CN108373465B

CN108373465B - Dabigatran etexilate impurity and preparation and detection methods thereof

Info

Publication number: CN108373465B
Application number: CN201810330431.2A
Authority: CN
Inventors: 杜振军; 王全刚; 郭培良; 陈翠翠; 葛志乐; 罗鸣; 黄浩喜; 李英富; 苏忠海
Original assignee: Chengdu Beite Pharmaceutical Co Ltd
Current assignee: Chengdu Beite Pharmaceutical Co Ltd
Priority date: 2018-04-13
Filing date: 2018-04-13
Publication date: 2020-04-07
Anticipated expiration: 2038-04-13
Also published as: CN108373465A

Abstract

The invention discloses a dabigatran etexilate impurity compound shown as a formula I and an impurity preparation and detection method, wherein the preparation method comprises the following steps: taking dabigatran etexilate and n-hexanol as raw materials, taking a mixture comprising an organic solvent and a buffer solution as a system solvent, reacting, and adjusting the pH of a reaction solution to 7.8-8.2, preferably 8.0 to obtain the compound; the detection method adopts high performance liquid chromatography for detection, and qualitatively or quantitatively detects impurities, and the detection conditions comprise: a chromatographic column: octadecylsilane chemically bonded silica gel column, detection wavelength: 210-400 nm, the mobile phase comprises a mobile phase A and a mobile phase B, and the mobile phase A: a water phase containing a buffer solution, the pH of which is 4.25 to 4.40, further 4.3; the mobile phase B mainly comprises methanol or acetonitrile; the mobile phase is eluted by a gradient elution method. The method provided by the invention improves the impurity control of dabigatran etexilate, further controls the product quality of dabigatran etexilate, and better meets the requirement of dabigatran etexilate consistency evaluation.

Description

Dabigatran etexilate impurity and preparation and detection methods thereof

Technical Field

The invention relates to the field of pharmacy, in particular to dabigatran etexilate impurities and preparation and detection methods thereof.

Background

Dabigatran etexilate (dabigatran etexilate) is known under the trade name pradaxa, developed by the company briringer berghein, germany. The initial marketing in germany and the uk in month 4 2008, month 10 2010, FDA approval of dabigatran etexilate for the prevention of stroke and AF disease in patients, month 4 2014 for the treatment of DVT and PE and reducing the risk of recurrent DVT and PE patients, and year 2015 for the prevention of DVT and PE in hip replacement patients. The product is a novel oral anticoagulant drug approved by FDA for 50 years after warfarin, and becomes a major breakthrough in the field of anticoagulation by virtue of the characteristics of oral administration, strong effect, no need of special medication monitoring, less drug interaction and the like, and has milestone significance. Due to the good market prospect, the compound is favored by a plurality of pharmaceutical companies.

After the national institute clearly points out that the consistency evaluation of the imitation drugs is accelerated 7-22 months after 2015, the CFDA immediately goes out of a series of matching policies, the consistency evaluation of 289 varieties is completed before 2018, the cancelled drugs are approved by the government if the 289 varieties do not pass, the product is off the shelf, the market is lost if the product is light, and the survival is influenced if the product is heavy. The challenge and the opportunity coexist, and who can firstly evaluate the consistency in the same variety can obtain the prior advantage to preempt the market.

In 2016, 5 months, the State administration of food and drug administration issued a notice about the implementation of the opinion on the quality and curative effect consistency evaluation of the imitation drugs in the office of State Council, that is, the state required the quality and curative effect of the imitation drugs to be consistent with those of the original medicines. Specifically, the impurity spectrum is required to be consistent, the stability is required to be consistent, the in vivo and in vitro dissolution rules are required to be consistent, in order to meet the higher and higher requirements on the quality of the medicine, the concept of quality source design (QbD) is required to be adhered to, the raw material medicine synthesis design and control are carried out from the source, and the important index, namely the impurity, is firstly required to be controlled when the raw material medicine synthesis design and control are carried out from the source. Particularly, the dabigatran etexilate structure contains two amides and ester bonds, the molecular structure is not stable, more unknown impurities are easily generated in the production process, and the quality of a final product is influenced. If unknown impurities generated in the process of preparing the dabigatran etexilate can be separated, structure confirmation is carried out, the reaction mechanism is further understood according to the impurity structure, the impurities are detected from the dabigatran etexilate, the content of the impurities is controlled according to the impurity detection result adjusting method, the process is effectively controlled from the source, the impurity control of the dabigatran etexilate is greatly improved, and the consistency evaluation requirement of the dabigatran etexilate is met.

In order to control dabigatran etexilate from the source and effectively improve the quality of a final product, enable the dabigatran etexilate to meet the consistency evaluation requirement and improve the enterprise competitiveness, the invention researches an impurity of the dabigatran etexilate and a preparation method thereof.

Disclosure of Invention

The dabigatran etexilate is unstable in structure, so that more impurities are easily generated in the preparation process, and if the structure of the generated impurities is not determined, the impurities bring greater risk to the quality control of finished products; only by determining the chemical structure of the impurities, understanding the generation mechanism of the impurities and further effectively controlling the reaction operation in the step can the dabigatran etexilate finished product meet the quality requirement.

Based on the structure of dabigatran etexilate, various documents (CN105348262, WO2013/144903, Journal of chemical Research,2016,40(8),461, WO2013/24384, WO2016/27077, Heterocycles,2013,87(8),1699, WO2014/167577) have taken the following synthetic routes: the compound 1 reacts with n-hexyl chloroformate under the alkaline condition to prepare the dabigatran etexilate.

Synthetic routes of dabigatran etexilate reported in various literatures

However, in the process research of the step, the inventor accidentally finds that the prepared dabigatran etexilate generates an unknown impurity, and once the unknown impurity is generated, the content of the unknown impurity exceeds the identification threshold value by 0.1 percent, and further complicates that the dabigatran etexilate is difficult to effectively remove by the purification process. The technical requirement of drug registration is clearly indicated by the international harmonization conference organization (ICH) in Q3A "impurities in new bulk drug", researchers should describe the structural characteristics of impurities with apparent amount greater than or equal to 0.1% actually existing in bulk drugs; more than or equal to 0.1% of impurities should be defined for repeated batches. The applicant finds the production rule of the unknown impurity by accident and effectively separates the unknown impurity, further, the structure of the unknown impurity is determined by characterization means such as LC-MS and NMR, the implementation of the optimization of the operation process of the step is guided by the applicant, and the production of the unknown impurity can be effectively controlled by controlling reaction parameters, so that the quality of the dabigatran etexilate is controlled.

Specifically, the inventor detects that the dabigatran etexilate contains unknown impurities of the compound shown in the formula I,

however, the presence of the compound of formula I is not reported at present, and even less found.

In further studies after the discovery of the compounds, the following mechanism of production was known:

under the alkaline condition, firstly, ester hydrolysis is carried out on the compound 1 to generate an intermediate state 1, then the intermediate state 1 reacts with n-hexyl chloroformate to form an unstable active ester intermediate state 2, and finally the active ester intermediate state reacts with n-hexyl alcohol for alcoholysis reaction to obtain a target product.

Mechanism for producing dabigatran etexilate impurity

Further, the inventors tried the conventional methods for preparing n-hexyl ester, such as an acyl chloride method and a condensation method, to prepare the impurity, but the dabigatran etexilate structure contains a structure which is easy to hydrolyze, so that the system obtained by the conventional method using acyl chloride has more impurities and cannot be purified. By using the traditional ester exchange method, the reaction conversion rate is extremely low and almost no new product appears due to n-hexyl steric hindrance and other reasons. The inventors have unexpectedly discovered that this impurity is produced by mixing the finished dabigatran etexilate with n-hexanol in a solvent comprising a buffer, and allowing to stand at room temperature, resulting in a simpler synthetic method for preparing this impurity.

In particular, a process for the preparation of a compound of formula I, said process comprising the steps of:

taking dabigatran etexilate and n-hexanol as raw materials, and carrying out the following operations: and (3) taking a mixture comprising an organic solvent and a buffer solution as a system solvent, reacting, and adjusting the pH of the reaction solution to 7.8-8.2, preferably 8.0.

Further, the amount ratio of the dabigatran etexilate to the hexanol substance is 1: 3-5, preferably 1:3, 1:4 or 1:5, and in one embodiment of the invention, the amount ratio of the substances is 1: 5.

Further, the volume ratio of the organic solvent to the buffer in the mixture is 3-5: 10, preferably 3:10, 4:10 or 5:10, and more preferably 4: 10.

Further, the pH value of the buffer solution is 4.6-4.8; preferably, the pH is 4.8.

Further, the buffer solution is an acetate-acetic acid buffer solution; the acetate is selected from one or a mixture of two of sodium acetate and ammonium acetate, preferably ammonium acetate, and further, the concentration of the ammonium acetate is 0.2-0.5 wt%, preferably 0.2 wt%.

Further, the organic solvent is selected from one or more of acetonitrile, acetone or ethanol.

Further, the preparation method comprises one or more of the following (1) to (4):

(1) the organic solvent is acetonitrile;

(2) the pH of the reaction solution is adjusted by adopting a 10% sodium bicarbonate aqueous solution;

(3) the reaction time is 36 h;

(4) the reaction temperature is 0-30 ℃.

Further, the preparation method also comprises the following steps: extracting and carrying out column chromatography; the solvent used for extraction is dichloromethane, the eluent used for column chromatography is a mixture of DCM and MeOH, the volume ratio of DCM to MeOH is 30:1, and further, the column chromatography is silica gel column chromatography.

On the basis of providing an impurity preparation method, the invention also provides a detection method of impurities in dabigatran etexilate, wherein the impurities are the compound shown in the formula I, the detection method adopts high performance liquid chromatography to detect, and qualitatively or quantitatively detects the impurities, and the detection conditions comprise:

a chromatographic column: octadecylsilane chemically bonded silica gel column, detection wavelength: 210-400 nm, the mobile phase comprises a mobile phase A and a mobile phase B, and the mobile phase A: a water phase containing a buffer solution, the pH of which is 4.25 to 4.40, further 4.3; the mobile phase B mainly comprises methanol or acetonitrile; the mobile phase is eluted by a gradient elution method, and the elution procedure is as follows:

the wavelengths to be detected in the present invention can be adjusted and selected by conventional means within the range disclosed above. When the optimal detection wavelength is found, the method can be carried out by using a full-wave-band scanning method and the like which are matched with an ultraviolet spectrophotometry method and HPLC, and then the appropriate detection wavelength is found by using the authorization of a conventional technology in combination with the detection effect (such as solvent interference avoidance and the like) of an HPLC detector. In one embodiment of the present invention, the detection wavelength is selected from 210 to 400nm, for example at 242nm, 310nm or/and 340 nm.

The detection step comprises:

(1) preparing a test solution and a reference solution;

(2) respectively injecting sample solution for test and reference solution for detection.

The test sample is the dabigatran etexilate to be detected.

The qualitative detection in the present invention can be performed by using conventional methods, such as corresponding analysis by external standard method with reference substance, or qualitative analysis by conventional identification means, such as mass spectrum, thin layer, ultraviolet, etc. after separating each component by HPLC.

In the invention, the content can be calculated by using the conventional methods such as an external standard method, an area normalization method and the like.

During quantitative analysis, if an external standard method is used, a standard curve is manufactured by a conventional method for calculation; however, in the qualitative analysis, a standard curve is not required to be prepared, and the determination can be made by the retention time.

Further, the detection conditions further include one or more of the following (1) to (6):

(1) the buffer solution of the mobile phase A is an acetate-acetic acid buffer solution; further, the acetate is ammonium acetate; furthermore, the concentration of ammonium acetate in the buffer solution is 0.2 wt% to 0.5 wt%, preferably 0.2 wt%.

(2) The mobile phase B mainly comprises acetonitrile;

(3) the size of the chromatographic column is 4.6 multiplied by 250mm and 5 mu m;

(4) the detection wavelength is 242nm, 310nm or/and 340 nm;

(5) the flow rate is 0.8ml/min to 1.2ml/min, preferably 1.0 ml/min;

(6) the column temperature is 28-40 ℃, and further 35 ℃.

In the invention, the parameters such as column temperature, flow rate, sample injection amount and the like can be selected in a common range.

The dabigatran etexilate impurity and the preparation and detection methods thereof have the following advantages:

1. by defining the structure and reaction mechanism of process impurities, the process parameter can be well controlled, so that the quality control of the product is better enhanced;

2. compared with the existing directional synthesis method, the method for destroying the finished product is adopted, excessive chemical reagents and post-treatment are not used, the reaction conversion rate is high, and the separation and preparation are easy;

3. the invention provides a new reference substance for impurity detection of dabigatran etexilate by synthesizing the compound shown in the formula I and providing a preparation method of the compound, which is more beneficial to development of a related substance detection method and further controls the product quality of dabigatran etexilate.

Drawings

FIG. 1 is a LC-MS diagram of the impurity of dabigatran etexilate prepared in example 5;

FIG. 2 shows the impurities of dabigatran etexilate prepared in example 5¹A HNMR map;

FIG. 3 shows the impurities of dabigatran etexilate prepared in example 5¹³A CNMR map;

FIG. 4 is an HPLC plot of the impurity of dabigatran etexilate prepared in example 5;

FIG. 5 is an HPLC plot of a sample of dabigatran etexilate prior to process optimization;

FIG. 6 is an HPLC chart of dabigatran etexilate samples after process optimization.

Detailed Description

Compound 1 is of the formula:

example 1

Dissolving the compound 1(1.25g, 2mmol) and n-hexanol (0.41g, 4mmol) in 20ml of dichloromethane, dropwise adding thionyl chloride (0.48g, 4mmol) at the temperature of 20-25 ℃, and then heating to reflux for 4 hours; TLC monitoring shows that a plurality of new points are generated in the reaction without main products, 20ml of water is added into the reaction liquid for quenching, organic phase is taken out after liquid separation, the pressure is reduced and concentration is carried out, 0.3g of oily matter is obtained, and the corresponding products are not obtained by column chromatography.

Example 2

Dissolving the compound 1(1.25g, 2mmol) and n-hexanol (0.41g, 4mmol) in 20ml of chloroform, adding DMAP (24mg, 0.2mmol), heating to 50-60 ℃, stirring for 12h, and monitoring by TLC to show that the raw material is not converted basically and has no obvious product point.

Example 3

Dabigatran etexilate (3.77g, 6mmol), n-hexanol (3.06g, 30mmol) were dissolved in 30ml acetonitrile and 7.5ml buffer (0.2% potassium dihydrogen phosphate solution, pH 3.20 adjusted with phosphoric acid) and stirred at room temperature for 24 hours; the reaction solution was adjusted to pH 8.0 with 10% sodium bicarbonate solution, and the residue obtained after concentration under reduced pressure was extracted with 60ml of dichloromethane; the residue after re-concentration has no excimer ion peak or fragment ion peak of the target product, as tested by LC-MS.

Example 4

Dissolving the finished dabigatran etexilate (3.77g, 6mmol) and n-hexanol (3.06g, 30mmol) in 30ml of acetonitrile and 7.5ml of buffer solution (0.2% potassium dihydrogen phosphate solution and 0.2% dipotassium hydrogen phosphate solution are mixed in equal proportion, and the pH value is 7.5), and stirring for 24 hours at room temperature; the reaction solution was adjusted to pH 8.0 with 10% sodium bicarbonate solution, and the residue obtained after concentration under reduced pressure was extracted with 60ml of dichloromethane; the residue after re-concentration has no excimer ion peak or fragment ion peak of the target product, as tested by LC-MS.

Example 5

Dissolving dabigatran etexilate finished product (5.02g, 8mmol) and n-hexanol (4.08g, 40mmol) in 40ml of acetonitrile and 10ml of buffer solution (0.2% ammonium acetate solution, pH value adjusted to 4.8 by glacial acetic acid), and stirring at room temperature for 36 hours; the reaction solution was adjusted to pH 8.0 with 10% sodium bicarbonate solution, and the residue obtained after concentration under reduced pressure was extracted with 80ml of dichloromethane; concentrating the organic phase, performing silica gel column chromatography (silica gel 100-200 mesh, eluent: DCM/MeOH: 30:1, color developing agent: 254nm ultraviolet + iodine fumigation), and collecting the product to obtain 3.42g of white solid with the yield of 62.4%.

LC-MS([M+H⁺])：ESI m/z＝684.3863.

HPLC purity: 99.073 percent.

¹H NMR(400MHz,d₆-DMSO)：δ9.132(1H,brs),8.652(1H,brs),8.390(1H,dd,J₁＝3.6Hz,J₂＝1.2Hz),7.803(2H,d,J＝8.8Hz),7.540(1H,td,J₁＝7.6Hz,J₂＝1.6Hz),7.476(1H,s),7.397(1H,d,J＝8.4Hz),7.138(2H,m),6.960(1H,t,J＝5.6Hz),6.885(1H,d,J＝8.0Hz),6.769(2H,d,J＝8.8Hz),4.597(2H,d,J＝5.2Hz),4.229(2H,t,J＝7.2Hz),3.950(4H,m),3.767(3H,s),2.695(2H,t,J＝7.2Hz),1.536(4H,m),1.298(11H,m),1.042(1H,d,J＝6.0Hz),0.853(6H,m).

¹³C NMR(100MHz,d₆-DMSO)：δ171.0,170.3,166.4,164.2,156.0,153.7,151.5,148.7,140.8,137.8,137.2,129.3,129.1,122.8,122.1,121.2,121.0,119.5,111.3,109.4,64.1,64.0,44.3,33.0,31.0,30.8,29.9,28.5,27.9,25.5,25.2,25.0,22.0,21.9,13.9,13.8.

Example 6

The following method is adopted for detecting impurities in dabigatran etexilate:

chromatographic conditions and system applicability test using octadecylsilane chemically bonded silica as filler (phenomenex gemini C18, 4.6 × 250mm, 5 μm); taking 0.2% ammonium acetate solution (adjusting pH value to 4.3 +/-0.1 by glacial acetic acid) as a mobile phase A, taking acetonitrile as a mobile phase B, and carrying out gradient elution according to the following table;

the column temperature was 35 ℃; the flow rate was 1ml per minute; the sample volume is 50 mul; the detection wavelengths are 242nm, 310nm and 340 nm.

The impurities of dabigatran etexilate prepared in example 5 were taken as a control, precisely weighed, and diluted with diluent 1[ acetonitrile: ethanol: water (12:3:10v/v) ] and diluted with diluent 2[ mobile phase a: mobile phase B (85:15v/v) is diluted to be about 0.3mg of solution in each 1ml, and is used as a reference solution; the dabigatran etexilate before and after process optimization is respectively taken as a test sample, precisely weighed and diluted by using a diluent 1[ acetonitrile: ethanol: water (12:3:10v/v) ] and diluted with diluent 2[ mobile phase a: and diluting the mobile phase B (85:15v/v) to prepare a solution containing about 0.3mg of dabigatran etexilate in each 1ml, wherein the dabigatran etexilate solution before process optimization is used as a test solution (1), and the dabigatran etexilate solution after process optimization is used as a test solution (2).

Precisely measuring 50 μ L of each of the test solution and the reference solution, injecting into a liquid chromatograph, and recording chromatogram; according to the detection result, the absolute retention time of the reference substance is 42.479min, and the content of the reference substance is 99.073 percent (shown in figure 4) by an area normalization method; the absolute retention time of the impurity of the present invention in the test article (1) was 42.270min, and its content was 0.2112% as measured by the area normalization method (see FIG. 5); the absolute retention time of the impurity of the present invention in the test article (2) was 43.098min, and its content was 0.043% by area normalization (see FIG. 6).

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

Claims

1. A compound of formula I:

2. a process for the preparation of a compound according to claim 1, comprising the steps of:

taking dabigatran etexilate and n-hexanol as raw materials, taking a mixture comprising an organic solvent and a buffer solution as a system solvent, reacting, and adjusting the pH of a reaction solution to 7.8-8.2 to obtain the dabigatran etexilate-n-hexanol composite material;

the buffer solution is an acetate-acetic acid buffer solution;

the acetate is selected from ammonium acetate;

the concentration of the ammonium acetate is 0.2 to 0.5 weight percent.

3. The method for producing a compound according to claim 2, wherein the reaction solution is adjusted to a pH of 8.0.

4. The method for preparing the compound according to claim 2, wherein the amount ratio of the dabigatran etexilate to the n-hexanol substance is 1: 3-5.

5. The method for preparing the compound according to claim 4, wherein the amount ratio of the dabigatran etexilate to the n-hexanol material is 1:3, 1:4 or 1: 5.

6. The method for preparing the compound according to claim 5, wherein the amount ratio of the dabigatran etexilate to the n-hexanol material is 1: 5.

7. The method for preparing the compound according to claim 2, wherein the volume ratio of the organic solvent to the buffer in the mixture is 3-5: 10.

8. The method of claim 7, wherein the volume ratio of the organic solvent to the buffer in the mixture is 3:10, 4:10, or 5: 10.

9. The method of claim 8, wherein the volume ratio of the organic solvent to the buffer in the mixture is 4: 10.

10. The method of claim 2, wherein the buffer has a pH of 4.6 to 4.8.

11. The method of claim 10, wherein the buffer has a pH of 4.8.

12. A process for the preparation of a compound according to claim 2, wherein the ammonium acetate concentration is 0.2 wt%.

13. A process for the preparation of a compound according to any one of claims 2, 7 or 10, wherein the organic solvent is selected from a mixture of one or more of acetonitrile, acetone or ethanol.

14. The method of claim 13, wherein the organic solvent is acetonitrile.

15. The method for preparing the compound according to claim 2, wherein the method further comprises the steps of extraction and column chromatography.

16. The method of claim 15, wherein the solvent used for the extraction is dichloromethane.

17. The process of claim 15, wherein the column chromatography is performed on silica gel using a mixture of DCM and MeOH as eluent, DCM: MeOH ═ 30:1 v/v.

18. A method for detecting impurities in dabigatran etexilate, wherein the impurities are the compound of claim 1, the method for detecting impurities qualitatively or quantitatively by high performance liquid chromatography comprises the following steps:

a chromatographic column: octadecylsilane chemically bonded silica gel column, detection wavelength: 210-400 nm, the mobile phase comprises a mobile phase A and a mobile phase B, and the mobile phase A: a water phase containing a buffer solution, wherein the pH value is 4.25-4.40; the mobile phase B mainly comprises methanol or acetonitrile; the mobile phase is eluted by a gradient elution method, and the elution procedure is as follows:

the detection step comprises:

(1) preparing a test solution and a reference solution;

19. The detection method according to claim 18, wherein the mobile phase a has a pH of 4.3.

20. The detection method according to claim 18, wherein the detection condition further includes one or more of the following (1) to (6):

(1) the buffer solution of the mobile phase A is an acetate-acetic acid buffer solution;

(2) the mobile phase B mainly comprises acetonitrile;

(4) the detection wavelength is 242nm, 310nm or/and 340 nm;

(5) the flow rate is 0.8ml/min to 1.2 ml/min;

(6) the column temperature is 28-40 ℃.

21. The assay of claim 20, wherein the acetate salt is ammonium acetate.

22. The detection method according to claim 21, wherein the concentration of ammonium acetate in the buffer solution is 0.2 to 0.5 wt%.

23. The assay of claim 22, wherein the ammonium acetate concentration in the buffer is 0.2 wt%.

24. The assay of claim 20, wherein the flow rate is 1.0ml/min and the column temperature is 35 ℃.