| Clarithromycin is one of drugs of the second generation of erythromycin and its good antibacterial properties and pharmacokinetic properties has been widespread concerned in medical. The impurities that had similar structure and properties to clarithromycin produced in clarithromycin industrial production are called relative substances. 16 kinds of relative substances were prescribed in European Pharmacopoeia and American pharmacopoeia while only total amount of impurities were controlled in Chinese Pharmacopoeia. It can be seen that there are still a large gap between Chinese and European Pharmacopoeia. The reason of this disparity is that relative substances sample libraries and databases haven't been established. Therefore, it has great significance to study the preparation and build the sample libraries and databases of the relative substances thus to improve the Chinese Pharmacopoeia standards of clarithromycin.6,11-Di-O-methylerythromycin A(EP-E) and 6,4''-Di-O-methylerythromycin A(EP-P)were synthesized and analysis method of erythromycin A(E)-9-oxime(EP-J), 3-Odecladinose-8,9,10,11-bisdehydration-6-O-methylerythromycin A 9,12-hemiketal(EP-K),3'-N-demethyl-6-O-methylerythromycin A(E)-9-oxime(EP-M) and 10,11-dehydration-6-O-methylerythromycin A(EP-N) were estsblished in this study. The multimethylated product of clarithromycin relative substances 6, 11-Di-O-methylerythromycin A(EP-E) was synthesized and the intermediates were separated and structures confirmed. In the synthesis of 6,11-Di-O-methylerythromycin A, clarithromycin was used as raw material, after silylated protection of 2'-OH and 4''-OH, methylation of 11-OH and deprotection of TMS groups, an overall yield of 75.8% and purity of 92.7% 6,11-Di-O-methylerythromycin A product was obtaind and its structure was confirmed by1H-NMR,13C-NMR and MS;temperature and time were important factors that affected the rate and efficiency of methylation; the reaction parameters waso ptimized and 1.5~2h in ice bath were selected as methylation conditions. In addotion, 6,4''-Di-O-methylerythromycin A was trying to be synthesized based on 2',4'-O-bis(trimethylsilyl)-6-O-methylerythromycin A 9-(1-ethoxy-1-methylethyl) oxime as raw material. 4''-OTMS was taken off in alkaline then through methylation and deprotection, the final products were mixture of 6,4''-Di-O-methylerythromycin A and 6,11,4''-Tri-O-methylerythromycin A and identified through 1H-NMR.Then HPLC analysis of 4 kinds of clarithromycin relative substances was studied. Acertain percentage of 4 kind of relative substances including erythromycin A(E)-9-oxime(EP-J), 3'-N-demethyl-6-O-methylerythromycin A(E) 9-oxime(EP-M), 10,11-dehydration-6-O-methylerythromycin A(EP-N) and 3-O-decladinosyl-8,9;10,11-bisdehydration-6-Omethylerythromycin A-9,12-hemiketal(EP-K) were mixed and detected by HPLC. The study was focused on the simple and convenient isocratic method and the analysis detection method of the mixture was optimized. 5 kinds of components were eluted within 25 min and completely separated(Degree of separation was greater than 1.2) with the mobile phase0.067 mol/L KH2PO4 solution(pH 4.0)/acetonitrile=60/40, a flow rate of 1.2 ml/min, the column temperature of 35 ? and the detection wavelength of 205 nm. The relevant peak area and sample concentration had a good linear relationship in the range of 20~240 mg/L;the peak area relative standard deviations of ??all components were less than 2.0% and met the accuracy requirement; the average recovery rates were between 87.80~121.17%and the relative standard deviations were less than 3.14% of all the relative substances. The method is easy, accurate and reliable to be used in simultaneously separation and detection of J, M, N and K in clarithromycin. |
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