CN114965723B - Detection method of 2-acylaminothiazole compounds - Google Patents
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- G01N30/02—Column chromatography
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- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
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Abstract
The invention relates to a detection method of 2-acylaminothiazole compounds, which is characterized in that the purity of the compound of the formula X is measured by selecting a proper mobile phase and controlling the conditions of the concentration and the flow rate of the mobile phase, the column temperature of a chromatographic column and the like, particularly by controlling a gradient elution program, the compound of the formula X and other impurities can be effectively separated, the separation degree is high, and the detection sensitivity is high, the specificity is good and the accuracy is high.
Description
Technical Field
The invention belongs to the technical field of medicine analysis, and particularly relates to a detection method of a 2-acylaminothiazole compound.
Background
Avatrobap (Avatrobopag) is an orally bioavailable small molecule thrombopoietin receptor agonist that stimulates proliferation and differentiation of megakaryocytes in bone marrow cells, thereby increasing platelet production. The product was marketed in FDA lot 5 month 2018.
At present, the synthesis route of the atorvastatin has been reported in related literature, and in the synthesis route reported in a compound patent CN1639157A, a class of intermediate-2-acylaminothiazole compounds is involved: ethyl 1- (3-chloro-5- ((4- (4-chlorothien-2-yl) -5- (4-cyclohexylpiperazin-1-yl) thiazol-2-yl) carbamoyl) pyridin-2-yl) piperidine-4-carboxylate (compound of formula X) having the structure:
the quality control of the intermediate plays an important role in reaction monitoring and yield improvement, and also influences the quality of the final product. Up to now, no detection method of the compound of the formula X is reported in the related literature and patent, but the quality control is carried out from the initial raw material to the final product comprehensively and systematically, particularly the intermediate in the synthesis process, and the method has important guiding significance for the quality research of the avascular sapara.
Disclosure of Invention
In one aspect, the invention provides a method for detecting a compound of formula X, characterized by: the method is high performance liquid chromatography, which adopts a reversed phase chromatographic column, takes a mixed solution of ammonium formate solution and solvent I as eluent, and performs isocratic or gradient elution, wherein the structural formula of the compound of the formula X isThe solvent I is acetonitrile or a mixed solution of acetonitrile and ethanol (50:50), preferably acetonitrile.
In some exemplary embodiments, mobile phase a and/or mobile phase B are used as eluent, wherein mobile phase a is an ammonium formate solution and mobile phase B is acetonitrile.
In some more typical embodiments, the mobile phase a and mobile phase B are eluted with a gradient according to the following procedure:
the method comprises the steps of carrying out a first treatment on the surface of the And in the gradient elution process, the sum of the proportion of the mobile phase A and the proportion of the mobile phase B is 100 percent; wherein the mobile phase A proportion refers to the percentage of the mobile phase A volume to the total volume of the eluent, and the mobile phase B proportion refers to the percentage of the mobile phase B volume to the total volume of the eluent.
In some embodiments, mobile phase a and/or mobile phase B are used as eluent, wherein mobile phase a is an ammonium formate solution and mobile phase B is a mixed solution of acetonitrile and ethanol (50:50), and the mobile phases a and B are eluted in gradient according to the following procedure:
the method comprises the steps of carrying out a first treatment on the surface of the And in the gradient elution process, the sum of the proportion of the mobile phase A and the proportion of the mobile phase B is 100 percent; wherein the mobile phase A proportion refers to the percentage of the mobile phase A volume to the total volume of the eluent, and the mobile phase B proportion refers to the percentage of the mobile phase B volume to the total volume of the eluent.
In some embodiments, the mobile phase a concentration is from 0.009mol/l to 0.011nmol/l; in some typical embodiments, the mobile phase A is at a concentration of 0.01mol/l.
In some embodiments, the pH of mobile phase a is 2.8 to 3.2; preferably 3.0.
In some embodiments, the eluent has a flow rate of 0.9 to 1.1ml/min; in some more typical embodiments, the eluent flow rate is 1ml/min.
In some embodiments, the reverse phase chromatography column employs a non-polar stationary phase as a packing; in some typical embodiments, the reverse phase chromatography column employs octadecylsilane chemically bonded silica as a packing; in some more typical embodiments, the reverse phase chromatography column is Agilent Eclipse Plus C18, which is 4.6mm by 100mm,3.5 μm in size.
In some embodiments, the detection method is performed on a high performance liquid chromatograph, employing a diode array detector or an ultraviolet detector; in some typical embodiments, the assay is performed on a high performance liquid chromatograph, using an ultraviolet detector, and having a detection wavelength of 245nm to 255nm; preferably 250nm.
In some embodiments, the column temperature of the reverse phase chromatography column is 25 to 55 ℃; in some typical embodiments, the column temperature of the reverse phase chromatography column is 30 to 40 ℃; in some more typical embodiments, the column temperature of the reverse phase chromatography column is 35 ℃.
In another aspect, the present invention provides a method for detecting a compound of formula X, characterized by: the method is a high performance liquid chromatography method, and adopts a reversed phase chromatographic column, wherein the detector is an ultraviolet absorption detector, and the detection wavelength is 250nm;
the column temperature is 35 ℃;
mobile phase A is 0.01mol/l ammonium formate solution;
mobile phase B is acetonitrile; gradient elution was performed according to the following table;
the flow rate is 1.0ml/min;
injecting X sample solution;
the purity of the compound of formula X in the test sample was determined by area normalization.
In some particular embodiments, the invention provides a method for detecting a compound of formula X, characterized by:
(1) Mobile phase A is 0.01mol/l ammonium formate solution; mobile phase B is acetonitrile;
(2) Preparing a test solution: taking a proper amount of a test sample of the compound of formula X, precisely weighing, adding tetrahydrofuran for dissolution, diluting with acetonitrile for constant volume, and shaking uniformly to obtain the compound;
(3) Test article determination: adopting a reversed phase chromatographic column, wherein the mobile phase A is 0.01mol/l ammonium formate solution, the mobile phase B is acetonitrile, the flow rate is adjusted to be 1.0ml/min, and the column temperature is 35 ℃; the detection wavelength is 250nm; the sample injection amount is 10 μl; measuring the solution of the compound X to be tested, injecting the solution into a liquid chromatograph, performing gradient elution according to the following table, and recording each chromatogram;
(6) The purity of the compound of formula X in the test sample was determined by area normalization.
Herein, unless otherwise indicated, a compound of formula X for "test configuration" includes, but is not limited to, a freshly prepared or stored bulk drug of a compound of formula X, a pharmaceutical composition comprising a compound of formula X.
In the present invention, the substances concerned are also expressed as impurities.
In the present invention, the appropriate amount means that the amount of each compound is within the detection limit or quantitative limit of the high performance liquid chromatograph for the purpose of the experiment.
According to the detection method of the compound of the formula X, provided by the invention, the purity of the compound of the formula X is measured by selecting a proper mobile phase and controlling the conditions of the concentration and the flow rate of the mobile phase, the column temperature of a chromatographic column and the like, particularly by controlling a gradient elution program, the compound of the formula X and other impurities can be effectively separated, the separation degree is high, and the detection sensitivity is high, the specificity is good and the accuracy is high.
Drawings
FIG. 1 System suitability spectrum of example 1
FIG. 2 System suitability spectrum of example 3
FIG. 3 System suitability spectrum of example 4
Detailed Description
The reagents, compounds of formula X, and impurity compounds used in the following examples of the invention are all commercially available.
In the following examples, the structures of impurities 1 to 5 are as follows:
example 1
1. Solution preparation:
1) Control stock solution:
taking a proper amount of compound reference substance of formula X, precisely weighing, adding tetrahydrofuran accounting for about 30% of the volume of a measuring flask, carrying out ultrasonic dissolution, cooling, and diluting with acetonitrile to prepare a solution containing about 50 mug of compound of formula X in each 1ml, wherein the solution is used as a stock solution of compound reference substance of formula X;
taking a proper amount of the impurity 1 reference substance, precisely weighing, dissolving with methanol, and diluting to prepare a solution containing about 15 mug in each 1ml, wherein the solution is used as an impurity 1 reference substance stock solution;
taking a proper amount of impurity 2 reference substances, precisely weighing, respectively adding DMSO accounting for about 10% of the volume of a measuring flask, carrying out ultrasonic treatment to dissolve, cooling, diluting with acetonitrile to prepare a solution containing about 0.4mg of each 1ml of the solution, and respectively serving as impurity 2-5 reference substance stock solutions;
taking a proper amount of each of the 3-5 reference substances of impurities, precisely weighing, respectively adding tetrahydrofuran accounting for about 30% of the volume of a measuring flask, carrying out ultrasonic dissolution, cooling, and diluting with acetonitrile to prepare a solution containing about 20 mug of each impurity in each 1ml, wherein the solution is used as a stock solution of the 3-5 reference substances of impurities;
2) Positioning solution: taking 1ml of each impurity reference substance stock solution, respectively placing into 20ml measuring flask, adding tetrahydrofuran-acetonitrile (30:70) to dilute to scale, shaking uniformly, and taking as positioning solution of each impurity;
3) System applicability solution: taking a proper amount of a compound of formula X as a reference substance, adding tetrahydrofuran accounting for about 30% of the volume of a measuring flask, carrying out ultrasonic dissolution, cooling, adding a proper amount of an impurity reference substance stock solution, and quantitatively diluting with acetonitrile to prepare a solution which contains about 0.5mg of the compound of formula X, 7.5 mug of the compound of formula X and 10 mug of the impurity 1 and the rest of impurities in each 1ml, wherein the solution is used as a system applicability solution;
4) Test solution: taking about 10mg of a compound of formula X to be tested, precisely weighing, placing the compound into a 20ml measuring flask, adding tetrahydrofuran accounting for about 30% of the volume of the measuring flask, ultrasonically dissolving, cooling, and diluting with acetonitrile to prepare a solution containing about 0.5mg of the compound of formula X per 1ml to be used as a solution of the test sample;
5) Control solution: precisely measuring a proper amount of sample solution, and diluting with a solvent to obtain a solution containing about 1 mug of main component in each 1ml as a control solution;
6) Blank solvent: tetrahydrofuran-acetonitrile (30:70)
2. Chromatographic conditions:
chromatographic column: agilent ZORBAX Eclipse Plus C18 (100 mm. Times.4.6 mm,3.5 μm)
Mobile phase a:0.01mol/l ammonium formate solution (pH 3.0 adjusted with formic acid)
Mobile phase B: methanol-acetonitrile (50:50)
Column temperature: 35 ℃;
gradient elution procedure:
flow rate: 1.0ml/min
Detection wavelength: 250nm
Column temperature: 35 DEG C
Sample injection volume: 10 μl of
3. 10 μl of the system applicability solution was taken and injected into a liquid chromatograph, and the chromatogram was recorded. The chromatogram is shown in FIG. 1.
The result shows that in the chromatogram of the system applicability solution, the purity of the main component (the compound shown in the formula X) is 89.29%, the purity index is 999.99, the retention time of the main component peak is about 28.872min, the retention time of the impurity peak is 16.755min at the earliest, the shape of the impurity peak is poor, the number of the chromatographic peaks in the system applicability solution is consistent with the theoretical value, the minimum separation degree between each known chromatographic peak and the adjacent chromatographic peak is 2.28 & gt1.5, the separation degree is good, and the method needs to be further optimized to shorten the retention time of the main component.
The inventors have made a search for mobile phase conditions, detection wavelengths and gradient elution conditions, see examples 2-4 below.
Example 2 optimization of detection wavelength
Based on the chromatographic conditions, the system-applicable solution is scanned in the wavelength range of 190 nm-400 nm by using a diode array detector. As can be seen from the scanning result graphs, the ultraviolet absorption spectrum of the compound in the formula IV and related impurities thereof has stronger absorption near 250nm, so 250nm is selected as the measurement wavelength of the present chromatographic condition.
Example 3 optimization of mobile phase
Mobile phase B was replaced with pure acetonitrile based on the chromatographic conditions of example 1, the other chromatographic conditions being unchanged.
10 μl of the above system applicability solution was injected into a liquid chromatograph, and the chromatogram was recorded, as shown in FIG. 2.
The results of FIG. 2 show that under the above chromatographic conditions, the purity of the main peak (compound of formula X) is 89.47%, the retention time of the main peak is 8.498min, the retention time of the impurity peak is 3.601min at the earliest, the peak shape of each impurity peak is good, the minimum degree of separation between each known chromatographic peak and the adjacent chromatographic peak is 4.11 > 1.5, and the method needs to be further optimized to properly adjust the retention time of the main peak.
Example 4 gradient elution procedure optimization
On the basis of the chromatographic conditions of example 3, the gradient elution procedure was optimized as follows, with the other chromatographic conditions unchanged.
And (3) taking proper amounts of the system applicability solution, the component positioning solution and the sample solution, respectively injecting into a liquid chromatograph, recording a chromatogram, and testing the result shown in table 1, wherein the chromatogram is shown in fig. 3.
Table 1 summary of the separation test results
Note that: "/" indicates that it is not detected
The result shows that the blank solvent has no interference to the detection of related substances of the product; in the chromatogram of the system applicability solution, the purity of the main peak (compound of formula X) is 89.59%, the purity index is 1000.00, which is better than that of the example 1, the retention time of the main peak is 17.891min, the retention time of the impurity peak is 13.063min at the earliest, the peak shape of each impurity peak is good, the minimum separation degree between each known chromatographic peak and the adjacent chromatographic peak is 3.83 & gt 1.5, namely, under the chromatographic condition, each known component peak can be effectively separated from the adjacent chromatographic peak, and the retention time of the main peak is better.
In combination with examples 1-4, optimal chromatographic conditions were determined:
chromatographic column: agilent ZORBAX Eclipse Plus C18 (100 mm. Times.4.6 mm,3.5 μm)
Mobile phase a:0.01mol/l ammonium formate solution (pH 3.0 adjusted with formic acid)
Mobile phase B: acetonitrile
Column temperature: 35 ℃;
gradient elution procedure:
flow rate: 1.0ml/min
A detector: ultraviolet detector
Detection wavelength: 250nm
Column temperature: 35 DEG C
Sample injection volume: 10 μl of
Example 5
The purity of the compound of formula X in the test sample was determined by area normalization based on the optimal chromatographic conditions determined in example 4. The specific test is as follows:
the compound of formula I (5.38 kg), the compound of formula II (3.06 kg) and triethylamine (1.94 kg) were added to a mixed solvent of DMSO/THF (38L/16L), the temperature was controlled to 70-80℃and the reaction was stirred at constant temperature for 3-4h. Cooling to below 60deg.C, controlling temperature to 30-60deg.C, and adding anhydrous ethanol (108L). After the addition, cooling to 0-10 ℃, preserving heat, stirring and crystallizing for 1h; filtering, washing the filter cake with absolute ethanol (16L), filtering, and vacuum drying at 55-65deg.C to obtain compound of formula X.
Taking about 10mg of a compound of formula X to be tested, precisely weighing, placing the compound into a 20ml measuring flask, adding tetrahydrofuran accounting for about 30% of the volume of the measuring flask, ultrasonically dissolving, cooling, and diluting with acetonitrile to prepare a solution containing about 0.5mg of the compound of formula X per 1ml to be used as a solution of the test sample;
based on the optimal chromatographic conditions determined in example 4, a test solution of the compound of formula X was measured, injected into a liquid chromatograph, subjected to gradient elution, and the chromatogram was recorded, and the purity of the compound of formula X in the test was measured by an area normalization method, and the purity of the compound of formula X was measured to be 99.80%.
In addition, the inventors performed a series of methodological validation experiments on this assay, see examples 6-10.
EXAMPLE 6 specificity test
The blank solvent, each impurity locating solution and system applicability solution described in the examples were measured precisely at 10. Mu.l each, and were injected into a liquid chromatograph, and the chromatograms were recorded. The results are shown in Table 2.
TABLE 2 results of specific experiments
Note that: "/" indicates that it is not detected
The results show that the blank solvent does not interfere with the determination of the relevant substances; the minimum value of the separation degree of the impurities from the main peak of the compound of the formula X is 3.22 and is more than 1.5, and the method has good specificity.
Example 7 quantitative limit and detection limit
Taking a compound reference substance of a formula X and a proper amount of each impurity reference substance, precisely weighing, adding tetrahydrofuran-acetonitrile (30:70) to dissolve to prepare a solution with a certain concentration, diluting step by step, precisely weighing 10 μl of each diluted solution, injecting into a liquid chromatograph, recording a chromatogram, taking a quantitative limit according to a signal-to-noise ratio S/N of not less than 10 and a detection limit according to a signal-to-noise ratio S/N of not less than 3, taking a quantitative limit solution, continuously sampling for 6 times, and calculating the RSD of a peak area. The results are shown in tables 3 and 4.
TABLE 3 quantitative limit results
TABLE 4 limit of detection results
Note that: "/" indicates no detection or detection
As can be seen from tables 3 and 4, the quantitative limit and the detection limit meet the verification requirement, and the method has good sensitivity.
Example 8 Linear test
The compound stock solution and the impurity stock solutions were precisely measured, respectively, and mixed and diluted to the limit level concentrations of 20%, 50%, 80%, 100%, 150% and 200% as linear solutions. Taking each horizontal linear solution, carrying out sample injection analysis by adopting the optimal chromatographic conditions determined in the embodiment 4, recording a chromatogram, taking the main component and each impurity concentration as an abscissa, taking the corresponding solution peak area as an ordinate, calculating each linear regression equation and the correlation coefficient r, wherein the correlation coefficient r of each impurity is larger than 99.9%, and the linear relation is good when each impurity and the compound of the formula X are at the limit level of quantitative limit-200%.
Example 9 accuracy test
Mixing an impurity stock solution: a proper amount of each impurity stock solution (except for the compound of formula X) was precisely measured, and a solution containing about 15. Mu.g of the compound of impurity 1 and 20. Mu.g of the remaining impurities per 1ml was prepared by dilution with a solvent, as a mixed impurity stock solution.
Recovery rate solution: taking about 10mg of a compound sample of the formula X, precisely weighing, placing into a 20ml measuring flask, adding tetrahydrofuran accounting for about 30% of the volume of the measuring flask, carrying out ultrasonic dissolution, cooling, precisely adding 0.5ml, 1.0ml and 1.5ml of mixed impurity stock solution, diluting to a scale with acetonitrile, shaking uniformly, and respectively taking the mixed impurity stock solution as 50%, 100% and 150% recovery rate solutions. (3 parts per concentration level were prepared in parallel with reproducibility testing)
Control solution: the sample solution and the recovery solution were precisely measured, and each appropriate amount of the sample solution and the recovery solution was diluted with a solvent to prepare solutions containing about 1. Mu.g of the compound of formula X per 1ml, respectively, as control solutions.
The blank solvent, each impurity stock solution, test sample solution, and control solution described in example 1 were measured precisely at a concentration of 10. Mu.l each, and injected into a liquid chromatograph to record a chromatogram. Recovery was calculated as peak area by the external standard method. The results are shown in Table 5.
TABLE 5 recovery test results
The results show that: in the range of 50% -150% of the limit concentration, the recovery rate of each impurity is 80% -120%, the recovery rate RSD is less than 10.0%, and the method is good in accuracy.
Example 10 durability test
On the basis of the optimal chromatographic conditions determined in example 4, the flow rate was finely adjusted to be + -20% of the flow rate for investigation, a proper amount of the system applicability solution was taken, the solution was injected into a liquid chromatograph, a chromatogram was recorded, and the test results are shown in Table 6.
Table 6 summary of durability results-system applicability solution
Note that: "/" indicates no detection
The result shows that when the +/-20% of the fine adjustment flow rate is inspected, the minimum separation degree between adjacent chromatographic peaks in the chromatogram of the system applicability solution is 3.55 & gt 1.5, the separation degree is better, and the method has better durability.
Claims (9)
1. A detection method of 2-acylaminothiazole compounds is characterized in that: the 2-acylaminothiazole compound comprises a compound of a formula X and impurities 1-5, and the method is high performance liquid chromatography, and adopts a reversed phase chromatographic column, wherein the reversed phase chromatography is adoptedThe column adopts octadecylsilane chemically bonded silica as filler, the column temperature of the reversed phase chromatographic column is 30-40 ℃, mobile phase A and mobile phase B are used as eluent, the mobile phase A is ammonium formate solution, the mobile phase B is acetonitrile or mixed solution of acetonitrile and methanol 50/50, the concentration of the mobile phase A is 0.009 mol/l-0.011 mol/l, the flow rate of the eluent is 0.9-1.1 ml/min, the detection wavelength is 245 nm-255 nm, the elution is carried out according to gradient, wherein, the structural formula of the compound of formula X and the impurity 1-5 is that Wherein the gradient elution scheme is as follows,
gradient elution scheme 1: mobile phase a is ammonium formate solution, mobile phase B is acetonitrile, mobile phase a and mobile phase B elute according to the following procedure gradient:
；
and in the gradient elution process, the sum of the proportion of the mobile phase A and the proportion of the mobile phase B is 100 percent; wherein the proportion of the mobile phase A is the percentage of the volume of the mobile phase A to the total volume of the eluent, and the proportion of the mobile phase B is the percentage of the volume of the mobile phase B to the total volume of the eluent;
or alternatively, the first and second heat exchangers may be,
gradient elution scheme 2:
mobile phase A is ammonium formate solution, mobile phase B is mixed solution of acetonitrile and methanol 50/50, and mobile phase A and mobile phase B are eluted according to the following program gradient:
；
and in the gradient elution process, the sum of the proportion of the mobile phase A and the proportion of the mobile phase B is 100 percent; wherein the mobile phase A proportion refers to the percentage of the mobile phase A volume to the total volume of the eluent, and the mobile phase B proportion refers to the percentage of the mobile phase B volume to the total volume of the eluent.
2. The method of detection according to claim 1, wherein: the concentration of the mobile phase A is 0.01mol/l.
3. The detection method according to any one of claims 1 or 2, wherein: the pH value of the mobile phase A is 2.8-3.2.
4. A method of detecting as claimed in claim 3, wherein: the pH value of the mobile phase A is 3.0.
5. The method of detection according to claim 1, wherein: the flow rate of the eluent is 1ml/min.
6. The method of detection according to claim 1, wherein: the reverse phase chromatography column was Agilent Eclipse Plus C, its specification was 4.6mm×100mm,3.5 μm.
7. The method of detection according to claim 1, wherein: the detection method is carried out on a high performance liquid chromatograph, an ultraviolet detector is adopted, and the detection wavelength is 250nm.
8. The method of detection according to claim 1, wherein: the column temperature of the reversed phase chromatographic column is 35 ℃.
9. The method of detection according to claim 1, wherein:
(1) Preparing a test solution: taking a proper amount of a test sample of the compound of formula X, precisely weighing, adding tetrahydrofuran for dissolution, diluting with acetonitrile for constant volume, and shaking uniformly to obtain the compound;
(2) Test article determination: the detection method according to claim 1, wherein the sample amount is 10 μl, the sample solution of the compound of formula X is measured, the sample solution is injected into a liquid chromatograph, gradient elution is performed, each chromatogram is recorded, and the purity of the compound of formula X in the sample is measured by an area normalization method.
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| JP2002053568A (en) * | 2000-08-10 | 2002-02-19 | Tokuyama Corp | Method for producing amidothiazole acetic acid derivative |
| WO2021021000A1 (en) * | 2019-08-01 | 2021-02-04 | Марат Феликсович ФАЗЫЛОВ | Method for producing avatrombopag |
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| WO2007129044A1 (en) * | 2006-05-03 | 2007-11-15 | Astrazeneca Ab | Thiazole derivatives and their use as anti-tumour agents |
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| JP2002053568A (en) * | 2000-08-10 | 2002-02-19 | Tokuyama Corp | Method for producing amidothiazole acetic acid derivative |
| WO2021021000A1 (en) * | 2019-08-01 | 2021-02-04 | Марат Феликсович ФАЗЫЛОВ | Method for producing avatrombopag |
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| Identification of 5-substituted 2-acylaminothiazoles that activate Tat mediated transcription in HIV-1 latency models;William Nguyen 等;Journal of Medicinal Chemistry;第62卷(第10期);第5148-5175页 * |
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