CN115417912A - Nemantevir related substance and preparation method and application thereof - Google Patents
Nemantevir related substance and preparation method and application thereof Download PDFInfo
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- CN115417912A CN115417912A CN202210920384.3A CN202210920384A CN115417912A CN 115417912 A CN115417912 A CN 115417912A CN 202210920384 A CN202210920384 A CN 202210920384A CN 115417912 A CN115417912 A CN 115417912A
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- nemadevir
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- nemadefovir
- nitrite
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/42—Salmonella
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2410/00—Assays, e.g. immunoassays or enzyme assays, involving peptides of less than 20 animo acids
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Abstract
The invention discloses a nemadefovir related substance shown in a formula (I) and a preparation method thereof, wherein the substance shown in the formula (I) is prepared by oxidizing the nemadefovir in an acidic solvent. The preparation method provided by the invention has the advantages of simple route, simple and reliable process, low comprehensive cost, high yield and high purity, and the prepared nemadevir related substances can be used as qualitative and quantitative analysis standard substances of nemadevir Wei Zazhi and used for quality research of nemadevir bulk drugs and related preparations thereof.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to a nemadevir related substance, and a preparation method and application thereof.
Background
Parro Wei De (Paxlovid), developed by Pfizer, USA, is a specific drug for treating new coronaviruses, and Parro Wei De consists of two parts, namely nelmavir (Nirmatrelvir) which inhibits one of SARS-CoV-2 proteins to prevent virus replication, and Ritonavir tablets (Ritonavir) which slows down the decomposition of nelmavir to help it maintain a higher concentration time in the body. The molecular formula of the nemadevir is C 23 H 32 F 3 N 5 O 4 The structural formula is as follows:
research shows that impurities are a key quality attribute of medicines and belong to key research objects in the whole chemical medicine research and development process. Among them, the presence of degradative impurities, especially suspected genotoxic impurities, greatly affects the safety of administration, and the genotoxic impurities (e.g., N-nitroso substituent) induce gene mutation at a very low concentration and cause chromosome breakage and rearrangement, and thus have potential carcinogenicity, and have been receiving much attention in recent years. Wherein N-nitroso substituents, e.g. N-dimethylnitrosamine (formula C) 2 H 6 N 2 O, structural formula (CH) 3 ) 2 NNO, molecular weight 74.08, abbreviated NDMA), which is one of the most toxic chemicals of the N-nitrosamines class of compounds, and is classified as a class 2A carcinogen by the international agency for research on cancer (IARC). In consideration of the harm of related impurities in medicines to human bodies, the European Union and the U.S. medicine regulatory agency issue guiding principles of genotoxic and carcinogenic impurities in succession. On the basis, ICH (International Congregation for registration of drugs for human use) also formulated M7 guidelines for genotoxic impurities. 5/8/2020, the drug evaluation center of the State drug administration of China issued "Zhongya chemical drugsTechnical guidelines (trial) for research of nitrosamine impurities in the pharmaceutical products require that pharmaceutical enterprises fully evaluate the risk of nitrosamine impurities in the pharmaceutical products and control the level of nitrosamine impurities below safety limits. At present, no patent of literature reports about research on nemadevir related nitrosamine substances, and a preparation method of the nemadevir related nitrosamine substances is also lacked in the market. Therefore, the method has important significance for effectively controlling suspected genotoxic impurities of the bulk drugs and related preparations thereof by directionally preparing the target impurities and establishing a corresponding analysis method.
During production and normal storage according to a given process, impurities which need to be controlled may inevitably be contained or generated. At present, few reports on the research on the degradation of impurities by nemadevir are known through inquiring documents and patent databases. Considering the huge use of nemadefovir in the treatment of new coronavirus, the research on the degradation impurities of nemadefovir is especially important at present. The applicant finds that a brand new compound exists in the degradation process of the nemadefovir, the content is more than 0.1 percent, and the requirement of the nemadefovir on impurity limit is exceeded. The chemical structure and the preparation method of the compound are confirmed, and the compound plays a vital role in establishing a detection method, improving and perfecting the quality standard of the nemadevir and monitoring the clinical medication safety.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a nemadevir related substance, a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a nemadevir related substance, which has a structure shown in a formula (I):
the invention also provides a preparation method of the nemadevir related substance, which comprises the following steps:
dissolving the nemadevir in an acidic solvent, adding a nitrite, and carrying out an oxidation reaction until the nemadevir disappears; then filtering, decompressing and removing the solvent to obtain a crude product; purifying to obtain the nemadevir related substances.
Preferably, the acidic solvent is glacial acetic acid or concentrated hydrochloric acid.
Preferably, the ratio of the nemadefovir to the acidic solvent is 10mL acidic solvent per 100mg of nemadefovir.
Preferably, the nitrite salt is sodium nitrite.
Preferably, the molar ratio of nitrite to nemadevir is 1:1.
Preferably, the oxidation reaction is carried out at 0 ℃.
Preferably, the specific steps of the purification are as follows: the crude product was purified by column chromatography using a system of volume ratios dichloromethane to methanol = 20.
The invention also provides application of the nemadefovir related substance as a reference substance in medicine quality control or medicine research.
The invention has the beneficial effects that:
(1) The invention provides a new nemadefovir related substance, which can be used as a qualitative and quantitative analysis standard substance of nemadefovir Wei Zazhi, is used for quality research of a nemadefovir raw material medicament and related preparations thereof, and provides an important substance guarantee for further improving the quality standard of the nemadefovir.
(2) The preparation method of the nemadevir related substances provided by the invention has the advantages of simple route, simple and reliable process, low comprehensive cost, high yield and high purity.
Drawings
FIG. 1 is a high performance liquid detection spectrum of the oxidative degradation impurities of nemadefovir in example 1.
FIG. 2 is a mass spectrometric detection of the oxidative degradation impurities of nemadefovir in example 1.
FIG. 3 is a high performance liquid chromatography detection spectrum of Compound (I) in example 2.
FIG. 4 is a drawing showing the preparation of Compound (I) in example 2 1 Detecting a spectrogram by HNMR.
Detailed Description
The present invention is further described in detail below with reference to specific examples so that those skilled in the art can more clearly understand the present invention. The examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention. In the examples of the present invention, all the raw material components are commercially available products well known to those skilled in the art, unless otherwise specified; unless otherwise specified, all technical means used are conventional means well known to those skilled in the art.
The progress of the reaction of the invention can be monitored by monitoring methods conventional in the art (e.g., TLC, LCMS or NMR), typically at the end of the reaction when the reaction substrate is eliminated.
In the following examples, the liquid phase conditions identified for the detection of the preparation compounds were: shimadzu LCMS2020, G1322A degasser, G1312 binary high pressure pump, G1329A autosampler, G1316A column oven, G4212B diode array detector. The column was Xbridge C18 (50 mm. Times.4.6 mm,5.0 μm), mobile phase A was deionized water and mobile phase B was acetonitrile containing 0.05% trifluoroacetic acid, and the gradient elution was performed as follows:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0.01 | 95 | 5 |
| 1.50 | 95 | 5 |
| 3.00 | 5 | 95 |
| 3.50 | 5 | 95 |
| 4 | 95 | 5 |
| 5 | 95 | 5 |
The flow rate was 1.5mL/min, the column temperature was 40 ℃ and the detection wavelength was 220nm.
Example 1
The applicant examines the stability of the nemadevir through an oxidation forced degradation experiment (experimental conditions: acetic acid as a solvent, sodium nitrite as an oxidant, and a reaction time of 1 hour at 25 ℃), and the following conditions are detected and found by a research team:
as shown in fig. 1, the content of impurity I with a peak-off time of 3.256min is more than 0.1%, which exceeds the impurity limit requirement of nemadevir.
As shown in the negative ion mass spectrum of fig. 2, the results show that: the molecular ion peak of impurity I is 529.40. By combining the reaction mechanism and mass spectrum results, the structure of the compound is presumed to be the compound shown in the formula (I) most probably.
Example 2
This example provides a nemadevir related substance represented by formula (I), the synthetic route of which is shown in the following reaction scheme:
the specific synthesis method of compound (I) of this example comprises the following steps:
s1, preparing a crude compound (I):
dissolving compound II (100mg, 200.19 μmml) in glacial acetic acid (10 mL), adding sodium nitrite (13.81mg, 200.19 μmml) in portions at 0 ℃, and stirring the reaction until the compound II disappears; the reaction solution was then filtered, and the solvent was removed under reduced pressure to give 101mg of crude compound A.
S2, purification:
the crude compound a obtained in step S1 was purified by column chromatography using a system of dichloromethane to methanol =20 in volume ratio to obtain compound I (91 mg) as a white solid in 86.01% purity of 98%.
The compound (I) prepared in this example was identified as follows:
ESI-MS(m/z):529.4；
1 H NMR(400MHz,DMSO-d6)δ9.03(d,J＝8.5Hz,1H),7.68(s,1H),4.98(td,J＝10.8,5.0Hz,1H),4.42(d,J＝8.4Hz,1H),4.16(s,1H),3.92(dd,J＝10.3,5.4Hz,1H),3.70(d,J＝10.4Hz,1H),3.15(t,J＝8.9Hz,1H),3.04(dd,J＝16.5,9.2Hz,1H),2.44–2.36(m,1H),2.18–2.05(m,2H),1.77–1.67(m,2H),1.58(s,1H),1.33(d,J＝7.7Hz,1H),1.03(s,3H),0.99(s,9H),0.86(s,3H).
meanwhile, the compound was subjected to liquid phase detection and localization under the same detection conditions as the high performance liquid phase in experiment 1, and the results are shown in fig. 3: the peak-off time of compound I was 3.256min, which is consistent with the peak-off time of impurity 1 in fig. 1, and the result further confirms that impurity 1 is the same compound as compound a.
Example 3
This example provides a nemadefovir related substance of formula (I), which is synthesized as shown in the following reaction scheme:
this example was conducted in a specific manner, with reference to example 2, except that the acetic acid solvent in example 2 was replaced with a concentrated hydrochloric acid solvent, to thereby obtain Compound I in a yield of 78% and a purity of 98%.
Example 4
Ames Salmonella reverse mutation assay
The Ames test is a short-term in vitro test method for primarily screening mutagenicity of chemical substances by utilizing the property of reversion of histidine auxotrophic mutant strains of salmonella typhimurium. The gene toxicity of the impurities can be defined through an Ames test, genetic toxicological danger identification is carried out, whether target compounds can cause damage to genetic materials is identified, and exposure is further controlled, so that risks are reduced.
The experimental procedure was as follows: 0.1ml of the cryopreserved bacterial solution (strains: TA98, TA97, TA100, TA102 and TA1535; source: moltox Molecular biology incorporated.) was inoculated into 10.0ml of the nutrient broth medium and cultured in a 37 ℃ constant temperature water bath at a rate of about 90 to 120 times per minute shaking for 10 hours. Then, 2ml of the melted top agar was added to a sterile test tube, and then 0.1ml of a compound (I) solution (500. Mu.g/dish, 1000. Mu.g/dish) or a solvent, 0.1ml of an enrichment medium, and 0.5ml of a phosphate buffer (pH 7.4,0.2 mol/L) or 0.5ml of an S9 mixture (activated metabolic system, S9 source: moltox Molecular biology Incorporated; composition of rat liver microsome enzyme homogenate mixture (S9-mix: S9.0 ml, mgCl2-KCl salt solution 2.0ml, phosphate buffer 25ml,1mol/L G-6-P0.5 ml,0.1mol/L NADP 3ml, and sterile distilled water 25ml, S9 mixture was prepared immediately before use) were added to the test tube. After the components in the suspension tube are shaken, the mixture is spread on the surface of a bottom culture medium plate. After the top agar solidified, the plate was placed upside down in an incubator and incubated at 37 ℃ for about 48 hours. And (4) confirming that the bacterial background lawn grows well under a microscope, and counting the retromorphous colonies under the condition of no obvious sparseness compared with a solvent control.
TABLE 1 Compound I Ames test results
Note: (1) a is the average of three plates;
(2) P <0.05 compared to vehicle control.
As can be seen from the results in Table 1, the significant increase in the number of the test strain TA97 and TA98 recurrent colonies was observed at the dose of 500 and 1000. Mu.g/dish, regardless of the presence of the S9 activation system, indicating that the compound (I) is mutagenic to Salmonella murine under the conditions of this test. The above results indicate that the presence of compound (I) has potential toxic side effects on the human body and that the impurities need to be controlled.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.
Claims (9)
1. A nemadevir related substance has a structure shown in a formula (I):
2. the process for the preparation of nemadevir related substances as claimed in claim 1, characterized by the steps of:
dissolving the nemadevir in an acidic solvent, adding a nitrite, and carrying out an oxidation reaction until the nemadevir disappears; then filtering, decompressing and removing the solvent to obtain a crude product; purifying to obtain the nemadevir related substance.
3. The method for preparing a nemadevir related substance as claimed in claim 2, wherein the acidic solvent is glacial acetic acid or concentrated hydrochloric acid.
4. The process for the preparation of nemadefovir related substance according to claim 2, characterized in that the ratio of nemadefovir to acidic solvent is 10mL acidic solvent per 100mg of nemadefovir.
5. The process according to claim 2, wherein the nitrite is sodium nitrite.
6. The process for the preparation of nemadevir related substance as claimed in claim 2, wherein the molar ratio of nitrite to nemadevir is 1:1.
7. The process for preparing nemadevir related substances according to claim 2, wherein the oxidation reaction is carried out at 0 ℃.
8. The method for preparing a nemadevir related substance as claimed in claim 2, wherein the purification comprises the following steps: the crude product was purified by column chromatography using a system of volume ratios dichloromethane to methanol = 20.
9. Use of a nemadevir related substance as described in claim 1 as a reference substance in drug quality control or drug research.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111517991A (en) * | 2020-04-24 | 2020-08-11 | 常州齐晖药业有限公司 | Preparation method of diclazuril impurity A |
| CN112946107A (en) * | 2021-01-28 | 2021-06-11 | 石药集团欧意药业有限公司 | Analysis method of N-nitrosodimethylamine and N-nitrosodiethylamine in argatroban bulk drug or preparation |
| WO2022126207A1 (en) * | 2020-12-20 | 2022-06-23 | Topelia Aust Limited (Acn 652 771 670) | Drugs, therapeutic combinations and methods for preventing viral and microbial infections and their sequelae |
| CN114716441A (en) * | 2022-04-08 | 2022-07-08 | 斯坦德标准技术研究(湖北)有限公司 | Impurity compound A in new crown treatment medicine palofurtide, preparation method and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111517991A (en) * | 2020-04-24 | 2020-08-11 | 常州齐晖药业有限公司 | Preparation method of diclazuril impurity A |
| WO2022126207A1 (en) * | 2020-12-20 | 2022-06-23 | Topelia Aust Limited (Acn 652 771 670) | Drugs, therapeutic combinations and methods for preventing viral and microbial infections and their sequelae |
| CN112946107A (en) * | 2021-01-28 | 2021-06-11 | 石药集团欧意药业有限公司 | Analysis method of N-nitrosodimethylamine and N-nitrosodiethylamine in argatroban bulk drug or preparation |
| CN114716441A (en) * | 2022-04-08 | 2022-07-08 | 斯坦德标准技术研究(湖北)有限公司 | Impurity compound A in new crown treatment medicine palofurtide, preparation method and application thereof |
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