Compound as well as preparation method, detection method and application thereof

Abstract

The invention provides a compound as well as a preparation method, a detection method and application thereof, and relates to the field of pharmaceutical analysis. The invention provides a compound, the chemical structural formula of the compound is # imgabs0 #, the invention also provides a preparation method and a detection method of the compound, and the quality safety and reliability of the compound are ensured by researching the compound; the compound can be used as a reference substance for detecting impurities in an eptazocine preparation and the content of the impurities.

Description

Technical Field

[0001] The present invention relates to the field of drug analysis, and in particular to a compound and a preparation method and a detection method thereof, and application thereof in the detection of impurities in eptazocine preparations. Background Art

[0002] Eptazocine hydrobromide was originally developed by Nihon Lyakuhin Kogyo Co., Ltd. and was launched in Japan in 1987. It is mainly used to treat postoperative pain and cancer pain. Eptazocine hydrobromide is a partial agonist of opioid receptors, acting on K receptors, using selective antagonists to block postsynaptic receptors and chemical messengers that transmit pain information. In terms of analgesia, the analgesic effect of Eptazocine is 1-2 times that of Pentazocine. The structural formula of Eptazocine hydrobromide is as follows:

[0003]

[0004] Patent EP0384917A1 discloses a method for synthesizing eptazocine hydrobromide. The method uses a compound 1-1,4-dimethyl-10-methoxy-2,3,4,5-tetrahydro-1,6-methylene-1H-4-benzoazoline-7(6H)-one, subjected to sodium borohydride reduction reaction and hydrogenation reduction reaction, and then reacts with hydrobromic acid to demethylate and form a salt to obtain eptazocine hydrobromide. The reaction route is:

[0005]

[0006] The requirements of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) and the European Pharmacopoeia require strict control of impurities in drugs: (1) Actual and potential impurities may be generated during the synthesis, refining and storage of APIs. If these impurities are not strictly controlled, they may have serious toxic side effects on the human body; (2) During the preparation of drugs, it is usually necessary to add certain excipients to improve the uniformity, solubility and stability of the API in the preparation. Excipients can improve the appearance, taste and preparation process of the product on the one hand, and improve the effectiveness of the drug on the other hand. Excipients are usually inactive ingredients, but they may also significantly affect various properties of the drug. The physical interaction between the API and the excipients can regulate the pharmacokinetic characteristics of the API, such as improving the bioavailability of the drug; while the chemical interaction between the two may cause the degradation of the drug. At the same time, the excipients may gradually degrade during the production or storage process, producing trace amounts of unknown impurities. These reactive impurities will affect the stability of the drug throughout its life cycle, thereby causing the drug to lose its effectiveness, lose its efficacy or form toxic degradation products.

[0007] Therefore, it is necessary to control and study the impurities in drugs. Summary of the invention

[0008] Based on this, the primary purpose of the present invention is to provide a compound, a method for preparing the compound, and an application of the compound in the detection of impurities in eptazocine preparations, so as to provide a reference for the quality research of eptazocine preparations and further ensure the safety and reliability of their quality.

[0009] The purpose of the present invention can be achieved through the following technical solutions:

[0010] The inventors studied the eptazocine preparation placed under high temperature conditions, separated and purified an unknown compound from the eptazocine preparation by means of high performance liquid chromatography, and performed structural identification on it. Finally, the structure of the compound was confirmed. After checking, no reports were found on the structure.

[0011] Based on this, the present invention provides a compound, the chemical name of the compound is (((1S,6S)-10-hydroxy-1,4-dimethyl-2,3,4,5,6,7-hexahydro-1H-1,6-methanobenzo[e]azonin-9-yl)methyl)glycine, and the chemical structural formula is:

[0012]

[0013] The present invention further studies the preparation method of the above-mentioned compound and explores the influence of temperature and time on its preparation. Based on this, the present invention provides a preparation method of the above-mentioned compound, that is, dissolving etazocine and glycine in water and reacting them to obtain the compound.

[0014] The reaction equation is as follows:

[0015]

[0016] During the implementation process, it was found that increasing the reaction temperature can accelerate the reaction speed and thus shorten the reaction time.

[0017] That is, the reaction temperature of the above reaction is greater than or equal to 0°C, and the reaction time is greater than or equal to 5 hours;

[0018] Preferably, the reaction temperature of the above reaction is 40-400° C., preferably 50-350° C., more preferably 80-320° C., more preferably 100-300° C., and more preferably 100-200° C. When the reaction is carried out at this reaction temperature, the reaction can proceed at the maximum reaction speed without reducing the content of the compound.

[0019] Preferably, the reaction time is 10-72h; preferably 12-64h; more preferably 15-60h;

[0020] More preferably, the reaction time is 20-50 hours, more preferably 25-40 hours, and even more preferably 30-40 hours. This reaction time can ensure that the reaction is fully and completely carried out.

[0021] The mass ratio of eptazocine to glycine is 1:1.5-15; preferably 1:2-13; more preferably 1:3-7; further preferably 1:4-6; further preferably 1:5-6.

[0022] The present invention also provides a separation and detection method for the above compound, which uses high performance liquid chromatography to measure the compound, and the chromatographic conditions are as follows:

[0023] Chromatographic column: A chromatographic column with octadecylsilane bonded silica gel as filler is used;

[0024] Mobile phase: consists of mobile phase A and mobile phase B;

[0025] Elution conditions: isocratic or gradient elution.

[0026] in,

[0027] Mobile phase A: Mobile phase A: glacial acetic acid aqueous solution; Mobile phase B: methanol;

[0028] During the isocratic elution, the volume ratio of mobile phase A to mobile phase B is 80-90:10-20, preferably 80:20.

[0029] The elution procedure during the gradient elution is:

[0030]

[0031] Preferably, the volume fraction of the glacial acetic acid aqueous solution is 1-3%, preferably 2%; the pH value of the glacial acetic acid aqueous solution is 2-6.

[0032] The preparation method of the glacial acetic acid aqueous solution is as follows: 970-990 mL of water is measured and placed in a measuring cylinder, 10-30 mL of glacial acetic acid is added, the mixture is shaken well, and the pH value is adjusted to 2-6 with triethylamine to obtain the solution.

[0033] As some preferred embodiments, the column temperature of the chromatographic column in the above detection method is 35-45°C; preferably 40°C.

[0034] As some preferred embodiments, the detection wavelength in the above detection method is 280 nm.

[0035] As some preferred embodiments, the flow rate of the mobile phase in the above detection method is 0.5-1.5 mL / min, and more preferably 1.0 mL / min.

[0036] As some preferred embodiments, the injection volume in the above detection method is 15-25 μl, and more preferably 20 μl.

[0037] As a preferred embodiment, the high performance liquid chromatography detection used in the detection method of the above-mentioned compound provided by the present invention is carried out under the following chromatographic conditions:

[0038] Chromatographic column: KINGFUN Eldath AQ-C18 4.6mm×250mm, 5μm;

[0039] Mobile phase: Mobile phase A: 2% glacial acetic acid aqueous solution, pH = 2.5; Mobile phase B: methanol;

[0040] Flow rate: 1.0 mL / min;

[0041] Column temperature: 40°C;

[0042] The detection wavelength is 280nm;

[0043] Injection volume: 20 μl;

[0044] Elution conditions: isocratic elution, the volume ratio of mobile phase A to mobile phase B was 80:20;

[0045] Or gradient elution, the elution program is:

[0046]

[0047] The present invention also provides the use of the above compound in the detection of impurities in eptazocine preparations. The application is to use the compound as a reference substance to detect impurities and their contents in eptazocine preparations.

[0048] The present invention also provides the use of the above compound in the detection of impurities in eptazocine preparations. The application is to use the compound as a reference substance to detect impurities and their contents in eptazocine preparations.

[0049] Preferably, the method of using the compound as a reference substance for detecting impurities in an eptazocine preparation comprises the following steps:

[0050] (1) Prepare test solution;

[0051] (2) preparing a reference solution;

[0052] (3) HPLC detection: Take the test sample solution and the reference sample solution, inject them into the liquid chromatograph respectively, record the chromatogram, and calculate according to the external standard method.

[0053] in,

[0054] The preparation method of the test solution described in step (1) above is:

[0055] Take the eptazocine preparation to be tested and dilute it with a solvent to prepare a solution containing about 1 mg of eptazocine per 1 ml;

[0056] The preparation method of the reference solution described in step (2) above is:

[0057] Take the compound prepared by the above method and dilute it with a solvent to make a solution containing about 1.5 μg per 1 ml;

[0058] The solvent is a mixture of mobile phase A and mobile phase B; the volume ratio of mobile phase A to mobile phase B is 75-85:25-15; preferably, the volume ratio of mobile phase A to mobile phase B is 80:20.

[0059] As a preferred embodiment, in the use of the compound in the above-mentioned eptazocine preparation, the method for detecting impurities in the eptazocine preparation using the compound as a reference substance comprises the following steps:

[0060] (1) Take the test preparation of eptazocine and quantitatively dilute it with 2% glacial acetic acid aqueous solution-methanol (80:20) at pH = 2.5 to prepare a solution containing about 1 mg of eptazocine per 1 ml, which is used as the test preparation to detect and further control impurities in the test preparation;

[0061] (2) Preparation of reference substances:

[0062] The compound eptazocine glycine prepared by the above method is diluted with a solvent to prepare a solution containing about 2 μg per 1 ml;

[0063] (3) Detection: High performance liquid chromatography detection: Take the test sample solution and the reference sample solution, inject them into the liquid chromatograph respectively, record the chromatogram, and calculate according to the external standard method.

[0064] Chromatographic column: KINGFUN Eldath AQ-C18 4.6mm×250mm, 5μm;

[0065] Mobile phase: Mobile phase A: 2% glacial acetic acid aqueous solution, pH = 2.5; Mobile phase B: methanol;

[0066] Flow rate: 1.0 mL / min;

[0067] Column temperature: 40°C;

[0068] The detection wavelength is 280nm;

[0069] Injection volume: 20 μl;

[0070] Elution conditions: isocratic elution, the volume ratio of mobile phase A to mobile phase B was 80:20.

[0071] The above-mentioned eptazocine preparations include but are not limited to eptazocine hydrobromide preparations.

[0072] Compared with the prior art, the present invention has the following beneficial effects:

[0073] 1. Filling the gap in the industry, the present invention provides a new compound and a method for detecting the new compound - eptazocine glycine, which can be used for the quality control of the purity of the active ingredient.

[0074] 2. The present invention further discovered a new compound, eptazocine glycine, in the eptazocine preparation by applying the inspection method of the present invention. The compound could not be found when the old inspection method was used before. Therefore, the new inspection method can effectively enhance the quality control of the eptazocine preparation and increase the safety of the drug.

[0075] 3. The present invention has determined that the new compound is eptazocine glycine through research, and has also continued to conduct systematic methodological wavelength detection verification. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 This is the spectrum of the sample of etazocin preparation placed under accelerated conditions for 6 months.

[0077] Figure 2 This is the DAD UV scan of eptazocine of the present invention;

[0078] Figure 3 This is the DAD UV scan of eptazocine glycine prepared in Example 1-2 of the present invention;

[0079] Figure 4 This is a positive ion mode mass spectrum of eptazocine glycine prepared in Example 1-2 of the present invention;

[0080] Figure 5 Negative ion mode mass spectrum of eptazocine glycine prepared in Example 1-2 of the present invention;

[0081] Figure 6 The separation spectrum of impurities in eptazocine preparations was performed using the compound as a reference substance. DETAILED DESCRIPTION

[0082] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0083] The experimental methods used in the following examples are all conventional methods unless otherwise specified; the water used is all ultrapure water; the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0084] The eptazocine preparation uses glycine as an auxiliary material, and the auxiliary material interferes with the detection of related substances or affects the production of related substances. The present invention mainly solves the technical problem of auxiliary material interference and effective detection of related substances in the product.

[0085] Example 1 Preparation of Compounds and Detection of Their Content

[0086] Example 1-1:

[0087] 15 mg of eptazocine and 40 mg of glycine were dissolved in water and refluxed at 400° C. for 10 hours under oil bath conditions to obtain the compound.

[0088] Embodiment 1-2:

[0089] 15 mg of eptazocine and 185 mg of glycine were dissolved in water and refluxed at 80° C. for 60 hours under oil bath conditions to obtain the compound.

[0090] Embodiment 1-3:

[0091] 15 mg of eptazocine and 100 mg of glycine were dissolved in water and refluxed at 90° C. under oil bath conditions for 70 hours to obtain the compound.

[0092] Embodiment 1-4:

[0093] 15 mg of eptazocine and 60 mg of glycine were dissolved in water and refluxed at 100° C. in an oil bath for 50 hours to obtain the compound.

[0094] The above compound samples were accurately weighed and added to a solution of 2% glacial acetic acid aqueous solution-methanol (80:20) at pH = 2.5 to prepare a test solution with a concentration of 1 mg / ml.

[0095] The compound content was determined by high performance liquid chromatography with the following chromatographic conditions:

[0096] Chromatography: High Performance Liquid Chromatography

[0097] Fluidity: Mobile phase A: 2% glacial acetic acid aqueous solution, pH = 2.5; Mobile phase B: methanol;

[0098] Chromatographic column: KINGFUN Eldath AQ-C18 4.6mm×250mm, 5μm

[0099] The flow rate is 1.0 ml per minute;

[0100] The column temperature was 40°C;

[0101] The detection wavelength is 280nm;

[0102] Injection volume 20 μl;

[0103] The programmed elution conditions were: isocratic elution, with a volume ratio of mobile phase A to mobile phase B of 80:20;

[0104] Or gradient elution, the elution program is:

[0105]

[0106] Using the above detection method, the contents of the compounds determined were 3.33%, 15.12%, 8.26% and 4.84%, respectively.

[0108] The prepared eptazocine preparation was placed under accelerated conditions for 6 months and then tested according to the above test method. Figure 1 It can be clearly seen that the eptazocine preparation contained the compound eptazocine glycine after being stored under accelerated conditions for 6 months.

[0109] Example 2 Structural Identification of Compounds

[0110] The samples after the reaction in Example 1-2 were taken and separated and purified by high performance liquid chromatography, and the fractions corresponding to the compounds were collected. The compounds were obtained after vacuum distillation and drying.

[0111] The compound was detected by high-resolution mass spectrometry, and the results were:

[0112] The mass-to-nuclear ratio in positive ion mode is 319.28, and that in negative ion mode is 317.35, resulting in a molecular weight of 318 (e.g. Figure 4 and Figure 5 ).

[0113] Combining the information of the compound's mass spectrum to identify its structure, the final structure of the compound is determined to be:

[0114]

[0115] Example 3 Compounds were used as reference substances to separate and detect impurities in eptazocine preparations

[0116] The steps are:

[0117] (1) Preparation of test solution: quantitatively dilute the eptazocine preparation with mobile phase A-methanol (80:20) to prepare a solution containing approximately 1 mg of eptazocine per 1 ml as the test solution;

[0118] (2) taking the compound eptazocine glycine prepared by the above method, diluting it with a solvent to prepare a solution containing about 2 μg per 1 ml;

[0119] (3) HPLC detection: Take the test sample solution and the reference sample solution, inject them into the liquid chromatograph respectively, record the chromatogram, and calculate according to the external standard method.

[0120] Chromatographic column: KINGFUN Eldath AQ-C18 4.6mm×250mm, 5μm;

[0121] Mobile phase: Mobile phase A: 2% glacial acetic acid aqueous solution, pH = 2.5; Mobile phase B: methanol;

[0122] Flow rate: 1.0 mL / min;

[0123] Column temperature: 40°C;

[0124] The detection wavelength is 280nm;

[0125] Injection volume: 20 μl;

[0126] The program elution conditions are: gradient elution, and the elution program is:

[0127]

[0128] According to the attached Figure 6 From the detection spectrum, it can be seen that the separation degree between eptazocine preparation and eptazocine in the separation spectrum is greater than 1.5, the separation effect is good, the peak shape is symmetrical, the peak time is moderate, and the main peak purity angle is less than the purity threshold, which meets the requirements.

[0129] A new impurity in eptazocine preparations was detected by this detection method. This impurity could not be found when the TLC method was used before. Therefore, this new detection method can effectively enhance the quality control of eptazocine preparations and increase the safety of the drug. After research, it was determined that this new impurity was eptazocine glycine, which was an impurity produced by eptazocine and the excipient glycine under high temperature conditions.

[0130] In order to strictly control the product quality of etazocin preparations, the following research was conducted on this new impurity. After developing and designing the above-mentioned new detection method, a systematic methodological verification was carried out.

[0131] 1. Determination of detection wavelength

[0132] Take the test solution obtained in Example 2 and perform spectral analysis using a diode array detector (DAD detector). Figure 2 Etazocine DAD UV scan and Figure 3 The DAD UV scanning diagram of glycine eptazocine is shown in Figure 2. The results are shown in Table 2.

[0133] Table 2 Summary of DAD UV scans (wavelength: nm)

[0134] Element Maximum absorption wavelength (nm) Etazocine 280.8 Etazocine Glycine 286.44

[0135] Conclusion: The maximum absorption wavelengths of eptazocine glycine and eptazocine are both around 280nm, which is 6nm different from the detection wavelength of 280nm of the new test method. The detection wavelength of 280nm can be selected.

[0136] 2. Screening of detection conditions

[0137] The RT retention time of the compound eptazocine glycine is about 8 minutes, the RRT relative retention time is 0.5, and the separation degree from the adjacent impurities is greater than 1.5. The separation is good, the peak shape is good, and it does not affect the detection of known impurities, such as the impurity M 2 g, impurity Ze, impurity M 4 The RRTs of impurity Za, impurity Zf and methyl eptazocine were 0.60, 0.86, 1.09, 1.14, 1.68 and 1.75 respectively. This data adopts the RRT data of each impurity under the basic conditions in the specificity test. The new detection method can meet the detection requirements of the compound glycine eptazocine. Therefore, the determination method of the compound glycine eptazocine selected this new test method, and the methodological verification related to glycine eptazocine was carried out.

[0138] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A compound, characterized in that: The chemical structural formula of the compound is:

2. A method for preparing the compound according to claim 1, characterized in that: Dissolving etazocine and glycine in water for reaction, thus obtaining the compound; The reaction equation is as follows:

3. The preparation method according to claim 2, characterized in that: The reaction temperature of the reaction is greater than or equal to 0°C.

4. The preparation method according to claim 2, characterized in that: The reaction time of the reaction is greater than or equal to 5 hours.

5. The preparation method according to claim 3 or 4, characterized in that: The reaction temperature of the reaction is 40-400°C; the reaction time is 10-72h.

6. A method for detecting the compound according to claim 1, characterized in that: The compounds were determined by high performance liquid chromatography, and the chromatographic conditions were as follows: Chromatographic column: A chromatographic column with octadecylsilane bonded silica gel as filler is used; Mobile phase: composed of phase A and phase B, wherein the mobile phase A is glacial acetic acid aqueous solution, and the mobile phase B is methanol; Elution conditions: gradient elution or isocratic elution.

7. The detection method according to claim 6, characterized in that: The volume fraction of the glacial acetic acid aqueous solution is 1-3%; the pH value of the glacial acetic acid aqueous solution is 2-6.

8. The detection method according to claim 6, characterized in that: During isocratic elution, the volume ratio of mobile phase A to mobile phase B is 80-90:10-20; The elution program for gradient elution is:

9. Use of the compound according to claim 1 in the detection of impurity compounds in eptazocine preparations, characterized in that: The compound was used as a reference substance to detect the impurities and their contents in eptazocine preparations.

10. The use according to claim 9, characterized in that: The following steps are involved: (1) Prepare test solution; (2) Preparation of reference solution: (3) High performance liquid chromatography detection: Take the test sample solution and the reference sample solution and inject them into the liquid chromatograph for detection.

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