β-Lactam抗生素之研究： I. β-環糊精-高效能液相層析法對β-lactam抗生素的同時含量分析 II.氟喹諾酮-頭孢菌素衍生物的合成與生物活性評估

Study on the β-Lactam Antibiotics： I. Simultaneous determination of β-lactam antibiotics by β-cyclodextrin-HPLC II. Synthesis and bioactivities of fluoroquinolonyl- cephalosporin derivatives

鄒台黎(Tai-Li Tsou)

指導教授 ： 陳素惠

高雄醫學大學/藥學院/藥學研究所/博士(2009年)

摘要

本研究論文共有兩部分，第一部分是利用β-cyclodextrin-HPLC層析法測定製劑或生物檢品中β-lactam抗生素及β-lactamase抑制劑之含量及確效分析。藉由新的層析思維，本方法可改進原有傳統方法的限制及缺失。研究內容包括三項子題：

子題一：由篩選模式發現β-cyclodextrin-HPLC法中，流動相的醋酸四乙基銨鹽(tetraethyl ammonium acetate, TEAA)試劑、pH値、甲醇濃度等是影響β-lactam混合劑成分物分離的重要因子。並結合等高線法及後管柱層析技術可完成ampicillin、sulbactam、cefoperazone同時含量測定及確效分析。層析條件是：β-cyclobond管柱 (Cyclobond I, 250×4.6 mm, 5 mm)為固定相，甲醇–5 mM TEAA＝35：65 (v/v)，pH 4.5為流動相，流速0.8 ml/min，UV偵測波長280 nm。本方法簡單、精密、回收率佳，故可作為Unasyn、Sulperazone等混合製劑在無菌水、沙林溶液和5%葡萄糖注射溶液中之常規性檢測。

子題二：以β-cyclodextrin-HPLC法結合Doehlert的三中心點分析模式，完成製劑中ticarcillin、clavulanic acid等抗生物質之同時含量測定。層析條件是：β-cyclobond管柱為固定相，甲醇–16 mM AA＝50：50 (v/v)，pH 6.0為流動相，流速0.8 ml/min，及UV偵測波長220 nm。本方法之確效是以專一性、耐韌性、準確度、精密度及回收率進行評估。本法與美國藥典法相比，主要的優點在於能以較低流速(0.8 mL/min)、較短滯留時間(<14 min)、極佳的解析度達到改進目的。

子題三：以β-cyclodextrin-HPLC法結合Doehlert的三中心點分析模式，完成piperacillin、tazobactam抗生物質在血漿中同時分離、含量測定及確效分析。層析條件是：以β-cyclobond管柱為固定相，甲醇–12 mM TEAA＝50：50 (v/v)，pH 3.4為流動相，流速0.8 ml/min，UV偵測波長225 nm。與四種文獻方法比較，本法採用內孔徑複合吸附及等度流動相機制，一則不需要使用複雜的梯度流動相；二則採用低流速(0.8 mL/min)及適當酸值(pH 3.4)的條件，故可方便省時，並降低對層析管柱之損害。

本研究第二部分是利用半合成方法研製一系列Fluoroquinolonyl cephalosporin (FQ-cephalosporin)衍生物。實驗時，FQ基團上之N-1位置先進行烷基化反應。再將酸基以HONSu活化成縮酯化合物，並與cefaclor、cephalexin之C-11a位置上的胺基進行醯胺化偶合，以製得12種終產物。終產物經物理化學特性及UV、FT-IR、1H-NMR, 1H-1H COSY,、FAB-MS等圖譜資料，判定出正確之結構式。In vitro抗菌活性試驗結果顯示：(1) 化合物5xa、5xb的抗菌活性與範圍較佳，可保有對抗金黃色葡萄球菌、鏈球菌及ampicillin抗藥性菌株之活性；但6xa~6xf衍生物之抗菌活性均較起始藥物差。(2) C-11a胺基位置上之FQ基團取代，可擴增對抗綠膿桿菌菌株的能力。(3) C-3位置上之基團取代，其抗菌活性顯示Cl > CH3。(4) 5xa、5xb衍生物會使短桿菌無法分裂而不斷地生長延長。In vivo急毒性試驗顯示，化合物5xa會造成小白鼠肝腎毒性及血尿；但化合物5xb卻無類似之病變產生。

關鍵字

β-環糊精-高效能液相層析法 ； 安匹西林 ； 西孚黴素 ； 舒巴坦 ； 泰卡黴素 ； 克拉維酸 ； 必力黴素 ； 他挫巴坦 ； 氟喹諾酮基-頭孢菌素 ； 合成 ； 結構鑑定 ； 生物活性

並列摘要

The present study includes two parts. In the part I, a new β-cyclodextrin-HPLC method
(β-CyD-HPLC) has been developed for the simultaneous determination and validation of
β-lactam antibiotics and β-lactamase inhibitor in pharmaceutical products and biological
samples. There are three subjects described as following:
1. An accurate and reproducible method for the simultaneous determination of ampicillin
(AMP), sulbactam (SUL), and cefoperazone (CFP) in pharmaceutical formulations by using
HPLC with β-CyD stationary phase was developed. It involved the use of the added
tetraethylammonium acetate (TEAA) reagent, pH, and methanol as the significant parameters
and the isothermal curve technique to find the optimal chromatographic condition. A high
resolution and selectivity of analytes was obtained by running the mobile phase in methanol–5
mM TEAA buffer = 35:65 (v/v, pH 4.5) at 280 nm. This new HPLC method is simple, rapid,
accurate, reproducible, and time-consuming for the routine assay of these components in
sterilized H2O, saline, or 5% dextrose injection solutions.
2. A new β-CyD-HPLC method has been developed and validated for the simultaneous
determination of ticarcillin (TIC) and clavulanic acid (CA) in pharmaceutical formulations. In
experiment, using the Doehlert design to find the optimal chromatographic condition. The
HPLC separation was achieved on a β-CyD column (Cyclobond I, 250×4.6 mm, 5 mm) with
methanol–16 mM AA buffer=50:50 (v/v, pH 6.0) mobile phase at a flow rate of 0.8 mL/min.
Detection was at 220 nm. The specificity, robustness, accuracy, and precision were performed
to evaluate the method validation. Compared with the U.S. Pharmacopoeia method, the
proposed method has the advantage of relatively low flow rate (0.8 mol/min) and short analysis
time (<14 min) for routine assay.
3. A new β-CyD-HPLC method combined with Doehlert design has been developed for the
simultaneous determination of piperacillin (PIP) and tazobactam (TAZ) in human plasma.
Samples were pretreated with acetonitrile and dichloromethane. HPLC separation and
quantitation were achieved on a β-CyD column (Cyclobond I, 250×4.6 mm, 5 mm) with running
the mobile phase in methanol-12 mM TEAA buffer=50:50 (v/v, pH 3.4), UV detection at 225 nm,
and a flow-rate of 0.8 mL/min. Compared with the four published procedures, the proposed
method give a different chromatographic mechanism and isocratic mobile composition. It is
considered that the proposed method is improved for the determination of PIP/TAZ in human
biological samples by the lower flow rate (0.8 mL/min), acceptable pH value, and suitable
retention time (<16 min).
In the part II, a series of fluoroquinolonyl cephalosporin (FQ-cephalosporin) derivatives have
been prepared to improve the antibacterial therapy. In the synthetic processes, the FQ
moieties are firstly modified at N-1 position for decreasing its polarity. Then, each intermediate
is activated by HONSu and coupled with cefaclor or cephalexin at C-11??-amino group to give
FQ-cephalosporin derivatives. After separation, structures of products were identified by the
UV, FT-IR, 1H-NMR, 1H-1H cosy, and FAB-MS spectral analyses. These derivatives were
evaluated the in vitro antibacterial activity by the paper disk and MIC methods. The results
indicate some facts: (1) In comparison with the parent drugs, compounds 5xa and 5xb are
active against Gram-positive and Gram-negative bacteria; but all of 6xa~6xf derivatives are
inferior to the starting material. (2) The FQ moieties that substituted at the C-11?? amino group
of cefaclor are extended the activity against Pseudomonas aeruginosa strains. (3) The activity
of substituents at C-3 position is Cl > CH3. (4) Compounds 5xa and 5xb cause extensive
filamentation in cells of bacterial strains at the concentration near the half of MIC value. The in
vivo preliminary test for acute toxicity was identified in mice with the selected candidates. It
exhibites that compound 5xa causes the liver and kidney damage and blood urine; but 5xb
shows much lower acute intraperitioneal toxicity as the parent drugs.

並列關鍵字

β-Cyclodextrin-HPLC ； β-Lactam ； Ampicillin ； Cefoperazone ； Sulbactam ； Ticarcillin ； Clavulanic acid ； Piperacillin ； Tazobactam ； Fluoroquinolonyl cephalosporin ； Synthesis ； Structural identification ； Bioactivity

參考文獻

Part I：

Abdel-Moety EM, Abounassif MA, Mohamed ME, Khattab NA (1989). Spectrophotometric determination of amoxycillin and clavulanic acid in pharmaceutical preparation. Talanta, 36(6): 683-685.

Abdel-Moety EM, Abounassif MA, Gad-Kariem RA, Khattab NA (1993). Simultaneous determination of amoxicillin and dicloxacillin in capsules by potentiometric titrimetry and high-performance liquid chromatography. Talanta, 40(6): 811-817

Abounassif MA, Abdel-Moety EM, Mohamed ME, Gad-Kariem RA (1991). Liquid chromatographic determination of amoxicillin and clavulanic acid in pharmaceutical preparations. J. Pharm. Biomed. Anal., 9: 731-735

Adam D (2002). Global antibiotic resistance in Streptococcus pneumoniae. J. Antimicrob.Chemother., 50(Suppl): 1-5.