[0066] The heating reaction step comprises: adding 10.00 g of compound I (0.03 mol) and 250 mL of dimethyl sulfoxide into a 500 mL four-necked bottle under nitrogen protection, stirring until dissolved, adding 6.41 g of sodium tert-butoxide, heating to 55° C., and reacting for 1 hour to obtain an alkaline heating reaction solution;

[0067] The steps of the substitution reaction include: cooling to 20° C., adding 27.44 g of compound II (0.07 mol), reacting for 2 to 3 hours, and obtaining a substitution reaction solution containing a compound represented by formula III;

[0068] The hydrolysis reaction step comprises: adding 160 mL of water and 16.81 g of lithium hydroxide monohydrate to a substitution reaction solution containing a compound represented by formula III, adjusting the pH to 12-13, stirring the reaction for 12 h, adjusting the pH to 2.75 with hydrochloric acid after the reaction is completed, a large amount of crystals precipitate, adding 200 mL of ethyl acetate, stirring until the crystals are dissolved, standing for phase separation, extracting the aqueous phase with 150 mL×3 times of ethyl acetate, filtering the aqueous phase, and obtaining a hydrolysis reaction solution containing a compound represented by formula IV; then subjecting the hydrolysis reaction solution containing the compound represented by formula IV to liquid chromatography separation and purification, and freeze-drying to obtain a compound represented by formula IV, with a mass of 9.55 g, a yield of 60%, and a purity of 99.78%; the diameter of the chromatographic column used in the liquid chromatography separation and purification process is 50 mm, the filler is Unisil8-120 C18, the mobile phase A is acetonitrile, the mobile phase B is 0.2% acetic acid aqueous solution, and the elution process is the same as that in Example 1.

[0069] The oxidation reaction step comprises: adding 1.50g of compound IV and 450mL of water into a 500mL four-necked bottle, stirring, adding 0.1N sodium hydroxide solution to adjust the pH to 7.09, completely dissolving the solid, cooling to 10°C, slowly adding 25mg of tetramethylpiperidinium oxide Tempo and 468mg of calcium hypochlorite, controlling the temperature at 10°C for reaction, and adding calcium hypochlorite during the reaction process to control the pH between 6.5 and 8.0; after the reaction is completed, the mother liquor is separated and purified by liquid chromatography, and freeze-dried to obtain a 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolite shown in formula V, with a mass of 0.94g, a yield of 61%, and a purity of 99.80%; the diameter of the chromatographic column used in the liquid chromatography separation and purification process is 50mm, the filler is Unisil 8-120 C18, the mobile phase A is acetonitrile, and the mobile phase B is 0.2% acetic acid aqueous solution for elution, and the elution process is the same as that in Example 1.

[0070] Experimental Example 1

[0071] Experimental Example 1 of the present application conducted an experimental test on the 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolite shown in Formula V prepared in Example 1, and the experiment included mass spectrometry, nuclear magnetic resonance hydrogen spectrum and nuclear magnetic resonance carbon spectrum analysis of the 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolite shown in Formula V.

[0072] The results of mass spectrometry, hydrogen nuclear magnetic resonance spectrum and carbon nuclear magnetic resonance spectrum analysis are as follows Figure 1-3 As shown, from Figure 1-3 It can be seen qualitatively that the preparation method provided by the present application successfully synthesizes 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolites, overcoming the current lack of 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolites, namely (2R,3R,4R,5S,6R)-6-((2-(4-carboxy-2-chlorophenyl)quinoline-6-yl)oxy)-3,4,5-trihydroxytetrahydropyran-2-carboxylic acid compounds; overcomes the current lack of synthesis methods for 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolites, and can prepare 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolites in large quantities, meeting the demand for drug detection reference substances for 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid.

[0073] At the same time, as described in Examples 1-3, the yield of the compound represented by formula IV prepared by substitution reaction and hydrolysis reaction in the present application is 59% to 69%, which indicates that in the process of the substitution reaction of the present application, the hydrogen at a specific position on the structure of the compound represented by formula I can be substituted, and the regional selectivity of the substitution reaction is good; and the compound represented by formula III obtained after the substitution reaction, in the process of the hydrolysis reaction, also converts most of the acetoxy groups into hydroxyl groups to obtain the compound represented by formula IV.

[0074] At the same time, as described in Examples 1-3, the yield of the 3-chloro-4-(6-hydroxy-quinoline-2-yl)-benzoic acid metabolite of formula V prepared by the Tempo oxidation reaction after the substitution reaction and the hydrolysis reaction is between 58% and 68%, which indicates that the present application successfully oxidized most of the compounds of formula IV during the oxidation reaction.

[0075] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing a 3-chloro-4-(6-hydroxy-quinolin-2-yl)-benzoic acid metabolite, characterized in that: Includes steps: Step S1, adding the compound 3-chloro-4-(6-hydroxy-quinolin-2-yl)-benzoic acid represented by Formula I and a first alkaline substance into an organic solvent for heating reaction to obtain an alkaline heating reaction solution; Step S2, adding the compound represented by formula II to an alkaline heated reaction solution cooled to room temperature to carry out a substitution reaction, thereby obtaining a substitution reaction solution containing the compound represented by formula III; Step S3, adding a second alkaline substance to the substitution reaction solution containing the compound represented by formula III to carry out a hydrolysis reaction to obtain a compound represented by formula IV; Step S4, adding tetramethylpiperidinyl oxide and a third alkaline substance to a solution of the compound represented by formula IV to carry out an oxidation reaction to obtain a 3-chloro-4-(6-hydroxy-quinolin-2-yl)-benzoic acid metabolite; The compound shown in formula II is: ； The compound shown in formula III is: ； The compound shown in formula IV is: 。 2. The method for preparing a 3-chloro-4-(6-hydroxy-quinolin-2-yl)-benzoic acid metabolite according to claim 1, characterized in that: In step S1, the heating reaction temperature is 45°C to 65°C, and the time is 0.5h to 2h.

3. The method for preparing a 3-chloro-4-(6-hydroxy-quinolin-2-yl)-benzoic acid metabolite according to claim 1, characterized in that: In step S2, the room temperature of the substitution reaction is 15°C to 25°C, and the time is 1h to 3h.

4. The method for preparing a 3-chloro-4-(6-hydroxy-quinolin-2-yl)-benzoic acid metabolite according to claim 1, characterized in that: In step S3, the pH value of the hydrolysis reaction is 12-13, and the time is 8h-16h.

5. The method for preparing a 3-chloro-4-(6-hydroxy-quinolin-2-yl)-benzoic acid metabolite according to claim 1, characterized in that: In step S4, the temperature of the oxidation reaction is 5°C to 15°C, and the pH value is 6.5 to 8.

0.

6. The method for preparing a 3-chloro-4-(6-hydroxy-quinolin-2-yl)-benzoic acid metabolite according to claim 1, characterized in that: In step S1 and step S2, the molar ratio of the compound represented by formula I to the compound represented by formula II is 1:1-3.

7. The method for preparing a 3-chloro-4-(6-hydroxy-quinolin-2-yl)-benzoic acid metabolite according to claim 1, characterized in that: Step S3 specifically includes: Step S31, adding a second alkaline substance to the substitution reaction solution to perform hydrolysis, acidification with hydrochloric acid, and extraction with ethyl acetate in sequence to obtain a hydrolysis reaction solution containing the compound represented by formula IV; Step S32, subjecting the hydrolysis reaction solution containing the compound represented by formula IV to separation and purification by liquid chromatography and freeze-drying in sequence to obtain the compound represented by formula IV.

8. The method for preparing a 3-chloro-4-(6-hydroxy-quinolin-2-yl)-benzoic acid metabolite according to claim 1, characterized in that: Step S4 specifically includes: Step S41, adding tetramethylpiperidinyl oxide and a third alkaline substance to a solution of the compound represented by formula IV to carry out an oxidation reaction, thereby obtaining an oxidation reaction solution containing the compound represented by formula V; Step S42, subjecting the oxidation reaction solution containing the compound of formula V to liquid chromatography separation and purification, and freeze-drying in sequence to obtain the compound of formula IV, 3-chloro-4-(6-hydroxy-quinolin-2-yl)-benzoic acid metabolite.

9. The method for preparing a 3-chloro-4-(6-hydroxy-quinolin-2-yl)-benzoic acid metabolite according to claim 1, characterized in that: In step S4, the third alkaline substance is selected from at least one of sodium bicarbonate, sodium hydroxide, calcium hypochlorite and calcium hydroxide.

10. Use of a 3-chloro-4-(6-hydroxy-quinolin-2-yl)-benzoic acid metabolite prepared by the preparation method according to any one of claims 1 to 9 in drug detection.

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