阅读说明：本技术 二碘异酞酰氯类化合物、其制备方法和应用 (Diiodoisophthaloyl chloride compound, preparation method and application thereof ) 是由 辛坤云 李壮 童耀 危军 吴灵静 王晶翼 于 2020-05-25 设计创作，主要内容包括：本发明涉及5-氨基-4-氯-2,6-二碘异酞酰氯和5-氨基-2-氯-4,6-二碘异酞酰氯、其制备方法及其在起始物料5-氨基-2,4,6-三碘异酞酰氯和/或碘普罗胺等造影剂的质量控制中的用途。其有利于进一步提高5-氨基-2,4,6-三碘异酞酰氯及其后续终产物的药物质量,减少可能导致的副作用,有利于提高用药安全性。(The invention relates to 5-amino-4-chloro-2, 6-diiodoisophthaloyl chloride and 5-amino-2-chloro-4, 6-diiodoisophthaloyl chloride, a preparation method thereof and application thereof in quality control of starting materials, namely 5-amino-2, 4, 6-triiodoisophthaloyl chloride and/or iopromide and other contrast agents. The method is favorable for further improving the medicine quality of the 5-amino-2, 4, 6-triiodo isophthalic acid chloride and subsequent final products thereof, reducing possible side effects and improving the medicine safety.)

1. A compound represented by the structure selected from:

2. a process for the preparation of compound 1 according to claim 1, which process is carried out according to the following reaction scheme:

wherein R is C_1-4An alkyl group; preferably, R is selected from methyl, ethyl and n-propyl;

the method comprises the following steps:

dissolving a compound 3 in an alcohol solvent, and carrying out an esterification reaction under the catalysis of a catalyst to obtain a compound 4; alternatively, Compound 3 is reacted with C under basic conditions_1-4Halogenated hydrocarbon or sulfate compound reacts to generate ester;

step b), dissolving the compound 4 in an organic solvent, and carrying out chlorination reaction under the action of a chlorination reagent to obtain a compound 5 and a compound 6;

step c), carrying out hydrolysis reaction on the compound 5 under an alkaline condition to obtain a compound 7;

step d), carrying out iodination reaction on the compound 7 under the action of an iodination reagent to obtain a compound 8;

and e), carrying out acyl chlorination reaction on the compound 8 under the action of an acyl chlorination reagent to generate a compound 1.

3. The process of claim 2, wherein the catalyst in step a) is an acid or a substance that can be hydrolyzed to an acid; preferably, the catalyst is selected from the group consisting of sulfuric acid, phosphorus trichloride and thionyl chloride;

the chlorinating agent in step b) is selected from N-chlorosuccinimide and chlorine;

the base in step c) is selected from the group consisting of lithium hydroxide, sodium hydroxide and potassium hydroxide;

the iodinating reagent in step d) is selected from the group consisting of iodine monochloride, N-iodosuccinimide, elemental iodine and a combination of potassium iodide and potassium iodate;

in step e) the acylchlorinating reagent is selected from thionyl chloride, oxalyl chloride and phosphorus trichloride.

4. The process according to claim 2, wherein the base in step a) is sodium bicarbonate, potassium bicarbonate or triethylamine;

said C is_1～4The halogenated hydrocarbon is selected from methyl iodide, ethyl iodide and propyl iodide;

the sulfate compound is selected from dimethyl sulfate, diethyl sulfate, dipropyl sulfate and dibutyl sulfate.

5. A process for the preparation of compound 2 according to claim 1, which process is carried out according to the following reaction scheme:

wherein the method comprises the following steps:

step f), hydrolyzing the compound 6 under an alkaline condition to obtain a compound 9;

step g), iodinating the compound 9 under the action of an iodinating reagent to obtain a compound 10;

and h) carrying out acyl chlorination on the compound 10 to generate a compound 2.

6. The process according to claim 5, wherein the base in step f) is selected from lithium hydroxide, sodium hydroxide and potassium hydroxide;

the iodinating reagent in step g) is selected from the group consisting of iodine monochloride, N-iodosuccinimide, elemental iodine and a combination of potassium iodide and potassium iodate;

in step h) the acylchlorinating agent is selected from thionyl chloride, oxalyl chloride and phosphorus trichloride.

7. Use of compound 1 and/or compound 2 according to claim 1 for quality control of a compound as shown below:

8. use of compound 1 and/or compound 2 according to claim 1 in quality control of non-ionic iodine contrast agents.

9. The use of claim 8, the non-ionic iodocontrast agent being selected from the group consisting of iopromide, iohexol, iopamidol, ioversol, iomeprol and iodixanol; preferably, the non-ionic iodine contrast agent is iopromide.

Technical Field

The invention relates to a diiodoisophthaloyl chloride compound, a preparation method and application thereof.

Background

X-ray contrast agents are an important diagnostic agent in X-ray diagnostics. Wherein the non-ionic iodine contrast agent comprises: iopromide, iohexol, iopamidol, ioversol, iomeprol, iodixanol, etc. are widely used in diagnostic medicine due to their low osmotic pressure, high contrast density, low toxicity, etc. The non-ionic iodine contrast agent can be applied to contrast examination of a plurality of pathological changes in urinary tract, blood vessel, ventricle, lymphatic channel, gastrointestinal contrast agent and CT examination, and has good application prospect.

The structural general formula of the iodine contrast agent is as follows: the triiodobenzene ring is taken as a mother nucleus, carboxylic acid groups at 1 and 3 positions of the benzene ring are connected with two amino alcohols in an amido bond mode, and amino at 5 position is connected with an alkyl chain through alkylation reaction or amidation reaction.

The 5-amino-2, 4, 6-triiodoisophthaloyl chloride has a parent nucleus structure of the nonionic monomer type iodine contrast agent, and the synthesis of the nonionic type iodine contrast agent from the 5-amino-2, 4, 6-triiodoisophthaloyl chloride only needs to be subjected to subsequent functional group conversion, so the 5-amino-2, 4, 6-triiodoisophthaloyl chloride is often used as a key starting material for synthesizing the nonionic type iodine contrast agent. For example: the patent US4364921, a compound of iopromide original research corporation, discloses the synthesis of iopromide by 3-step amidation reaction using 5-amino-2, 4, 6-triiodoisophthaloyl chloride as starting material. In both Chinese patents CN201410155481.3 and CN201410157384.8, 5-amino-2, 4, 6-triiodoisophthaloyl chloride is used as a starting material for synthesizing iopromide. Furthermore, in the iopamidol synthesis method reported in british patent GB1472050, 5-amino-2, 4, 6-triiodoisophthaloyl chloride was also used as starting material; the synthesis of iohexol is also reported in US5705692 using 5-amino-2, 4, 6-triiodoisophthaloyl chloride as starting material.

At present, 5-amino-2, 4, 6-triiodo isophthaloyl chloride is generally synthesized by taking isophthalic acid as a starting material and carrying out nitration, reduction, iodination and acyl chlorination on the isophthalic acid. Iodine monochloride is mostly adopted as an iodine reagent in the iodination reaction, and the iodine monochloride and organic matters are subjected to iodination and chlorination at the same time, so that chlorinated compounds are inevitably generated, and the existence of the chlorinated compounds is detected in the research process. The main chlorinated compounds are 5-amino-4-chloro-2, 6-diiodoisophthaloyl chloride (compound 1) and 5-amino-2-chloro-4, 6-diiodoisophthaloyl chloride (compound 2).

The 5-amino-4-chloro-2, 6-diiodoisophthaloyl chloride (compound 1) and the 5-amino-2-chloro-4, 6-diiodoisophthaloyl chloride (compound 2) have similar structures and relatively similar chemical properties with the 5-amino-2, 4, 6-triiodoisophthaloyl chloride, are difficult to remove in the preparation and purification process, and can enter subsequent intermediate products and final products along with the flow of a process route, so that the quality of the final product is influenced, and even the clinical safety risk exists.

Taking the synthesis process of iopromide as an example, the method disclosed in patent US4364921 is usually adopted to synthesize iopromide, and the synthetic route is shown as follows:

the process route is that 5-amino-2, 4, 6-triiodoisophthaloyl chloride is used as a starting material to perform amidation reaction with methoxyacetyl chloride to generate an intermediate 1. And then, carrying out amidation reaction on the intermediate 1 and 2, 3-dihydroxypropylamine under the action of an acid-binding agent to obtain an intermediate 2. And finally, carrying out amidation reaction on the intermediate 2 and 3-methylamino-1, 2-propylene glycol under the action of an acid-binding agent to generate iopromide.

In the synthesis process of iopromide, corresponding side reactions of the compound 1 and the compound 2 in the starting material occur, and the side reactions remain in the subsequent intermediates and the final product along with the flow of the synthesis steps, and the impurity flows are as follows:

flow direction of compound 1:

the compound 1 reacts with methoxyacetyl chloride to generate an impurity A, then the impurity A and aminopropanediol undergo amidation reaction to generate an impurity B, and the impurity B reacts with methylaminopropanediol to generate an impurity C.

Compound 2 also undergoes the above reaction to form the corresponding impurity D, impurity E and impurity F, as shown in the following reaction:

furthermore, patent GB1472050 discloses a synthesis process for iopamidol, also starting from 5-amino-2, 4, 6-triiodoisophthaloyl chloride, the synthetic route of which is shown below:

the route starts from 5-amino-2, 4, 6-triiodoisophthaloyl chloride, and an intermediate 3 is obtained through amidation reaction with (L) -2-acetoxypropionyl chloride, and then the intermediate 3 is subjected to amidation reaction with 2-amino-1, 3-propylene glycol, and hydrolysis deprotection is carried out to obtain iopamidol.

In the synthesis process of iopamidol, the compound 1 and the compound 2 also have corresponding side reactions, which affect the quality safety of subsequent products and final products. In addition to the above-described process, other non-ionic iodine contrast agents prepared starting from 5-amino-2, 4, 6-triiodoisophthaloyl chloride also run the risk of remaining such material.

Therefore, it is necessary to provide a high-purity compound 1 and/or compound 2 as a reference substance by directional synthesis, which is used for qualitative and quantitative analysis of impurities in 5-amino-2, 4, 6-triiodoisophthaloyl chloride or subsequent bulk drugs, and has important significance for ensuring the quality safety and effectiveness of non-ionic iodine contrast agents such as iopromide and the like.

Disclosure of Invention

To this end, the present application provides, in a first aspect, compounds 1 and 2 of the formula,

the chemical name of the compound 1 is 5-amino-4-chloro-2, 6-diiodoisophthaloyl chloride; the chemical name of the compound 2 is 5-amino-2-chloro-4, 6-diiodoisophthaloyl chloride.

In a second aspect, the present application provides a process for the preparation of compound 1, as follows:

wherein R is C_1-4An alkyl group; preferably, R is selected from methyl, ethyl and n-propyl;

the method comprises the following steps:

dissolving a compound 3 in an alcohol solvent, and carrying out an esterification reaction under the catalysis of a catalyst to obtain a compound 4; alternatively, Compound 3 is reacted with C under basic conditions_1-4Halogenated hydrocarbon or sulfate compound reacts to generate ester;

step b), dissolving the compound 4 in an organic solvent, and carrying out chlorination reaction under the action of a chlorination reagent to obtain a compound 5 and a compound 6;

step c), carrying out hydrolysis reaction on the compound 5 under an alkaline condition to obtain a compound 7;

step d), carrying out iodination reaction on the compound 7 under the action of an iodination reagent to obtain a compound 8;

and e), carrying out acyl chlorination reaction on the compound 8 under the action of an acyl chlorination reagent to generate a compound 1.

In some embodiments, the catalyst in step a) is an acid or a substance that can be hydrolyzed to an acid, preferably, the catalyst is selected from the group consisting of sulfuric acid, phosphorus trichloride, thionyl chloride, and the like;

in some embodiments, the alcoholic solvent in step a) is selected from methanol, ethanol, propanol and benzyl alcohol;

in some embodiments, the chlorinating agent in step b) is selected from the group consisting of N-chlorosuccinimide and chlorine gas;

in some embodiments, the base in step c) is selected from the group consisting of lithium hydroxide, sodium hydroxide, and potassium hydroxide;

in some embodiments, the iodinating agent in step d) is selected from the group consisting of iodine monochloride, N-iodosuccinimide, elemental iodine, and a combination of potassium iodide and potassium iodate;

in some embodiments, the acid chloride reagent in step e) is selected from thionyl chloride, oxalyl chloride and phosphorus trichloride.

In some embodiments, the base in step a) is sodium bicarbonate, potassium bicarbonate, or triethylamine;

in some embodiments, said C in step a)_1～4The halogenated hydrocarbon is selected from methyl iodide, ethyl iodide and propyl iodide;

in some embodiments, the sulfate-based compound in step a) is selected from the group consisting of dimethyl sulfate, diethyl sulfate, dipropyl sulfate, and dibutyl sulfate.

In a third aspect, the present application provides a process for the preparation of compound 2, as follows:

wherein the method comprises the following steps:

step f), hydrolyzing the compound 6 under an alkaline condition to obtain a compound 9;

step g), iodinating the compound 9 under the action of an iodinating reagent to obtain a compound 10;

and h) carrying out acyl chlorination on the compound 10 to generate a compound 2.

In some embodiments, the base in step f) is selected from lithium hydroxide, sodium hydroxide, potassium hydroxide, and the like;

in some embodiments, the iodinating agent in step g) is selected from the group consisting of iodine monochloride, N-iodosuccinimide, elemental iodine, and a combination of potassium iodide and potassium iodate;

in some embodiments, the acid chloride reagent in step h) is selected from thionyl chloride, oxalyl chloride, phosphorus trichloride, and the like.

In a third aspect of the invention there is provided the use of compound 1 and/or compound 2 in the quality control of a non-ionic contrast agent and/or 5-amino-2, 4, 6-triiodoisophthaloyl chloride.

In some embodiments, the present application provides the use of compound 1 and/or compound 2 in the quality control of iopromide.

In some embodiments, the present application provides the use of compound 1 and/or compound 2 in the quality control of a compound as shown below:

the compound 1 and the compound 2 have potential health threat risks to organisms, and the compound 1 and the compound 2 are prepared and provided as reference substances, are applied to quality control of contrast agents or starting materials thereof, are beneficial to establishing more complete quality standards, can detect and control the content of the contrast agents in the whole preparation and purification process, and have important significance for further improving the quality of the raw material medicines and preparations thereof of the contrast agents and the medication safety.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the specific embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

Various aspects of the invention are further illustrated by the following specific examples, and various advantages of the invention are shown by the following examples.

The English abbreviated compound of the invention corresponds to the Chinese name as follows:

DMSO, DMSO: dimethyl sulfoxide NCS: chlorosuccinimide

SOCl₂: thionyl chloride (or thionyl chloride)

1. LC-MS liquid-mass spectrometry assay

The tests were carried out with high resolution mass spectrometry (Agilent Technologies 1260 Infinity II liquid chromatography) in positive and negative ion mode, ESI ionization source, scanning range 50-1000 m/z.

2、¹H-NMR nuclear magnetic resonance measurement

The assay was carried out using a Qone-WNMR-I-AS400 NMR spectrometer, solvent d6-DMSO, at a test temperature of 28 ℃.

3. The detection method of the compounds 1 and 2 in the 5-amino-2, 4, 6-triiodoisophthaloyl chloride comprises the following steps:

detecting a compound 1 and a compound 2 in 5-amino-2, 4, 6-triiodo isophthalic acid chloride serving as a starting material by an HPLC (high performance liquid chromatography) method, wherein the specific detection method comprises the following steps:

example 1: synthesis of dimethyl 5-aminoisophthalate (Compound 4A)

Weighing 5-aminoisophthalic acid (15.00g,82.64mmol) in a reaction bottle, adding 75mL of methanol for dissolving, slowly dropwise adding thionyl chloride (24.57g,206.61mmol) into the system under stirring, heating the system to 65 ℃ after the completion of the addition, carrying out reflux reaction for 7h until the raw material is completely converted. Cooling the reaction solution to room temperature, adding 500mL of water, stirring for dissolving, adjusting the pH value to 8 by using a saturated sodium bicarbonate solution, precipitating a large amount of solid in the system, stirring for 0.5h, filtering, washing a filter cake by using 20mL of water, and drying in a 50 ℃ forced air drying oven to obtain a compound 4A, 15.76g of white solid, wherein the yield is as follows: 91%, LCMS [ M + H ]⁺]⁺:210.05。

Example 2: synthesis of 5-amino-4-chloroisophthalic acid dimethyl ester (Compound 5A) and 5-amino-2-chloroisophthalic acid dimethyl ester (Compound 6A)

Adding 5-aminoisophthalic acid dimethyl ester (15.76g,75.41mmol) into a reaction bottle, adding 200mL of acetonitrile, stirring to dissolve, adding chlorosuccinimide (10.07g,75.41mmol) into the system in three batches, and reacting for 3 hours at room temperature until the raw materials are basically unchanged. Cooling the reaction solution to room temperature, adding 500mL of ethyl acetate and 500mL of water, stirring uniformly, standing for layering, concentrating the organic phase, separating the obtained crude product by using a preparative chromatographic column (the elution system is 30% of water and acetonitrile), and obtaining 7.93g (yield: 43%) of a light yellow solid of the compound 5A and 2.07g (yield: 11%) of a light yellow solid of the compound 6A;

compound 5A:

LCMS[M+H⁺]⁺:244.1,

¹H NMR(400MHz,CDCl₃)δ7.78(d,J＝2.0Hz,1H),7.54(d,J＝2.0Hz,1H),4.44(s,2H),3.91(s,3H),3.88(s,3H).

compound 6A:

LCMS[M+H⁺]⁺:243.95.

¹H NMR(400MHz,CDCl₃)δ7.06(s,2H),3.90(s,6H).

example 3: synthesis of 5-amino-4-chloroisophthalic acid (Compound 7)

5-amino-4-chloroisophthalic acid dimethyl ester (2.50g,10.28mmol) is put into a reaction bottle, 20mL of methanol and 5mL of water are added for dissolution, and after sodium hydroxide (1.60g,41.10mmol) is added, the reaction is carried out for 4h at room temperature until the conversion of the raw material is complete. The methanol was removed by rotary evaporation, the residue was cooled in an ice water bath, 2N HCl was added dropwise to adjust the pH to 3, after stirring for 0.5 hour, suction filtration was carried out, and the obtained solid was dried to obtain compound 7 in a yield of 84% as a white solid, 1.87 g. LCMS [ M-H ]⁺]^-:213.95.

Example 4: synthesis of 5-amino-4-chloro-2, 6-diiodoisophthalic acid (Compound 8)

Iodine chloride (4.03g,24.69mmol) and potassium chloride (5.75g,39.50mmol) were placed in a reaction flask, dissolved in 12mL of water, stirred for 5min and filtered to obtain the filtrate for use. Adding 5-amino-4-chloroisophthalic acid into a reaction bottle, adding 30mL of 1mol/L hydrochloric acid for dissolving, slowly dripping the filtrate obtained by filtering into a reaction system for 1 hour, completely dripping, heating to 60 ℃, and reacting for 3 days until the raw materials are completely converted. Cooling at room temperature, cooling to 0 ℃ in an ice water bath, stirring for 0.5h, performing suction filtration, washing a filter cake with 10mL of water, and drying to obtain a compound 8, 3.70g of yellow solid, wherein the yield is as follows: 96 percent. LCMS [ M + NH ]₄ ⁺]⁺:484.7.

Example 5: synthesis of 5-amino-4-chloro-2, 6-diiodoisophthaloyl chloride (Compound 1)

5-amino-4-chloro-2, 6-diiodoisophthalic acid (0.74g,1.59mmol) is weighed into a reaction bottle, 7.4mL of thionyl chloride is added, and the mixture is heated to 80 ℃ for reflux reaction for 5 hours until the reaction is complete. Cooling to room temperature, removing thionyl chloride by rotary evaporation, adding 20mL of ethyl acetate to dissolve, washing with 15mL of saturated sodium bicarbonate and 15mL of saturated sodium chloride solution in sequence, drying the organic phase with anhydrous sodium sulfate, filtering, and concentrating to obtain compound 1, 0.77g of yellow solid, yield: 96% and purity 96.18%.

LCMS[M-H⁺]^-:501.5,

¹³C NMR(100MHz,DMSO-d6)δ169.89,167.17,147.48,145.69,142.03,112.70,78.64,68.87.

Example 6: synthesis of 5-amino-2-chloro-4, 6-diiodoisophthalic acid (Compound 10)

5-amino-2-chloroisophthalic acid dimethyl ester (0.77g,3.16mmol) is put into a reaction bottle, 6.2mL of methanol and 1.5mL of water are added for dissolution, sodium hydroxide (0.51g,12.66mmol) is added for reaction at room temperature for 3h until the conversion of the raw material is complete. The pH was adjusted to 3 with 1N HCl and concentrated, and the crude product was used for the next reaction without further treatment.

Iodine chloride (1.55g,9.48mmol) and potassium chloride (2.21g,15.17mmol) are put into a reaction bottle, 5mL of water is added for dissolving, the mixture is stirred for 5min and then filtered, and the filtrate is taken for standby. And (3) adding 10mL of 1N HCl into the crude product to dissolve the crude product, and heating to 60 ℃ to react for 45 hours until the raw material is completely converted. Cooling the reaction solution to room temperature, transferring to ice water bath for cooling, filtering after 0.5H, oven drying to obtain compound 10, 1.00g gray solid (two-step yield: 68%), LCMS [2M-H ]⁺]^-:932.45.

Example 7: synthesis of 5-amino-2-chloro-4, 6-diiodoisophthaloyl chloride (Compound 2)

Weighing 5-amino-2-chloro-4, 6-diiodoisophthalic acid (1.00g,2.14mmol) into a reaction bottle, adding 10mL of thionyl chloride, heating to 80 ℃ after the addition, and carrying out reflux reaction for 8h until the reaction is complete. Cooling to room temperature, removing thionyl chloride by rotary evaporation, adding 25mL of ethyl acetate to dissolve, washing with 20mL of saturated sodium bicarbonate and 20mL of saturated sodium chloride solution in sequence, drying the organic phase with anhydrous sodium sulfate, filtering, and concentrating to obtain compound 2, 1.05g of yellow solid, yield: 98%, purity: 92.83 percent.

LCMS[M-H⁺]^-:501.5.

¹³C NMR(100MHz,DMSO-d6)δ167.12(2C),143.55,143.10(2C),121.78,98.44(2C).

Test example 1 detection of Compound 1 and Compound 2 in starting Material

Starting materials (5-amino-2, 4, 6-triiodoisophthaloyl chloride) of 5 different manufacturers are purchased respectively, the content of the compound 1 and the compound 2 in the starting materials is detected by an HPLC method, the purity of the starting materials and the content of the compound 1 and the compound 2 are calculated by an area normalization method, and the measurement results are shown in the following table 1.

TABLE 1 initial Material test results

Manufacturer of the product	Purity of 5-amino-2, 4, 6-triiodo isophthaloyl chloride%	Content of Compound No. 1%	Content of Compound 2%
				A	96.05	0.21	0.07
B	93.72	0.34	0.33
				C	94.93	0.43	0.08
D	96.12	0.53	0.09
				F	95.45	0.25	0.12

As can be seen from the data in the table above, the starting material 5-amino-2, 4, 6-triiodoisophthaloyl chloride contains a large amount of compound 1 and compound 2, and has quality safety risk for subsequent products.

Test example 2 Ames experiment (bacterial reverse mutation experiment)

Standard Ames experiments were performed on Compound 1 prepared in example 5, as follows:

1) preparation of bacterial liquid

After the frozen strains are melted in water bath at 37 ℃, 50 mu l of frozen strain liquid is inoculated into 5ml of nutrient broth culture solution, and the mixture is subjected to 120rpm dark shaking culture for about 16 to 18 hours.

2) Experimental procedure

Adopting a pre-culture method, wherein 0.5ml of S9 mixed solution is added under the condition of metabolic activation, 0.5ml of phosphate buffer solution (0.2mol/L, pH 7.2-7.4) is added under the condition of non-metabolic activation, and 0.1ml of bacterial solution, 0.1ml of test solution or solvent reference solution, or 0.1ml of positive reference solution are respectively added into a centrifuge tube, and after being uniformly mixed, the mixture is cultured for 20min at 37 ℃.

After the culture is finished, adding about 2ml of top-layer culture medium into the centrifuge tube, after the top-layer culture medium is fully and uniformly mixed, quickly pouring the top-layer culture medium onto the bottom-layer culture medium, rotating the plate to uniformly distribute the top-layer culture medium on the bottom layer, inverting the culture dish after the culture dish is flatly placed and solidified, carrying out shading culture at 37 ℃ for 48-72 h, and counting the number of retrogradation colonies of each dish.

3) Results of the experiment

The experimental result shows that the compound has mutation-causing effect on TA97a, TA98, TA100 and TA1535 strains under the condition of existence or nonexistence of a metabolic activation system, and the experimental result is positive.