A method for determining non-deuterated substances in the deuterated drug deuterated remdesivir hydrobromide

Abstract

The present invention provides a method for determining non-deuterated substances in a deuterated drug deuterated remidevir hydrobromide, which fills the technical gap that there is currently no corresponding non-deuterated substance quantitative detection method for deuterated remidevir hydrobromide; and compared with other methods for detecting non-deuterated substances in deuterated compounds, the method provided by the present invention has the advantages of no satellite peak interference, similar relaxation time of two quantitative peaks and chemical environment, reduced quantitative peak integration error, high accuracy (recovery rate 98.5%~101.1%), high sensitivity (quantitative limit less than 0.05%), simple operation, no need for accurate weighing, no need for any standard substance, and strong universality.

Description

Technical Field

[0001] The present invention relates to the field of drug analysis, and in particular to a method for determining non-deuterated substances in a deuterated drug deuterated remdevir hydrobromide. Background Art

[0002] Deuterium remdevir hydrobromide, also known as VV116, has a chemical name of (2 R ,3 R ,4 R ,5 R )-2-(4-aminopyrrolo[2,1- f [1,2,4]triazine-7-yl-5-deuterio)-2-cyano-5-[(isobutyryloxy)methyl]tetrahydrofuran-3,4-diyl bis(2-methylpropionate) hydrobromide is a viral RNA-dependent RNA polymerase inhibitor that has been approved for marketing for the treatment of mild to moderate novel coronavirus infection (COVID-19) in adults.

[0003] Deuterium remidevir hydrobromide is a deuterated drug formed by replacing hydrogen (H) at a specific metabolic site in the compound structure with deuterium (D). This deuterated modification can inhibit its oxidation and ring-opening degradation in the body, thereby improving its bioavailability and antiviral activity. According to the literature (Cell Research, 2021, 31(11): 1212-1214) and patent CN114516875A, its deuterated synthesis strategy is an isotope exchange method in which the synthetic intermediate is deuterated by an iodine-deuterium exchange reaction under the action of a metal catalyst and a deuterium source. The reaction conditions are harsh, the deuteration efficiency is limited, and the corresponding non-deuterated products that are not deuterated will inevitably be generated during the reaction. Therefore, the content of the corresponding non-deuterated products in deuterium remidevir hydrobromide is a key quality attribute that affects its effectiveness and safety.

[0004] However, the physical and chemical properties of deuterated drugs and their corresponding non-deuterated products are almost the same. When using analytical techniques such as chromatography to determine the content or impurity inspection of deuterated drugs, deuterated drugs and corresponding non-deuterated products cannot be distinguished. Therefore, it is of great significance to develop a quantitative analysis method for the corresponding non-deuterated products in deuterated remdesivir hydrobromide for its production quality control.

[0005] The literature (Journal of Pharmaceutical and Biomedical Analysis, 2023, 228:115340.) and the literature (Journal of Chromatography B, 2024, 1235:124038.) both used liquid chromatography-mass spectrometry to qualitatively and quantitatively analyze the metabolites of VV116 in plasma, but neither involved non-deuterated substances. Patent CN112630345A proposes a method for detecting the isotopic distribution and abundance of deuterated compounds using quadrupole orbital trap high-resolution mass spectrometry, which can determine the content of non-deuterated substances, but this method has high requirements on the resolution and instrument quality deviation of the mass spectrometer, so the detection is difficult and has poor universality. Patent CN112305007A proposes a method for determining the deuterium isotope abundance of deuterium-labeled compounds using nuclear magnetic resonance hydrogen spectrum or deuterium spectrum, but this technical solution can only determine the isotope abundance of deuterated compounds (deuterium atomic percentage atom%D), and requires accurate addition of standard substances as internal standards, and the detection process is complicated. The literature (Chinese Journal of Hospital Pharmacy, 2023, 43(16): 1828-1832.) proposes a hydrogen nuclear magnetic resonance spectroscopy relative quantitative method for determining the content of non-deuterated impurities in another deuterated drug, donafenib tosylate, but the pulse sequence zg used in this method will have obvious satellite peaks that affect the accuracy of integration and the identification of quantitative peaks, and cannot be used for the determination of non-deuterated substances in deuterated remdesivir hydrobromide; and the relaxation time and chemical environment of the two quantitative peaks are quite different, which further increases the integration error.

[0006] Therefore, in order to ensure the safety and effectiveness of deuterated remdesivir hydrobromide, it is urgently necessary to develop a method for detecting the residual non-deuterated content in deuterated remdesivir hydrobromide. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a method for detecting the content of non-deuterated substances in deuterated remdevir hydrobromide, and the method is simple to operate and does not require any standard substances.

[0008] The technical solution of the present invention to solve the above technical problems is as follows:

[0009] A method for determining non-deuterated substances in the deuterated drug deuterated remdevir hydrobromide, comprising the following steps:

[0010] (1) Take an appropriate amount of deuterated remdevir hydrobromide sample and dissolve it in an appropriate amount of deuterated methanol as the test solution;

[0011] (2) Use a nuclear magnetic resonance spectrometer to collect the hydrogen spectrum of the test solution, select the pulse sequence noesyigld1d, and collect the hydrogen spectrum after automatically adjusting the gain;

[0012] (3) Perform phase adjustment, baseline correction and chemical shift peak calibration on the collected hydrogen spectrum, integrate the signal peaks at chemical shifts δ 7.5 and δ 7.1, and calculate the content of non-deuterated substances in the deuterated remdesivir hydrobromide sample;

[0013] The chemical structures of the deuterated remdevir hydrobromide and the corresponding non-deuterated product are shown below:

[0014] .

[0015] Furthermore, some atoms of deuterated remdevir hydrobromide and the corresponding non-deuterated products are labeled as follows:

[0016] .

[0017] Furthermore, a method for determining non-deuterated substances in the deuterated drug deuterated remdesivir hydrobromide comprises the following steps:

[0018] S1. Take a deuterated remidevir hydrobromide sample and dissolve it in deuterated methanol to make the mass concentration of deuterated remidevir hydrobromide 80-120 mg / mL, specifically 100 mg / mL.

[0019] S2. Use a nuclear magnetic resonance spectrometer to collect the hydrogen spectrum of the test solution. Select the pulse sequence noesyigld1d, and collect the hydrogen spectrum after automatically adjusting the gain;

[0020] S21, the relaxation delay time (D1) of the nuclear magnetic resonance spectrometer is not less than 15 s, specifically 15 s; the number of scans (NS) is not less than 32 times, specifically 32 times;

[0021] S22, NMR spectrometer test temperature is 293~303 K, specifically 298 K, pulse excitation angle is 90°, decoupling power is 0.5 W, number of empty scans (DS) is 4 times, spectrum width (SWH) is 20 ppm, sampling time (AQ) is 3.28 s, sampling point number (TD) is 64 K;

[0022] S3. The collected hydrogen spectrum was phase adjusted and baseline corrected, and the chemical shifts of other signal peaks were marked with reference to the deuterated methanol solvent peak chemical shift δ of 3.3. The signal peaks at chemical shifts δ 7.5 and δ 7.1 were integrated, and the content of non-deuterated substances in the deuterated remidevir hydrobromide sample was calculated according to the following formula.

[0023] ,

[0024] in, P is the percentage of non-deuterated substances (%), A sis the integral value of the signal peak at chemical shift δ 7.5, A r is the integral value of the signal peak at the chemical shift δ 7.1.

[0025] When the nuclear magnetic resonance spectroscopy method is used to determine the trace amount of non-deuterated substances in the sample, the detection sensitivity is a challenge. Through solvent screening, the inventors found that the deuterated hydrobromide remidevir sample has a large solubility in deuterated methanol and deuterated dimethyl sulfoxide, and the sensitivity can be improved by increasing the sample concentration. However, when deuterated dimethyl sulfoxide is used as a solvent, due to the presence of NH2 and HBr active hydrogen in the compound structure, the baseline is greatly disturbed, which seriously affects the accuracy of integration. By the commonly used method of adding heavy water, when using deuterated dimethyl sulfoxide-heavy water mixed solvent, although active hydrogen interference can be eliminated, the signal peak resolution at δ 7.5 is significantly deteriorated, and the signal-to-noise ratio is low; and when the deuterated methanol selected in the present invention is used as a solvent, not only the detection sensitivity can be increased by increasing the sample concentration, but also the interference of active hydrogen on integration can be eliminated, and the signal peak at δ 7.5 has a higher resolution and signal-to-noise ratio.

[0026] In the present invention, the signal peak (7 H signal peaks on the structural formula, bimodal) at the chemical shift δ 7.5 that the inventor has found can significantly distinguish the deuterium hydrobromide remidevir and the corresponding non-deuterated substance in the sample. And the other signal peak (δ 7.1, 8 H signal peaks on the structural formula) that the inventor has found can not only be used as the total signal peak of deuterium hydrobromide remidevir and the corresponding non-deuterated substance, and the signal peak is tested experimentally, and its longitudinal relaxation time is shorter, only 2.701 s, so the D1 value required is smaller, reducing the sampling time. In addition, the signal peak is close to the signal peak longitudinal relaxation time (2.736 s) at δ 7.5 and the chemical environment, and the integral error between the two can be reduced.

[0027] Nuclear magnetic resonance spectroscopy is often quantified by adding a standard substance as an internal standard. The accuracy of quantification depends on the selection and accurate weighing of the internal standard, and the operation is relatively cumbersome. In the present invention, the inventors use the signal peak at the chemical shift δ 7.5 to represent the signal peak of the non-deuterated substance (the 7-position H signal peak in the structural formula, a double peak), and the integral value is recorded as A s ; The signal peak at the chemical shift of δ7.1 represents the total signal peak of deuterated remdesivir hydrobromide and non-deuterated product (the 8th H signal peak in the structural formula), and the integral value is recorded as Ar. The relative molecular masses of non-deuterated product and deuterated remdesivir hydrobromide are 582 and 583, respectively. Then: P = A s ×582 / [ A s ×582+( A r -A s )×583]=582 A s / (583 A r - A s ). Therefore, the technical solution provided by the present invention can directly calculate the percentage of non-deuterated substances in the sample based on the integral value of the signal peaks at the chemical shifts δ 7.5 and δ 7.1, without relying on any standard substance, without the need to select an internal standard, and without the need for accurate weighing.

[0028] The pulse sequences commonly used for quantitative NMR spectroscopy are zg or zg30. 13 C decoupling, the corresponding hydrogen spectrum will be 13 C- 1 The H coupling effect produces satellite peaks. When a trace amount of non-deuterated substances in a sample is measured, the signal peak of the non-deuterated substance will not show satellite peaks, while the total signal peak of deuterated remdesivir hydrobromide and the non-deuterated substance will show obvious satellite peaks due to the high concentration of deuterated remdesivir hydrobromide, thereby increasing the integration error. The pulse sequence noesyigld1d and the decoupling power of 0.5 W selected by the present invention can completely remove the satellite peaks, improve the integration accuracy of the signal peak, and accurately identify the δ 7.5 quantitative peak.

[0029] If there is any conflict between the Chinese name and the structural formula of the compound in the present invention, the structural formula shall prevail.

[0030] The beneficial effects of the present invention are: the present invention provides a method for determining non-deuterated substances in the deuterated drug deuterated remidevir hydrobromide, which makes up for the technical gap that there is currently no corresponding non-deuterated substance quantitative detection method for deuterated remidevir hydrobromide; and the method provided by the present invention has no satellite peak interference, two quantitative peak relaxation times and similar chemical environments compared to other deuterated compounds. The quantitative peak integration error is reduced, the accuracy is high (recovery rate 98.5%~101.1%), the sensitivity is high (quantitative limit is less than 0.05%), the operation is simple, no accurate weighing is required, no standard substance is required, and the advantages of strong universality are provided. It also provides a reference for the determination of non-deuterated substances in other deuterated drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a global graph of the test spectrum of the deuterium remdevir hydrobromide sample in Example 1;

[0032] Figure 2 This is a partial enlarged view of the test spectrum of the deuterium remidevir hydrobromide sample in Example 1;

[0033] Figure 3It is a comparison chart of the test spectra of the samples of Comparative Example 1, Comparative Example 2 and Example 1;

[0034] Figure 4 It is a comparison chart of the test spectra of the samples of Comparative Example 3 and Example 1. DETAILED DESCRIPTION

[0035] The present invention is described below with reference to examples, but the present invention is not limited thereto. In the art, simple replacement or improvement made by technicians in the field to the present invention belongs to the technical solution protected by the present invention.

[0036] Embodiment 1:

[0037] Weigh about 50 mg of the sample, add 0.5 mL of deuterated methanol, shake to dissolve, and transfer to a 5 mm NMR tube as the test solution.

[0038] The NMR tube was placed in the NMR spectrometer, and the noesyigld1d pulse sequence was selected with a pulse excitation angle of 90°. The sampling parameters were set as follows: relaxation delay time (D1) 15 s, number of scans (NS) 32 times, decoupling power 0.5 W, test temperature 298 K, number of empty scans (DS) 4 times, spectrum width (SWH) 20 ppm, sampling time (AQ) 3.28 s, sampling points (TD) 64 K, and the hydrogen spectrum was collected after automatically adjusting the gain.

[0039] The collected hydrogen spectrum was phase adjusted and baseline corrected in turn, and then the chemical shifts of other signal peaks were marked with reference to the deuterated methanol solvent peak chemical shift δ of 3.3. The signal peaks at chemical shifts δ 7.5 and δ 7.1 were integrated, and the content of non-deuterated substances in the deuterated remidevir hydrobromide sample was calculated according to the following formula.

[0040] ,

[0041] in, P is the percentage of non-deuterated substances (%), A s is the integral value of the signal peak at chemical shift δ 7.5, A r is the integral value of the signal peak at the chemical shift δ 7.1.

[0042] Embodiment 2:

[0043] Weigh about 40 mg of the sample, add 0.5 mL of deuterated methanol, shake to dissolve, and transfer to a 5 mm NMR tube as the test solution.

[0044] The NMR tube was placed in the NMR spectrometer, and the noesyigld1d pulse sequence was selected with a pulse excitation angle of 90°. The sampling parameters were set as follows: relaxation delay time (D1) 20 s, number of scans (NS) 64 times, decoupling power 0.5 W, test temperature 303 K, number of empty scans (DS) 4 times, spectrum width (SWH) 20 ppm, sampling time (AQ) 3.28 s, sampling points (TD) 64 K, and the hydrogen spectrum was collected after automatically adjusting the gain.

[0045] The collected hydrogen spectrum was phase adjusted and baseline corrected in turn, and then the chemical shifts of other signal peaks were marked with reference to the deuterated methanol solvent peak chemical shift δ of 3.3. The signal peaks at chemical shifts δ 7.5 and δ 7.1 were integrated, and the content of non-deuterated substances in the deuterated remidevir hydrobromide sample was calculated according to the following formula.

[0046] ,

[0047] in, P is the percentage of non-deuterated substances (%), A s is the integral value of the signal peak at chemical shift δ 7.5, A r is the integral value of the signal peak at the chemical shift δ 7.1.

[0048] Test Example 1: Methodology Validation

[0049] Linear range: Accurately weigh an appropriate amount of deuterated remidevir hydrobromide non-deuterated reference substance, dissolve it in deuterated methanol and dilute it to prepare a series of solutions with concentrations of 0.2, 0.5, 1.0, 1.5, and 2.0 mg / ml [equivalent to 0.2%, 0.5%, 1.0%, 1.5%, and 2.0% of the sample concentration (100 mg / mL), respectively], transfer 0.5 mL to a 5 mm nuclear magnetic tube, and measure it according to the method of Example 1. With the concentration of the non-deuterated substance as the horizontal coordinate and the integral value ratio of the δ 7.5 signal peak to the solvent peak as the vertical coordinate, linear regression was performed by the least squares method. As a result, the linear equation of the δ 7.5 signal peak is y = 0.016 x - 0.0003 ( r = 0.9999). This indicates that the signal peak intensity of δ 7.5 has a good linear relationship with the concentration in the range of 0.2% to 2.0% for non-deuterated substances.

[0050] Limit of quantitation: 0.5 mL of a 0.05 mg / mL deuterated remdesivir hydrobromide non-deuterated reference solution was added to a 5 mm NMR tube and determined according to the method of Example 1. The signal-to-noise ratio of the signal peak at δ 7.5 was greater than 10, indicating that the method limit of quantitation was less than 0.05%, which had a high sensitivity.

[0051] Precision: Take the same batch of samples, prepare 6 test solutions in parallel according to the method of Example 1, and then measure the results. The average value is 0.99% and the RSD is 0.27% ( n = 6), indicating that the method has high precision.

[0052] Accuracy: About 50 mg of the same batch of samples was accurately weighed and added to the non-deuterated reference solution to prepare spiked sample solutions with concentrations of 0.5, 1.0, and 2.0 mg / mL (equivalent to 0.5%, 1.0%, and 2.0% of the sample concentration, respectively). Three replicates were prepared for each concentration and determined according to the method of Example 1. The recovery of the non-deuterated substance was calculated and the results were in the range of 98.53% to 101.06%, indicating that the method has high accuracy.

[0053] Solution stability: Take the same sample solution and place it at room temperature for 0, 2, 4, 8, 16, and 24 h, respectively, and then measure it according to the method in Example 1. The RSD of the measurement results is 0.42%, indicating that the sample solution is stable at room temperature for at least 24 h.

[0054] Comparative Example 1: Determination of non-deuterated substances in deuterated remdevir hydrobromide using deuterated dimethyl sulfoxide as solvent.

[0055] Weigh about 50 mg of the sample, add 0.5 mL of deuterated dimethyl sulfoxide, shake to dissolve, and transfer to a 5 mm NMR tube as the test solution. Place the NMR tube in a NMR spectrometer and measure according to the method in Example 1.

[0056] The comparison diagram of the local enlarged diagram of the sample test spectrum obtained by the results collection and the local enlarged diagram of the sample test spectrum collected in Example 1 is shown in Figure 3 It can be seen that the baseline of the sample in Comparative Example 1 fluctuates severely due to the interference of active hydrogen and cannot be accurately integrated; while the baseline of the sample in Example 1 is stable.

[0057] Comparative Example 2: Determination of non-deuterated substances in deuterated remdevir hydrobromide using a deuterated dimethyl sulfoxide-heavy water mixed solvent.

[0058] Weigh about 50 mg of the sample, add 0.5 mL of a mixed solvent of deuterated dimethyl sulfoxide-heavy water (10:1), shake to dissolve, and transfer to a 5 mm NMR tube as the test solution. Place the NMR tube in a NMR spectrometer and measure according to the method in Example 1.

[0059] The comparison diagram of the local enlarged diagram of the sample test spectrum obtained by the results collection and the local enlarged diagram of the sample test spectrum collected in Example 1 is shown in Figure 3It can be seen that the peak resolution of the δ 7.5 signal in Comparative Example 2 is poor, and the signal-to-noise ratio is 56; while the peak resolution of the δ 7.5 signal in Example 1 is high, and the signal-to-noise ratio is 484, which is much greater than that in Comparative Example 2.

[0060] Comparative Example 3: Determination of non-deuterated substances in deuterated remdesivir hydrobromide using a zg pulse sequence and no decoupling power.

[0061] Take the test solution of Example 1, place it in a nuclear magnetic resonance spectrometer, select the zg pulse sequence, do not set the decoupling power, and keep other sampling parameters the same as in Example 1.

[0062] The comparison diagram of the local enlarged diagram of the sample test spectrum obtained by the results collection and the local enlarged diagram of the sample test spectrum collected in Example 1 is shown in Figure 4 It can be seen that in Example 1, the satellite peaks near the high-intensity total signal peak are completely removed, which can make the integration of the δ 7.1 signal peak more accurate and the identification of the δ 7.5 signal peak simpler.

[0063] The above is only a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for determining non-deuterated substances in the deuterated drug deuterated remdesivir hydrobromide, characterized in that: The steps include: (1) Take an appropriate amount of deuterated remdevir hydrobromide sample and dissolve it in an appropriate amount of deuterated methanol as the test solution; (2) Use a nuclear magnetic resonance spectrometer to collect the hydrogen spectrum of the test solution, select the pulse sequence noesyigld1d, and collect the hydrogen spectrum after automatically adjusting the gain; (3) performing phase adjustment, baseline correction, and chemical shift peak calibration on the collected hydrogen spectrum, integrating the signal peaks at chemical shifts δ 7.5 and δ 7.1, and calculating the content of non-deuterated substances in the deuterated remdesivir hydrobromide sample; The chemical structures of the deuterated remdevir hydrobromide and the corresponding non-deuterated product are shown below: 。 2. The method according to claim 1, characterized in that The mass concentration of deuterated remdesivir hydrobromide in the test solution in step (1) is 80-120 mg / mL.

3. The method according to claim 2, characterized in that The mass concentration of deuterated remdesivir hydrobromide in the test solution in step (1) is 100 mg / mL.

4. The method according to claim 1, characterized in that: In the step (2), the relaxation delay time of the nuclear magnetic resonance spectrometer is not less than 15 s; and the number of scans is not less than 32 times.

5. The method according to claim 4, characterized in that In the step (2), the relaxation delay time of the nuclear magnetic resonance spectrometer is 15 s; and the number of scans is 32 times.

6. The method according to claim 1, characterized in that In the step (2), the test temperature of the nuclear magnetic resonance spectrometer is 293-303 K, the pulse excitation angle is 90°, the decoupling power is 0.5 W, the number of empty scans is 4 times, the spectrum width is 20 ppm, the sampling time is 3.28 s, and the number of sampling points is 64 K.

7. The method according to claim 6, characterized in that The test temperature of the nuclear magnetic resonance spectrometer in step (2) is 298 K.

8. The method according to claim 1, characterized in that The step (3) specifically comprises: performing phase adjustment and baseline correction on the collected hydrogen spectrum, taking the deuterated methanol solvent peak chemical shift δ of 3.3 as a reference, marking the chemical shifts of other signal peaks; integrating the signal peaks at chemical shifts δ 7.5 and δ 7.1, and calculating the content of non-deuterated substances in the deuterated remidevir hydrobromide sample according to the following formula: ， in, P is the percentage of non-deuterated substances, A s is the integral value of the signal peak at chemical shift δ 7.5, A r is the integral value of the signal peak at the chemical shift δ7.1.

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