Method for preparing plerixafor and method for determining potential ring-opening impurities in plerixafor

Abstract

The present invention relates to a method for preparing plerixafor and a method for determining potential ring-opening impurities in plerixafor. On the one hand, it relates to a method for determining potential ring-opening impurities in plerixafor using high performance liquid chromatography, wherein the impurities are selected from impurities A, impurities B, impurities C and impurities D, and the method comprises the following steps and conditions: using a sodium heptane sulfonate solution whose pH value is adjusted to 1.6 with perchloric acid as mobile phase A, and acetonitrile as mobile phase B; preparing an impurity reference solution and a test solution with a solvent, using a chromatographic column with octadecylsilane bonded silica gel as a filler, using mobile phase A and mobile phase B to perform gradient elution according to an elution program, recording a chromatogram, and calculating the amount of each impurity in the test sample by peak area according to an external standard method. It also relates to a method for preparing high-quality plerixafor, in which the method for determining potential ring-opening impurities in plerixafor using high performance liquid chromatography of the present invention is used to determine the potential impurities therein. The methods of various aspects of the present invention present excellent effects.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and relates to plerixafor, and also relates to a method for preparing high-quality plerixafor. Further, the present invention also relates to a method for determining potential ring-opening impurities in plerixafor using high-performance liquid chromatography, and quality control by using the method for determining potential ring-opening impurities in plerixafor using high-performance liquid chromatography, thereby preparing high-quality plerixafor. Background Art

[0002] The hematopoietic stem cell mobilizer plerixafor publicly launched by GENZYME has an English name: Plerixafor, and the commercial injection solution is called Mozobil. Its English chemical name is 1,4-Bis((1,4,8,11-tetraazacyclotetradecan-1-yl)methyl)benzene, and its Chinese chemical name is 1,4-bis((1,4,8,11-tetraazacyclotetradecan-1-yl)methyl)benzene or 1,1'-[1,4-phenylene (methylene)]-di-1,4,8,11-tetraazacyclotetradecan. Its molecular formula is C28H54N8, molecular weight is 502.79, and its chemical structure is as follows:

[0003] .

[0004] Plerixafor is a white to off-white crystalline solid that is hygroscopic with a typical melting point in the range of 125-131°C; the partition coefficient between 1-octanol and pH 7 aqueous buffer is <0.1.

[0005] Plerixafor is a hematopoietic stem cell activator and CXCR4 chemokine antagonist. Its injection, Mozobil, was approved by the U.S. FDA in December 2008 for autologous transplantation in patients with non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM). Plerixafor can be used in combination with granulocyte colony-stimulating factor (G-CSF, filgrastim) to block the binding of CXCR4 to SDF-1α, drive the release of CD34+ cells into the peripheral blood, and quickly and effectively increase the number of hematopoietic stem cells in the blood circulation of NHL and MM patients.

[0006] Due to various factors (such as storage time, storage conditions, etc.), one or more of the following four potential open-ring impurities may exist in the API or its preparations:

[0007] Plerixafor Impurity A: ,

[0008] Chemical name: N1-(2-((4-(1,4,8,11-tetraazacyclotetradecane-1-yl)methyl)benzyl)amino)ethyl)-N3-(2-aminoethyl)propane-1,3-diamine;

[0009] Plerixafor Impurity B: ,

[0010] Chemical name: N1-(4-(1,4,8,11-tetraazacyclotetradecane-1-yl)methyl)benzyl)-N1-(2-(3-aminopropyl)amino)ethyl)propane-1,3-diamine;

[0011] Plerixafor Impurity C: ,

[0012] Chemical name: N1-(4-(1,4,8,11-tetraazacyclotetradecane-1-yl)methyl)benzyl)-N1,N3-bis(2-aminoethyl)propane-1,3-diamine;

[0013] Plerixafor Impurity D: ,

[0014] Chemical name: N1-(4-(1,4,8,11-tetraazacyclotetradecane-1-yl)methyl)benzyl)-N3-2-((3-aminopropyl)amino)ethyl)propane-1,3-diamine.

[0015] In this article, the above-mentioned plerixafor impurity A, plerixafor impurity B, plerixafor impurity C, and plerixafor impurity D may also be referred to as impurity A, impurity B, impurity C, and impurity D, respectively, and they may also be collectively referred to as open-ring impurities.

[0016] In industrial production such as the preparation of plerixafor API, product quality control is an important task. To improve work efficiency, it is very meaningful to use HPLC to simultaneously determine plerixafor impurities A, B, C, and D in plerixafor API. However, the existing technology does not provide any solution to this technical requirement.

[0017] Therefore, those skilled in the art are eager to have an HPLC method that can simultaneously determine the plerixafor impurities A, B, C, and D in the plerixafor API. Summary of the invention

[0018] The object of the present invention is to provide a method for preparing high-quality plerixafor. Alternatively, the object of the present invention is to provide a method for determining potential ring-opening impurities in plerixafor using high performance liquid chromatography, or the object of the present invention is to monitor the quality of the potential ring-opening impurities in plerixafor using high performance liquid chromatography, thereby preparing high-quality plerixafor.

[0019] To this end, the first aspect of the present invention provides a method for determining potential ring-opening impurities in plerixafor using high performance liquid chromatography, wherein the potential ring-opening impurities are selected from:

[0020] Plerixafor impurity A: N1-(2-((4-(1,4,8,11-tetraazacyclotetradecane-1-yl)methyl)benzyl)amino)ethyl)-N3-(2-aminoethyl)propane-1,3-diamine,

[0021] Plerixafor impurity B: N1-(4-(1,4,8,11-tetraazacyclotetradecane-1-yl)methyl)benzyl)-N1-(2-(3-aminopropyl)amino)ethyl)propane-1,3-diamine,

[0022] Plerixafor impurity C: N1-(4-(1,4,8,11-tetraazacyclotetradecane-1-yl)methyl)benzyl)-N1,N3-bis(2-aminoethyl)propane-1,3-diamine,

[0023] Plerixafor impurity D: N1-(4-(1,4,8,11-tetraazacyclotetradec-1-yl)methyl)benzyl)-N3-2-((3-aminopropyl)amino)ethyl)propane-1,3-diamine;

[0024] The method comprises the following steps:

[0025] (1) Liquid preparation

[0026] Mobile phase: 0.005 mol / L sodium heptane sulfonate solution adjusted to pH 1.6 with perchloric acid was used as mobile phase A, and acetonitrile was used as mobile phase B;

[0027] Impurity reference solution: Take appropriate amount of plerixafor impurity A reference, plerixafor impurity B reference, plerixafor impurity C reference, and plerixafor impurity D reference, accurately weigh, dissolve in solvent and quantitatively dilute to prepare reference solution containing about 2 μg of each in 1 ml;

[0028] Test solution: Take an appropriate amount of plerixafor, dissolve it in solvent and quantitatively dilute it to make a solution containing about 2 mg of the test solution per 1 ml;

[0029] Each test solution (test solution, impurity reference solution, and other solutions used for HPLC testing) was stored at room temperature until the determination;

[0030] (2) Chromatographic conditions

[0031] Use octadecylsilane bonded silica gel as a chromatographic column as filler, and use mobile phase A and mobile phase B to perform gradient elution as shown in the following table:

[0032] ;

[0033] The flow rate was 0.8 ml per minute, the detection wavelength was 210 nm, the column temperature was 28°C to 35°C, and the injection volume was 10 μl;

[0034] (3) Determination

[0035] Inject the mixed impurity reference solution and the test sample solution into the liquid chromatograph respectively, record the chromatogram, and calculate the amount of each impurity in the test sample by peak area according to the external standard method.

[0036] According to the method of the first aspect of the present invention, the column temperature used in the chromatographic conditions is 30°C.

[0037] According to the method of the first aspect of the present invention, each test solution is stored at 20-25° C. until the measurement.

[0038] According to the method of the first aspect of the present invention, appropriate amounts of four reference substances are taken respectively, accurately weighed, and solvents are used to prepare four impurity reference substance solutions containing approximately 2 μg of each impurity in each ml, which are used for qualitative determination of the four impurities; for example, the solvent is prepared by the following method: 0.08 mol / L succinic acid aqueous solution and acetonitrile are mixed in a volume ratio of 95:5.

[0039] According to the method of the first aspect of the present invention, the specification of the chromatographic column is 4.6×250 mm.

[0040] According to the method of the first aspect of the present invention, the particle size of the chromatographic column filler is 5 μm.

[0041] According to the method of the first aspect of the present invention, the chromatographic column is an Agilent ZORBAX SB-C18 chromatographic column.

[0042] According to the method of the first aspect of the present invention, the chromatographic column is a chromatographic column with a specification of 4.6×250 mm, a filler particle size of 5 μm, and a brand of Agilent ZORBAX SB-C18.

[0043] According to the method of the first aspect of the present invention, the temperature of the sample plate during chromatographic determination is room temperature.

[0044] According to the method of the first aspect of the present invention, the temperature of the sample plate during chromatographic determination is 20-25°C.

[0045] According to the method of the first aspect of the present invention, the separation degrees among impurities A, B, C and D are all greater than 3.0.

[0046] According to the method of the first aspect of the present invention, the separation degree between the main component plerixafor and its closest impurity is greater than 5.0.

[0047] Furthermore, the second aspect of the present invention provides a method for preparing plerixafor, comprising the following steps:

[0048] (1) 10.5 g of 1,4,8,11-tetraaza-5,7,12-trioxocyclotetradecane was dissolved in 150 mL of anhydrous acetonitrile, and 2.5 g of terephthaloyl chloride was dissolved in 10 mL of triethylamine. The two solutions were injected into a reactor at a molar ratio of cyclotetradecane compound to terephthaloyl chloride of 4:1, and reacted at 80° C. for 30 min. The effluent was collected, filtered, dried, and recrystallized with DMSO / ethanol in a volume ratio of 1 / 5 to obtain phthaloyl-substituted bis[1,4,8,11-tetraaza-5,7,12-trioxocyclotetradecane];

[0049] (2) 5.0 g of the product of the previous step and 9.0 g of sodium borohydride were added to 150 mL of dioxane at room temperature, 15 g of glacial acetic acid was added and the mixture was reacted for 45 h, the pH value was adjusted to 2 with 10% hydrochloric acid, the solvent was removed under reduced pressure, water was added to the residue and stirred to dissolve, 0.05 M sodium hydroxide solution was added to adjust the pH value to 12, the solvent was recovered under reduced pressure, the mixture was extracted with dichloromethane, and the mixture was concentrated under reduced pressure. The residue was recrystallized with isopropanol / ethyl acetate in a volume ratio of 1 / 5 to obtain white crystals of plerixafor;

[0050] (3) Determine the area normalized purity of the product plerixafor in step (2) and the content of potential open-ring impurities, i.e., impurity A, impurity B, impurity C, and impurity D, using the method described in any one of the first aspects.

[0051] The method of the present invention exhibits excellent effects. For example, the HPLC method of the present invention for determining potential open-ring impurities of plerixafor has relatively low limits of quantitation and detection, excellent linearity, repeatability of determination results and recovery rate for four impurities, and the product has excellent purity when plerixafor is prepared by the method of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 : Liquid chromatogram of impurity reference solution.

[0053] Figure 2 : Liquid chromatogram of plerixafor test solution.

[0054] Figure 3 : Liquid chromatogram of plerixafor-impurity mixed solution.

[0055] Figure 4 : Liquid chromatogram of impurity quantification limit solution.

[0056] Figure 5 : Liquid chromatogram of impurity detection limit solution. DETAILED DESCRIPTION

[0057] In order to make the purpose, technical solution and advantages of the method more clear, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0058] The application principle of the present invention is described in detail below with reference to examples.

[0059] Example 1: Method for determining potential ring-opening impurities in plerixafor

[0060] This example uses high performance liquid chromatography to determine potential open-ring impurities in plerixafor, which specifically includes the following steps.

[0061] (1) Liquid preparation

[0062] Mobile phase: 0.005 mol / L sodium heptane sulfonate solution adjusted to pH 1.6 with perchloric acid was used as mobile phase A, and acetonitrile was used as mobile phase B;

[0063] Solvent: Mix 0.08 mol / L succinic acid aqueous solution and acetonitrile in a volume ratio of 95:5 to obtain;

[0064] Impurity reference solution: Take appropriate amount of Plerixafor Impurity A Reference, Plerixafor Impurity B Reference, Plerixafor Impurity C Reference, and Plerixafor Impurity D Reference, accurately weigh them, dissolve them in solvent, and quantitatively dilute them to prepare reference solution containing about 2 μg of each in 1 ml (in addition, take appropriate amount of four reference substances, accurately weigh them, and prepare four impurity reference solutions containing about 2 μg of each in 1 ml in the same way, which can be used to qualitatively determine the four impurities);

[0065] Test solution: Take an appropriate amount of plerixafor, dissolve it in solvent and quantitatively dilute it to make a solution containing about 2 mg of the test solution per 1 ml;

[0066] All test solutions (test solution, impurity reference solution, etc., solutions used for HPLC testing) were stored at room temperature (20-25°C) until determination.

[0067] (2) Chromatographic conditions

[0068] Use a chromatographic column filled with octadecylsilane bonded silica gel (Agilent ZORBAX SB-C18, 4.6×250mm, 5μm) and mobile phase A and mobile phase B for gradient elution according to the elution program shown in the following table:

[0069] ;

[0070] The flow rate was 0.8 ml per minute, the detection wavelength was 210 nm, the column temperature was 28°C to 35°C (specifically 30°C), the injection volume was 10 μl, and the sample tray temperature was room temperature (20 to 25°C);

[0071] (3) Determination

[0072] Inject the mixed impurity reference solution (and the reference solution prepared for each impurity) and the test solution into the liquid chromatograph respectively, record the chromatogram, and calculate the amount of each impurity in the test sample by peak area using the external standard method.

[0073] The retention time of the reference solution prepared for each impurity was used to determine the attribution of each peak in the four impurity mixed solutions;

[0074] The above method was used to determine the bulk drug of Plerixafor, and the results were as follows:

[0075] The HPLC chromatogram of the reference solution containing four impurities is typically as follows: Figure 1 As shown, the four impurities were prepared separately with a single reference solution, and the peak order was determined by the same method: impurity A (retention time about 39 min), impurity B (retention time about 42 min), impurity C (retention time about 44 min), impurity D (retention time about 46 min);

[0076] The HPLC chromatogram of the test solution of Plerixafor (lot number LPlsf-006) is typically as follows: Figure 2 As shown, it is shown that there are trace amounts of impurities A and B in this batch of plerixafor raw materials. According to the external standard method, the contents of impurity A (rrt = 1.45) and impurity B (rrt = 1.55) are 0.05% and 0.03%, respectively, and impurity C and impurity D are not detected. Unless otherwise specified herein, the plerixafor involved is the LPlsf-006 batch. The method of this embodiment 1 was used to measure two batches of homemade plerixafor with batch numbers LPlsf-008 and LPlsf-009. As a result, the contents of impurity A, impurity B, and impurity D of LPlsf-008 were 0.05%, 0.02%, and 0.03%, respectively, and impurity C was not detected. The contents of impurity A and impurity B of LPlsf-009 were 0.04% and 0.02%, respectively, and impurity C and impurity D were not detected.

[0077] In addition, the method of Example 1 of the present invention can also be used to determine the area normalized purity of plerixafor. For example, the area normalized purity of the above three batches of plerixafor was greater than 99%.

[0078] In addition, referring to the preparation method of the reference solution and the test solution in Example 1, an appropriate amount of the four impurities of plerixafor and plerixafor were taken, dissolved in a solvent and quantitatively diluted to prepare a mixed solution containing about 2 mg of plerixafor and 2 μg of each of the four impurities per 1 ml, as a plerixafor-impurity mixed solution; using the method of Example 1, the mixed solution was injected into a liquid chromatograph and a chromatogram was recorded. A typical chromatogram is shown in FIG. Figure 3 As shown, after calculation, the separation between impurities A, B, C and D is greater than 3.6, for example, the separation between impurities B and C is 3.65, and the separation between the main component plerixafor and its closest impurity is greater than 5.0, indicating that the separation between the components of the HPLC method of the present invention is excellent. Figure 3 Taking the main component plerixafor as the benchmark, the relative retention times of the four impurities were calculated to be impurity A = 1.43 (38.817 / 27.063), impurity B = 1.54 (41.559 / 27.063), impurity C = 1.60 (43.334 / 27.063), and impurity D = 1.70 (45.980 / 27.063).

[0079] (4) Limit of quantification and detection limit

[0080] The reference solution containing the four impurities was diluted step by step, and the solution with S / N ≥ 10 was used as the quantitative limit solution, and the solution with S / N ≥ 3 was used as the detection limit solution.

[0081] The method described in "(2) Chromatographic conditions" and "(3) Determination" of this embodiment was used to continuously inject 6 injections of the quantitative limit solution and 1 injection of the detection limit solution, and the chromatogram was recorded. Figure 4 and Figure 5 shown.

[0082] result:

[0083] The limit of quantification of impurity A of plerixafor was 5.58 ng, the signal-to-noise ratio was greater than 10, and the RSD value of the peak area was 8.1%, less than 10%, equivalent to 0.03% of the test sample concentration; the limit of detection was 2.79 ng, the signal-to-noise ratio was greater than 3, equivalent to 0.01% of the test sample concentration;

[0084] The limit of quantification of impurity B of plerixafor was 5.53 ng, the signal-to-noise ratio was greater than 10, and the RSD value of the peak area was 7.7%, less than 10%, equivalent to 0.03% of the test sample concentration; the detection limit was 2.77 ng, the signal-to-noise ratio was greater than 3, equivalent to 0.01% of the test sample concentration;

[0085] The limit of quantification of impurity C of plerixafor was 5.34 ng, the signal-to-noise ratio was greater than 10, and the RSD value of the peak area was 9.7%, less than 10%, equivalent to 0.03% of the test sample concentration; the detection limit was 2.67 ng, the signal-to-noise ratio was greater than 3, equivalent to 0.01% of the test sample concentration;

[0086] The quantification limit of plerixafor impurity D was 5.27 ng, the signal-to-noise ratio was greater than 10, and the RSD value of the peak area was 8.3%, which was less than 10%, equivalent to 0.03% of the test sample concentration; the detection limit was 2.63 ng, and the signal-to-noise ratio was greater than 3, equivalent to 0.01% of the test sample concentration.

[0087] The above results show that the HPLC method of the present invention has good sensitivity when determining the four potential impurities in plerixafor.

[0088] (5) Linear investigation

[0089] Take appropriate amounts of plerixafor impurity A, plerixafor impurity B, plerixafor impurity C and plerixafor impurity D reference substances, accurately weigh them, dissolve them in solvent and quantitatively dilute them to prepare a solution containing about 200 μg of each impurity per 1 ml to obtain stock solution A.

[0090] Accurately measure an appropriate amount of stock solution A and dilute it with solvent to make solutions with concentrations of approximately 0.5μg / ml, 1μg / ml, 3μg / ml, 6μg / ml, 10μg / ml, 15μg / ml, and 20μg / ml, respectively.

[0091] The method of "(2) Chromatographic conditions" and "(3) Determination" in this example was used to perform linear regression with the peak area (y) and concentration (x) of each impurity. The results are as follows:

[0092] For impurity A of plerixafor, in the concentration range of 0.55-20.39 μg / ml, the linear relationship between peak area and concentration (μg / ml) is y=9778.8762x+958.7084, r=0.9999;

[0093] The linear relationship between peak area and concentration (μg / ml) of plerixafor impurity B in the concentration range of 0.58-99.74 μg / ml is y=9683.4887x-541.1228, r=0.9999;

[0094] The linear relationship between peak area and concentration (μg / ml) of plerixafor impurity C in the concentration range of 0.53~20.07μg / ml is y=7685.8213x+66.4514, r=1.0000;

[0095] In the concentration range of 0.53~20.03μg / ml, the linear relationship between peak area and concentration (μg / ml) of plerixafor impurity D is y=9424.7086x-447.3962, r=0.9999.

[0096] The above results show that the linear relationship is good.

[0097] (6) Repeatability

[0098] The six spiked test sample solutions (used in the present invention) were measured using the methods of "(2) Chromatographic Conditions" and "(3) Determination" in this example. Figure 3 The repeatability of the method was investigated by measuring the content of plerixafor impurity A, plerixafor impurity B, plerixafor impurity C, and plerixafor impurity D in the spiked test solution. The RSD of the repeatable determination of plerixafor impurity A was 2.2%, and the RSD value of plerixafor impurity B was 1.3%; the RSD value of plerixafor impurity C was 1.5%; and the RSD value of plerixafor impurity D was 1.6%. The determination results met the general requirements for repeatability determination in this field.

[0099] (7) Sample recovery rate

[0100] Take appropriate amounts of Plerixafor Impurity A, Plerixafor Impurity B, Plerixafor Impurity C and Plerixafor Impurity D, accurately weigh them, dissolve them in solvent and quantitatively dilute them to prepare a solution containing about 2 μg of each impurity per 1 ml, as reference stock solution 1.

[0101] Take appropriate amounts of plerixafor impurity A, plerixafor impurity B, plerixafor impurity C and plerixafor impurity D, accurately weigh them, dissolve them in solvent and quantitatively dilute them to prepare a solution containing about 8 μg of each impurity per 1 ml, as reference stock solution 2.

[0102] 30% recovery solution: Take about 20 mg of plerixafor test sample, weigh accurately, place in a 10 ml volumetric flask, add appropriate amount of solvent to dissolve, accurately add 3 ml of reference substance stock solution 1, dilute to the scale with solvent, shake well, as the 30% recovery solution, prepare 3 copies in parallel.

[0103] 100% recovery solution: Take about 20 mg of plerixafor test sample, accurately weigh it, put it in a 10 ml volumetric flask, add an appropriate amount of solvent to dissolve it, accurately add 2.5 ml of reference substance stock solution 2, dilute to the scale with solvent, shake well, and use this as the 100% recovery solution. Prepare 3 copies in parallel.

[0104] 160% recovery solution: Take about 20 mg of plerixafor test sample, accurately weigh it, put it in a 10 ml volumetric flask, add an appropriate amount of solvent to dissolve it, accurately add 4 ml of reference substance stock solution 2, dilute to the scale with solvent, shake well, and use this as the 160% recovery solution. Prepare 3 copies in parallel.

[0105] The recovery rate was determined using the methods described in "(2) Chromatographic conditions" and "(3) Determination" of this embodiment. The results are shown in the following table.

[0106]

[0107] The methodological performance of Example 1 was investigated by using the performance parameters such as limit of quantitation, limit of detection, linearity, repeatability, and recovery rate of sample addition, indicating that the method of Example 1 of the present invention is excellent and can fully meet the requirements for the determination of potential open-ring impurities in plerixafor.

[0108] In fact, in some experiments in the early stage of the research, the inventors found that when the solvent prepared in "(1) solution preparation" is not succinic acid but other acids or the amount of succinic acid is changed, the four open-ring impurities show unstable results in the solution. The typical phenomenon is that the peak areas detected for the four impurities are significantly reduced when the prepared test solution is left for 12 hours, and this reduction decreases over time. These results are specifically obtained in the following supplementary experiments. Supplementary test a: A mixed solution containing 2 mg / ml of plerixafor and 6 μg / ml of each of the four impurities was prepared using a mixed solution of 0.02 mol / L hydrochloric acid aqueous solution and acetonitrile in a volume ratio of 95:5 as a solvent. A mixed solution a1 containing 2 mg / ml of plerixafor and 6 μg / ml of each of the four impurities was prepared using a mixed solution of 0.06 mol / L hydrochloric acid aqueous solution and acetonitrile in a volume ratio of 95:5 as a solvent. A mixed solution a2 containing 2 mg / ml of plerixafor and 6 μg / ml of each of the four impurities was prepared using a mixed solution of 0.15 mol / L hydrochloric acid aqueous solution and acetonitrile in a volume ratio of 95:5 as a solvent. / ml mixed solution a3; the three mixed solutions were placed at room temperature (20-25°C) and measured at 0h, 2h, 6h and 12h according to the method of "(2) Chromatographic conditions" and "(3) Measurement" of Example 1; for impurity A in mixed solution a1, the peak area obtained by sampling and measuring at 0h was taken as the relative calculation basis and calculated as 100%, and the peak areas at the three sampling time points of 2h, 6h and 12h were calculated as the relative percentages of the peak area of ​​impurity A at 0h of the test mixed solution (which can be referred to as relative peak area in the present invention, and the peak area of ​​each impurity of each mixed solution at each sampling time point is the relative percentage of the peak area of ​​each impurity of each mixed solution at each sampling time point). The relative peak area can be calculated in the same way at any time. For example, the relative peak area of ​​impurity A in mixed solution a1 at 6h is the percentage value obtained by dividing the peak area of ​​impurity A measured at 6h by the peak area of ​​impurity A measured at 0h, and then multiplying the quotient by 100%. The results are 97.3%, 92.7% and 84.1% respectively. Impurities B, C and D in mixed solution a1 also show a similar phenomenon of decreasing peak area over time during the storage process. The relative peak areas of the three at 12h are 86.2%, 85.4% and 83.4% respectively. For impurities A, Impurity B, impurity C and impurity D also showed a phenomenon of decreasing peak area over time during the storage process, and the relative peak areas of the four at 12 hours were 85.7%, 88.4%, 87.4% and 84.5%, respectively; for impurity A, impurity B, impurity C and impurity D in the mixed solution a3, the peak area also decreased over time during the storage process, and the relative peak areas of the four at 12 hours were 81.3%, 84.2%, 84.4% and 82.7%, respectively; the higher relative peak area in 0.06 mol / L hydrochloric acid indicates that the impurities in the mixed solution prepared with this concentration of acid are relatively more stable.Supplementary test b: A mixed solution of 0.02 mol / L acetic acid aqueous solution and acetonitrile in a volume ratio of 95:5 was used as a solvent to prepare a mixed solution b1 containing 2 mg / ml of plerixafor and 6 μg / ml of each of the four impurities; a mixed solution of 0.1 mol / L acetic acid aqueous solution and acetonitrile in a volume ratio of 95:5 was used as a solvent to prepare a mixed solution b2 containing 2 mg / ml of plerixafor and 6 μg / ml of each of the four impurities; a mixed solution of 0.25 mol / L acetic acid aqueous solution and acetonitrile in a volume ratio of 95:5 was used as a solvent to prepare a mixed solution b3 containing 2 mg / ml of plerixafor and 6 μg / ml of each of the four impurities; the relative humidity of the four impurities A, impurity B, impurity C and impurity D in the three mixed solutions at 12 h was determined and calculated by referring to the method of supplementary test a. For the peak area, the results were as follows: the peak areas of the four impurities in the mixed solution b1 decreased with the passage of time, and the relative peak areas of the four impurities at 12 h were 78.6%, 82.3%, 81.5% and 79.6%, respectively; the peak areas of the four impurities in the mixed solution b2 decreased with the passage of time, and the relative peak areas of the four impurities at 12 h were 82.2%, 84.8%, 85.4% and 81.3%, respectively; the peak areas of the four impurities in the mixed solution b3 decreased with the passage of time, and the relative peak areas of the four impurities at 12 h were 79.8%, 82.1%, 82.7% and 80.3%, respectively; the higher relative peak area at 0.1 mol / L acetic acid indicated that the impurities in the mixed solution prepared with this acid concentration were more stable.Supplementary test c: A mixed solution containing 2 mg / ml of plerixafor and 6 μg / ml of each of the four impurities was prepared using a mixed solution of 0.03 mol / L succinic acid aqueous solution and acetonitrile in a volume ratio of 95:5 as a solvent. A mixed solution c1 containing 2 mg / ml of plerixafor and 6 μg / ml of each of the four impurities was prepared using a mixed solution of 0.08 mol / L succinic acid aqueous solution and acetonitrile in a volume ratio of 95:5 as a solvent. A mixed solution c2 containing 2 mg / ml of plerixafor and 6 μg / ml of each of the four impurities was prepared using a mixed solution of 0.2 mol / L succinic acid aqueous solution and acetonitrile in a volume ratio of 95:5 as a solvent. The mixed solution c3 was prepared with 2 mg / ml of impurities and 6 μg / ml of each of the four impurities; the relative peak areas of impurities A, B, C and D in the three mixed solutions at 12 h were determined and calculated by referring to the method of supplementary test a. The results showed that the peak areas of the four impurities in the mixed solution c1 decreased slightly over time, and the relative peak areas of the four impurities at 12 h were 96.5%, 98.4%, 97.7% and 96.8%, respectively; the peak areas of the four impurities in the mixed solution c2 remained basically unchanged over time. The relative peak areas of the four impurities at 12 h were 99.8%, 100.6%, 100.3% and 99.6%, respectively. The peak areas of the four impurities in the mixed solution c3 remained basically unchanged or slightly decreased over time, and the relative peak areas of the four impurities at 12 h were 98.6%, 99.3%, 99.7% and 97.2%, respectively. The higher relative peak area at 0.08 mol / L succinic acid indicated that the impurities in the mixed solution prepared with this concentration of acid were more stable. In addition, the mixed solution c2 was further tested for 2 h at room temperature. The relative peak areas of the four impurities at 4h, 36h and 48h showed that the relative peak areas of the four impurities at 24h were 99.6%, 99.9%, 100.4% and 99.4%, respectively; the relative peak areas of the four impurities at 36h were 98.8%, 99.6%, 99.7% and 99.3%, respectively; the relative peak areas of the four impurities at 48h were 98.5%, 99.5%, 99.2% and 98.7%, respectively, indicating that the test solution made with a solvent prepared with 0.08mol / L succinic acid has relatively better stability.Supplementary test d: A mixed solution containing 2 mg / ml of plerixafor and 6 μg / ml of each of the four impurities was prepared using a mixed solution of 0.05 mol / L butyric acid aqueous solution and acetonitrile in a volume ratio of 95:5 as a solvent to prepare a mixed solution d1 containing 2 mg / ml of plerixafor and 6 μg / ml of each of the four impurities; a mixed solution containing 2 mg / ml of plerixafor and 6 μg / ml of each of the four impurities was prepared using a mixed solution of 0.1 mol / L butyric acid aqueous solution and acetonitrile in a volume ratio of 95:5 as a solvent to prepare a mixed solution d3 containing 2 mg / ml of plerixafor and 6 μg / ml of each of the four impurities; the relative peak values ​​of the four impurities A, B, C and D in the three mixed solutions at 12 h were determined and calculated according to the method of supplementary test a. Area, results: the four impurities in the mixed solution d1 showed a phenomenon of decreasing peak area over time during the storage process, and the relative peak areas of the four at 12 h were 84.5%, 88.4%, 85.2% and 83.7%, respectively; the four impurities in the mixed solution d2 showed a phenomenon of decreasing peak area over time during the storage process, and the relative peak areas of the four at 12 h were 86.5%, 89.2%, 87.6% and 85.4%, respectively; the four impurities in the mixed solution d3 showed a phenomenon of decreasing peak area over time during the storage process, and the relative peak areas of the four at 12 h were 82.7%, 86.4%, 83.8% and 81.4%, respectively; the higher relative peak area at 0.1 mol / L butyric acid indicated that the impurities in the mixed solution prepared with this concentration of acid were relatively more stable.Supplementary test e: A mixed solution of 0.005 mol / L trifluoroacetic acid aqueous solution and acetonitrile in a volume ratio of 95:5 (equivalent to a trifluoroacetic acid concentration of about 0.05% w / v in the mixed solution) was used as a solvent to prepare a mixed solution e1 containing 2 mg / ml of plerixafor and 6 μg / ml of each of the four impurities; a mixed solution of 0.02 mol / L trifluoroacetic acid aqueous solution and acetonitrile in a volume ratio of 95:5 was used as a solvent to prepare a mixed solution e2 containing 2 mg / ml of plerixafor and 6 μg / ml of each of the four impurities; a mixed solution of 0.06 mol / L trifluoroacetic acid aqueous solution and acetonitrile in a volume ratio of 95:5 was used as a solvent to prepare a mixed solution e3 containing 2 mg / ml of plerixafor and 6 μg / ml of each of the four impurities; the impurities A, B, C and The relative peak areas of the four impurities in impurity D at 12 h. The results are as follows: the peak areas of the four impurities in the mixed solution e1 decreased with the passage of time during the storage process, and the relative peak areas of the four impurities at 12 h were 78.4%, 78.8%, 79.5% and 77.8%, respectively; the peak areas of the four impurities in the mixed solution e2 decreased with the passage of time during the storage process, and the relative peak areas of the four impurities at 12 h were 81.3%, 83.1%, 80.8% and 79.3%, respectively; the peak areas of the four impurities in the mixed solution e3 decreased with the passage of time during the storage process, and the relative peak areas of the four impurities at 12 h were 77.5%, 79.6%, 88.4% and 76.5%, respectively; the higher relative peak areas at 0.02 mol / L trifluoroacetic acid indicate that the impurities in the mixed solution prepared with this acid concentration are relatively more stable.Supplementary test f: A mixed solution f1 containing 2 mg / ml of plerixafor and 6 μg / ml of each of the four impurities was prepared using a mixed solution of 0.005 mol / L trifluoroacetic acid aqueous solution and acetonitrile in a volume ratio of 95:5 as a solvent (equivalent to a trifluoroacetic acid concentration of about 0.05% w / v in the mixed solution). A mixed solution f2 containing 2 mg / ml of plerixafor and 6 μg / ml of each of the four impurities was prepared using a mixed solution of 0.02 mol / L trifluoroacetic acid aqueous solution and acetonitrile in a volume ratio of 95:5 as a solvent. A mixed solution f3 containing 2 mg / ml of plerixafor and 6 μg / ml of each of the four impurities was prepared by mixing a mixture of 1% acetonitrile and 1% acetonitrile in a volume ratio of 95:5 as a solvent; the three mixed solutions were placed in an environment of 5°C, and measured at 0 h, 24 h, and 48 h according to the methods of "(2) Chromatographic conditions" and "(3) Measurement" in Example 1, and the relative peak areas of the four impurities A, B, C, and D in the three mixed solutions at 24 h and 48 h were calculated according to the method of Supplementary Test a. The results showed that the four impurities in the mixed solution f1 showed an increase in the relative peak areas during the storage process. The peak areas of the four impurities in the mixed solution f2 showed a slight decrease over time, and the relative peak areas of the four impurities at 48 h were 94.5%, 96.7%, 95.3% and 94.8%, respectively. The peak areas of the four impurities in the mixed solution f3 showed a slight decrease over time, and the relative peak areas of the four impurities at 48 h were 95.6%, 97.1%, 94.7% and 95.4%, respectively. The peak areas of the four impurities in the mixed solution f3 showed a slight decrease over time, and the relative peak areas of the four impurities at 48 h were 93.2%, 95.3%, 94 .1% and 93.6%; the relative peak area is higher when the concentration of trifluoroacetic acid is 0.02mol / L, indicating that the impurities in the mixed solution prepared with this concentration of acid are more stable. In addition, it is shown that when an appropriate amount of trifluoroacetic acid is added to the solvent for preparing the test solution, the stability of the obtained test solution at 5°C is significantly better than that at room temperature. However, since it is difficult for the liquid chromatograph to keep the sample plate at a low temperature of 5°C for a long time, and there is a huge difference between the low temperature of 5°C and the column temperature of about 30°C of the chromatographic column, storing the test solution at 5°C is not conducive to the automatic analysis test of high-performance liquid chromatography. From the results of this article, even if the test solution is stored at 5°C, its stability is still not as good as the stability of the test solution prepared with succinic acid at room temperature.

[0109] In Example 1 of the present invention, an appropriate amount of succinic acid is added to the solvent used to prepare the high-performance liquid phase detection sample. In step (1) to step (7) of Example 1, it is shown that the method exhibits excellent results. The above supplementary test shows that the detection sample prepared by using the solvent containing succinic acid is stable for 12 hours or even up to 48 hours, which is of positive significance. The reason is that in the high-performance liquid chromatography detection, a large number of samples need to be prepared manually, and then these samples are placed on the high-performance liquid chromatograph with automatic injection for automatic sampling and measurement without manual intervention. This measurement process is very time-consuming, such as Figure 2 As shown, it takes about 75 minutes to test one sample. The time that the prepared test sample is placed on the chromatographic instrument varies greatly. It may need to be placed for several hours, 24 hours, or even longer, up to 48 hours before sampling and measurement. Therefore, it is very important that the prepared test sample solution remains stable before sampling.

[0110] Example 2: Preparation of Plerixafor

[0111] In this example, plerixafor was prepared by referring to the prior art method.

[0112] Dissolve 10.5 g of 1,4,8,11-tetraaza-5,7,12-trioxocyclotetradecane in 150 mL of acetonitrile (anhydrous), and dissolve 2.5 g of terephthaloyl chloride in 10 mL of triethylamine. Inject the two solutions into a microreactor using a metering pump at a molar ratio of cyclotetradecane compound to terephthaloyl chloride of 4:1. React at 80°C for 30 min, collect the effluent, filter, dry, and recrystallize with DMSO / ethanol (1 / 5) to obtain the product phthaloyl-substituted bis[1,4,8,11-tetraaza-5,7,12-trioxocyclotetradecane]. Then, 5.0 g of the product of the previous step and 9.0 g of sodium borohydride were added to 150 mL of dioxane at room temperature, 15 g of glacial acetic acid was added, and the reaction was allowed to proceed for 45 h, pH = 2 was adjusted with 10% hydrochloric acid, the solvent was removed under reduced pressure, 100 mL of water was added to the residue and stirred to dissolve, 0.05 M sodium hydroxide solution was added to adjust pH = 12, the solvent was recovered under reduced pressure, extracted with dichloromethane, concentrated under reduced pressure, and the residue was recrystallized with isopropanol / ethyl acetate (volume ratio 1 / 5) to obtain white crystals of plerixafor, mp = 126.3 ~ 127.5 ° C. Using the method of Example 1 of the present invention, the area normalized purity of the plerixafor obtained above was 99.92%, impurity A was 0.036%, impurity B was 0.023%, and impurity C and impurity D were not detected.

[0113] In the supplementary test, referring to the method of Example 2 above, the only difference is that ethanol / acetone (volume ratio 1:3) is used as the solvent in the final recrystallization, and the obtained product has mp=125.4~127.7°C, and the melting range is significantly longer; measured by the method of Example 1 of the present invention, the area normalized purity of plerixafor is 97.86%, impurity A is 0.383%, impurity B is 0.227%, impurity C is 0.113%, impurity D is 0.174%, and significant amounts of other unknown impurities are also detected. This shows that the use of the recrystallization solvent of Example 2 has a significantly better effect.

[0114] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining potential ring-opening impurities in plerixafor using high performance liquid chromatography, wherein the potential ring-opening impurities are selected from four impurities having the following structures: Plerixafor Impurity A: , Plerixafor Impurity B: , Plerixafor Impurity C: , Plerixafor Impurity D: ; The method comprises the following steps: (1) Liquid preparation Mobile phase: 0.005 mol / L sodium heptane sulfonate solution adjusted to pH 1.6 with perchloric acid was used as mobile phase A, and acetonitrile was used as mobile phase B; Solvent: Mix 0.08 mol / L succinic acid aqueous solution and acetonitrile in a volume ratio of 95:5 to obtain; Impurity reference solution: Take appropriate amount of plerixafor impurity A reference, plerixafor impurity B reference, plerixafor impurity C reference, and plerixafor impurity D reference, accurately weigh, add solvent to dissolve and quantitatively dilute to make reference solution containing 2 μg of each in 1 ml; Test solution: Take an appropriate amount of plerixafor, dissolve it in solvent and quantitatively dilute it to make a test solution containing 2 mg per 1 ml; Each test solution was stored at room temperature until the measurement; (2) Chromatographic conditions Use a chromatographic column with a size of 4.6×250 mm, a filler particle size of 5 μm, and a brand of Agilent ZORBAX SB-C18, and use mobile phase A and mobile phase B to perform gradient elution according to the elution program shown in the following table: ； The flow rate was 0.8 ml per minute, the detection wavelength was 210 nm, the column temperature was 28°C to 35°C, and the injection volume was 10 μl; (3) Determination Inject the mixed impurity reference solution and the test sample solution into the liquid chromatograph respectively, record the chromatogram, and calculate the amount of each impurity in the test sample by peak area according to the external standard method.

2. The method according to claim 1, wherein the column temperature used in the chromatographic conditions is 30°C.

3. The method according to claim 1, wherein each solution to be tested is stored at 20-25°C until determination.

4. The method according to claim 1, wherein the sample pan temperature during chromatographic determination is room temperature.

5. according to the method for claim 1, wherein the separation degree between impurity A, impurity B, impurity C and impurity D is greater than 3.

0.

6. The method according to claim 1, wherein the resolution between the main component plerixafor and its closest impurity is greater than 5.

0.

7. A method for preparing plerixafor, comprising the steps of: (1) 10.5 g of 1,4,8,11-tetraaza-5,7,12-trioxocyclotetradecane was dissolved in 150 mL of anhydrous acetonitrile, and 2.5 g of terephthaloyl chloride was dissolved in 10 mL of triethylamine. The two solutions were injected into a reactor at a molar ratio of cyclotetradecane compound to terephthaloyl chloride of 4:1, and reacted at 80° C. for 30 min. The effluent was collected, filtered, dried, and recrystallized with DMSO / ethanol in a volume ratio of 1 / 5 to obtain phthaloyl-substituted bis[1,4,8,11-tetraaza-5,7,12-trioxocyclotetradecane]; (2) 5.0 g of the product of the previous step and 9.0 g of sodium borohydride were added to 150 mL of dioxane at room temperature, 15 g of glacial acetic acid was added and the mixture was reacted for 45 h, the pH value was adjusted to 2 with 10% hydrochloric acid, the solvent was removed under reduced pressure, water was added to the residue and stirred to dissolve, 0.05 M sodium hydroxide solution was added to adjust the pH value to 12, the solvent was recovered under reduced pressure, the mixture was extracted with dichloromethane, and the mixture was concentrated under reduced pressure. The residue was recrystallized with isopropanol / ethyl acetate in a volume ratio of 1 / 5 to obtain white crystals of plerixafor; (3) Using the method according to any one of claims 1 to 6, the area normalized purity of the product plerixafor in step (2) and the content of potential open-ring impurities, i.e., plerixafor impurity A, plerixafor impurity B, plerixafor impurity C, and plerixafor impurity D, are determined.

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