周 宇，胡麗娜，羅劍飛，俞世沖，吳秋業(yè)

(1.解放軍第82醫(yī)院，江蘇 淮安 223001； 2. 第二軍醫(yī)大學(xué)有機(jī)化學(xué)教研室，上海 200433)

芒果苷衍生物的合成及其PTP1B酶抑制活性研究

周 宇1，胡麗娜2，羅劍飛2，俞世沖2，吳秋業(yè)2

(1.解放軍第82醫(yī)院，江蘇 淮安 223001； 2. 第二軍醫(yī)大學(xué)有機(jī)化學(xué)教研室，上海 200433)

目的設(shè)計(jì)合成一系列芒果苷衍生物并進(jìn)行體外蛋白酪氨酸磷酸酶1B(PTP1B)抑制活性實(shí)驗(yàn)。方法利用親核取代反應(yīng)在芒果苷上引入疏水芐基，設(shè)計(jì)合成8個(gè)新化合物4～11，采用比色法對(duì)化合物進(jìn)行PTP1B抑制活性研究。結(jié)果設(shè)計(jì)合成的8個(gè)化合物對(duì)PTP1B酶都有一定的抑制作用。結(jié)論芒果苷衍生物的活性明顯好于芒果苷本身的活性，芐基的對(duì)位取代活性要優(yōu)于鄰位和間位取代，且芐基上氯原子取代的衍生物要高于其它原子取代的化合物活性。

芒果苷；蛋白酪氨酸磷酸酶1B；化學(xué)合成；抑制活性

1 前言

芒果苷(mangiferin)，又名芒果素，知母寧，是從百合科植物知母中提取的天然多酚類化合物，分子式為C19H18O11。芒果苷有多種重要生理活性，如抗氧化[1]、抗腫瘤[2]、抗炎[3]、神經(jīng)保護(hù)[4]以及ACAT酶抑制作用[5]等。近來(lái)研究發(fā)現(xiàn)芒果苷通過(guò)抑制蛋白酪氨酸磷酸酶1B(PTP1B)表現(xiàn)出較好的抗糖尿病活性[6,7]。

PTP1B是胰島素轉(zhuǎn)導(dǎo)通路關(guān)鍵的負(fù)調(diào)控因子，它可以控制通路中信號(hào)蛋白(包括胰島素受體) 的去磷酸化，拮抗激酶催化的磷酸化反應(yīng)，從而對(duì)生物體產(chǎn)生影響[8～10]。臨床研究發(fā)現(xiàn)PTP1B酶抑制劑可以通過(guò)降低PTP1B濃度來(lái)增強(qiáng)胰島素的活性，PTP1B是治療Ⅱ型糖尿病和肥胖癥的有效靶點(diǎn)[11,12]，因此關(guān)于PTP1B抑制劑的研究也成為國(guó)際研究的熱點(diǎn)之一。

芒果苷本身溶解性不好，限制了進(jìn)一步的應(yīng)用。廖洪利等[13]其進(jìn)行結(jié)構(gòu)改造，引入烷基和芐基以提高其溶解性，并且發(fā)現(xiàn)結(jié)果化合物2和3有良好的 PTP1B酶抑制活性。基于以上研究，筆者設(shè)計(jì)合成了一系列芐基取代的芒果苷衍生物，并且對(duì)這些化合物進(jìn)行PTP1B酶抑制活性測(cè)試。

2 合成實(shí)驗(yàn)

熔點(diǎn)用YRT-3熔點(diǎn)測(cè)定儀(溫度未經(jīng)校正)測(cè)定；質(zhì)譜用安捷倫1100型質(zhì)譜儀測(cè)定；核磁共振氫譜用Varian INOVA-600型核磁共振儀(DMSO-d6為溶劑，TMS為內(nèi)標(biāo))測(cè)定。所用試劑均為市售分析純。

2.1目標(biāo)化合物4-11的合成 將芒果苷(420 mg, 1 mmol)溶于干燥的DMF (20 ml)，加入碳酸鉀(100 mg), 攪拌20 min后加入取代的氯芐(4 mmol)，攪拌，油浴加熱60 ℃, 10 h后，TLC顯示反應(yīng)完全。將反應(yīng)混合液真空濃縮，柱層析，二氯甲烷/甲醇為洗脫劑，得到438 mg淡黃色粉末，為目標(biāo)化合物4。其它目標(biāo)化合物5-11均按此法合成，合成路線如圖1，其結(jié)構(gòu)、產(chǎn)率、熔點(diǎn)、MS和1H NMR數(shù)據(jù)見表1。

圖1 目標(biāo)化合物的合成路線

表1目標(biāo)化合物的結(jié)構(gòu)、熔點(diǎn)和光譜數(shù)據(jù)

化合物R收率(%)mp(℃)MS(M+1)+1HNMR(DMSO-d6,δppm)458.7139～142747.6213.49(1H,s),7.80-7.19(14H,m),6.82(1H,s),5.40-5.27(6H,m),4.78(2H,b),4.70(1H,m),4.53(1H,b),4.37(1H,b),4.32(1H,m),3.96(1H,m),3.48(1H,m),3.24-3.15(3H,m).564.1143～144735.6813.47(1H,s),7.58(1H,s),7.46-7.18(12H,m),6.72(1H,s),5.31-5.17(6H,m),4.77(2H,b),4.69(1H,m),4.47(1H,b),4.34(1H,b),3.97(1H,m),3.76(1H,m),3.52(1H,m),3.27-3.13(3H,m),2.48(9H,s).667.6138～140783.6213.53(1H,s),7.58(1H,s),7.57-6.92(13H,m),6.72(1H,s),5.25-5.13(6H,m),4.82(2H,b),4.69(1H,s),4.67(1H,b),4.66(1H,b),4.64(1H,m),4.57(1H,m),3.79-3.72(9H,s),3.29(1H,m),3.20-2.49(3H,m).765.4134～136797.5113.50(1H,s),7.64(1H,s),7.60-7.35(13H,m),6.80(1H,s),5.41-5.27(6H,m),4.82(2H,b),4.70(1H,m),4.47(1H,b),4.34(1H,b),4.00(1H,m),3.75(1H,m),3.50(1H,m),3.21-3.11(3H,m).867.5137～139797.4913.52(1H,s),7.63-7.37(13H,m),7.30(1H,s),6.67(1H,s),5.38-5.19(6H,m),4.87(2H,b),4.70(1H,m),4.62(1H,b),4.49(1H,b),3.90(1H,m),3.75(1H,m),3.50(1H,m),3.25-3.14(3H,m).958.3145～147735.6813.56(1H,s),7.68-7.16(14H,m),6.85(1H,s),5.34-5.18(6H,m),4.71(2H,b),4.70(1H,m),4.47(1H,b),4.34(1H,b),3.99(1H,m),3.75(1H,m),3.40(1H,m),3.25-3.15(3H,m),2.36-2.32(9H,s).1065.2124～126890.2713.50(1H,s),7.91(1H,s),7.64-7.35(10H,m),7.39(1H,s),6.72(1H,s),5.42～5.27(6H,m),4.72(2H,b),4.70(1H,m),4.47(1H,b),4.34(1H,b),3.99(1H,m),3.75(1H,m),3.45(1H,m),3.28-3.20(3H,m)1156.6144～146747.6113.47(1H,s),7.82-7.25(14H,m),6.82(1H,s),5.40[5.27(6H,m),4.79(2H,b),4.72(1H,m),4.51(1H,b),4.38(1H,b),4.35(1H,m),3.94(1H,m),3.44(1H,m),3.26-3.18(3H,m).

3 藥理實(shí)驗(yàn)

根據(jù)Goldstein等人的方法測(cè)定化合物的酶抑制活性[14]。將化合物用DMSO配制成濃度分別為20 μg/ml和4 μg/ml的供試品溶液，以p-NPP為底物，實(shí)驗(yàn)混合液包含10 mmol/L p-NPP，50 mmol/L HEPES緩沖液 (pH 7.0)以及1 mmol/L EDTA和DTT。當(dāng)向體系中加入500 ml 0.1 mol/L NaOH時(shí)，反應(yīng)停止，測(cè)定410 nm時(shí)的光吸收度。不含酶實(shí)驗(yàn)組用來(lái)校正非酶反應(yīng)和反應(yīng)體系中產(chǎn)生的對(duì)硝基苯酚鹽離子濃度，用摩爾消光系數(shù)1.78×104來(lái)測(cè)定體系中對(duì)硝基苯酚鹽離子濃度，釩酸鈉為對(duì)照組(IC50, 2 mmol/L)。該類化合物的PTP1B酶抑制活性測(cè)試實(shí)驗(yàn)結(jié)果如表2所示。

從藥理活性篩選結(jié)果發(fā)現(xiàn)：該類化合物表現(xiàn)出良好的PTP1B酶抑制活性。在4 mg/ml的濃度時(shí)，化合物8, 10和11的PTP1B酶抑制活性分別為24.1%, 37.6%, 40.9%。芒果苷本身的PTP1B酶抑制活性較弱，但是將芒果苷的C-3, C-6, C-7酚羥基取代可以顯著增強(qiáng)抑制活性。構(gòu)效關(guān)系表明:帶有氯原子取代芐基的化合物活性要優(yōu)于其他基團(tuán)取代芐基的化合物。通過(guò)比較化合物4, 5和10, 11的活性，還發(fā)現(xiàn)：芐基的對(duì)位取代要優(yōu)于鄰位和間位取代的活性。

4 結(jié)論

目前國(guó)際上通過(guò)天然產(chǎn)物分離或化學(xué)合成的PTP1B酶抑制劑，由于電負(fù)性高、細(xì)胞膜通透性差及專一性差等原因，離最終成為臨床藥物還有一定距離。本課題以天然產(chǎn)物芒果苷為先導(dǎo)化合物，設(shè)計(jì)合成了一系列新衍生物4-11，并對(duì)其進(jìn)行體外PTP1B 酶抑制活性測(cè)定，結(jié)果表明該類化合物表現(xiàn)出一定的PTP1B酶抑制活性，為以后對(duì)芒果苷進(jìn)一步的結(jié)構(gòu)修飾和構(gòu)效關(guān)系研究奠定了基礎(chǔ)。

表2目標(biāo)化合物的體外PTP1B酶抑制活性

化合物RInh(%)20μg/ml4μg/ml4a-5a2.46a-745.4-8a24.19a-1061.937.611a40.9

注：a加入緩沖液后樣品出現(xiàn)沉淀。

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2011-01-06

[修回日期] 2011-03-10

SynthesisandPTP1Bactivityofenzymeinhibitionofmangiferinderivates

ZHOU Yu1, HU Li-na2, LUO Jian-fei2, YU Shi-chong2,WU Qiu-ye2

(1.NO. 82 Hospital of PLA, Huaian 223001，China; 2. Department of Organic Chemistry，School of Pharmacy，Second Military Medical University，Shanghai 200433，China)

ObjectiveTo synthetize and conduct PTP1B inhibitory activity experiment of series of novel mangiferin derivates.MethodsDifferent benzyl groups were introduced to mangiferin framework by nucleophilic substitution reaction. All the compounds were screened against protein tyrosine phosphatase 1B(PTP1B)with the colorimetrie assay.ResultsEight compounds were synthesized and all the compounds exhibited PTP1B inhibitory activity to some extent.ConclusionsDerivates of mangiferin remarkably enhanced the activity compared with mangiferin itself. In addition, the benzylated derivates with chloro atom had better inhibitory activity than other substitution groups. Furthermore,theparaposition of the benzyl was a better place for introducing substituent thanmetaandorthoposition.

mangiferin; protein tyrosine phosphatase 1B; chemical synthesis; inhibition

上海市科委基礎(chǔ)重點(diǎn)項(xiàng)目 ( 08JC1405500).

周 宇(1962-)，女，副主任藥師.

吳秋業(yè). Tel: (021)81871225, E-mail: wuqy6439@sohu.com.

R914.5

A

1006-0111(2011)03-0193-04