Method for detecting content of key intermediate of maleic acid olaratumib by high performance liquid chromatography
Patent Information
- Application Number
- CN202611075488.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-07-20
- Publication Date
- 2026-10-27
AI Technical Summary
[0003]目前马来酸奥拉替尼的合成工艺存在以下技术难点：（1）中间体质量控制难度大：合成路线涉及9步反应、多个关键中间体，各中间体的纯度直接影响终产品质量
[0025]与现有技术相比，本发明一套方法覆盖全部关键中间体，通过两套梯度程序，可分别检测7个关键中间体，操作简便；有效分离关键杂质，方法灵敏度高，检测限可达0.05μg/ml，定量限可达0.15μg/ml；方法耐用性好，在不同品牌色谱柱、不同仪器间均表现稳定。
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of pharmaceutical analytical chemistry, specifically relating to a method for detecting the content of key intermediates of olalatinib maleate using high performance liquid chromatography. Background Technology
[0002] Olapinib maleate is a JAK inhibitor used to control itching associated with canine atopic dermatitis and allergic dermatitis. The original drug, Ibrance, was developed by Zoetis Corporation in the United States and launched in China in 2019. With the rapid development of the pet economy, market demand for this product is strong.
[0003] The synthesis process of olatinib maleate currently faces the following technical challenges: (1) Difficulty in controlling the quality of intermediates: The synthesis route involves 9 steps and multiple key intermediates, and the purity of each intermediate directly affects the quality of the final product. In the existing technology, the detection methods for different intermediates are not uniform and the operation is cumbersome; (2) High requirements for isomer control: The process uses trans-(4-hydroxymethyl)cyclohexylcarbamate tert-butyl ester as the starting material. The conversion of cis and trans isomers must be strictly controlled during the synthesis process, otherwise the efficacy will be affected; (3) Complex impurity spectrum: The process uses a variety of reagents such as p-toluenesulfonyl chloride, potassium thioacetate, formic acid / hydrogen peroxide, thionyl chloride, and methylamine, which may produce a variety of impurities such as p-toluenesulfonate esters, thionyl / sulfones, chlorinated byproducts, and nitrosamines.
[0004] Therefore, there is an urgent need to establish a specific, sensitive, and reproducible HPLC method for detecting the content of key intermediates in olatinib maleate, thereby controlling the quality of these key intermediates. Summary of the Invention
[0005] To address the aforementioned technical problems, this invention provides a method for detecting the content of key intermediates in olalatinib maleate using high-performance liquid chromatography (HPLC). This invention utilizes a single chromatographic platform and can detect the content of multiple key intermediates simply by adjusting the gradient program. This invention can effectively separate process impurities, degradation products, and cis-trans isomers from each key intermediate and achieve accurate determination of their content.
[0006] To achieve the above objectives, the present invention adopts the following technical solution:
[0007] A method for determining the content of key intermediates in olalatinib maleate using high-performance liquid chromatography (HPLC) employs octadecylsilane-bonded silica gel as the packing material; gradient elution is performed using an aqueous phosphoric acid solution or an aqueous trifluoroacetic acid solution as mobile phase A and acetonitrile as mobile phase B; the key intermediate is at least one of ALTN-II, ALTN-III, ALTN-IV, ALTN-V, ALTN-VI, and ALTN-VII, and the structural formulas of each key intermediate are as follows:
[0008] .
[0009] The aforementioned key intermediates are all generated during the synthesis of olalatinib maleate, as detailed below:
[0010] .
[0011] Among them, ALTN-II: (1r,4r)-4-{[methyl(7-toluenesulfonyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]methyl}cyclohexylmethanol;
[0012] ALTN-III: (1r,4r)-4-{[methyl(7-toluenesulfonyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]methyl}cyclohexylmethyl 4-methylbenzenesulfonate;
[0013] ALTN-IV: Thioacetic acid S-[(1r,4r)-4-{[methyl(7-toluenesulfonyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]methyl}cyclohexyl]methyl ester;
[0014] ALTN-V: 1-{(1r,4r)-4-{[methyl(7-toluenesulfonyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]methyl}cyclohexyl}methanesulfonic acid;
[0015] ALTN-VI: 1-{(1r,4r)-4-{[methyl(7-toluenesulfonyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]methyl}cyclohexyl}methanesulfonyl chloride;
[0016] ALTN-VII: N-methyl-1-{(1r,4r)-4-{[methyl(7-toluenesulfonyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]methyl}cyclohexyl}methanesulfonamide.
[0017] The chromatographic column has a length of 150~250mm, an inner diameter of 4.0~4.6mm, and a packing particle size of 3~5μm, preferably Agilent Zorbax SB-C18 (250×4.6mm, 5μm).
[0018] The volume concentration of the trifluoroacetic acid aqueous solution is 0.04%~0.1%, and the volume concentration of the phosphoric acid aqueous solution is 0.1%.
[0019] When the key intermediates are ALTN-II, ALTN-V, ALTN-VI, and ALTN-VII, the gradient elution program is as follows: from 0 min to 12 min, the volume percentage of mobile phase B increases linearly from 5% to 95%; from 12 min to 16 min, mobile phase B is maintained at 95%; from 16 min to 16.1 min, mobile phase B decreases from 95% to 5%; and from 16.1 min to 25 min, mobile phase B is maintained at 5%.
[0020] When the key intermediate is ALTN-III or ALTN-IV, the gradient elution program is as follows: 0 min to 6 min, the volume percentage of mobile phase B increases linearly from 0% to 95%; 6 min to 16 min, mobile phase B is maintained at 95%; 16 min to 16.1 min, mobile phase B decreases from 95% to 0%; 16.1 min to 25 min, mobile phase B is maintained at 0%.
[0021] The specific steps are as follows:
[0022] (1) Prepare a test solution of the key intermediate of olatinib maleate; the test solution is prepared with methanol, acetonitrile or methanol-water mixture, and the mass concentration of the test solution is 0.5~2.0 mg / ml.
[0023] (2) Inject the test solution into the liquid chromatograph, record the chromatogram, and calculate the content of olatinib maleate intermediate in the test solution by the area normalization method.
[0024] System suitability requirements: The theoretical plate number calculated based on the main peak is not less than 10,000; the resolution between the main peak and adjacent impurity peaks is not less than 1.5; the tailing factor is between 0.8 and 1.5; continuous injection of 5 needles, the main peak retention time RSD is ≤1.0%, and the peak area RSD is ≤2.0%.
[0025] Compared with existing technologies, the present invention provides a single method that covers all key intermediates. Through two gradient programs, seven key intermediates can be detected separately. The method is simple to operate, effectively separates key impurities, has high sensitivity with a detection limit of 0.05 μg / ml and a quantitation limit of 0.15 μg / ml, and exhibits good robustness, remaining stable across different brands of chromatographic columns and instruments. Attached Figure Description
[0026] Figure 1 Here is the HPLC chromatogram of the ALTN-II intermediate;
[0027] Figure 2 HPLC chromatogram of ALTN-III intermediate;
[0028] Figure 3 Here is the HPLC chromatogram of the ALTN-IV intermediate;
[0029] Figure 4 Here is the HPLC chromatogram of the ALTN-V intermediate;
[0030] Figure 5 Here is the HPLC chromatogram of the ALTN-VI intermediate;
[0031] Figure 6 This is the HPLC chromatogram of the ALTN-VII intermediate. Detailed Implementation
[0032] The present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.
[0033] Example 1: Purity detection of ALTN-II intermediate
[0034] Preparation of test solution: Take 10 mg of ALTN-II intermediate (batch number: 260204-01), dissolve in methanol and bring the volume to 10 ml.
[0035] Chromatographic conditions: Agilent Zorbax SB-C18 (250 × 4.6 mm, 5 μm), mobile phase A was 0.1% phosphoric acid aqueous solution, mobile phase B was acetonitrile, flow rate was 1.0 mL / min, column temperature was 40 °C, detection wavelength was 254 nm, injection volume was 10 μL, and the gradient elution program was as follows: 0 min to 12 min, the volume percentage of mobile phase B linearly increased from 5% to 95%; 12 min to 16 min, mobile phase B was maintained at 95%; 16 min to 16.1 min, mobile phase B decreased from 95% to 5%; 16.1 min to 25 min, mobile phase B was maintained at 5%.
[0036] Measurement results (see) Figure 1 The main peak retention time was 6.866 min, with an area percentage of 98.83%. Impurity 1: RT 6.657 min, area 0.16%; Impurity 2: RT 7.673 min, area 0.63%; Impurity 3: RT 8.113 min, area 0.28%; Impurity 4: RT 15.469 min, area 0.10%. The total impurity was 1.17%, which meets the requirement of intermediate II purity ≥97%.
[0037] Example 2: Purity detection of ALTN-III intermediate
[0038] Preparation of test solution: Take 10 mg of ALTN-III intermediate (batch number: 260210-01-3), dissolve in methanol and bring the volume to 10 ml.
[0039] Chromatographic conditions: Agilent Zorbax SB-C18 (250 × 4.6 mm, 5 μm), mobile phase A was 0.1% aqueous phosphoric acid solution, mobile phase B was acetonitrile, gradient elution program was as follows: 0 min to 6 min, the volume percentage of mobile phase B linearly increased from 0% to 95%; 6 min to 16 min, mobile phase B was maintained at 95%; 16 min to 16.1 min, mobile phase B decreased from 95% to 0%; 16.1 min to 25 min, mobile phase B was maintained at 0%; the rest was the same as in Example 1.
[0040] Measurement results (see) Figure 2 The main peak retention time was 10.145 min, and the area percentage was 94.00%. Major impurities were: RT 3.189 min, 0.98%; RT 5.740 min, 0.84%; RT 9.471 min, 2.39%; RT 9.662 min, 0.35%; RT 10.396 min, 1.44%. Total impurities were 6.00%, meeting the requirement of intermediate III purity ≥93%.
[0041] Example 3: Purity detection of ALTN-IV intermediates
[0042] Preparation of test solution: Take 10 mg of ALTN-IV intermediate (batch number: 260211-01-3), dissolve in methanol and bring the volume to 10 ml.
[0043] Chromatographic conditions: Same as in Example 2.
[0044] Measurement results (see) Figure 3 The main peak retention time was 10.309 min, with an area percentage of 98.91%. Impurities were: RT 9.147 min, 0.09%; T 9.410 min, 0.27%; RT 9.600 min, 0.06%; RT 11.094 min, 0.66%. The total impurities were 1.09%, which meets the requirement of intermediate IV purity ≥ 97%.
[0045] Example 4: Purity detection of ALTN-V intermediates
[0046] Preparation of test solution: Take 10 mg of ALTN-V intermediate (batch number: 260225-01), dissolve in methanol and bring the volume to 10 ml.
[0047] Chromatographic conditions: Same as in Example 1.
[0048] Measurement results (see) Figure 4The main peak retention time was 9.787 min, and the area percentage was 97.82%. The main impurities were: RT8.757 min, 1.10%, RT9.306 min, 0.06%, RT9.451 min, 0.07%, RT11.504 min, 0.29%, and RT11.637 min, 0.35%. The total impurities were 2.18%, which meets the requirement that the purity of intermediate V is ≥96%.
[0049] Example 5: Purity detection of ALTN-VI intermediates
[0050] Preparation of test solution: Take 10 mg of ALTN-VI intermediate (batch number: 260227-01), dissolve in methanol and bring the volume to 10 ml.
[0051] Chromatographic conditions: Same as in Example 1.
[0052] Measurement results (see) Figure 5 The main peak retention time was 12.065 min, and the area percentage was 97.32%. Major impurities included: Impurity 1 (RT 9.008 min, 0.30%), Impurity 2 (RT 9.863 min, 0.32%), RT 1 (RT 0.316 min, 0.10%), RT 1 (RT 0.616 min, 0.48%), RT 1 (RT 11.253 min, 0.12%), and RT 1 (RT 11.466 min, 0.31%). Other process impurities ranged from RT 13.188 to 16.427 min, totaling 0.78%. The total impurity content was 2.68%, meeting the requirement of intermediate VI purity ≥96%.
[0053] Example 6: Purity detection of ALTN-VII intermediate
[0054] Preparation of test solution: Take 10 mg of ALTN-VII intermediate (batch number: 260228-01), dissolve in methanol and bring the volume to 10 ml.
[0055] Chromatographic conditions: Same as in Example 1.
[0056] Measurement results (see) Figure 6 The main peak retention time was 7.203 min, with an area percentage of 98.05%. Impurities 1 (RT 6.390 min, 0.22%), 2 (RT 6.624 min, 0.31%), and 3 (RT 6.875 min, 0.29%) were all unknown process impurities. The hydrolysis products (RT 7.501 min, 0.41%; RT 7.742 min, 0.06%; RT 7.840 min, 0.07%; RT 8.003~15.439 min, totaling 0.56%) were present. The total impurities were 1.95%, which meets the requirement of intermediate VII purity ≥97%.
[0057] Example 7: System Suitability Test
[0058] The ALTN-VII intermediate test solution was injected six times consecutively, and the retention time and peak area of the main peak were recorded. Results: The retention time RSD was 0.15%, and the peak area RSD was 0.42%; the theoretical plate number calculated based on the main peak was 15478; the tailing factor was 1.05–1.15; and the resolutions between the main peak and adjacent impurities were 2.34 and 2.01, respectively. This indicates that the system suitability meets the requirements.
[0059] Example 8: Method Robustness
[0060] The ALTN-VII intermediate test solution was tested under the following fine-tuning conditions: mobile phase A: 0.04% trifluoroacetic acid (replacing 0.1% phosphoric acid); column temperature: 38℃, 42℃; flow rate: 0.9 ml / min, 1.1 ml / min; different brands of C18 columns (Agilent, Waters, YMC). The results showed that the retention time of the main peak fluctuated within ±5%, the relative retention times of each impurity were basically consistent, the resolution was greater than 1.5, and the RSD of the area normalization result was less than 2.0%, indicating that the method has good robustness.
[0061] Example 9: Determination of Limit of Quantitation and Limit of Detection
[0062] Accurately weigh an appropriate amount of ALTN-VII reference standard, dissolve it in methanol, and quantitatively dilute it to prepare a reference standard stock solution containing approximately 1 mg of ALTN-VII per ml. Using a stepwise dilution method, the stock solution was successively diluted with methanol to prepare a series of solutions of different concentrations, which were then injected for analysis. The concentration at which the signal-to-noise ratio (S / N) ≈ 3 was used as the limit of detection (LOD), and the concentration at which S / N ≈ 10 was used as the limit of quantitation (LOQ). The LOD was 0.05 μg / ml, the LOQ was 0.15 μg / ml, and the consecutive injection RSD (n=6) of the LOQ concentration was 1.8%, which met the requirements.
[0063] Example 10: Recovery Test
[0064] A certain amount of the major impurity reference standard ALTN-VI was added to the intermediate of ALTN-VII with known content, and the spiked recovery rate was calculated. The recovery rate of ALTN-VI was between 95% and 105%, and the RSD was ≤2.0%.
[0065] Example 11: Suitability Test of ALTN-III Intermediate System
[0066] Take an appropriate amount of ALTN-III intermediate, dissolve it in methanol, and quantitatively dilute it to prepare a test solution containing approximately 1.0 mg per ml. Simultaneously, take appropriate amounts of ALTN-III intermediate and its adjacent impurity reference standards, dissolve them in methanol, and prepare a mixed reference solution. Inject and analyze according to the chromatographic conditions described in Example 2. Inject the ALTN-III test solution six times consecutively, recording the retention time and peak area of the main peak. The RSD of the main peak retention time was 0.18%, and the RSD of the peak area was 0.53%; the theoretical plate number calculated based on the ALTN-III main peak was 16892; the tailing factor was between 1.02 and 1.12; the resolutions between the main peak and adjacent impurity peaks were 2.51 and 2.23, respectively, both greater than 1.5. Injecting the mixed reference solution showed that the resolution between the ALTN-III main peak and the cis-trans isomer impurity peak was 2.36, and the resolution with the p-toluenesulfonate impurity peak was 3.02; the resolution between each impurity peak was greater than 1.8. The above results indicate that this system is suitable for impurity separation and purity determination of ALTN-III intermediates, and the system's applicability meets the requirements.
[0067] Example 12: Determination of the limit of quantitation and limit of detection of ALTN-V intermediates
[0068] Accurately weigh an appropriate amount of ALTN-V reference standard, dissolve it in methanol, and quantitatively dilute it to prepare a reference standard stock solution containing approximately 1.0 mg of ALTN-V per ml. Using a stepwise dilution method, the stock solution was successively diluted with methanol to prepare a series of solutions of different concentrations (concentration range: 0.01 μg / ml to 10 μg / ml), and each solution was injected for analysis. The limit of quantitation (LOQ) concentration was injected six times consecutively. The limit of detection (LOD) for ALTN-V was 0.06 μg / ml, the limit of quantitation (LOQ) concentration was 0.20 μg / ml, the peak area RSD was 1.9%, and the retention time RSD was 0.22%, indicating that this method has good precision and reproducibility at the LOQ concentration.
[0069] Intermediates ALTN-II / V / VI / VII belong to one category, and ALTN-III / IV belong to another category. A complete set of validation data is provided for each. Since the structures of each intermediate are related and the testing platform is consistent, polarity coverage at both ends and typical intermediates (such as ALTN-VII and ALTN-III) are selected for full validation. The remaining intermediates are validated for key items, and the results all meet the requirements.
[0070] While the specific embodiments of the present invention have been described above, they are not intended to limit the scope of protection of the present invention. Based on the technical solutions of the present invention, various modifications or variations that can be made by those skilled in the art without creative effort are still within the scope of protection of the present invention.
Claims
1. A method for determining the content of key intermediates of olatinib maleate using high performance liquid chromatography, characterized in that, The column was packed with octadecylsilane-bonded silica gel; gradient elution was performed using an aqueous solution of phosphoric acid or an aqueous solution of trifluoroacetic acid as mobile phase A and acetonitrile as mobile phase B; the intermediate was at least one of ALTN-II, ALTN-III, ALTN-IV, ALTN-V, ALTN-VI, and ALTN-VII, and the structural formulas of each intermediate are as follows: 。 2. The method for detecting the content of key intermediates of olalatinib maleate using high performance liquid chromatography according to claim 1, characterized in that, The flow rate was 0.8~1.2 ml / min, the column temperature was 30~45℃, the injection volume was 5~20 μl, and the detection wavelength was 254 nm.
3. The method for detecting the content of key intermediates of olalatinib maleate using high performance liquid chromatography according to claim 1, characterized in that, The chromatographic column has a length of 150~250mm, an inner diameter of 4.0~4.6mm, and a packing particle size of 3~5μm.
4. The method for detecting the content of key intermediates of olalatinib maleate using high performance liquid chromatography according to claim 1, characterized in that, The volume concentration of the trifluoroacetic acid aqueous solution is 0.04%~0.1%, and the volume concentration of the phosphoric acid aqueous solution is 0.1%.
5. The method for detecting the content of key intermediates of olalatinib maleate using high performance liquid chromatography according to claim 1, characterized in that, When the intermediates are ALTN-II, ALTN-V, ALTN-VI, or ALTN-VII, the gradient elution program is as follows: 0 min to 12 min, the volume percentage of mobile phase B increases linearly from 5% to 95%; 12 min to 16 min, mobile phase B is maintained at 95%; 16 min to 16.1 min, mobile phase B decreases from 95% to 5%; 16.1 min to 25 min, mobile phase B is maintained at 5%.
6. The method for detecting the content of key intermediates of olatinib maleate using high performance liquid chromatography according to claim 1, characterized in that, When the intermediate is ALTN-III or ALTN-IV, the gradient elution program is as follows: 0 min to 6 min, the volume percentage of mobile phase B increases linearly from 0% to 95%; 6 min to 16 min, mobile phase B is maintained at 95%; 16 min to 16.1 min, mobile phase B decreases from 95% to 0%; 16.1 min to 25 min, mobile phase B is maintained at 0%.
7. The method for detecting the content of key intermediates of olatinib maleate using high performance liquid chromatography according to claim 1, characterized in that, The specific steps are as follows: (1) Prepare the test solution of olatinib maleate intermediate; (2) Inject the test solution into the liquid chromatograph, record the chromatogram, and calculate the content of olatinib maleate intermediate in the test solution by the area normalization method.
8. The method for detecting the content of key intermediates of olatinib maleate using high performance liquid chromatography according to claim 7, characterized in that, The mass concentration of the test solution is 0.5~2.0 mg / ml.
9. The method for detecting the content of key intermediates of olatinib maleate using high performance liquid chromatography according to claim 7, characterized in that, The test solution is prepared using methanol, acetonitrile, or a methanol-water mixture.
10. The application of the method according to any one of claims 1 to 9 in the synthesis of olatinib maleate.