CN107976497B

CN107976497B - Method for determining synthesis reaction degree of ticagrelor intermediate 1 and application thereof

Info

Publication number: CN107976497B
Application number: CN201711184173.3A
Authority: CN
Inventors: 陈静; 唐舒棠; 唐朝军
Original assignee: Chongqing Huapont Pharm Co Ltd
Current assignee: Chongqing Huapont Pharm Co Ltd
Priority date: 2017-11-23
Filing date: 2017-11-23
Publication date: 2020-11-10
Anticipated expiration: 2037-11-23
Also published as: CN107976497A

Abstract

The invention belongs to the field of analytical chemistry, and particularly relates to a method for determining the degree of synthesis reaction of a ticagrelor intermediate 1. The measurement was carried out by quantitatively detecting the starting material SM2 in the reaction solution, and the measurement was carried out by high performance liquid chromatography equipped with an evaporative light scattering detector. The method comprises the steps of firstly establishing a calculation model for setting the content of SM2, wherein the calculation model is as follows: lgY ═ algX + b, where Y is the peak area detected by the evaporative light scattering detector, X is the content of SM2, and a, b are constants. Substituting the measured peak area of SM2 into the linear equation to obtain the content of SM2 in the test solution, and further obtaining the content of SM2 in the reaction solution, namely obtaining the synthesis reaction degree of ticagrelor intermediate 1. The method for measuring the synthesis reaction degree of the ticagrelor intermediate 1 provided by the invention has extremely important significance for improving the process yield of ticagrelor, controlling the quality and ensuring the safety.

Description

Method for determining synthesis reaction degree of ticagrelor intermediate 1 and application thereof

Technical Field

The invention belongs to the field of analytical chemistry, and particularly relates to a method for measuring the synthesis reaction degree of a ticagrelor intermediate 1 by measuring the content of an initial raw material SM2 and application thereof.

Background

Ticagrelor is an anti-platelet aggregation drug and can be used for patients with acute coronary syndrome (unstable angina, non-ST elevation myocardial infarction or ST elevation myocardial infarction), including patients receiving drug therapy and Percutaneous Coronary Intervention (PCI) therapy, to reduce the incidence of thrombotic cardiovascular events.

The raw material drug ticagrelor produced by the company is prepared by carrying out substitution reaction on an initial raw material SM1 and an initial raw material SM2 to generate an intermediate 1, carrying out cyclization reaction on the intermediate 1 and sodium nitrite to generate an unseparated intermediate 2, carrying out substitution reaction on the unseparated intermediate 2 and SM3 to generate an unseparated intermediate 3, hydrolyzing the unseparated intermediate 3 with hydrochloric acid to obtain an intermediate 4, namely a crude product, and refining the intermediate 4 with acetonitrile to obtain a finished product of ticagrelor. The synthesis route of ticagrelor is as follows:

as can be seen from the above-mentioned synthetic route,

the molecular formula of SM1 is: 4, 6-dichloro-2- (propylsulfanyl) -5-aminopyrimidine of the formula:

the molecular formula of SM2 is: 2- [ [ (3aR,4S,6R,6aS) -6-aminotetrahydro-2, 2-dimethyl-4H-cyclopenta-1, 3-dioxan-4-yl ] oxy ] -ethanol (2R,3R) -2, 3-dihydroxysuccinate having the following chemical structure:

the formula of the ticagrelor intermediate 1 is as follows: 2- ((3aR,4S,6R,6aS) -6- (5-amino-6-chloro-2- (propylmercapto) -pyrimidin-4-ylamino) -2, 2-dimethyl-tetrahydro-3 aH-cyclopenta [ d ] [1,3] dioxolan-4-yloxy) ethanol: the chemical structural formula is as follows:

from the structural formula, SM2 has no chromophoric group, and in consideration of production cost, when ticagrelor intermediate 1 is prepared, SM1 is an excessive reactant, so that the intermediate 1 can be generated to the maximum extent by ensuring the full reaction in the reaction step, and the yield of the finished product is finally ensured, and the reaction in the step needs quantitative detection on SM 2. Considering that SM2 has no chromophoric group and no ultraviolet absorption, the conventional high performance liquid chromatography is provided with an ultraviolet detector, and the area normalization method cannot monitor the residual quantity of SM2 in the reaction process, namely cannot effectively monitor the proceeding degree of the reaction in the step in real time. The conventional gas chromatography is used for monitoring the reaction process of the intermediate 1, and is easy to generate interference on the target object SM2 to be monitored due to more solvents in a reaction solution, and in addition, if the high performance liquid chromatography-equipped with a differential detector is adopted, the analysis is complicated due to the interference of a solvent front peak, a base line is unstable due to extreme sensitivity to temperature, and in addition, the response sensitivity of the differential detector is limited. That is, the prior art lacks an effective method for quantitative determination of SM2 used to synthesize ticagrelor intermediate 1.

To date, no published method has been reported to be effective for the quantitative determination of SM2 used in the synthesis of ticagrelor intermediate 1. Therefore, the method for determining SM2 used for synthesizing ticagrelor intermediate 1 is beneficial to realizing control over the reaction process, and has extremely important significance for realizing improvement of yield of ticagrelor process, quality control and safety guarantee.

Disclosure of Invention

In view of the above, an object of the present invention is to provide a method for determining the degree of synthesis reaction of ticagrelor intermediate 1, which is performed by determining the content of SM2 using high performance liquid chromatography with an evaporative light scattering detector.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the reaction formula of the synthesis reaction of ticagrelor intermediate 1 is as follows:

in order to reduce the cost, SM1 is an excessive reactant in production, so the determination method is completed by quantitative determination of SM2 in the reaction liquid, and high performance liquid chromatography-equipped with an evaporative light scattering detector is used for determination. High performance liquid chromatography-equipped with an evaporative light scattering detector with liquid chromatography as the separation system and an evaporative light scattering detector as the detection system. Separating the sample on a chromatographic column by flowing phase, atomizing the eluent of the column to form aerosol, evaporating the solvent in a heated drift tube, and finally detecting the remained particles of the nonvolatile solute in a light scattering detection cell.

The measuring method comprises the following steps:

1) diluting the reaction solution by a diluent to obtain a test solution;

2) taking a sample solution, passing through a high performance liquid chromatography column, eluting a mobile phase to complete separation, and detecting by an evaporative light scattering detector to obtain a peak area Y';

3) and (3) calculating:

and substituting the logarithmic lg Y ' of the measured peak area Y ' of the SM2 into a linear equation obtained by an external standard method to obtain the logarithmic concentration lgX ' of the SM2 in the test solution, and obtaining the concentration X ' of the SM2 in the test solution according to lgX ', so as to obtain the content of the SM2 in the reaction solution.

The diluent is an aqueous solution of acetonitrile.

Wherein the content of the SM2 is the content of the SM in a free state, and the theoretical mass number of the SM2 in the free state is 0.59 g.

The response of the scattered light from an evaporative light scattering detector is related to the particle mass as follows:

I＝kmb

taking logarithm:

lgI＝blgm+lgk

wherein:

m: the sum of the particle masses of the components to be measured;

i: the intensity of scattered light generated by the component to be measured;

k, b: a constant;

it can be known that when the sample amount is the same, the logarithmic concentration lgX is linearly related to the logarithmic peak area lgY, so a log/log double-log method needs to be used to generate a linear curve, and linear regression is performed according to more than three corresponding values to obtain a linear equation and a correlation coefficient r, and the calculation of the linear equation comprises the following steps:

1) taking more than three reference substance solutions and diluent solutions with concentrations, separating by using a high performance liquid chromatography column, and detecting by using an evaporative light scattering detector to obtain a peak area Y;

2) linear curves were generated using the log/log method: and (3) performing linear regression on the logarithmic concentration lgX of the control solution with different concentrations on the logarithmic peak area lgY, and calculating to obtain a linear equation and a correlation coefficient r, wherein r is more than 0.999.

Preferably, the volume content of acetonitrile in the diluent is 50-80%.

Preferably, the high performance liquid chromatography is a column using silica gel as a matrix and bonded with an alkylamide-based filler.

Furthermore, the particle size of the chromatographic column is 2-5 μm, the column length is 150-250mm, and the column temperature is 25-55 ℃.

Preferably, a mixed solution of an ammonium acetate solution containing trifluoroacetic acid and an organic solvent is used as a mobile phase, wherein the volume content of the organic solvent is 50-80%.

Further, preferably, the organic solvent is acetonitrile, the concentration of the ammonium acetate solution is 0.01 mol/L-0.03 mol/L, and the volume content of trifluoroacetic acid in the ammonium acetate solution is 0.1% -0.5%.

Preferably, the flow rate of the mobile phase is 0.5ml/min to 1.5 ml/min.

The detection principle of the evaporative light scattering detector is that firstly column eluent is atomized to form aerosol, then a solvent is evaporated in a heated drift tube, and finally the remaining non-volatile solute particles are detected in a light scattering detection pool.

1. Atomizing:

the liquid mobile phase is converted into fine liquid drops in the atomizing chamber under the action of the carrier gas pressure, so that the solvent is easier to evaporate. The evaporative light scattering detector ensures that a narrow droplet size distribution is formed in the atomization chamber by accurately controlling the air pressure and temperature, so that the temperature required by droplet evaporation is greatly reduced.

2. And (3) evaporation:

the carrier gas carries the droplets from the atomization chamber to the drift tube for evaporation. In the drift tube, the solvent is removed, leaving droplets of fine particles or pure solute. The evaporative light scattering detector adopts a low-temperature evaporation mode, and particle uniformity is maintained.

3. And (3) detection:

the light source adopts laser, and solute particles enter the light detection pool after coming out of the drift tube and pass through the laser beam. Light scattered by solute particles is collected by a photomultiplier tube.

Preferably, the temperature of the drift tube of the evaporative light scattering detector is 40-115 ℃, preferably 80-100 ℃.

Preferably, the carrier gas flow rate is 1.5ml/min to 3.0 ml/min.

Preferably, the striker is On.

The second purpose of the present invention is to provide an application of the above-mentioned determination method in the determination of the synthesis reaction degree of ticagrelor intermediate 1, which has the advantages of high accuracy, simple operation and capability of quickly and accurately determining the synthesis reaction degree of ticagrelor intermediate 1.

determination of the extent of the synthesis reaction of ticagrelor intermediate 1 was accomplished by measuring the amount of SM2 using high performance liquid chromatography with an evaporative light scattering detector. Substituting the measured peak area of SM2 into a linear equation obtained by an external standard method to obtain the content of SM2 in the test solution, and further obtaining the content of SM2 in the reaction solution, namely obtaining the synthesis reaction degree of the ticagrelor intermediate 1.

The invention has the beneficial effects that: the invention provides a high performance liquid chromatography-analysis method for determining the residual amount of SM2 used for synthesizing a ticagrelor intermediate 1 by an evaporative light scattering detector, which comprises the steps of firstly establishing a calculation model for setting the SM2 content, calculating the SM2 content by the model accurately and simply, and rapidly and accurately determining the synthesis reaction degree of the ticagrelor intermediate 1. The method has extremely important significance for realizing the improvement of the process yield of ticagrelor, the quality control and the safety guarantee.

Drawings

FIG. 1 is a high performance liquid chromatogram of a diluent.

Figure 2 is a high performance liquid chromatogram of a control linear solution of SM 2.

Fig. 3 is a high performance liquid chromatogram for determining the remaining SM2 content in synthetic ticagrelor intermediate 1.

Fig. 4 is a linear regression equation.

Detailed Description

Hereinafter, preferred embodiments of the present invention will be described in detail (with reference to the accompanying drawings). The experimental methods of the preferred embodiments, which do not indicate specific conditions, are generally performed according to conventional conditions, and the examples are given for better illustration of the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.

Example 1

Determination of content of residual SM2 in synthetic ticagrelor intermediate 1

1. Apparatus and conditions

The instrument comprises the following steps: high performance liquid chromatograph SHIMADZU 20a (SHIMADZU);

a chromatographic workstation: LcSolution;

a detector: an evaporative light scattering detector;

a chromatographic column: a chromatographic column (GL Sciences Inertsil Amide 4.6X 250mm, 5 μm) with silica gel as a matrix and bonded with alkylamide filler;

mobile phase (V: V): 0.02mol/L ammonium acetate solution (containing trifluoroacetic acid with volume content of 0.15%): acetonitrile 30: 70;

diluent (V: V): acetonitrile: 70:30 of water

Column temperature: 30 ℃;

column flow rate: 1.0 ml/min;

sample introduction amount: 30 mu l of the mixture;

detector (ELSD): temperature of the drift tube: 90 ℃; gas flow rate: 2.5 ml/min;

striker (Impact): on.

2. Experimental procedure

(1) Control stock solution: precisely weighing about 21.2mg of SM2 as control, placing into a 25ml measuring flask, adding diluent to dissolve and dilute to scale, and shaking to obtain the final product (concentration is about 500 μ g/ml based on free SM 2).

(Note that the theoretical mass number of the free state in 1g of SM2 is 0.59 g.)

(2) Control linear solution: taking a proper amount of the reference stock solution, quantitatively diluting with a diluent to prepare a group of solutions with linear gradient concentration (in an SM2 free state), and shaking up to obtain the final product. The concentrations of SM2 in the resulting control linear solutions were 20.28. mu.g/ml, 50.69. mu.g/ml, 101.39. mu.g/ml, and 202.77. mu.g/ml, respectively.

(3) Test solution: and (3) adding a diluent to dissolve a proper amount of reaction liquid for synthesizing the ticagrelor intermediate 1, diluting the reaction liquid by a certain multiple n (such as 80 times and 40 times) according to the volume, shaking up, and filtering to obtain the ticagrelor intermediate (the requirement that the peak area of free SM2 in the prepared test solution is required to be within the linear range of a reference substance).

(4) The control linear solution, the sample solution, and the solvent (acetonitrile: water: 70:30) were injected into a liquid chromatograph, measured under the above chromatographic conditions, and the chromatogram was recorded, and the results are shown in fig. 1 to 3.

3. The result of the detection

A30. mu.l portion of the control linear solution was injected, chromatograms were recorded, and linear regression was performed on the log peak area (lgY) at log concentration (lgX), corresponding values are shown in Table 1. The linear equation lgY is 1.5967lgX-2.2098 and the correlation coefficient r is 0.9999, and the linear equation is shown in fig. 4.

TABLE 1 concentration X and Peak area Y and their logarithmic counterparts

SM2 control concentration X (μ g/ml)	20.48	51.19	102.38	204.75
					lgX	1.3113	1.7092	2.0102	2.3112
SM2 reference substance determination peak area Y	0.769	3.228	10.337	29.829
					lgY	-0.1141	0.5089	1.0144	1.4746

Sampling 30 mu l of test solution, recording chromatogram, substituting the peak area (Y ') of the free SM2 into the linear equation to obtain the concentration (X') of the free SM2 in the test solution, and then obtaining the residual amount (in free state percent) of SM2 in the reaction solution according to the dilution times and the feeding ratio of the reaction solution, wherein the results are shown in Table 2.

The calculation formula is as follows:

in the formula: p-residual amount of free SM2 in the reaction solution (in free form),%;

x is the concentration of free SM2 in the test solution, mu g/ml;

n is the dilution multiple of the reaction solution;

v is the total volume of the reaction solution, ml;

m-actual charge of free SM2, g;

0.59 to 1g of free SM2 contains 0.59g of free theoretical mass.

Table 2 results of SM2 residues in the samples shown in fig. 3

Batch number	Residual amount of SM2
		01	4.0％
02	2.5％

It can be seen that the extent of the synthesis reaction of ticagrelor intermediate 1 in the samples of lots 01 and 02 was 96% and 97.5%, respectively.

The method provided by the invention can rapidly and accurately measure the synthesis reaction degree of the ticagrelor intermediate 1 through the obtained linear equation, and is convenient for monitoring the reaction process of synthesizing the ticagrelor intermediate 1. The method has extremely important meanings for realizing the improvement of the process yield of ticagrelor, the quality control and the safety guarantee.

Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.

Claims

1. A method for determining the synthesis reaction degree of a ticagrelor intermediate 1 is disclosed, wherein the reaction formula of the synthesis reaction of the intermediate 1 is as follows:

the method is characterized in that the determination is completed by quantitative determination of SM2 in the reaction liquid, and SM2 content determination is carried out by adopting high performance liquid chromatography and an evaporative light scattering detector, wherein the content of SM2 is the content of the free state of the SM 2; the chromatographic column of the high performance liquid chromatography takes alkylamido bonded silica gel as a filler; the mobile phase adopted by the high performance liquid chromatography is a mixed solution of 0.02mol/L ammonium acetate solution and acetonitrile, and the volume ratio is 30: 70, the ammonium acetate solution contains trifluoroacetic acid with the volume content of 0.15%.

2. The assay method according to claim 1, comprising the steps of:

1) diluting the reaction solution by a diluent to obtain a test solution;

2) the sample solution passes through a high performance liquid chromatography column, the separation is completed by elution of mobile phase liquid, and the peak area Y' of SM2 is obtained by detection of an evaporative light scattering detector;

3) and (3) calculating:

firstly, taking more than three SM2 solutions with the concentration diluted by a diluent and the diluent to pass through a high performance liquid chromatography column to complete separation, and detecting by an evaporative light scattering detector to obtain a peak area Y;

second, linear curves are generated using a log/log double logarithm approach: carrying out linear regression on logarithmic concentration lgX logarithmic peak area lgY of SM2 solutions with different concentrations, and calculating to obtain a linear equation and a correlation coefficient r, wherein r is more than 0.999;

substituting the measured peak area Y 'of SM2 into the linear equation obtained by an external standard method to obtain the concentration X' of SM2 in the test solution, and further obtaining the content of SM2 in the reaction solution.

3. The method according to claim 2, wherein the column temperature of the high performance liquid chromatography is 25 to 55 ℃.

4. The method of claim 2, wherein the drift tube temperature of the evaporative light scattering detector is 80-100 ℃.

5. The method according to claim 2, wherein the diluent is an aqueous solution of acetonitrile.

6. The method according to claim 5, wherein the acetonitrile is contained in an amount of 50 to 80% by volume.

7. Use of the assay of claim 1 in the determination of the extent of a synthesis reaction of ticagrelor intermediate 1.