型小分子類肽氨肽酶N抑制劑的設(shè)計、合成與初步活性評價 精靈論

1、.新型小分子類肽氨肽酶 N 抑制劑的設(shè)計、合 成與初步活性評價李荀（山東大學(xué)藥學(xué)院，濟南 250012）摘要：目的設(shè)計合成 L-異谷氨酰胺類小分子類肽化合物，體外篩選其氨肽酶 N (APN)及 MMP-2 的抑制活性，研究構(gòu)-效關(guān)系。方法通過“拼接原理”，經(jīng)縮合等反應(yīng)合成小分子類肽 衍生物，采用體外抑酶活性實驗測定目標(biāo)化合物抑制 APN 與 MMP-2 的活性。結(jié)果合成了22 個未見文獻報道的化合物，結(jié)構(gòu)經(jīng)紅外光譜、核磁共振氫譜、質(zhì)譜確證。結(jié)論這類化合 物的氨肽酶 N 抑制活性顯著高于 MMP-2，為選擇性較高的氨肽酶 N 抑制劑。其中，化合物8 (IC50 = 10.2 0.9 M)的活性顯

2、著高于陽性對照藥 Bestatin (IC50 = 13.1 0.7 M),有望 成為先導(dǎo)物做進一步的研究。關(guān)鍵詞： 藥物化學(xué)；APN/CD13; 抑制劑; Peptidomimetic; AnticancerDesign, Synthesis and Preliminary Activity Evaluation ofNovel Peptidomimetics as Aminopeptidase N/CD13InhibitorsLi Xun(College of Pharmacy, Shandong University, Jinan250012)Abstract: Aim To desig

3、n and synthesize novel peptidomimetic analogues evaluated their inhibitory activities against aminopeptidase N (APN/CD13) and matrix metalloproteinase-2 (MMP-2). Methods According to the “combination principles”, peptidomimetic analogues were synthesized through condensation reaction, and their enzy

4、matic inhibitory activities were assayed in vitro. Results 22 peptidomimetic APN inhibitors were prepared which have not been reported in literatures. Their structures were confirmed by IR, 1H-NMR, and MS. Conclusion Most of the compounds displayedselective inhibition against APN as compared with MM

5、P-2, with IC50 values in micromole range. Within this series, compound 8 (IC50 = 10.2 0.9 M) demonstrated comparable APN inhibitory activities as compared with the positive control bestatin (IC50 = 13.1 0.7 M), which may providea promising lead for further molecular optimizations.Key words: Medicina

6、l Chemistry; APN/CD13; inhibitor; Peptidomimetic; Anticancer0引言氨肽酶 N (APN, EC 3.4.11.2)是一種含鋅離子的膜結(jié)合型外肽酶, 已經(jīng)證明其在腫瘤細(xì) 胞表面大量表達，對腫瘤的侵襲和血管生成有重要的作用1,2。此外，它還能夠表達于抗原 遞呈細(xì)胞表面，降解多種免疫活性物質(zhì)，使機體免疫力下降，削弱巨噬細(xì)胞和 NK 細(xì)胞對腫 瘤細(xì)胞的識別和殺傷能力3,4。該酶還可降解細(xì)胞外基質(zhì)（ECM）的主要成分，促進腫瘤細(xì) 胞的生長和轉(zhuǎn)移5,6。因此，抑制該酶的活性就可對腫瘤的侵襲和轉(zhuǎn)移以及血管生成進行有 效地控制，同時增強粒細(xì)胞的趨化性

7、，提高機體的免疫能力，進而可能成為有效的抗癌或抗 炎藥物7-9。截至目前，已經(jīng)有多種 APN 抑制劑類藥物進入臨床10，其中，Bestatin 作為抗 白血病的治療藥物已經(jīng)在日本上市多年11，本實驗將采用其作為陽性對照12?；痦椖浚翰┦奎c青年基金（20070422061）；山東省自然基金（Y2008C01）；山東大學(xué)自主創(chuàng)新基金(2009TS113)作者簡介：李荀（1976.7），女，副教授，抗腫瘤藥物的設(shè)計合成. E-mail: tjulx2004.;傳統(tǒng)的以酶為靶點的肽類化合物有很多缺點，例如對蛋白水解酶不穩(wěn)定、生物利用度低、 作用時間短而排泄快等。因而目前大多數(shù)報道的 APN 抑制劑采

8、用了模擬肽（peptidemimics） 的結(jié)構(gòu)。它是一種利用人工合成的物質(zhì)來模擬天然多肽的結(jié)構(gòu)，或者利用構(gòu)象型模板誘導(dǎo)相 鄰的多肽序列而形成的具特異性結(jié)構(gòu)的化合物。因此，設(shè)計合成模擬肽類化合物是發(fā)現(xiàn)酶抑 制劑類藥物的理想策略13,14。本文根據(jù) APN 三維結(jié)構(gòu)特征，結(jié)合計算機輔助藥物設(shè)計，通過藥物設(shè)計中的“拼接原 理”，將具有抗腫瘤活性的沒食子酸和天然 L-異谷氨酸胺利用縮合反應(yīng)進行對接定向合成， 再引入各種天然氨基酸片段，合成了 22 個未見文獻報道的新型小分子類肽化合物，合成路 線見圖 1。該法以 DCC 為縮合劑，反應(yīng)條件溫和、操作簡單。1合成熔點用 X-4 型數(shù)字顯微熔點儀測定，溫

9、度未校正。IR 譜采用 FTIT-8400 型紅外分光光 度計測定，KBr 壓片。1H-NMR 譜采用 Bruker400 型核磁共振譜儀測定，DMSO-d6 為溶劑， TMS 為內(nèi)標(biāo)。生物活性測定用試劑如 APN，MMP-2，L-亮氨酰對硝基苯胺均購自 Sigma 公 司，使用前超低溫冷藏保存?zhèn)溆?。實驗中其余試劑均為市售分析純或化學(xué)純，薄層色譜用硅 膠 GF254 由青島海洋生產(chǎn)。OHOOHH3COOOHH3COOClH3COOCOOHNCOOHabcHHOH3COOHOCH3H3COOCH3H3COOCH31234COOH3 O OH3COOdN1HOH3COH3COeH3COOR1NR2

10、NHOCOOCH3OCH3various nucleophilic agentsOCH356a-r, 7HO N COOH OH H H OH3CO H3CON HOCH3N R2 fO COOCH3H3CO H3CON HOCH3N OH NH O R26a R2=H6b R2=CH38 R2=H9 R2=CH3圖 1 目標(biāo)化合物的反應(yīng)路線示意圖Fig. 1 Reaction route of target compounds: a) Me2SO4, 40 % NaOH, then 1 N HCl. b) SOCl2 in benzene. c)L-Glu-Na, Na2CO3, then

11、2N HCl. d) Ac2O, 5560 °C. e) L-amino acid methyl ester hydrochloride, anhydrousCH2Cl2, Et3N, rt. f) NH2OK in anhydrous CH3OH, then acetic acid (two steps).1.1 3,4,5-三甲氧基苯甲酸(2)的制備室溫下，將沒食子酸(1, 5g, 29.4 mmol)溶于 50 mL 4 N NaOH 溶液中，滴加 (CH3)2SO4 (8.9 g, 71 mmol)，在 30-40 °C 繼續(xù)反應(yīng) 20 分鐘, 繼續(xù)加入沒食子酸(5

12、 克) 的 50 mL 4 N NaOH 溶液。將此溶液緩慢升溫至 90 °C 反應(yīng) 1 h 后回流 2h，冷卻至室溫，用 2 N HCl 酸化至 pH 2, 過濾,用蒸餾水洗滌沉淀。粗品再用 50% EtOH 重結(jié)晶的白色針狀晶體的目標(biāo)化合 物 2 (40.8 g, 65%)：熔點 168-171 °C. 1H NMR 3.97 (s, 9H), 7.23 (s, 2H), 12.11 (s, 1H).1.23,4,5-三甲氧基苯甲酰氯(3)將上述化合物 2 (21.2 g, 100 mmol)溶于 320 mL 苯中，逐滴滴加 SOCl2 (40 mL, 548 mmo

13、l)，回流反應(yīng) 3 h, 減壓蒸除溶劑得到黃色油狀物 3，繼續(xù)加入 50 mL 苯后減壓整除過量 的 SOCl2，所得的粗品不經(jīng)純化直接用于下一步反應(yīng)中。1.3(R)-2-(3,4,5-三甲氧基苯甲酰氨基)戊二酸(4)-5 °C 下，將無水 Na2CO3 (19.6 g, 185 mmol)、L-谷氨酸(16.9 g, 115 mmol) 溶于 150 mL 蒸餾水中，逐滴加入 化合物 3 的 200 mL 苯溶液， 1 h 滴加完畢。反應(yīng)約 5 小時后，用 2 N HCl 酸化至 pH 5-6, 水相用 2 N HCl 酸化至 pH 3-4, 在冰箱中將有機相（氯仿）放置過夜， 將

14、析出的白色固體用蒸餾水洗滌三次，干燥后的目標(biāo)化合物 4 (28.6 g, 89.3%)。熔點 120-121°C, IR (KBr, cm-1): 3299.7 & 3255.4 (NH), 2942.5 (CH), 1744.7 & 1699.5 (C=O), 1500.7 ( NH),1235.5 & 1127.7 (C-O). 1H NMR (DMSO-d6, ppm): 12.41 (s, 2H, 2-COOH), 8.53 (d, 1H, J =7.2 Hz, NH), 7.22 (s, 2H, Ar-H), 4.41 (m, 1H, CH), 3.

15、83 (s, 6H, 2-OCH3), 3.71 (s, 3H, OCH3),2.35 (m, 2H, CH2), 2.21 (m, 1H, CH), 1.96 (m, 1H, CH). ESI-MS: m/z (rel intensity) 341.8.1.4(R)-N-(2,6-二氧-四氫-2H-吡喃-3-基)-3,4,5-三甲氧基苯甲酰胺(5)在 55-60°C 下，將 10 g (2.9 mmol)化合物 4 和 80 mL 重蒸過的醋酸酐置于 250-mL 的圓 底燒瓶中反應(yīng) 5 h。 然后快速趁熱過濾掉體系中的不溶物，然后加入 10 mL 無水乙醚，濾 液冷卻至室溫，析出

16、白色固體，過濾，干燥后得化合物 5 (5.2 g, 55%). 熔點 150-152 °C, IR (KBr, cm-1): 3310.0 (NH), 2945.1 (CH), 1777.0 & 1640.7 (O=C-O-C=O), 1504.2 ( NH), 1239.6& 1129.6 (C-O). 1H NMR (DMSO-d6, ppm): 6.99 (s, 2H, Ar-H), 8.13 (d, 1H, J = 7.2 Hz, NH),4.45 (m, 1H, CH), 3.73 (s, 9H, 3-OCH3), 1.96-2.25 (m, 2H, CH2

17、), 2.18-2.28 (m, 2H, CH2). ESI-MS: m/z (rel intensity) 323.8.1.5L-氨基酸甲酯鹽酸鹽以甘氨酸甲酯鹽酸鹽為例：0°C 下，甘氨酸(15.0 g, 200 mmol)和 150 mL 無水甲醇的混 懸液通入干燥的 HCl 氣體直至混懸液澄清。將此溶液置于冰箱中過夜，蒸出溶劑并加入少 量無水甲醇帶走多余的 HCl 氣體，將所得的白色固體用 1:4 的無水甲醇/乙醚重結(jié)晶得純品20g, 80%。熔點 175-176 °C, IR (KBr, cm-1): 3215.0-3523.0 (m, NH), 2935.2 (C

18、H), 1676.3 (C=O),1129.3 (C-O).1.6(R)-5-(2-甲氧基-2-氧基乙氨基)-5-氧-4-(3,4,5-三甲氧基苯甲酰胺基)戊酸(6a)將化合物 5 (3.23 g, 10 mmol) 和氨基酸甲酯鹽酸鹽(2.5 g, 20 mmol)溶于 30 mL CH2Cl2 中，加入 mL Et3N，室溫下反應(yīng)直至反應(yīng)物消失，蒸出溶劑，所得物倒入 50 mL 冰水中， 用 2 N HCl 酸化至 pH 2。置于冰箱中過夜，過濾出所得的白色鱗片狀晶體，用冰水洗滌， 干燥，得化合物 6a (0.94 g, 74%). 熔點 169-172 °C, IR (KBr,

19、 cm-1): 3325.1 (NH), 2939.6 (CH),1745.7 & 1714.5 (O=C-NH), 1637.8 (O=C-O), 1584.3 (C=C), 1131.3 (C-O). 1H NMR (DMSO-d6, ppm): 8.45 (d, 1H, J = 7.5 Hz, NH), 8.38 (t, 1H, J = 5.4 Hz, NH), 7.23 (s, 2H, Ar-H), 4.47 (m,1H, CH), 3.88 (d, 2H, J = 5.4 Hz, CH2), 3.83 (s, 6H, 2-OCH3), 3.69 (s, 3H, OCH3),

20、3.62 (s, 3H, COOCH3), 2.34 (t, 2H, J = 8.0 Hz, CH2), 2.06, 1.92 (m, 2H, CH2). ESI-MS: m/z (rel intensity)412.8.1.7(R)-5-(S)-1-甲氧基-1-氧基丙烷-2-氨基)-5-氧-4-(3,4,5-三甲氧基苯甲酰胺基)戊酸(6b)制備方法同 6a，產(chǎn)率 35.0%, 熔點 153-155 °C, IR (KBr, cm-1): 3304.8 (NH), 2933.5 (CH),1751.1 & 1719.9 (O=C-NH), 1647.7 (O=C-O), 15

21、84.2 (C=C), 1129.9 (C-O). 1H NMR (DMSO-d6, ppm): 8.74 (d, 1H, J = 7.8 Hz, NH), 8.33 (d, 1H, J = 6.0 Hz, NH), 7.24 (s, 2H, Ar-H), 4.36 (m,1H, CH), 4.23 (m, 1H, CH), 3.85 (s, 6H, 2-OCH3), 3.73 (s, 3H, OCH3), 3.60 (s, 3H, COOCH3),2.23 (t, 2H, J = 7.8 Hz, CH2), 2.06, 1.83 (m, 2H, CH2), 1.23 (d, 3H, J =

22、7.2 Hz, CH3). ESI-MS:m/z (rel intensity) 425.1.1.8(R)-5-(S)-1-甲氧基-1-氧基丙烷-2-氨基)-5-氧-4-(3,4,5-三甲氧基苯甲酰胺基)戊酸(6c)制備方法同 6a，產(chǎn)率 28.5%, 熔點 105-107 °C, IR (KBr, cm-1): 3305.2 (NH), 2942.2 (CH),1746.1 & 1717.9 (O=C-NH), 1644.7 (O=C-O), 1586.7 (C=C), 1127.6 (C-O). 1H NMR (DMSO-d6, ppm): 8.73 (d, 1H, J

23、= 7.8 Hz, NH), 8.37 (d, 1H, J = 6.0 Hz, NH), 7.00 (s, 2H, Ar-H), 4.36 (m,1H, CH), 4.27 (m, 1H, CH), 3.73 (s, 6H, 2-OCH3), 3.67 (s, 3H, OCH3), 3.82 (s, 3H, COOCH3),2.23 (t, 2H, J = 7.0 Hz, CH2), 2.06, 1.83 (m, 2H, CH2), 1.92 (m, 2H, CH2), 0.98 (d, 3H, J = 7.2 Hz, CH3). ESI-MS: m/z (rel intensity) 439

24、.4.1.9(R)-5-(S)-1-甲氧基-1-氧基丙烷-2-氨基)-5-氧-4-(3,4,5-三甲氧基苯甲酰胺基)戊酸(6d)制備方法同 6a，產(chǎn)率 23.4%, 熔點 110-112 °C, IR (KBr, cm-1): 3307.8 (NH), 2940.6 (CH),1755.4 & 1718.1 (O=C-NH), 1640.9 (O=C-O), 1589.2 (C=C), 1125.5 (C-O). 1H NMR (DMSO-d6, ppm): 8.88 (d, 1H, J = 7.8 Hz, NH), 8.24 (d, 1H, J = 8.1 Hz, NH),

25、 6.98 (s, 2H, Ar-H), 4.33 (m,1H, CH), 4.53 (m, 1H, CH), 3.73 (s, 6H, 2-OCH3), 3.62 (s, 3H, OCH3), 3.72 (s, 3H, COOCH3),2.10 (t, 2H, J = 15.3 Hz, CH2), 2.11, 1.79 (m, 2H, CH2), 1.32 (m, 2H, CH2), 1.90 (m, 2H, CH2),0.97 (d, 3H, J = 8.4 Hz, CH3). ESI-MS: m/z (rel intensity) 453.3.1.10(R)-5-(S)-1-甲氧基-1-

26、氧基丙烷-2-氨基)-5-氧-4-(3,4,5-三甲氧基苯甲酰胺 基)戊酸(6e)制備方法同 6a，產(chǎn)率 61.2%, 熔點 99-101 °C, IR (KBr, cm-1): 3321.4 & 3256.9 (NH),2956.2 (CH), 1724.1 (O=C-NH), 1647.6 (O=C-O), 1584.3 (C=C), 1130.5 (C-O). 1H NMR (DMSO-d6, ppm): 7.82 (d, 1H, J = 7.6 Hz, NH), 7.15 (s, 1H, Ar-H), 7.12 (s, 1H, Ar-H), 6.44 (d,1H, J

27、 = 7.2 Hz, NH), 4.93 (m, 1H, CH), 4.60 (m, 1H, CH), 3.92 (s, 6H, 2-OCH3), 3.80 (s, 3H,OCH3), 3.68 (s, 3H, COOCH3), 2.28 (t, 2H, J = 7.2 Hz, CH2), 2.06 (m, 2H, CH2), 1.92 (m, 2H, CH2), 1.29 (br, 4H, 2-CH2), 0.87 (t, 3H, J = 7.8 Hz, CH3). ESI-MS: m/z (rel intensity) 467.1.1.11 (R)-5-(S)-1-甲氧基-1-氧基-3-苯

28、基丙烷-2-氨基)-5-氧-4-(3,4,5-三甲氧基苯甲 酰胺基)戊酸(6f)制備方法同 6a，產(chǎn)率 56.3%, 熔點 141-144 °C, IR (KBr, cm-1): 3416.0 & 3303.7 (NH),2939.9 (CH), 1743.7 & 1718.8 (O=C-NH), 1646.4 (O=C-O), 1584.1 & 1536.7 (C=C), 1128.9 (C-O). 1H NMR (DMSO-d6, ppm): 8.69 (d, 1H, J = 4.3 Hz, NH), 8.38 (d, 1H, J = 6.8 Hz, NH

29、),7.24 (m, 2H, Ar-H), 7.18 (m, 5H, Ar-H), 4.44 (m, 1H, CH), 4.34 (m, 1H, CH), 3.85 (s, 6H,2-OCH3), 3.70 (s, 3H, OCH3), 3.57 (s, 3H, COOCH3), 2.18 (t, 2H, J = 7.6 Hz, CH2), 2.11 (d, 2H, J= 7.4 Hz, CH2), 1.98, 1.76 (2m, 2H, CH2). ESI-MS: m/z (rel intensity) 501.1.1.12(R)-5-(S)-3-(4-氟苯基)-1-甲氧基-1-氧基丙烷-2

30、-氨基)-5-氧-4-(3,4,5-三甲氧 基苯甲酰胺基)戊酸(6g)制備方法同 6a，產(chǎn)率 61.4%, 熔點 107-111 °C, IR (KBr, cm-1): 3296.3 (NH), 2942.1 (CH),1743.0 & 1720.4 (O=C-NH), 1648.6 (O=C-O), 1584.2 & 1537.3 (C=C), 1127.6 (C-O). 1H NMR (DMSO-d6, ppm): 7.78 (d, 1H, J = 6.6 Hz, NH), 7.26 (s, 2H, Ar-H), 7.12 (m, 2H, Ar-H), 6.95

31、(m,2H, Ar-H), 6.47 (d, 1H, J = 8.0 Hz, NH), 4.78 (m, 1H, CH), 4.57 (m, 1H, CH), 3.93 (s, 6H,2-OCH3), 3.86 (s, 3H, OCH3), 3.68 (s, 3H, COOCH3), 3.10 (d, 2H, J = 8.2 Hz, CH2), 2.27 (t, 2H, J= 7.2 Hz, CH2), 1.89 (m, 2H, CH2). ESI-MS: m/z (rel intensity) 519.2.1.13(R)-5-(S)-3-(4-氯苯基)-1-甲氧基-1-氧基丙烷-2-氨基)-

32、5-氧-4-(3,4,5-三甲氧 基苯甲酰胺基)戊酸(6h)制備方法同 6a，產(chǎn)率 67.2%, 熔點 143-145 °C, IR (KBr, cm-1): 3300.1 (NH), 2942.3 (CH),1742.9 & 1718.6 (O=C-NH), 1647.0 (O=C-O), 1584.0 & 1536.9 (C=C), 1129.0 (C-O). 1H NMR (DMSO-d6, ppm): 7.74 (d, 1H, J = 6.4 Hz, NH), 7.29 (s, 2H, Ar-H), 7.22 (m, 2H, Ar-H), 7.10 (m,2H

33、, Ar-H), 6.54 (d, 1H, J = 7.8 Hz, NH), 4.79 (m, 1H, CH), 4.55 (m, 1H, CH), 3.93 (s, 6H,2-OCH3), 3.89 (s, 3H, OCH3), 3.72 (s, 3H, COOCH3), 3.12 (d, 2H, J = 7.6 Hz, CH2), 2.31 (t, 2H, J= 7.6 Hz, CH2), 1.95 (m, 2H, CH2). ESI-MS: m/z (rel intensity) 535.1.1.14(R)-5-(S)-3-(4-溴苯基)-1-甲氧基-1-氧基丙烷-2-氨基)-5-氧-4

34、-(3,4,5-三甲氧 基苯甲酰胺基)戊酸(6i)制備方法同 6a，產(chǎn)率 50.7%, 熔點 166-168 °C, IR (KBr, cm-1): 3303.4 (NH), 2946.1 (CH),1744.2 & 1719.0 (O=C-NH), 1647.4 (O=C-O), 1583.8 & 1534.6 (C=C), 1128.7 (C-O). 1H NMR (DMSO-d6, ppm): 7.77 (d, 1H, J = 6.8 Hz, NH), 7.26 (s, 2H, Ar-H), 7.18 (m, 2H, Ar-H), 7.05 (m,2H, Ar-

35、H), 6.62 (d, 1H, J = 4.5 Hz, NH), 4.83 (m, 1H, CH), 4.57 (m, 1H, CH), 3.91 (s, 6H,2-OCH3), 3.80 (s, 3H, OCH3), 3.77 (s, 3H, COOCH3), 3.01 (d, 2H, J = 8.4 Hz, CH2), 2.39 (t, 2H, J= 5.8 Hz, CH2), 1.98 (m, 2H, CH2). ESI-MS: m/z (rel intensity) 580.3.1.15(R)-5-(S)-3-(4-氫苯基)-1-甲氧基-1-氧基丙烷-2-氨基)-5-氧-4-(3,4

36、,5-三甲氧 基苯甲酰胺基)戊酸(6j)制備方法同 6a，產(chǎn)率 49.0%, 熔點 87-89 °C, IR (KBr, cm-1): 3355.2 & 3300.7 (NH), 2952.3 (CH), 1744.1 (O=C-NH), 1647.7 (O=C-O), 1585.9 & 1498.7 (C=C), 1257.7 & 1126.5 (C-O). 1H NMR (DMSO-d6, ppm): 8.72 (d, 1H, J = 7.0 Hz, NH), 8.33 (d, 1H, J = 6.4 Hz, NH), 7.23 (d, 2H, J = 3

37、.4 Hz, Ar-H), 6.95 (m, 2H, Ar-H), 6.64 (m, 2H, Ar-H), 4.34 (m, 1H, CH), 3.83 (s, 6H,2-OCH3), 3.70 (s, 3H, OCH3), 3.56 (s, 3H, COOCH3), 3.49 (m, 1H, CH), 2.20 (t, 2H, J = 7.5 Hz, CH2), 2.11 (d, 2H, J = 6.8 Hz, CH2), 2.00, 1.78 (2m, 2H, CH2). ESI-MS: m/z (rel intensity) 517.1.1.16(R)-5-(S)-3-羥基-1-甲氧基-

38、1-氧基丙烷-2-氨基)-5-氧-4-(3,4,5-三甲氧基苯 甲酰胺基)戊酸(6k)制備方法同 6a，產(chǎn)率 60.0%, 熔點 85-87 °C, IR (KBr, cm-1): 3266.1 (NH), 2941.7 (CH),1744.2 (O=C-NH), 1632.2 (O=C-O), 1584.3 (C=C), 1129.3 (C-O). 1H NMR (DMSO-d6, ppm): 8.76 (d, 1H, J = 6.5 Hz, NH), 8.26 (d, 1H, J = 6.8 Hz, NH), 7.24 (m, 2H, Ar-H), 5.02 (m, 1H, CH

39、),4.33 (m, 1H, CH), 3.82 (s, 6H, 2-OCH3), 3.71 (s, 3H, OCH3), 3.61 (s, 3H, COOCH3), 3.05 (d, 2H, J = 5.5 Hz, CH2), 2.28 (t, 2H, J = 6.0 Hz, CH2), 2.05, 1.83 (2m, 2H, CH2). ESI-MS: m/z (rel intensity) 441.1.1.17(R)-5-(S)-1-甲氧基-4-(甲基硫基)-1-氧基丁基-2-氨基)-5-氧-4-(3,4,5-三甲氧 基苯甲酰胺基)戊酸(6l)制備方法同 6a，產(chǎn)率 59.1%, 熔點

40、124-127 °C, IR (KBr, cm-1): 3289.3 (NH), 2941.8 (CH),1750.9 & 1717.8 (O=C-NH), 1647.2 (O=C-O), 1584.5 (C=C), 1129.7 (C-O). 1H NMR (DMSO-d6, ppm): 8.72 (d, 1H, J = 5.7 Hz, NH), 8.32 (d, 1H, J = 7.3 Hz, NH), 7.24 (s, 2H, Ar-H), 4.47 (m,1H, CH), 4.37 (m, 1H, CH), 3.78 (s, 6H, 2-OCH3), 3.70 (s,

41、 3H, OCH3), 3.61 (s, 3H, COOCH3),2.26 (t, 2H, J = 7.8 Hz, CH2), 2.13 (t, 2H, J = 7.4 Hz, CH2), 2.06 (m, 4H, 2-CH2), 1.83 (s, 3H, CH3). ESI-MS: m/z (rel intensity) 486.1.1.18(R)-5-(R)-1-甲氧基-1-氧基-3-(苯基硫基)丙烷-2-氨基)-5-氧-4-(3,4,5-三甲氧 基苯甲酰胺基)戊酸(6m)制備方法同 6a，產(chǎn)率 54.0%, 熔點 77-79 °C, IR (KBr, cm-1): 3272.9

42、 (NH), 2940.6 (CH),1746.5 & 1722.0 (O=C-NH), 1648.5 (O=C-O), 1584.0 (C=C), 1128.4 (C-O). 1H NMR (DMSO-d6, ppm): 8.77 (d, 1H, J = 6.8 Hz, NH), 8.46 (d, 1H, J = 7.2 Hz, NH), 7.34 (d, 2H, J = 4.1 Hz, Ar-H),7.30 (m, 5H, Ar-H), 4.47 (m, 1H, CH), 4.20 (m, 1H, CH), 3.83 (s, 6H, 2-OCH3), 3.75 (s, 3H, OC

43、H3), 3.60 (s, 3H, COOCH3), 3.32 (s, 2H, CH2), 3.06 (d, 2H, J = 6.8 Hz, CH2), 2.26 (t, 2H, J =7.8 Hz, CH2), 2.07 & 1.84 (2m, 2H, CH2). ESI-MS: m/z (rel intensity) 547.1.1.19(R)-5-(S)-5-(芐氧羰基)-1-甲氧基-1-氧基戊基-2-氨基)-5-氧-4-(3,4,5-三甲氧 基苯甲酰胺基)戊酸(6n)制備方法同 6a，產(chǎn)率 43.0%, 熔點 108-112 °C, IR (KBr, cm-1): 3

44、333.2 & 3276.9 (NH),2945.9 (CH), 1745.0 & 1718.6 (O=C-NH), 1688.4, 1584.1 (C=C), 1128.2 (C-O). 1H NMR (DMSO-d6, ppm): 8.74 (d, 1H, J = 5.6 Hz, NH), 8.28 (d, 1H, J = 6.9 Hz, NH), 7.34 (m, 5H, Ar-H), 7.24 (s, 2H, Ar-H), 4.99 (s, 2H, CH2), 4.36 (m, 1H, CH), 4.19 (m, 1H, CH), 3.83 (s, 6H,2-OCH3)

45、, 3.71 (s, 3H, OCH3), 3.59 (s, 3H, COOCH3), 2.96 (t, 2H, J = 5.2 Hz, CH2), 2.25 (t, 2H, J= 7.8 Hz, CH2), 2.06 & 1.83 (2m, 2H, CH2), 1.66 & 1.54 (2m, 2H, CH2), 1.45 (m, 2H, CH2). ESI-MS: m/z (rel intensity) 602.1.1.20(R)-5-(S)-6-(芐氧羰基)-1-甲氧基-1-氧基己基-2-氨基)-5-氧-4-(3,4,5-三甲氧 基苯甲酰胺基)戊酸(6o)制備方法同 6a

46、，產(chǎn)率 48.0%, 熔點 147-150 °C, IR (KBr, cm-1): 3325.6 (NH), 2940.1 (CH),1720.9 (O=C-NH), 1648.8 (O=C-O), 1584.8 & 1498.2 (C=C), 1234.6 & 1126.5 (C-O). 1H NMR (DMSO-d6, ppm): 8.72 (t, 1H, J = 6.0 Hz, NH), 8.24 (t, 1H, J = 7.2 Hz, NH), 7.33 (m, 5H, Ar-H), 7.24 (s, 2H, Ar-H), 7.20 (s, 1H, Ar-H)

47、, 4.99 (s, 2H, CH2), 4.37 (m, 1H, CH), 4.17 (m, 1H, CH), 3.83 (s, 6H, 2-OCH3), 3.74 (s, 3H, OCH3), 3.61 (s, 3H, COOCH3), 2.94 (t, 2H, J = 6.0 Hz, CH2), 2.27 (t, 2H, J = 7.8 Hz, CH2), 2.13 (s, 3H, CH3), 2.05 (m, 1H, CH), 1.85 (m, 1H, CH), 1.56 (m, 2H, CH2), 1.36 (m, 2H, CH2), 1.26 (m, 2H, CH2). ESI-M

48、S: m/z (rel intensity) 616.1.1.21(R)-5-(S)-1-甲氧基-5-(3-硝基胍基)-1-氧基戊基-2-氨基)-5-氧-4-(3,4,5-三甲 氧基苯甲酰胺基)戊酸(6p)制備方法同 6a，產(chǎn)率 52.2%, 熔點 123-126 °C, IR (KBr, cm-1): 3307.4 (NH), 2943.8 (CH),1739.8 (O=C-NH), 1649.1 (O=C-O), 1584.7 & 1499.7 (C=C), 1236.4 & 1127.5 (C-O). 1H NMR (DMSO-d6, ppm): 8.66 (d

49、, 1H, J = 6.6 Hz, NH), 8.22 (t, 1H, J = 6.0 Hz, NH), 7.24 (s, 2H, Ar-H),4.37 (m, 1H, CH), 4.24 (m, 1H, CH), 3.84 (s, 6H, 2-OCH3), 3.72 (s, 3H, OCH3), 3.61 (s, 3H,COOCH3), 3.13 (d, 2H, J = 5.4 Hz, CH2), 2.27 (t, 2H, J = 7.2 Hz, CH2), 2.13 (s, 2H, CH2), 2.07(m, 1H, CH), 1.85 (m, 1H, CH), 1.71 (m, 1H,

50、CH), 1.58 (m, 2H, CH2), 1.51 (m, 1H, CH). ESI-MS:m/z (rel intensity) 555.1.1.22(R)-5-(S)-3-(1H-吲哚-3-基)-1-甲氧基-1-氧基丙基-2-氨基)-5-氧-4-(3,4,5-三 甲氧基苯甲酰胺基)戊酸(6q)制備方法同 6a，產(chǎn)率 44.1%, 熔點 167-169 °C, IR (KBr, cm-1): 3362.5 (NH), 2948.5 (CH),1739.8 (O=C-NH), 1652.4 (O=C-O), 1584.7 & 1497.1 (C=C), 1233.3 &

51、amp; 1126.5 (C-O). 1H NMR (DMSO-d6, ppm): 8.14 (s, 1H, NH), 8.09 (s, 1H, OH), 7.91 (d, 1H, J = 6.1 Hz, NH), 7.84 (d, 1H, J = 6.3 Hz, NH), 7.51 (t, 2H, J = 8.4 Hz, Ar-H), 7.16 (s, 2H, Ar-H), 7.14 (s, 2H, Ar-H), 7.08 (m,1H, =CH), 4.76 (m, 1H, CH), 4.55 (m, 1H, CH), 3.92 (s, 6H, 2-OCH3), 3.90 (s, 3H, O

52、CH3), 3.71 (s, 3H, COOCH3), 2.41 (m, 1H, CH), 2.27 (m, 2H, CH2), 2.20 (m, 2H, CH2), 1.96 (m, 1H, CH). ESI-MS: m/z (rel intensity) 540.1.1.23(R)-5-(S)-3-(1-(芐氧羰基)-1H-咪唑-4-基)-1-甲氧基-1-氧基丙基-2-氨基)-5-氧-4-(3,4,5-三甲氧基苯甲酰胺基)戊酸(6r)制備方法同 6a，產(chǎn)率 56.3%, 熔點 112-116 °C, IR (KBr, cm-1): 3241.5 (NH), 2947.2 (CH)

53、,1744.7 (O=C-NH), 1661.9 (O=C-O), 1584.4 & 1498.5 (C=C), 1233.9 & 1125.8 (C-O). 1H NMR (DMSO-d6, ppm): 8.40 (d, 1H, J = 5.7 Hz, NH), 8.27 (d, 1H, J = 6.0 Hz, NH), 7.35 (s, 2H, Ar-H), 7.34 (s, 1H, =CH), 7.52 (d, 1H, J = 5.3 Hz, Ar-H), 7.22 (d, 1H, J = 2.6 Hz, Ar-H), 7.12 (d,1H, J = 6.6 Hz, Ar

54、-H), 6.67 (s, 1H, =CH), 5.03 (m, 2H, Ar-CH2), 4.74 (m, 1H, CH), 4.59 (m, 1H, CH), 3.92 (s, 6H, 2-OCH3), 3.91 (s, 3H, OCH3), 3.89 (s, 3H, COOCH3), 3.07 (m, 2H, CH2), 2.53 (m, 2H, CH2), 2.46 (m, 2H, CH2). ESI-MS: m/z (rel intensity) 581.2.1.24(R)-5-(2-甲氧基-2-氧基乙基)甲胺基-5-氧基-4-(3,4,5-三甲氧基苯甲酰胺基)戊酸(6s)制備方法同

55、 6a，產(chǎn)率 60.6%, 熔點 127-129 °C, IR (KBr, cm-1): 3295.9 (NH), 2940.9 (CH),1750.8 & 1729.9 (O=C-NH), 1683.3 & 1646.9 (O=C-O), 1582.9 (C=C), 1123.5 (C-O). 1H NMR (DMSO-d6, ppm): 8.70 (d, 1H, J = 6.8 Hz, NH), 7.22 (s, 2H, Ar-H), 4.38 (m, 1H, CH), 3.87 (s,6H, 2-OCH3), 3.71 (s, 3H, OCH3), 3.62 (

56、s, 3H, COOCH3), 2.99 (s, 2H, CH2), 2.56 (s, 3H, N-CH3),2.12 (t, 2H, J = 6.7 Hz, CH2), 2.06 & 1.85 (2m, 2H, CH2). ESI-MS: m/z (rel intensity) 426.9.1.25(4R)-5-(4-(苯甲氧基)-2-(甲氧羰基)吡咯烷-1-基-5-氧基-4-(3,4,5-三甲氧基苯 甲酰胺基)戊酸(7)制備方法同 6a，產(chǎn)率 65.4%, 熔點 98-101 °C, 1H NMR (DMSO-d6, ppm): 8.66 (d, 1H, J= 4.0 H

57、z, NH), 7.26-7.32 (m, 5H, Ar-H), 7.23 (s, 1H, Ar-H), 4.50 (s, 2H, CH2), 4.39 (m, 1H, CH),4.32 (t, 1H, J = 7.8 Hz, CH), 3.83 (s, 6H, 2-OCH3), 3.72 (s, 3H, OCH3), 3.61 (s, 3H, COOCH3),3.23 (s, 1H, CH), 2.39 (m, 2H, CH2), 2.32 (m, 1H, CH), 2.12 (d, 1H, J = 7.8 Hz, CH2), 2.06 (m,1H, CH), 1.98 (m, 1H, CH

58、), 1.87 (m, 1H, CH). ESI-MS: m/z (rel intensity) 557.1.1.26(R)-N-(5-羥氨基-1-(2-羥氨基-2-氧基乙氨基-1,5-二氧基戊烷-2-基)-3,4,5-三 甲氧基苯甲酰胺(8)將 KOH (5.6 g, 100 mmol) 溶于熱的 15 mL 無水甲醇中，逐滴加入鹽酸羥胺(4.67 g, 67.2 mmol)的 25 mL 熱無水甲醇溶液, 將此混合液在 35°C 下反應(yīng) 5 小時后，加入化合物 6a (0.5 g,1.2 mmol)的 10 mL 無水甲醇溶液，繼續(xù)在室溫下反應(yīng) 4 小時，乙酸酸化至 pH 5-6，而后用 二氯甲烷萃取三次，蒸除有機溶劑后得化合物 8 (0.4 g, 80%)。熔點 176-179 °C, IR (KBr,