CN113376301B

CN113376301B - Detection method of sodium 2-amino-3-chloropyridine-4-mercaptide

Info

Publication number: CN113376301B
Application number: CN202011566069.2A
Authority: CN
Inventors: 樊天宇; 陈晓冬; 刘学军
Original assignee: Shanghai Yaotan Pharmaceutical Research And Development Co ltd
Current assignee: Shanghai Yaotan Pharmaceutical Research And Development Co ltd
Priority date: 2020-12-25
Filing date: 2020-12-25
Publication date: 2022-11-15
Anticipated expiration: 2040-12-25
Also published as: CN113376301A

Abstract

The invention discloses a detection method of 2-amino-3-chloropyridine-4-sodium mercaptide, which adopts high performance liquid chromatography, wherein a mobile phase A is acetonitrile, and a mobile phase B is phosphate buffer solution with pH of 6.6-7; in the high performance liquid chromatography, the filler is octadecylsilane chemically bonded silica; the preparation of the phosphate buffer solution comprises the following steps: mixing phosphate solution and alkali solution to adjust the pH value; the alkali solution comprises a sodium hydroxide solution and/or a potassium hydroxide solution. The main peak obtained by the detection method of the invention has good symmetry and stability, and the accuracy of detecting the purity of the product is high.

Description

Detection method of sodium 2-amino-3-chloropyridine-4-mercaptide

Technical Field

The invention relates to a detection method of 2-amino-3-chloropyridine-4-sodium mercaptide.

Background

The tyrosine phosphatase SHP2 consists of two N-terminal Src homology 2 domains (N-SH 2 and C-SH 2) and a protein tyrosine phosphatase catalytic domain (PTP). At the cellular level, SHP2 is involved in multiple tumor cell signaling pathways, as well as in programmed death receptor 1 (PD 1) mediated immune system suppression. SHP2 is capable of dephosphorylating antigen receptor pathway proteins in T cells, and thus, inhibition of SHP2 activity can reverse immunosuppression in the tumor microenvironment.

SHP2 is an important member of the protein tyrosine phosphatase family, is related to various diseases of human beings, and can be used as a novel druggable target. WO2016/203406 reports that a pyrimido-ring system compound as a brand-new SPH2 inhibitor shows good inhibitory activity on tumor cells, and has a broad drug development prospect. The intermediate 2-amino-3-chloropyridine-4-sodium mercaptide is a very important intermediate for synthesizing the SHP2, an analytical method with high accuracy and good stability is found to be of great significance for the synthesis of the SHP2, and the method is a technical problem to be solved in the field of SHP2 synthesis at present.

The structure of the 2-amino-3-chloropyridine-4-sodium mercaptide is as follows:

disclosure of Invention

The invention mainly aims to overcome the defects and provides a detection method of 2-amino-3-chloropyridine-4-sodium mercaptide. The main peak obtained by the detection method of the invention has good symmetry and stability, and the accuracy of detecting the purity of the product is high.

In the development process, the inventors have appeared an unusual phenomenon in the characterization and analysis method of sodium 2-amino-3-chloropyridine-4-mercaptide, and only based on common knowledge (as can be seen from the chemical structural formula of sodium 2-amino-3-chloropyridine-4-mercaptide, which contains amino groups, is easily decomposed in an acidic solution and is generally suitable for analysis and detection of neutral and alkaline mobile phases), one mobile phase is selected, for example, a buffer solution in the mobile phase is selected to be a system with a pH of 7.4, and the obtained HPLC chromatogram shows that the product contains a hetero peak and is not a product with high purity. But actually, the purity is higher when the NMR spectrum is used. Therefore, the difference of the mobile phase has great influence on the peak type and the detection result of the obtained chromatogram. When the acetonitrile-water system is used, although the system has a good symmetrical peak pattern, the impurity content becomes large after a long-term storage. Therefore, the inventor explores a high performance liquid chromatography detection method with high accuracy, good repeatability and good stability aiming at the problems.

The invention mainly solves the technical problems through the following technical scheme.

The invention provides a detection method of 2-amino-3-chloropyridine-4-sodium mercaptide, which adopts high performance liquid chromatography, wherein a mobile phase A is acetonitrile, and a mobile phase B is phosphate buffer solution with pH of 6.6-7; in the high performance liquid chromatography, the filler is octadecylsilane chemically bonded silica;

the preparation of the phosphate buffer solution comprises the following steps: mixing phosphate solution and alkali solution to adjust the pH value; the alkali solution comprises a sodium hydroxide solution and/or a potassium hydroxide solution.

In the present invention, the sodium 2-amino-3-chloropyridine-4-thiol generally refers to a compound represented by formula I:

in the present invention, the acetonitrile may be one conventionally understood in the art, and generally means acetonitrile having a purity of 99.9% or more.

In the present invention, the pH of the mobile phase B is preferably 6.7, 6.8 or 6.9.

As known to those skilled in the art, the pH value will generally have an error of. + -. 0.02 during the actual operation. The pH referred to in the present invention is a pH value measured by a pH meter at the time of formulation. The temperature of the pH test was 25 ℃.

In the present invention, the molar concentration of the phosphate solution is, for example, 0.15 to 0.25mol/L, preferably 0.2mol/L.

In the present invention, the molar concentration of the sodium hydroxide solution is, for example, 0.15 to 0.25mol/L, preferably 0.2mol/L.

In the present invention, the ratio of the molar concentration of the phosphate solution to the molar concentration of the sodium hydroxide solution is, for example, 1:1.

In the present invention, the phosphate solution is preferably potassium dihydrogen phosphate solution and/or sodium dihydrogen phosphate.

In the present invention, the solvent in the phosphate solution or the alkali solution is generally referred to as water, as known to those skilled in the art. The water is typically deionized water.

In the present invention, the mixing is generally performed by adding the alkali solution to the phosphate solution, as is known from the phosphate buffer solution.

In the present invention, the volume ratio of the phosphate solution to the alkali solution is mainly required to be such that the pH of the phosphate buffer solution can be in the range of 6.6 to 7, and is not particularly limited. The volume ratio will be adjusted, as known to those skilled in the art, according to the pH required for the actual formulation of the phosphate buffer solution. The volume ratio may be 250: (100 to 200), specifically, for example, 250 to 118.

As known to those skilled in the art, the phosphate buffer solution is diluted with water according to actual conditions after being prepared according to the above conditions, so that the pH value of the phosphate buffer solution reaches a target value.

In one embodiment of the present invention, the preparation of the phosphate buffer solution comprises the following steps: 118mL of 0.2mol/L sodium hydroxide solution is added into 250mL of 0.2mol/L potassium dihydrogen phosphate solution, diluted to 1000mL by water and shaken up to obtain the potassium dihydrogen phosphate.

In the present invention, the high performance liquid chromatography can be generally performed by gradient elution.

Wherein, the elution gradient can adopt the setting of the elution gradient commonly used in the field. The procedure for the gradient elution is preferably as shown in the following table:

time (min)	Mobile phase A (% v/v)	Mobile phase B (% v/v)
			0	5	95
18	80	20

. % v/v refers to the volume of the mobile phases in the eluent as a proportion of the total volume of the eluent.

As known to those skilled in the art, the elution conditions refer to that the initial elution at 0min is 5% by volume of mobile phase A and 95% by volume of mobile phase B; at the end of the 18min elution, the volume ratio of mobile phase A in the eluent was 80%, and the volume ratio of mobile phase B was 20%.

Wherein, the conversion rate of the mobile phase A from 5% to 80% is a uniform change process within 0-18 min. Accordingly, the transition rate of the mobile phase B from 95% to 20% is also a uniform process.

Wherein, the skilled person knows that said 0min generally refers to the initial time, the initial preparation time is generally negligible, for example, it takes 0.01min to inject 5% mobile phase A by volume into the chromatographic column.

The inventor also makes proper adjustment to the elution condition in the experimental process, if the peak time of the product is delayed by changing the setting of the elution gradient, but the accuracy of the analysis is not affected.

Wherein, preferably, the eluent with the retention time of 4-6 min is collected, namely the 2-amino-3-chloropyridine-4-sodium mercaptide.

In the invention, the detection wavelength of the sodium 2-amino-3-chloropyridine-4-mercaptide is terminal absorption, so that the detection wavelength in the high performance liquid chromatography can be 200-230 nm.

In the present invention, the high performance liquid chromatography may be carried out at a column temperature conventional in the art, for example, 25 to 35 ℃ and preferably 30 ℃.

In the present invention, the high performance liquid chromatography may be performed at a flow rate which is conventional in the art, for example, 0.9 to 1.1mL/min, preferably 1.0mL/min.

In the present invention, the amount of the feed in the high performance liquid chromatography may be generally 3 to 8. Mu.L, for example, 5. Mu.L.

In the invention, in the high performance liquid chromatography, the 2-amino-3-chloropyridine-4-sodium mercaptide is generally prepared into a solution form for injection.

Wherein, the concentration of the solution of the sodium 2-amino-3-chloropyridine-4-mercaptide in the sample injection can be generally 0.5 to 1.5mg/mL, for example, 1mg/mL.

The solvent in the solution of sodium 2-amino-3-chloropyridine-4-mercaptide is, for example, a 50% by volume aqueous solution of acetonitrile at the time of the sample injection.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows: according to the detection method of the high performance liquid chromatography, the peak patterns of the chromatogram acquired by the 2-amino-3-chloropyridine-4-sodium mercaptide are symmetrical, and the chromatogram cannot be decomposed after being separated and placed for a long time, so that the stability is good.

Drawings

FIG. 1 is a nuclear magnetic hydrogen spectrum of sodium 2-amino-3-chloropyridine-4-mercaptide examined in examples 1 to 4 and comparative examples 1 to 4.

FIG. 2 is an HPLC chromatogram obtained by the detection method of example 1.

FIG. 3 is an HPLC chromatogram obtained by the detection method of comparative example 1.

FIG. 4 is an HPLC chromatogram obtained by the detection method of comparative example 1 and an HPLC chromatogram obtained by re-detecting the separated product after leaving for a certain period of time.

FIG. 5 is an HPLC chromatogram obtained by the detection method of comparative example 2.

FIG. 6 is an HPLC chromatogram obtained by the detection method of comparative example 3.

FIG. 7 is an HPLC chromatogram obtained by the detection method of comparative example 4.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the invention thereto. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

FIG. 1 shows nuclear magnetic hydrogen spectra of sodium 2-amino-3-chloropyridine-4-mercaptide to be detected in examples 1 to 4 and comparative examples 1 to 4 described below.

In the nuclear magnetic hydrogen spectrogram, a group of peaks (1.87,3.71) are solvent peak shifts of tetrahydrofuran, a peak with a chemical shift of 5.5 is a solvent peak shift of dichloromethane, a peak with a chemical shift of 1.18 is an ethanol solvent peak shift, and the rest shifts conform to chemical shifts of all H in the structure of the sodium 2-amino-3-chloropyridine-4-mercaptide, and have no obvious impurity peak, so that the purity of the sodium 2-amino-3-chloropyridine-4-mercaptide can be calculated to be more than 98 percent by the nuclear magnetic hydrogen spectrogram.

The following examples 1 to 4 and comparative examples 1 to 4 all used the same batch of sodium 2-amino-3-chloropyridin-4-thiolate.

Example 1

A chromatographic column: chromatographic column using octadecylsilane chemically bonded silica as filler

Mobile phase: phase A is acetonitrile (purity of acetonitrile is above 99.9%), and phase B is buffer solution with pH of 6.8.

preparation of phosphate buffer solution with pH 6.8: 250mL of 0.2mol/L potassium dihydrogen phosphate solution is transferred, 118mL of 0.2mol/L sodium hydroxide solution is added, the solution is diluted to 1000mL by water, and the mixture is shaken up to obtain the potassium dihydrogen phosphate. The temperature of formulation was 25 ℃.

The gradient elution procedure was as follows:

time (min)	Mobile phase A (% v/v)	Mobile phase B (% v/v)
			0	5	95
18	80	20

Wherein, the conversion rate of the mobile phase A from 5% to 80% is a process of uniform change within 0-18 min. Accordingly, the transition rate of the mobile phase B from 95% to 20% is also a uniform process.

Column temperature: 30 ℃;

sample preparation and sample quantity: weighing 2-amino-3-chloropyridine-4-sodium mercaptide, adding acetonitrile water solution with the volume ratio of 50 percent, carrying out ultrasonic dissolution, and diluting to obtain a solution of 1.0 mg/mL. Precisely transferring 5 mu L of the solution to a liquid chromatograph;

flow rate: 1.0mL/min; detection wavelength: 200nm.

As shown in FIG. 2, it is an HPLC chromatogram obtained by the detection method of this example. As can be seen from the figure, the eluent with the retention time of 5-5.5 min is collected to be the sodium 2-amino-3-chloropyridine-4-mercaptide, and the peak corresponding to the time period is the main peak; the main peak has symmetrical shape and moderate peak position, and the purity of the detected product is more than 98 percent and is consistent with the result of nuclear magnetic hydrogen spectrum detection. And after being placed for 2 hours at the temperature of between 15 and 26 ℃, the height of an impurity peak is unchanged and the content of the impurities is not increased when the method is adopted again for detection.

Example 2

Detection wavelength: 230nm, the settings for the remaining detection methods are the same as in example 1. By adopting the detection method of the embodiment, the obtained peak is symmetrical, and the purity of the detected product is more than 98 percent and is consistent with the detection result of the nuclear magnetic hydrogen spectrum. And after being placed for 2 hours at the temperature of 15-26 ℃, when the scheme is adopted again for detection, the peak type is unchanged, the height of the impurity peak is unchanged, and the content of the impurities is not increased.

Example 3

Flow rate: 0.9mL/min; detection wavelength: 200nm, the settings for the remaining detection methods are the same as in example 1. By adopting the detection method of the embodiment, the obtained peak is symmetrical, and the purity of the detected product is more than 98 percent and is consistent with the detection result of the nuclear magnetic hydrogen spectrum. And after being placed for 2 hours at the temperature of between 15 and 26 ℃, when the method is adopted again for detection, the peak type is unchanged, the height of the impurity peak is unchanged, and the content of the impurities is not increased.

Example 4

Flow rate: 1.1mL/min; detection wavelength: 200nm, the settings for the remaining detection methods are the same as in example 1. By adopting the detection method of the embodiment, the obtained peak is symmetrical, and the purity of the detected product is more than 98 percent and is consistent with the detection result of the nuclear magnetic hydrogen spectrum. And after being placed for 2 hours at the temperature of between 15 and 26 ℃, when the method is adopted again for detection, the peak type is unchanged, the height of the impurity peak is unchanged, and the content of the impurities is not increased.

Comparative example 1

The mobile phase B was water and the rest of the elution conditions were set as in example 1.

FIG. 3 shows the HPLC chromatogram obtained by the detection method of comparative example 1. As can be seen from the figure, the peak corresponding to the retention time of 5-6 min is the main peak, and the peak pattern is symmetrical.

However, when the sample is again examined in the mobile phase system of this comparative example after being left at 15 to 26 ℃ for 2 hours, the examination results are shown in FIG. 4, in which the upper peak in FIG. 4 is a chromatographic peak enlarged by a certain factor in FIG. 3, the lower peak is a chromatographic peak after being left for 2 hours, and the arrow in the figure shows an enlarged impurity peak. As can be seen from the figure, with the detection conditions in comparative example 1, after being left for 2 hours, the impurity peak is significantly increased, which means that the impurity content is significantly increased and the stability is poor.

Comparative example 2

The mobile phase B was a 1% aqueous triethylamine solution, and the rest of the elution conditions were set to the same conditions as in example 1.

FIG. 5 shows the HPLC chromatogram obtained by the detection method of this comparative example. As can be seen from the figure, the peak corresponding to the retention time of 12.5-15 min is the main peak, the peak type of the main peak is asymmetric, so that the purity of the product cannot be accurately represented, and the peak emergence time is 12.5-15 min, the peak emergence position is not moderate, which is not favorable for judging the purity of the product.

Comparative example 3

Mobile phase B: a buffer at pH 7.4, prepared as follows: adding 79mL of 0.1mol/L sodium hydroxide solution into 1.36g of monopotassium phosphate, and diluting to 200mL by using water to obtain the potassium phosphate. The rest of the elution conditions were set to the same conditions as in example 1.

FIG. 6 shows the HPLC chromatogram obtained by the detection method of this comparative example. As can be seen from the figure, the peak corresponding to the retention time of 4-5 min is the main peak, the peak pattern is asymmetric, and the impurity peak at the main peak is higher, so that the purity of the product cannot be accurately judged.

Comparative example 4

The B phase in the mobile phase is phosphate buffer solution with pH 6.8, and the phosphate buffer solution is prepared as follows: 3.4g of potassium dihydrogen phosphate is weighed out and dissolved in 1000mL of water, and the pH value is adjusted to 6.8 with sodium hydroxide. The rest of the elution conditions were set to be the same as in example 1.

As shown in FIG. 7, in the HPLC chromatogram obtained by the detection method of this comparative example, the retention time of 3.5 to 4min is the product peak, as can be seen from the main peak indicated in the figure. In the figure, the side of the main peak has obvious mixed peaks, and the main peak has asymmetric peak types, so that the purity of the product cannot be accurately detected and judged finally.

The same batch of sodium 2-amino-3-chloropyridine-4-thiolate was examined in each of examples 1 to 4 and comparative examples 1 to 4, and the injection volumes and concentrations of the samples were identical. However, in comparative examples 1 to 4, different mobile phase systems were used, and the purity and stability of the product shown in the chromatograms were different. For example, in comparative example 3 and comparative example 4, a clear hetero-peak is appeared beside the peak of the product, which may be caused by instability of the product in the mobile phase system or by the mobile phase system itself, and the detection and judgment of the purity of the product are seriously influenced.

Claims

1. A detection method of 2-amino-3-chloropyridine-4-sodium mercaptide is characterized in that the detection method adopts high performance liquid chromatography, a mobile phase A is acetonitrile, and a mobile phase B is a phosphate buffer solution with the pH value of 6.6 to 7;

in the high performance liquid chromatography, the filler is octadecylsilane chemically bonded silica;

the preparation of the phosphate buffer solution comprises the following steps: mixing phosphate solution and alkali solution to regulate the pH value;

the molar concentration of the phosphate solution is 0.15 to 0.25mol/L;

the alkali solution comprises a sodium hydroxide solution and/or a potassium hydroxide solution.

2. The assay of claim 1, wherein the mobile phase B has a pH of 6.7, 6.8, or 6.9.

3. The assay of claim 1, wherein the phosphate solution has a molarity of 0.2mol/L;

and/or the molar concentration of the alkali solution is 0.15 to 0.25mol/L;

and/or the ratio of the molar concentration of the phosphate solution to the molar concentration of the alkali solution is 1:1.

4. the detection method according to claim 3, wherein the molar concentration of the alkali solution is 0.2mol/L.

5. The method of claim 1, wherein the phosphate solution is a potassium dihydrogen phosphate solution and/or a sodium dihydrogen phosphate solution.

6. The assay of claim 1, wherein the solvent in the phosphate solution is water;

and/or the solvent in the alkali solution is water.

7. The assay of claim 1, wherein the volume ratio of phosphate solution to alkaline solution is 250:100 to 200.

8. The assay of claim 7, wherein the volume ratio of phosphate solution to base solution is 250:118.

9. the assay of claim 1, wherein the phosphate buffer solution is formulated to comprise the steps of: adding 118mL of 0.2mol/L sodium hydroxide aqueous solution into 250mL of 0.2mol/L potassium dihydrogen phosphate aqueous solution, diluting with water to 1000mL, and shaking up to obtain the final product.

10. The assay of any one of claims 1~7 wherein the high performance liquid chromatography uses gradient elution.

11. The detection method according to claim 10, wherein the gradient elution is performed by:

in the high performance liquid chromatography, the time of gradient elution is changed from 0min of mobile phase to 18min of mobile phase, which is a process of uniform speed change.

12. The detection method according to claim 8, wherein in the high performance liquid chromatography, the detection wavelength is 200 to 230nm;

in the high performance liquid chromatography, the column temperature is 25 to 35 ℃;

in the high performance liquid chromatography, the flow rate of elution is 0.9 to 1.1mL/min.

13. The detection method according to claim 12,

in the high performance liquid chromatography, the column temperature is 30 ℃;

in the high performance liquid chromatography, the flow rate of elution is 1.0mL/min.

14. The detection method according to claim 8, wherein in the high performance liquid chromatography, the feeding amount is 3~8 μ L;

and/or the sodium 2-amino-3-chloropyridine-4-mercaptide is injected in the form of a solution.

15. The detection method according to claim 14, wherein in the high performance liquid chromatography, the feed amount is 5 μ L;

during sample injection, the concentration of the solution of the 2-amino-3-chloropyridine-4-sodium mercaptide is 0.5 to 1.5mg/mL;

when the sample is injected, the solvent in the solution of the 2-amino-3-chloropyridine-4-sodium mercaptide is a 50% acetonitrile aqueous solution in volume ratio.

16. The detection method according to claim 14, wherein the concentration of the solution of sodium 2-amino-3-chloropyridine-4-mercaptide at the time of sample injection is 1mg/mL.