Seven-membered heterocyclic compound or salt thereof, and preparation method and application thereof
阅读说明：本技术 一种七元杂环化合物或其盐、其制备方法及应用 (Seven-membered heterocyclic compound or salt thereof, and preparation method and application thereof ) 是由 刘潍源 周伟澄 林快乐 周亭 吕训磊 臧金鹏 王成成 于 2020-04-01 设计创作，主要内容包括：本发明公开了一种七元杂环化合物或其盐、其制备方法及应用。本发明提供了一种如式7a所示的七元杂环化合物或其盐。本发明还提供了如式7a所示的七元杂环化合物或其盐作为艾拉普林的杂质标准品的应用。通过本发明的如式7a所示的七元杂环化合物作为对照品,来建立艾拉普林质量控制的分析方法,从而可提高艾拉普林临床应用的安全性和有效性。(The invention discloses a seven-membered heterocyclic compound or a salt thereof, and a preparation method and application thereof. The invention provides a seven-membered heterocyclic compound shown as a formula 7a or a salt thereof. The invention also provides application of the seven-membered heterocyclic compound shown as the formula 7a or the salt thereof as an impurity standard substance of the elaprine. The analysis method for controlling the quality of the elaprine is established by using the seven-membered heterocyclic compound shown as the formula 7a as a reference substance, so that the safety and the effectiveness of the elaprine in clinical application can be improved.)
1. A seven-membered heterocyclic compound represented by the formula 7a or a salt thereof,
2. a process for the preparation of a seven-membered heterocyclic compound of formula 7a according to claim 1, comprising the steps of:
subjecting elaprine or a salt thereof containing the seven-membered heterocyclic compound represented by the formula 7a or a salt thereof as claimed in claim 1 to chromatographic separation; the chromatographic column for chromatographic separation is a reverse phase silica gel column; the mobile phase for chromatographic separation is potassium dihydrogen phosphate buffer solution and acetonitrile.
3. The method according to claim 2,
the chromatographic column for chromatographic separation is octadecylsilane chemically bonded silica;
and/or, the mobile phase of the chromatographic separation, mobile phase a: 2mmol/L potassium dihydrogen phosphate buffer solution; mobile phase B: acetonitrile;
and/or the detection wavelength of the chromatographic separation is 220 nm;
and/or the temperature of the chromatographic separation column is 25-40 ℃;
and/or the flow rate of the chromatographic separation is 0.1 mL/min-2.0 mL/min;
and/or the sample injection volume of the chromatographic separation is 1-120 mu L.
4. The method according to claim 3, wherein the chromatographic column is YMC-pack ODS-AQ, 150X 3.0mm,3 μm silica;
and/or, the mobile phase A and the mobile phase B are subjected to gradient elution according to the following proportion:
the percentage is volume percentage accounting for the total volume;
and/or the temperature of the chromatographic separation column is 30-40 ℃;
and/or the flow rate of the chromatographic separation is 0.6mL/min to 1.0 mL/min.
5. Use of the seven-membered heterocyclic compound represented by the formula 7a or a salt thereof according to claim 1 as an impurity standard for elaprine or a salt thereof.
6. A bulk drug of elaprine or its salt, characterized in that it contains 0.01-1.0% of the heptatomic heterocyclic compound or its salt shown in formula 7a in claim 1.
7. The use of claim 6, wherein said percentage is determined by HPLC using a reverse phase silica gel column as the HPLC column; the mobile phase for chromatographic separation is potassium dihydrogen phosphate buffer solution and acetonitrile.
8. The method according to claim 7,
the chromatographic column for chromatographic separation is octadecylsilane chemically bonded silica;
and/or, the mobile phase of the chromatographic separation, mobile phase a: 2mmol/L potassium dihydrogen phosphate buffer solution; mobile phase B: acetonitrile;
and/or the detection wavelength of the chromatographic separation is 220 nm;
and/or the temperature of the chromatographic separation column is 25-40 ℃;
and/or the flow rate of the chromatographic separation is 0.1 mL/min-2.0 mL/min;
and/or the sample injection volume of the chromatographic separation is 1-120 mu L.
9. The method according to claim 8, wherein the chromatographic column is YMC-pack ODS-AQ, 150X 3.0mm,3 μm silica;
and/or, the mobile phase A and the mobile phase B are subjected to gradient elution according to the following proportion:
the percentage is volume percentage accounting for the total volume;
and/or the temperature of the chromatographic separation column is 30-40 ℃;
and/or the flow rate of the chromatographic separation is 0.6mL/min to 1.0 mL/min.
10. A preparation of elaprine or a salt thereof, which comprises the raw material drug of elaprine or a salt thereof as claimed in claim 6 and a pharmaceutical excipient.
Technical Field
The invention relates to a seven-membered heterocyclic compound or a salt thereof, and a preparation method and application thereof.
Background
Elaprine (Iclaprim) is a dihydrofolate reductase inhibitor developed by Motif Bio corporation, which was marketed to the us FDA as a therapeutic agent for the indication of acute bacterial skin and skin structure infections (abssi) in 2018. Iclaprim is currently in phase II clinical trials as a drug for the treatment of hospital-acquired bacterial pneumonia (HABP). In addition, staphylococcus aureus lung infections are currently in preclinical development as a drug for the treatment of cystic fibrosis patients. In regulatory terms, Iclaprim has been awarded by the FDA in the united states for qualified infectious disease product Qualification (QIDP) and rapid passage. In addition, the FDA has also awarded Iclaprim the orphan drug status for the treatment of staphylococcus aureus lung infections in cystic fibrosis patients. The chemical name is as follows: 5- [ (2-cyclopropyl-7, 8-dimethoxy-2H-1-benzopyran-5-yl) methyl ] -2, 4-pyrimidine-diamine, the structural formula of which is shown as compound 7:
the invention patent CN2019111264400 provides an elaprine synthesis method:
in the optimization of the HPLC purity detection method for the elaprine synthesis method reported in the invention patent CN2019111264400, a new impurity peak with higher content is found.
It is known to those skilled in the art that the research of related substances is a very important step in the development of drugs.
Further isolation and structural identification were not performed, considering that the impurity was not found in CN 2019111264400. In order to better study and control the quality of Iclaprim, it is necessary to confirm the structure of the relevant substances generated in the production process and prepare the relevant substances to obtain corresponding standard substances, so that the relevant substances are normatively studied and controlled within a reasonable limit range, which is not only beneficial to understanding of the synthesis process of the raw material medicines, study of the product quality, and establishment of quality standards of raw materials, auxiliary materials, intermediates and finished products, but also directly related to the quality and safety of Iclaprim.
Disclosure of Invention
The invention provides a seven-membered heterocyclic compound or salt thereof, a preparation method and application thereof, aiming at overcoming the defects of uncertain impurity source and structure and lack of preparation method in the elaprine product in the prior art; the synthetic method of the seven-membered heterocyclic compound provided by the invention has the advantages of simple steps, mild reaction conditions and strong operability, and a product with higher purity can be obtained through simple purification; the Icalaprim derivative can be used as an Icalaprim related substance, is beneficial to Icalaprim process research and quality standard formulation, effectively reduces research and development cost, and has important significance for drug development.
The present invention solves the above-mentioned problems by the following technical means.
The invention provides a seven-membered heterocyclic compound shown as a formula 7a or salt thereof,
the invention also provides a preparation method of the seven-membered heterocyclic compound shown in the formula 7a, which comprises the following steps: subjecting the elaprine or salt thereof containing the seven-membered heterocyclic compound or salt thereof shown in the formula 7a to chromatographic separation; the chromatographic column for chromatographic separation is a reverse phase silica gel column; the mobile phase for chromatographic separation is potassium dihydrogen phosphate buffer solution and acetonitrile.
The chromatographic column for chromatographic separation may be a reverse phase silica gel column conventional in the art, such as octadecylsilane bonded silica; preferably, the model is YMC-pack ODS-AQ, 150X 3.0mm,3 μm silica.
The mobile phase of the chromatographic separation, preferably mobile phase a: 2mmol/L potassium dihydrogen phosphate buffer solution; mobile phase B: acetonitrile;
more preferably, said mobile phase a and said mobile phase B are eluted with a linear gradient according to the following table;
time (minutes)
Phase A (%)
Phase B (%)
0
95
5
5
95
5
6
75
25
14
75
25
15
95
5
25
95
5
The percentage is volume percentage of the total volume.
In the preparation method, other chromatographic conditions except for the mobile phase and the chromatographic column can be the chromatographic conditions conventional in the field, and the following chromatographic conditions are preferred in the invention: the detection wavelength is 220 nm; the column temperature is 25-40 ℃ (for example, 30-40 ℃); the flow rate is 0.1mL/min to 2.0mL/min (preferably 0.6mL/min to 1.0 mL/min); the injection volume is 1-120 mu L (preferably 3-10 mu L).
The elaprine or salt thereof may be prepared by methods conventional in the art, for example according to the method of CN 2019111264400.
The invention also provides an application of the seven-membered heterocyclic compound shown in the formula 7a or the salt thereof as an impurity standard substance of the elaprine or the salt thereof.
The invention also provides a raw material drug of the elaprine or the salt thereof, which contains the seven-membered heterocyclic compound shown in the formula 7a or the salt thereof (such as 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8% and 0.9%) in percentage of 0.01-1.0%. The content percentage is determined by HPLC and is calculated by peak area.
The HPLC detection method specifically comprises the following steps: the detection wavelength is 220 nm; the column temperature is 25-40 ℃ (for example, 30-40 ℃); the flow rate is 0.1mL/min to 2.0mL/min (preferably 0.6mL/min to 1.0 mL/min); the injection volume is 1-120 mu L (preferably 3-10 mu L). The column model was YMC-pack ODS-AQ 150X 3.0mm,3 μm silica.
Mobile phase A: 2mmol/L potassium dihydrogen phosphate buffer solution; mobile phase B: acetonitrile; more preferably, the mobile phase A and the mobile phase B are subjected to gradient elution in the following ratio:
time (minutes)
Phase A (%)
Phase B (%)
0
95
5
5
95
5
6
75
25
14
75
25
15
95
5
25
95
5
The percentage is volume percentage of the total volume.
The invention also provides a preparation of the elaprine or the salt thereof, which comprises the bulk drug and the pharmaceutical excipients of the elaprine or the salt thereof.
The salt of the "elaprine or salt thereof" is the same as the salt of the "seven-membered heterocyclic compound represented by the formula 7a or salt thereof", and examples thereof include trifluoroacetic acid, p-toluenesulfonic acid and sulfuric acid.
The term "salt" denotes a salt formed from a suitable organic acid, inorganic acid, organic base or inorganic base and a compound. The organic acid may be any of various organic acids capable of forming a salt, which are conventional in the art, such as one or more of methanesulfonic acid, p-toluenesulfonic acid, maleic acid, fumaric acid, citric acid, tartaric acid, malic acid, lactic acid, formic acid, acetic acid, propionic acid, trifluoroacetic acid, oxalic acid, succinic acid, benzoic acid, isethionic acid, naphthalenesulfonic acid, and salicylic acid. The inorganic acid may be any of various inorganic acids capable of forming a salt, such as one or more of hydrochloric acid, sulfuric acid, and phosphoric acid, which are conventional in the art. Preferably one or more of trifluoroacetic acid, p-toluenesulfonic acid monohydrate and sulfuric acid.
The term "drug substance" means a substance consisting of the main pharmaceutical active ingredient, as well as impurities in controlled amounts.
The term "preparation" denotes a particular variety of pharmaceutical application forms (dosage forms) consisting of the main pharmaceutical active ingredient, as well as impurities and/or adjuvants of controlled content, prepared to meet the needs of clinical treatment or prevention according to the criteria approved by pharmacopoeia or the drug administration.
The term "impurity" means any substance that affects the purity of a drug. The impurities are generally classified into three categories according to their physicochemical properties: organic impurities, inorganic impurities and residual solvents. Depending on their origin, impurities can be classified into process impurities (including reactants and reagents, intermediates, by-products, etc., which are not completely reacted in the synthesis), degradation products, impurities mixed from reactants and reagents, and the like.
The above preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention without departing from the common general knowledge in the art.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows: by the reference substance of the elaprine heptatomic ring impurity, an analysis method for controlling the quality of elaprine can be established, so that the safety and the effectiveness of the elaprine in clinical application can be improved.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
EXAMPLE 1 preparation of (5- [ (2-cyclopropyl-7, 8-dimethoxy-2H-1-benzopyran-5-yl) methyl ] -2, 4-pyrimidine-diamine)
Step (1) (preparation of N, N' - (5- (2-acetyl-3, 4, 5-trimethoxybenzyl) pyrimidine-2, 4-diyl) diethylamide)
Adding compound 1 (trimethoprim) (10.02g, 34.52mmol), acetic anhydride (17.06g, 167.11mmol) and 100ml chloroform into a 250ml reaction bottle, adding tin tetrachloride (8.00ml, 68.36mmol) under stirring, carrying out reflux reaction for 1h, detecting that raw materials basically disappear by TLC, cooling to room temperature, pouring the reaction solution into 50ml ice water, stirring for 6mins, carrying out liquid separation, washing an organic phase for 3 times by 5ml water, combining aqueous phases, extracting the aqueous phase for 3 times by 5ml chloroform, combining the organic phases, adjusting the pH of saturated sodium carbonate aqueous solution to 7-8, carrying out liquid separation, washing the organic phase for 1 time by 5ml water, drying with anhydrous sodium sulfate, filtering, carrying out vacuum concentration, and recrystallizing ethylene glycol monomethyl ether to obtain 13.30g of a product, wherein the yield is 92.63%. mp.203-205 ℃; purity by HPLC was 96.72%.1H-NMR(300MHz,CDCl3)δ(ppm):10.03(s,1H),9.20(s,1H),8.41,(s,1H),6.34(s,1H),3.95(s,3H),3.84(s,3H),3.76(s,2H),3.68(s,3H),2.60(s,3H),2.48(s,3H),2.19(s,3H)；MS(ESI+):m/z,417([M+H]+).
Step (2) preparation of (1- (6- ((2, 4-diaminopyrimidin-5-yl) methyl) -2,3, 4-trimethoxyphenyl) ethanone)
To a reaction flask were added compound 2(65.00g, 156.25mmol), potassium carbonate (15.090g, 109.35mmol) and 600ml methanol, stirred at reflux for 1.5h, and the addition was stoppedHeating, cooling to room temperature, placing in an ice bath, stirring slowly, stirring, crystallizing, filtering, washing with water, and drying to obtain 47.00g of white solid compound 3 with yield of 90.5% and mp.121-123 deg.C; purity by HPLC was 97.22%.1H-NMR(400MHz,CDCl3)δ(ppm):7.28(s,1H),6.66(s,1H),6.12,(s,2H),5.70(s,2H),3.82(s,3H),3.75(d,6H,4Hz),3.42(s,2H),2.29(s,3H).
Step (3) preparation of (1- (6- ((2, 4-diaminopyrimidin-5-yl) methyl) -2-hydroxy-3, 4-dimethoxyphenyl) ethanone)
Adding the compound 3(30.00g, 90.36mmol) and 600ml of dichloromethane into a 1L reaction bottle, cooling to-6 ℃ in an ice salt bath, slowly dropwise adding 135.5ml of boron tribromide dichloromethane solution of 1mol/L, and raising the temperature to room temperature for reaction for 5 h. Cooling to 0 deg.C, quenching with 300ml methanol, stirring for 1h, vacuum concentrating the dry solvent, and recrystallizing with ethanol to obtain 25.50g white solid compound 4 with yield 88.74%. mp.217 ℃; purity was 96.10% by HPLC.1H-NMR(300MHz,CDCl3)δ(ppm):9.66(s,1H),8.31(s,1H),7.91(s,1H),7.54(s,2H),7.14(s,1H),6.46(s,1H),3.77(s,3H),3.69(s,3H),3.48(s,2H),2.42(s,3H)；MS(ESI+):m/z,319([M+H]+).
Step (4) preparation of (2-cyclopropyl-5- ((2, 4-diaminopyrimidin-5-yl) methyl) -7, 8-dimethoxychroman-4-one)
Compound 4(4.01g, 12.58mmol), cyclopropanecarboxaldehyde (1.07g, 15.29mmol) and 40ml acetonitrile were added to a 100ml reaction flask, and pyrrolidine (1.34g, 18.87mol) and acetic acid (1.13g, 18.83mmol) were slowly added dropwise with stirring, stirred at room temperature for 36h, filtered with suction and dried to give 4.57g of acetate as a white-like solid compound 5, yield 84.28%, mp168-171 ℃.1H-NMR(400MHz,CDCl3)δ(ppm):7.13(s,1H),6.49(s,1H),6.31(s,2H),5.92(s,2H),3.89-4.00(m,2H),3.78-3.86(m,4H),3.71(s,3H),2.78-2.85(m,1H),2.64-2.69(m,1H),1.89(s,3H),1.19-1.25(m,1H)0.53-0.63(m,2H),0.45-0.51(m,1H),0.37-0.43(m,1H)；MS(ESI+):m/z,373([M+H]+).
Step (5) preparation of (2-cyclopropyl-5- ((2, 4-diaminopyrimidin-5-yl) methyl) -7, 8-dimethoxychroman-4-ol)
Adding the compound 5(4.00g, 10.81mmol) and 80ml of methanol into a reaction bottle, cooling to 4 ℃ in an ice bath, adding sodium borohydride (0.21g, 5.56mmol), reacting at room temperature for 2.5h, evaporating the methanol to dryness, adding 40ml of water, stirring for 10mins, filtering, adding methanol into a filter cake, refluxing for 3h, concentrating in vacuum, and recrystallizing ethanol and water (the volume ratio of ethanol to water is 5:1) to obtain 3.88g of a white solid compound 6, wherein the yield is 96.48%. mp.211-213 deg.C; the purity of the product is 99.23% by HPLC detection;1H-NMR(400MHz,CDCl3).δ(ppm):7.49(s,1H),6.17-6.2(d,3H),5.58-5.64(m,3H),4.93-4.94(d,1H),3.78-3.82(d,1H),3.63(s,3H),3.62(s,3H),3.53-3.57(m,1H),2.24-2.28(m,1H),1.96-2.02(m,1H),1.34-1.37(m,1H)，0.49-0.1(d,2H),0.31-0.36(m,2H)；MS(ESI+):m/z,373([M+H]+)。
step (6) (5- [ (2-cyclopropyl-7, 8-dimethoxy-2H-1-benzopyran-5-yl) methyl ] -2, 4-pyrimidine-diamine) preparation
Adding compound 6(2.01g, 5.40mmol, HPLC purity 99.23%) and 20ml tetrahydrofuran into a reaction bottle, adding p-toluenesulfonic acid monohydrate (1.54g, 8.10mmol) under stirring, heating and refluxing for 1h, detecting by TLC that no raw material is remained basically, cooling to room temperature, precipitating a large amount of white solid, filtering, and drying to obtain 2.54g of p-toluenesulfonic acid salt of compound 7 as a white solid with yield of 89.27%; mp.208 ℃; the purity of the product is 99.91% by HPLC detection;1H-NMR(400MHz,CDCl3).δ(ppm):8.06(b,2H),7.45-7.49(m,3H),7.10-7.12(d,2H),6.85(s,1H),6.47(s,1H),6.39-6.42(m,1H),5.75-5.78(m,1H),4.24-4.27(m,1H),3.74(s,3H),-3.72(s,3H),3.57(b,2H),2.28(s,3H),1.12-1.20(m,1H),0.29-0.54(m,4H)，MS(ESI+):m/z,355([M+H]+). P-toluenesulfonate (2.54g, 4.83mmol) of the compound 7 was stirred in a saturated sodium carbonate solution for 1 hour, suction filtered, and dried to give 1.71g of the compound 7 as a white solid in a total yield of 89.40%. mp.215 ℃. The purity of the product is 99.96% by HPLC detection;1H-NMR(400MHz,CDCl3).δ(ppm):7.07(s,1H),6.45-6.46(d,1H),6.42(s,1H),6.19(s,2H),5.70-5.72(m,1H),5.68(s,2H),4.24-4.26(m,1H),3.70(s,3H),3.71(s,3H),3.52(d,2H),1.11-1.15(m,1H),0.43-0.51(m,2H),0.35-0.39(m,1H),0.30-0.33(m,1H)；MS(ESI+):m/z,355([M+H]+)。
the HPLC assay described above was carried out using method 1: the high performance liquid chromatography column is YMC-pack ODS-AQ (150X 3.0mm,3 μm silica); taking 0.1% formic acid buffer solution as a mobile phase A and acetonitrile as a mobile phase B, and carrying out linear gradient elution according to the following table; the detection wavelength is 220 nm; flow rate: 0.6 mL/min; sample introduction volume: 3-10 μ L; the column temperature was 40 ℃.
Time (minutes)
Phase A (%)
Phase B (%)
0
95
5
5
95
5
6
75
25
16
75
25
17
95
5
27
95
5
The percentage is volume percentage of the total volume.
Table 1 the HPLC test results are shown in the following table:
our investigators found a new higher impurity peak in the optimization of the HPLC analytical method for the Iclaprim product described above.
The specific HPLC detection adopts the following method 2: the high performance liquid chromatography column is YMC-pack ODS-AQ (150X 3.0mm,3 μm silica); taking 2mmol/L potassium dihydrogen phosphate buffer solution as a mobile phase A and acetonitrile as a mobile phase B, and performing linear gradient elution according to the following table; the detection wavelength is 220 nm; flow rate: 0.6 mL/min; sample introduction volume: 3-10 μ L; the column temperature was 40 ℃.
Time (minutes)
Phase A (%)
Phase B (%)
0
95
5
5
95
5
6
75
25
14
75
25
15
95
5
25
95
5
The percentage is volume percentage of the total volume.
Table 2 the optimized HPLC assay results are shown in the following table:
the peak time of the impurity 7a is 13.009mins, and the peak time of Iclairm is 12.212 mins. The time to peak of impurity 7a relative to drug Iclaprim was 1.07. The liquid phase method can effectively separate the drug from the impurities.
In view of the fact that the content of the above-mentioned impurity 7a in the Iclaprim product is high by the above-mentioned newly developed HPLC method, the content of the impurity exceeds the standards stipulated by pharmacopoeia, the quality of the Iclaprim product is seriously affected, and a great risk is brought to safe administration of patients.
As known to those skilled in the art, in the process of drug development, research on related substances is a very important link, and in order to better perform quality research and control on Iclaprim, it is very necessary to confirm related substances generated in the production process and research the preparation methods of the related substances to obtain corresponding standard substances, so that the related substances are researched normatively and controlled within a reasonable limit range, which is not only beneficial to understanding of the synthesis process of raw material drugs, research on product quality, and establishment of quality standards of raw and auxiliary materials, intermediates and finished products, but also more directly related to quality and safety of Iclaprim.
Therefore, the inventor uses the optimized HPLC conditions to perform enrichment separation through a preparative chromatographic column, and researches the structure confirmation of the impurity.
The structure identification data are as follows: mp.202-204 ℃; MS (ESI +): M/z,355([ M + H ]]+)。
1H-NMR(600MHz,DMSO-d6).δ(ppm):7.51(s,1H),6.90(s,1H),6.52(s,1H),5.55(s,2H),5.06-5.05(d,1H),4.40-4.38(d,1H),3.74(s,3H),3.63(s,3H),3.32-3.30(m,1H),3.16-3.13(d,2H),2.27-2.24(d,1H),1.87-1.82(m,1H),1.10-1.03(m,1H),0.63-0.58(m,2H),0.52-0.48(m,1H),0.46-0.42(m,1H)；13C NMR(100MHz,DMSO-d6)δ162.05,161.73,155.43,151.87,147.58,135.81,135.16,115.35,103.96,103.71,75.10,59.90,55.81,42.87,33.44,32.22,15.06,3.40,1.25.
From the above analytical data, the chemical structure of the impurity was determined to be a compound represented by the following formula 7 a:
however, no related research on the impurities exists at present, and a standard substance is lacked, so that the pharmacological and toxicological effects of the impurities cannot be further researched, and the content of the impurities in the product and a detection method are also lacked with more accurate and reasonable standards. Therefore, in order to ensure safety and effectiveness of Iclaprim, i have conducted studies on synthesis of the above-mentioned impurity 7a according to the above-mentioned structure. The details are shown in the following examples.
Example 2
Adding compound 6(1.69g, 4.54mmol) and 20ml tetrahydrofuran into a reaction bottle, adding trifluoroacetic acid (1.56g, 13.68mmol) under stirring, heating and refluxing for 15h, detecting by TLC that no raw material is left basically, adjusting the pH value of saturated sodium carbonate to about 7, evaporating tetrahydrofuran, adjusting the pH value of an organic phase to 10 by using a saturated sodium carbonate solution, stirring for 1h, carrying out suction filtration, and drying to obtain 1.59g of compound 7, wherein the yield of a crude product is 98.87%. Compound 7 was found (as in example 1) to be 97.01% pure by HPLC, wherein HPLC showed the impurity level to be 2.10% and we isolated 0.021g by preparative column enrichment.
The structure identification data are as follows:
mp.202-204℃；1H-NMR(600MHz,DMSO-d6).δ(ppm):7.50(s,1H),6.91(s,1H),6.53(s,1H),5.56(s,2H),5.05-5.04(d,1H),4.41-4.39(d,1H),3.73(s,3H),3.64(s,3H),3.33-3.30(m,1H),3.17-3.13(d,2H),2.26-2.23(d,1H),1.86-1.82(m,1H),1.11-1.03(m,1H),0.64-0.59(m,2H),0.52-0.48(m,1H),0.45-0.41(m,1H)；MS(ESI+):m/z,355([M+H]+)。
on this basis, further validation by HPLC (HPLC method 2) was performed.
Table 3 HPLC result data for 7a prepared in example 2
It can be seen that the peak time is also the same as in example 1 above.
Example 3
Adding the compound 6(2.00g, 5.38mmol) and 20ml of tetrahydrofuran into a reaction bottle, adding p-toluenesulfonic acid monohydrate (1.54g, 8.10mmol) under stirring, heating and refluxing for 1h, detecting by TLC that almost no raw material remains, cooling to room temperature, adjusting the pH to about 7 with saturated sodium carbonate, evaporating off tetrahydrofuran, adjusting the pH to 10 with saturated sodium carbonate solution, stirring for 1h, filtering, and drying to obtain 1.81g of crude compound 7 with the total yield of 95.10%. Compound 7 was 98.25% pure by HPLC (HPLC method 2) and 1.02% impurity 7 a.
The analytical identification data (including the time to peak HPLC) were the same as in example 1 above.
Example 4:
adding compound 6(2.00g, 5.38mmol) and 20ml tetrahydrofuran into a reaction bottle, adding concentrated sulfuric acid (0.63g, 6.43mmol) while stirring, heating and refluxing for 4h, cooling to room temperature, adjusting the pH of saturated sodium carbonate solution to about 7, evaporating the solvent, adjusting the pH of saturated sodium carbonate solution to about 10, stirring for 1h, filtering, and drying to obtain 1.88g of white solid compound 7 with the total yield of 98.78%. mp.215 ℃; HPLC assay (HPLC method 2) compound 7 content 97.10% and impurity 7a content 1.53%.
The analytical identification data (including the time to peak HPLC) were the same as in example 1 above.
Therefore, the impurity 7a can be prepared by adopting the chromatographic method and can be used as an impurity standard substance, the quality of the elaprine is further improved, the safety and the effectiveness of the elaprine are really ensured, and the use risk is reduced.