Purity determination method of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate

Abstract

The invention discloses a method for determining the purity of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxyethyl] carbamate, which comprises a capillary column with (5%-phenyl)-95% methyl polysiloxane copolymer as a stationary liquid as a gas chromatography column, and a temperature program is used to calculate the purity of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxyethyl] carbamate by a peak area normalization method after injection. The invention adopts a gas chromatography program temperature method to detect the purity of Boc-Fapi-a, which can avoid the influence of weak UV response signals and different response intensities of each impurity component, makes up for the technical gap of the current lack of an effective chromatographic detection method for the substance, realizes convenient quality control, and meets the detection requirements.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a purity detection method for tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate. Background Art

[0002] Tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate, referred to as Boc-Fapi-a, CAS No.: 1448440-50-3, molecular formula: C 12 H 17 F 2 N 3 O 3 , molecular weight: 289.28, its structural formula is:

[0003]

[0004] When tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate is used as the starting material for the reaction, if its purity is low, it will seriously affect the reaction yield and the purity of the subsequent products. Therefore, it is necessary to control the purity of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate, for example, the following reaction process with Boc-Fapi-a as the reactant:

[0005] Step 1: Synthesis of Fapi-a.TsOH

[0006]

[0007] Step 2: Synthesis of H7ND-a

[0008]

[0009] Through literature search and data investigation, it is found that there is no public effective method for detecting the purity of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate. At present, various suppliers mainly control its purity by high performance liquid chromatography (HPLC). However, in the reaction process of synthesizing Boc-Fapi-a, the chromophore UV response signal of most materials is weak, so it is difficult to accurately determine its related impurities by high performance liquid chromatography. The specific synthesis reaction process is as follows:

[0010] Summary of the invention

[0011] The purpose of the present invention is to provide a purity detection method for tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate, and the purity is determined by gas chromatography (GC).

[0012] To achieve the above purpose, the technical solution provided by the present invention is:

[0013] A purity detection method for tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxyethyl]carbamate adopts gas chromatography, and a capillary column with (5%-phenyl)-95% methyl polysiloxane copolymer as a stationary liquid is used as a gas chromatography column; the heating program is as follows:

[0014] From 0 min to 2~3 min, the temperature was maintained at 78~83°C;

[0015] From 2~3min to 10~13min, the temperature changed linearly from 78~83℃ to 300~310℃;

[0016] From 10~13min to 25~30min, the temperature was maintained at 300~310℃;

[0017] Inlet temperature: 275-285°C;

[0018] Detector: Hydrogen flame ionization detector FID;

[0019] Detector temperature: 300℃-320℃;

[0020] After injection, the purity of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate was calculated by peak area normalization method.

[0021] In order to optimize the above technical solutions, the specific limitations adopted also include:

[0022] The flow rate is 2.0ml / min-3.0ml / min.

[0023] The injection volume is 2μl-10μl.

[0024] The test solution is prepared by taking tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate, dissolving it with a solvent and diluting it.

[0025] Furthermore, the solvent is anhydrous ethanol or anhydrous methanol.

[0026] Further, the solution was diluted to a concentration ranging from 1 mg / ml to 10 mg / ml.

[0027] Preferably, a capillary tube with a size of 30 m×0.32 mm and a specification of 0.25 μm and a (5%-phenyl)-95% methyl polysiloxane copolymer as the stationary liquid is used as the chromatographic column.

[0028] Preferably, the heating program is as follows:

[0029] From 0 min to 2 min, the temperature was maintained at 80 °C for 2 min;

[0030] From 2 min to 11 min, the temperature changed linearly from 80 °C to 305 °C, with a heating rate of 25 °C / min;

[0031] From 11 min to 26 min, the temperature was maintained at 305° C. for 15 min.

[0032] Preferably, the injection port temperature is 280°C and the detector temperature is 310°C.

[0033] Preferably, the flow rate is 2.5 ml / min and the injection volume is 5 μl.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The invention provides a method for detecting the purity of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate (Boc-Fapi-a), and adopts gas chromatography to realize quality control thereof.

[0036] The high performance liquid chromatography (HPLC) determination of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate is easily affected by interference peaks, the UV response signals of the chromophores of most materials are weak, the determination is difficult, and suitable HPLC determination conditions are difficult to obtain; the present invention adopts a gas chromatography programmed temperature method, which can avoid the influence of weak UV response signals of various impurity components and different response intensities, fills the technical gap of the current lack of an effective chromatographic detection method for the substance, realizes convenient quality control, and meets the detection needs.

[0037] The method of the present invention is simple to operate, and has been verified to have good specificity, detection limit and quantification limit, linearity and precision. The solution is stable and can be used for purity detection of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate (Boc-Fapi-a). BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is the HPLC chromatogram of the test solution in Comparative Example 1.

[0039] Figure 2The gas chromatogram of the test solution in Example 1.

[0040] Figure 3 This is the gas chromatogram of the specific mixed solution in Example 2.

[0041] Figure 4 This is the gas chromatogram of the detection limit in Example 3.

[0042] Figure 5 It is the gas chromatogram of the quantitative limit in Example 3.

[0043] Figure 6 It is the linear graph in Example 4.

[0044] Figure 7 This is an overlay of the gas chromatograms from the precision test in Example 5.

[0045] Figure 8 This is an overlay of the gas chromatograms from the stability test of the test solution in Example 6.

[0046] Fig. 9 It is the gas chromatogram of the test solution in Comparative Example 2.

[0047] Fig.10 This is the gas chromatogram of the test solution in Comparative Example 3. DETAILED DESCRIPTION

[0048] The above contents of the present invention are further described in detail below in the form of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments, and all technologies realized based on the above contents of the present invention belong to the scope of the present invention.

[0049] The test methods used in the following examples are all conventional methods unless otherwise specified, and the reagents, methods and equipment used are all conventional reagents, methods and equipment in the art unless otherwise specified.

[0050] Comparative Example 1

[0051] HPLC detection method:

[0052] Instrument and chromatographic conditions:

[0053] Instrument: High performance liquid chromatography;

[0054] Detector: UV detector;

[0055] Column: YMC Triart C18, 4.6 mm × 250 mm, 3 μm;

[0056] Flow rate: 0.8ml / min;

[0057] Column temperature: 30°C;

[0058] Detection wavelength: 200nm;

[0059] Injection volume: 5 μl;

[0060] Running time: 70 minutes

[0061] Mobile phase A: 10 mM ammonium formate aqueous solution;

[0062] Mobile phase B: acetonitrile.

[0063] Perform gradient elution according to Table 1:

[0064] Table 1 Gradient elution conditions

[0065]

[0066] Preparation of test solution: Take an appropriate amount of Boc-Fapi-a, weigh accurately, dissolve in 40% acetonitrile and quantitatively dilute to make a solution containing about 1 mg per 1 ml.

[0067] Test results are shown in Figure 1 The impurities in the mobile phase have a large interference when the UV detector absorbs at the end, and the UV response signal of the chromophore of most materials is weak, so the HPLC method cannot accurately determine the relevant impurities.

[0068] The boiling point of Boc-fapi-a is about 400° C. and it can be vaporized under existing GC conditions. Therefore, the GC method is selected for purity determination, as shown in Example 1, Comparative Example 1, and Comparative Example 2.

[0069] Example 1

[0070] Using gas chromatography:

[0071] A gas chromatography detection method for determining the purity of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate (Boc-Fapi-a) comprises the following steps: injecting a sample solution, performing detection according to gas chromatography conditions, and recording a chromatogram.

[0072] The chromatographic conditions are as follows: a capillary column with (5%-phenyl)-95% methyl polysiloxane copolymer as the stationary liquid; the heating program is as follows:

[0073] 0min→2min, maintain at 80℃ for 2min;

[0074] 2min→11min, 80℃ linearly changes to 305℃, heating rate is 25℃ / min;

[0075] 11min→26min, maintain at 305℃ for 15min;

[0076] Chromatographic conditions also include:

[0077] Inlet temperature: 280°C;

[0078] Detector: flame ionization detector (FID);

[0079] Detector temperature: 310°C;

[0080] Column flow rate: 2.5ml / min;

[0081] Split ratio: 20;

[0082] Injection volume: 5 μl;

[0083] Chromatographic column selection: Agilent HP-5, 30m×0.32mm, 0.25μm;

[0084] The test solution is prepared by the following process: take an appropriate amount of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate (Boc-Fapi-a) to be tested, add methanol to dissolve and dilute, shake well, and obtain; concentration: 5 mg / ml.

[0085] Results Figure 2 The main peak has a good shape and the theoretical plate number is 36942.

[0086] Comparative Example 2

[0087] Using gas chromatography:

[0088] The chromatographic column used was a highly polar chromatographic column, model DB-WAX, with a capillary using polyethylene glycol 20M as the stationary liquid, with specifications of 30m×0.32mm, 0.5µm, and other measurement conditions were the same as in Example 1.

[0089] Test results are shown in Fig. 9 The main peak response is small and is poorly separated from the solvent peak. The theoretical plate number is 495.

[0090] Comparative Example 3

[0091] Using gas chromatography:

[0092] The chromatographic column used was a non-polar chromatographic column, model DB-1, with a capillary using methyl polysiloxane as the stationary liquid, with specifications of 30m×0.32mm, 0.25µm, and other measurement conditions were the same as in Example 1.

[0093] Test results are shown in Fig.10 The main peak front is serious and the theoretical plate number is 16764.

[0094] Through comparative example tests, it is concluded that the method of Example 1 has the best determination effect on tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate.

[0095] The above comparative examples are not exhaustive. The present application studies various experimental conditions and parameters and ultimately concludes that the method of Example 1 is the most suitable gas chromatography detection method for purity determination of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate, and conducts methodological verification on it, as shown in Examples 2-6.

[0096] Example 2 Specificity Test

[0097] This example conducts a specificity test on the scheme established in Example 1.

[0098] Blank solvent: methanol;

[0099] Fapi-a-SM1 stock solution: Accurately weigh about 12.5 mg of Fapi-a-SM1 into a 5 ml volumetric flask, dissolve it with solvent and make up to the mark;

[0100] Fapi-a1 stock solution: Accurately weigh about 12.5 mg of Fapi-a1 into a 5 ml volumetric flask, dissolve it with solvent and make up to the mark;

[0101] Fapi-a2 stock solution: Accurately weigh about 12.5 mg of Fapi-a2 into a 5 ml volumetric flask, dissolve it with solvent and make up to the mark;

[0102] Fapi-a3 stock solution: Accurately weigh about 12.5 mg of Fapi-a3 into a 5 ml volumetric flask, dissolve it with solvent and make up to the mark;

[0103] Fapi-a-SM2 stock solution: Accurately weigh about 12.5 mg of Fapi-a-SM2 into a 5 ml volumetric flask, dissolve it with solvent and make up to the mark;

[0104] Pyridine stock solution: Accurately weigh about 12.5 mg of pyridine into a 5 ml volumetric flask, dissolve it with solvent and make up to the mark;

[0105] Dioxane stock solution: Accurately weigh about 12.5 mg of dioxane into a 5 ml volumetric flask, dissolve it with solvent and make up to the mark;

[0106] Fapi-a-SM1 positioning solution: accurately pipette 1 ml of Fapi-a-SM1 stock solution into a 10 ml volumetric flask, dissolve it with solvent and make up to the mark;

[0107] Fapi-a1 positioning solution: accurately transfer 1 ml of Fapi-a1 stock solution into a 10 ml volumetric flask, dissolve it with solvent and make up to the mark;

[0108] Fapi-a2 positioning solution: accurately transfer 1 ml of Fapi-a2 stock solution into a 10 ml volumetric flask, dissolve it with solvent and make up to the mark;

[0109] Fapi-a3 positioning solution: accurately transfer 1 ml of Fapi-a3 stock solution into a 10 ml volumetric flask, dissolve it with solvent and make up to the mark;

[0110] Fapi-a-SM2 positioning solution: accurately transfer 1 ml of Fapi-a-SM2 stock solution into a 10 ml volumetric flask, dissolve it with solvent and make up to the mark;

[0111] Pyridine positioning solution: accurately transfer 1 ml of pyridine stock solution into a 10 ml volumetric flask, dissolve it with solvent and make up to the mark;

[0112] Dioxane positioning solution: Accurately transfer 1 ml of dioxane stock solution into a 10 ml volumetric flask, dissolve it with solvent and make up to the mark;

[0113] Mixed positioning solution: accurately pipette 1 ml of each of Fapi-a-SM1 stock solution, Fapi-a1 stock solution, Fapi-a2 stock solution, Fapi-a3 stock solution, Fapi-a-SM2 stock solution, pyridine stock solution, and dioxane stock solution into a 10 ml volumetric flask, dissolve with solvent and make up to the mark;

[0114] Mixed solution: Accurately weigh about 10 mg of Boc-Fapi-a into a 2 ml volumetric flask, dissolve it with the mixed positioning solution and make up to the mark.

[0115] The determination conditions were the same as those in Example 1. Accurately measure 5 μl of each of the above solutions and inject them into the gas chromatograph to record the chromatogram.

[0116] Results: The solvent did not have an interfering peak at the Boc-Fapi-a position, and each positioning solution was well separated from Boc-Fapi-a and did not interfere with the determination, and the specificity met the requirements. The specific results are shown in Table 2. Figure 3 .

[0117] Table 2 Specificity test results

[0118]

[0119] Example 3 Limit of Quantitation and Limit of Detection

[0120] This example investigates the limit of quantitation and limit of detection of the scheme established in Example 1.

[0121] Preparation of quantitative limit and detection limit solutions: Accurately weigh an appropriate amount of Boc-Fapi-a, dilute it step by step with methanol, and inject 5 μl. The quantitative limit is established according to the signal-to-noise ratio S / N ≥ 10, and the detection limit is established according to the signal-to-noise ratio S / N ≥ 3.

[0122] The determination conditions were the same as those in Example 1. Accurately measure 5 μl of each of the above solutions and inject them into the gas chromatograph to record the chromatogram.

[0123] Results: The detection limit of Boc-Fapi-a was 12.66 μg / ml, equivalent to 0.3% of the test sample concentration, and the S / N value was 7.5, greater than 3; the quantification limit was 25.31 μg / ml, equivalent to 0.5% of the test sample concentration, less than 50% of the limit concentration, and the S / N value was between 10.6 and 19.8, all greater than 10, and the quantitative limit peak area RSD was 4.2% (n=6), less than 10.0%, all meeting the requirements. The specific results are shown in Table 3. Figure 4 and Figure 5 .

[0124] Table 3 Results of determination of limit of quantitation and limit of detection

[0125]

[0126] Example 4 Linearity Test

[0127] This example conducts a linear test on the scheme established in Example 1.

[0128] Preparation of linear solution: Take about 100 mg of Boc-Fapi-a, weigh accurately, place in a 10 ml volumetric flask, dissolve and dilute to the scale with solvent, mix well, and use as stock solution.

[0129] Accurately measure 0.2 ml of the stock solution into a 2 ml volumetric flask and dilute the solvent to the mark to obtain linear solution 1 (20%).

[0130] Accurately measure 0.4 ml of the stock solution into a 2 ml volumetric flask and dilute the solvent to the mark to obtain linear solution 2 (40%).

[0131] Accurately measure 0.6 ml of the stock solution into a 2 ml volumetric flask and dilute the solvent to the mark to obtain linear solution 3 (60%).

[0132] Accurately measure 0.8 ml of the stock solution into a 2 ml volumetric flask and dilute the solvent to the mark to obtain linear solution 4 (80%).

[0133] Accurately measure 1.0 ml of the stock solution into a 2 ml volumetric flask and dilute the solvent to the mark to obtain linear solution 5 (100%).

[0134] Accurately measure 1.2 ml of the stock solution into a 2 ml volumetric flask and dilute the solvent to the mark to obtain linear solution 6 (120%).

[0135] Accurately measure 50 μl of the linear solution into a 5-10 ml volumetric flask and dilute the solvent to the mark to obtain the quantitative limit solution (0.5%).

[0136] The determination conditions were the same as in Example 1. 5 μl of each linear solution was accurately measured and injected into the gas chromatograph to record the chromatogram. The Boc-Fapi-a peak area was recorded, with the concentration C (μg / ml) as the abscissa and the peak area A as the ordinate. The regression equation and correlation coefficient were calculated using the least squares method. The results are shown in Table 4 and Figure 6 As shown, when the concentration of Boc-Fapi-a is in the range of 25.1μg / ml to 6015.6μg / ml, the linear regression equation is y=2348.2x-513263, and the correlation coefficient is r is 0.999; the ratio of the Y-axis intercept to the 100% limit response value is 4.6%, and the linear relationship is good.

[0137] Table 4 Linear relationship results

[0138]

[0139] Example 5 Precision Test

[0140] This example conducts a precision test on the scheme established in Example 1.

[0141] Test solution: Take an appropriate amount of Boc-Fapi-a, weigh accurately, dissolve in methanol and quantitatively dilute to make a solution containing about 5 mg per 1 ml. Prepare six replicates in parallel.

[0142] The determination conditions were the same as in Example 1. 5 μl of each test solution was accurately measured and injected into the gas chromatograph to record the chromatogram. The purity of Boc-Fapi-a in the test solution was determined by the peak area normalization method. The results are shown in Table 5. Figure 7 As shown, the precision test results are good.

[0143] Table 5 Precision test results

[0144]

[0145] Example 6 Solution stability test

[0146] This example investigates the solution stability of the solution established in Example 1.

[0147] The test solution was placed at room temperature, and the test solution was measured at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h, 26h, 28h, 30h, 32h, 34h, 36h, 38h, 40h, 42h, 44h, 46h, 48h, 50h, 52h, 54h, respectively. The measurement conditions were the same as those in Example 1. 5μl of each solution was accurately measured and injected into the gas chromatograph. The chromatogram was recorded and compared with 0 hour. The difference in the impurity content of the test solution and the change in purity were calculated respectively. The results show that the test solution has good stability within 54 hours at room temperature. The results are shown in Table 6. Figure 8 shown.

[0148] Table 6 Solution stability results

[0149]

[0150] Summary: This application provides a gas chromatography method that can accurately detect the purity of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate (Boc-Fapi-a). The method is easy to operate, has good specificity, detection limit and quantification limit, linearity and precision, and the solution is stable. The purity can be calculated by using the peak area normalization method, which can effectively control the purity of Boc-Fapi-a and meet the current detection needs.

[0151] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, according to the technical essence of the present invention, any simple modification, equivalent replacement and improvement made to the above embodiment still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for determining the purity of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate, characterized in that: Gas chromatography was used, and a capillary column with a 30m×0.32mm, 0.25μm specification (5%-phenyl)-95% methyl polysiloxane copolymer as the stationary liquid was used as the chromatographic column; the heating program was as follows: From 0 min to 2 min, the temperature was maintained at 80 °C for 2 min; From 2 min to 11 min, the temperature changed linearly from 80 °C to 305 °C, with a heating rate of 25 °C / min; From 11min to 26min, the temperature was maintained at 305°C for 15min; Flow rate is 2.0ml / min-3.0ml / min; Inlet temperature: 275-285°C; Detector: Hydrogen flame ionization detector FID; Detector temperature: 300℃-320℃; The test solution is prepared by taking tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate, dissolving and diluting it with a solvent; the solvent is anhydrous ethanol or anhydrous methanol; The purity of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate was calculated by peak area normalization method after direct injection.

2. The method for determining the purity of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate according to claim 1, characterized in that: The injection volume is 2μl-10μl.

3. The method for determining the purity of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate according to claim 1, characterized in that: Dilute to a concentration range of 1 mg / ml-10 mg / ml.

4. The method for determining the purity of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate according to claim 1, characterized in that: Inlet temperature: 280°C; detector temperature: 310°C.

5. The method for determining the purity of tert-butyl-(S)-[2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl]carbamate according to claim 1, characterized in that: Flow rate: 2.5 ml / min; injection volume: 5 μl.

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