酒石酸系列氨肽酶抑制剂的设计合成与活性研究
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摘要
氨肽酶N(APN,又称CDl3)是含锌离子的一种膜结合型外肽酶,为依赖锌离子的金属蛋白酶系列,在哺乳动物的小肠肾脏、中枢神经系统等组织中有广泛表达。APN大量表达于肿瘤细胞表面,对肿瘤的血管生成、侵害具有重大的作用。该酶降解细胞外基质的主要成分,从而促进肿瘤细胞在人体的转移和生长；另还能够对许多免疫活性物质进行降解,削弱NK细胞和巨噬细胞识别和杀伤肿瘤细胞的能力,降低机体的免疫力。故抑制APN的活性可以控制对肿瘤肿瘤细胞血管生成,同时增强粒细胞的趋化性,使机体的免疫力得以提高。APN已被证明作为人体冠状病毒TEGV和229E病毒的受体。所有这些发现使APN的研究成为一个关注的目标,同时,近年来抗肿瘤药物研发的热点趋向于开发高选择性、高效的的APN抑制剂。
基于结构基础的药物设计原理和对已知的类肽类APN抑制剂结构特征的分析与总结,化合物与APN的结合部位可分为三个部分,A部分是同Bestatin的苯环相互作用的疏水口袋,B部分是锌结合组(ZBG型),C部分是另一侧疏水口袋。通过对计算机辅助药物设计软件的应用设计新的药物,结合酶的晶体结构,本研究设计并合成了酒石酸、苹果酸和烟酸三个系列共40个结构全新的化合物。其化学结构由波谱分析(IR、ESI-MS、1H NMR)确认,所合成的目标化合物
经查阅文献证实,未见文献报道,均为新化合物。
对所合成化合物进行了体外抑酶实验,并对其活性作出初步评价。选择APN来对合成物进行筛选,从而确定其酶活性的大小。其结果表明：本实验设计合成的化合物中的大多数都对APN有抑制作用。实验结果表明,L-酒石酸系列化合物对APN抑制活性最高,其半数抑制浓度(IC50)达到微摩尔级,具有较高抑酶作用。
本实验选用人红细胞白血病K562细胞对所合成的化合物进行抗肿瘤活性筛选,利用MTT法间接测定各目标产物作用后肿瘤细胞增殖的变化,并计算出肿瘤细胞的抑制率。并且,从所合成化合物选出四个肿瘤细胞抑制率较好的化合物(A1、B1、C1、D2),计算出它们的半数抑制浓度(IC50),筛选出体外活性较好的化合物D2。
Aminopeptidase N (APN/identical to CD 13) belongs to a family of zinc depen-dent exopeptidase has become an attractive target for structure-based drug designing in the last decades. It was originally used as a marker for the myeloid differentiation antigen.Aminopeptidase N (APN) is expressed on various mammalian cells including monocytes, myeloid, epithelial cells of the intestine and kidney, fibroblasts, endoth-elial cells and tumor cells. APN has been also proved to behave as a receptor for corona-viruses TEGV and 229E in humans.More impotantly, many researches have shown that APN is involved in the regulation of tumour cell invasion and metastasis. In addition, APN is involved in the processes of immunological regulation, and as a result, many immunologic active materials have been degradated and the ability of macrophage and NK cells to identify and kill tumor cells have been weakened. All these findings make this enzyme an interesting target for possible anti-tumor drugs research, which require the development of potent and more selective inhibitors.
Based on the infrastructure of design principles, analysis and summary the structural features of peptide-like APN inhibitors which have been synthesized. Studies have discovered that the binding site of the APN with compouns can be divided to three parts, part A is a hydrophobic pocket which interacts with the phenyl group of Bestatin; part B is the zinc binding group (ZBG) and part C is a hydrophobic pocket on the other side. In this thesis, by the combination of crystal structure of the enzyme and application of computer-aided drug design software, we designed and synthesized three series of 40 novel peptide-like derivatives:tartaric acid derivatives, malic acid derivatives and nicotine acid derivatives. Their structures are confirmed by IR, ESI-MS and 1H NMR. All the targeted compounds are novel without any report by now.
Preliminary bioactivity assays are carried out in vitro. The target compounds are evaluated for inhibitory activities toward APN. As a result, most of these newly syn-thesized compounds show good inhibitory activities on APN. The results of experi- ment show that L-tartaric acid derivatives are the most activity inhibitors toward APN, their half inhibitory concentration (IC50) achieved to micromolar range, these new compounds have potent and selective inhibitory activities toward APN.
The newly synthesized compounds were evaluated for their in vitro cytotoxicity by growth-inhibition studies using erythrocyte leukeemic cell line (K562). MTT assay was used of them and calculate the rate of inhibition against the tumor cells. Of these compounds, compounds A1,B1,C1 and D2 are the most active compounds. So we choose them as experimental subject, continued to obtain their cytotoxic activity IC50 and we found compound D2 have the best inhibitory activity toward K562 cells.
基于结构基础的药物设计原理和对已知的类肽类APN抑制剂结构特征的分析与总结,化合物与APN的结合部位可分为三个部分,A部分是同Bestatin的苯环相互作用的疏水口袋,B部分是锌结合组(ZBG型),C部分是另一侧疏水口袋。通过对计算机辅助药物设计软件的应用设计新的药物,结合酶的晶体结构,本研究设计并合成了酒石酸、苹果酸和烟酸三个系列共40个结构全新的化合物。其化学结构由波谱分析(IR、ESI-MS、1H NMR)确认,所合成的目标化合物
经查阅文献证实,未见文献报道,均为新化合物。
对所合成化合物进行了体外抑酶实验,并对其活性作出初步评价。选择APN来对合成物进行筛选,从而确定其酶活性的大小。其结果表明：本实验设计合成的化合物中的大多数都对APN有抑制作用。实验结果表明,L-酒石酸系列化合物对APN抑制活性最高,其半数抑制浓度(IC50)达到微摩尔级,具有较高抑酶作用。
本实验选用人红细胞白血病K562细胞对所合成的化合物进行抗肿瘤活性筛选,利用MTT法间接测定各目标产物作用后肿瘤细胞增殖的变化,并计算出肿瘤细胞的抑制率。并且,从所合成化合物选出四个肿瘤细胞抑制率较好的化合物(A1、B1、C1、D2),计算出它们的半数抑制浓度(IC50),筛选出体外活性较好的化合物D2。
Aminopeptidase N (APN/identical to CD 13) belongs to a family of zinc depen-dent exopeptidase has become an attractive target for structure-based drug designing in the last decades. It was originally used as a marker for the myeloid differentiation antigen.Aminopeptidase N (APN) is expressed on various mammalian cells including monocytes, myeloid, epithelial cells of the intestine and kidney, fibroblasts, endoth-elial cells and tumor cells. APN has been also proved to behave as a receptor for corona-viruses TEGV and 229E in humans.More impotantly, many researches have shown that APN is involved in the regulation of tumour cell invasion and metastasis. In addition, APN is involved in the processes of immunological regulation, and as a result, many immunologic active materials have been degradated and the ability of macrophage and NK cells to identify and kill tumor cells have been weakened. All these findings make this enzyme an interesting target for possible anti-tumor drugs research, which require the development of potent and more selective inhibitors.
Based on the infrastructure of design principles, analysis and summary the structural features of peptide-like APN inhibitors which have been synthesized. Studies have discovered that the binding site of the APN with compouns can be divided to three parts, part A is a hydrophobic pocket which interacts with the phenyl group of Bestatin; part B is the zinc binding group (ZBG) and part C is a hydrophobic pocket on the other side. In this thesis, by the combination of crystal structure of the enzyme and application of computer-aided drug design software, we designed and synthesized three series of 40 novel peptide-like derivatives:tartaric acid derivatives, malic acid derivatives and nicotine acid derivatives. Their structures are confirmed by IR, ESI-MS and 1H NMR. All the targeted compounds are novel without any report by now.
Preliminary bioactivity assays are carried out in vitro. The target compounds are evaluated for inhibitory activities toward APN. As a result, most of these newly syn-thesized compounds show good inhibitory activities on APN. The results of experi- ment show that L-tartaric acid derivatives are the most activity inhibitors toward APN, their half inhibitory concentration (IC50) achieved to micromolar range, these new compounds have potent and selective inhibitory activities toward APN.
The newly synthesized compounds were evaluated for their in vitro cytotoxicity by growth-inhibition studies using erythrocyte leukeemic cell line (K562). MTT assay was used of them and calculate the rate of inhibition against the tumor cells. Of these compounds, compounds A1,B1,C1 and D2 are the most active compounds. So we choose them as experimental subject, continued to obtain their cytotoxic activity IC50 and we found compound D2 have the best inhibitory activity toward K562 cells.
引文
[1]张涛Ahpa类氨肽酶N抑制剂的设计、合成与初步活性评价[D].山东大学,2005.
[2]Rawlings ND, Barrett AJ. Evolutionary families of peptidases[J]. Biochem J,1993,290 (Pt 1): 205-218.
[3]Hooper NM. Families of zinc metalloproteases[J]. FEBS Letters,1994,354 (1):1-6.
[4]Bauvois B, Dauzonne D. Aminopeptidase N/CD13 (EC 3.4.11.2) inhibitors:chemistry, biolo-gical evaluations, and therapeutic prospects[J]. Med Res Rev,2006,26 (1):88-130.
[5]Mizutani S, Shibata K, Kikkawa F, et al. Essential role of placental leucine aminopeptidase in gynecologic malignancy[J]. Expert Opin Ther Targets,2007,11 (4):453-461.
[6]Saiki I, Fujii H, Yoneda J, et al. Role of aminopeptidase N (CD13) in tumor-cell invasion and extracellular matrix degradation[J]. Int J Cancer,1993,54 (1):137-143.
[7]Menrad A, Speicher D, Wacker J, et al. Biochemical and functional characterization of amino-peptidase N expressed by human melanoma cells[J]. Cancer Res,1993,53(6):1450-1455.
[8]陈茂营,徐文方.蛋白酶体与蛋白酶体抑制剂[J].中国药物化学杂志,2007,17(4)：249-253
[9]Pauly RP, Demuth HU, Rosche F, et al. Improved glucose tolerance in rats treated with the dipeptidyl peptidase IV (CD26) inhibitor Ile-thiazolidide[J]. Metabolism,1999,48 (3):385-389.
[10]Rawlings ND, Barrett AJ. MEROPS:the peptidase database[J]. Nucleic Acids Res,1999,27 (1):325-331
[11]Xu W, Li Q. Progress in the development of aminopeptidase N (APN/CD13) inhibitors[J]. Curr Med Chem Anticancer Agents,2005,5 (3):281-301.
[12]Sjostrom H, Noren O, Olsen J. Structure and function of aminopeptidase N[J]. Adv Exp Med Biol,2000,477(8):25-34.
[13]Beate F, Marco A, Karin,et al. Estracellular cysteines define ectopeptidase (APN,CD13) esp-ression and function[J]. Free Radic. Biol.Med,2002,32(7):584-595.
[14]Ito K, Nakajima Y, Onohara Y, et al. Crystal structure of aminopeptidase N (proteobacteria alanyl aminopeptidase) from Escherichia coli and conformational change of methionine 260 involved in substrate recognition[J]. J Biol Chem,2006,281 (44):33664-33676.
[15]Jiang, M, Bond, JS, Families of metalloendopeptidases and their relationships[J]. FEBS Lert, 1992,312(2-3):110-114.
[16]Riemann D, Kehlen A, and Langner J. CD13-Not just a marker in leukemia typing[J]. Imm-unol Today,1999,20 (2):83-88.
[17]Teruki S, Keuji T, Kaoyoko H, et al. CD 13/Aminopeptidase N-Induced lymphocyte involvement in inflamed jionts of patients with rheumatoid arthritis[J]. Arthritis Rheum, 2002,46 (9):2330-2338.
[18]Emre S, Levent M, Senturk, et al. Expression of aminopeptidase N in human endom trium and regulation of its activity by estrogen[J]. Fertil Steril,2001,75 (6):1172-1176.
[19]Santos O A, Botbol V,Bestatin as experimental tool in mammals[J]. Curr Drug Metab,2001, 2(1):67-85.
[20]Santos AN, Langner J, Herrmann M, et al. Aminopeptidase N/CD13 is directly linked to sig-nal transduction pathways in monocytes[J]. Cell Immunol,2000,201 (1):22-32.
[21]Takeo H, Masahiro K, Kuniki K, et al. Enhancement of sensitivity by bestatin of acute promyelocytic leukemia NB4cell to all-trans retinoic acid[J]. Leukemia Res,2002,26 (12): 1097-1103.
[22]Lochter A. Bissell MJ. An odyssey from breast to bone:muftistep control ofmammary meta-stases and osteolysis by matrix metalloproteinases[J]. APMIS,1999,107(1):128-136.
[23]Folkman J. Seminars in Medicine of the Beth Israel Hospital, Boston. Clinical applications of research on angiogenesis[J]. N Engl J Med 1995,333(26):1757-1763.
[24]Martin DC, Fowlkes JL, Babic B, et al. Insulin-like growth factor Ⅱ signaling in neoplastic proliferation is blocked by transgenic expression of the metalloproteinase inhibitor TIMP-1 [J]. J Cell Biol,1999,146 (4):881-892.
[25]David E, Wentworth, Kathryn VH, Molecular determinants of species specificity in the coro-navirus receptor aminopeptidase N(CD13):influence of N-linked glycosylation[J]. J Virol, 2001,75 (20):9741-9752.
[26]Hanahan D, Folkman J,Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis[J]. Cell,1996,86 (3):353-364.
[27]R Pasqualini, E.Koivunen, R.Kain, et al. Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis[J]. Cancer Res,2000,60(12):722-727.
[28]Shim JS, Kim JH, Cho HY, et al. Irreversible inhibition of CD13/aminopeptidase N by the antiangiogentic agent curcumin[J]. Chem Biol,2003,10 (8):695-704.
[29]Alexander NS, Juergen L, Manfred H, et al. Aminopeptadase N/CD13 is directly linked to signal transduction pathways in monocytes[J]. Cellular Immunology,2000,201:22-32.
[30]Zetter BR, Angiogensis and tumor metastasis[J]. Annu Rev Med,1998,49(3):407-424.
[31]Shripad V, Bhagwat, Nenad P, et al. The angiogenic regulator CD13/APN is a transcriptional target of Ras signaling pathways in endothelial morphogenesis[J]. Blood,2003,101(5): 1818-1826.
[32]Ikeda N, NakajimaY, Tokuhara T, et al. Clinical significance of aminopeptidase N/CD13 exp-ression in human pancreatic carcinoma[J]. Clin Cancer Res,2003,9(4):1503-1508.
[33]Bhagwat SV, Lahdenranta J, Giordano R, et al. CD13/APN is a activated by angiogenic signals and is essential for capillary tube formation[J]. Blood,2001,97(3):652-659.
[34]Hiroki H, Arimichi T, Michiyuki K, Aminopeptidase N is involved in cell motility angio-gensis:Its clinical significance in human colon cancer[J]. Gastroenterology,2002,122(2): 376-386.
[35]Krakoff IH, Magill GB, Effects of 2-ethylamino-1,3,4 thiadiazole HCI on uric acid produc- tion in man[J]. Proc Soc Exp Biol Med,1956,91 (3):470-472.
[36]Elson PJ, Kvols LK, Vogl SE, et al. Phase Ⅱ trials of 5 day vinblastine infusion (NSC 49842), L-alanosine (NSC 153353), acivicin (NSC 163501), and aminothiadiazole (NSC 4728) in patients with recurrent or metastatic renal cell carcinoma[J]. Invest New Drugs,1988,6 (2): 97-103.
[37]Locker GY, Kilton L, Khandekar JD, et al. High-dose aminothiadiazole in advanced color-ectal cancer. An Illinois Cancer Center phase Ⅱ trial[J]. Invest New Drugs,1994,12 (4):299-301.
[38]Asbury RF, Wilson J, Blessing JA, et al. Aminothiadiazole (NSC 4728) in patients with advanced ovarian carcinoma. A phase Ⅱ study of the Gynecologic Oncology Group[J]. Am J Clin Oncol,1986,9 (4):334-336.
[39]Asbury RF, Blessing JA, McGuire WP, et al. Aminothiadiazole (NSC 4728) in patients with advanced carcinoma of the endometrium. A phase Ⅱ study of the Gynecologic Oncology group[J]. Am J Clin Oncol,1990,13(1):39-41.
[40]李少华,李刚,黄惠明,等.1,3,4-噻二唑衍生物的合成及初步活性测定[J].南昌大学学报,2009,33(3)：261-263.
[41]刘玉婷,晏会新,尹大伟,等.2,5-二取代-1,3,4-噻二唑类化合物的合成机应用研究[J].精细化工中间体,2009,39(01)：8-12.
[42]宋宝安,陈才俊,杨松,等.2-取代硫醚-5-(3,4,5-三甲氧基苯基)-1,3,4-噻二唑类化合物的合成、结构与体外抗癌活性[J].化学学报,2005,63(18)：1720-1726.
[43]杨锐生,胡国强,谢松强,等.2-(5-取代-1,3,4-噁二唑-2-亚甲硫)-5-吡啶-3-基-1,3,4-噻二唑的合成与抗癌活性[J].中国药学杂志,2008,43(5)：388-390.
[44]郑开波,何俊,张杰.含1,3,4-噻二唑的-5-氟尿嘧啶衍生物的合成及其抗肿瘤活性[J].华西药学杂志,2008,23(5)：528-530.
[45]蒋庆琳,吴林艳,吴勇.N1-(芳)烷酰氧亚甲基-5-氟尿嘧啶的合成及其初步抗肿瘤活性的研究[J].华西药学杂志,2005,20(1)：12-14.
[46]石德清,陈琦,李中华.含氨基酸希夫碱的5-氟尿嘧啶衍生物的合成及其抗肿瘤活性[J].有机化学,2005,25(5)：549-553.
[47]Masao Tada, Antineoplastic Agents.The preparation of 5-fluorouracil-l-acetic acid derivat ives[J].Bull Chem Soc Japan,1975,48 (11):3427-3428.
[48]Rzeski W, Matysiak J, Kandefer-Szerszeh M.Anticancer,neuroprotective activities and com-putation studies of 2-amino-1,3,4-thiadiazole based compound [J]. Bioorg Med Chem,2007, 15(9):3201-3207.
[49]Barve V, Ahmed F, Adsule S, et al.Synthesis, molecular charaterization, and biological acti-vity of chromen-4-one in human cancer cell[J].J Med Chem,2006,49(13):3800-3808.
[50]胡国强,毋晓魁,王新,等.氟喹诺酮C3杂环取代衍生物的合成及抗肿瘤活性研究(Ⅰ)：环丙沙星噻二唑希夫碱[J].药学学报,2008,43(11)：1112-1115.
[51]张耀洲,王海燕,王新,等.噻二唑硫醚双腙的合成及抗肿瘤活性[J].化学试剂,2009,31(9)：682-684.
[52]Umezawa H, Aoyagi T, Suda H, et al. Bestatin, an inhibitor of aminopeptidase B, produced by actinomycetes[J]. J Antibiot (Tokyo),1976,29 (1):97-99.
[53]Pulido Cejudo G, Conway B, Proulx P, et al. Bestatin-mediated inhibition of leucine amino-peptidase may hinder HIV infection[J]. Antiviral Res,1997,36 (3):167-177.
[54]Mathe G. Bestatin, An aminopeptidase inhibitor with a multi-pharmacological function[J]. Biomed Pharmacother,1991,45 (2-3):49-54.
[55]Ota K, Kurita S, Yamada K, et al. Immunotherapy with bestatin for acute non-lymphocytic leukemia in adults[J]. Cancer Immunol Immunother,1986,23 (1):5-10.
[56]Ino K, Bierman PJ, Varney ML, et al. Monocyte activation by an oral immuno-modulator (bestatin) in lymphoma patients following autologous bone marrow transplantation[J]. Cancer Immunol Immunother,1996,43 (4):206-212.
[57]Aoyagi T, Yoshida S, Nakamura Y, et al. Probestin, a new inhibitor of aminopeptidase M, produced by Streptomyces azureus MH663-2F6. I. Taxonomy, production, isolation, phy-sico chemical properties and biological activities[J]. J Antibiot,1990,43(2):143-148.
[58]Repic Lampret B, Kidric J, Kralj B, et al. Lapstatin, a new aminopeptidase inhibitor prod-uced by Streptomyces rimosus, inhibits autogenous aminopeptidases[J]. Arch Microbiol, 1999,171(6):397-404.
[59]Nagai M, Kojima F, Naganawa H, et al. Phebestin, a new inhibitor of aminopeptidase N, produced by Streptomyces sp. MJ716-m3[J]. J Antibiot,1997,50(1):82-84
[60]Aoyagi T, Tobe H, Kojima F, et al. Amastatin, an inhibitor of aminopeptidase A, produced by actinomycetes[J]. J Antibiot,1978,31(6):636-638.
[61]Rich DH, Moon BJ, Harbeson S Inhibition of aminopeptidases by amastatin and bestatin derivatives. Effect of inhibitor structure on slow-binding processes [J]. J Med Chem,1984, 27(4):417-422.
[62]Umezawa H, Aoyagi T, Tanaka T, et al. Production of actinonin, an inhibitor of amino-peptidase M, by actinomycetes[J]. J Antibiot,1985,38(11):1629-1630.
[63]Sui Z., Salto R, Li J, et al. Inhibition of the HIV-1 and HIV-2 proteases by curcumin and curcumin boron complexes[J]. Bioorg Med Chem,1993,1(6):415-420.
[64]Shim JS, Kim JH, Cho HY, et al. Irreversible inhibition of APN/aminopeptidase N by the antiangiogenic agent curcumin[J]. Chem Biol,2003,10(8):695-704.
[65]Egan ME, Pearson M, Weiner SA, et al. Curcumin, a major constituent of turmeric, corrects cystic fibrosis defects[J]. Science,2004,304(5670):600-602.
[66]Shin JS, Lee HS, Shin J, et al. Psammaplin A, a marine natural product, inhibits amino-peptidase N and suppresses angiogenesis in vitro[J]. Cancer Lett,2004,203(2):163-169.
[67]Melzig MF, Bormann H, Betulinic acid inhibits aminopeptidase N activity[J]. Planta Med, 1998,64(7):655-657.
[68]Aoyagi T, Yoshida S, Matsuda N, et al. Leuhistin, a new inhibitor of aminopeptidase M, produced by Bacillus laterosporus BMI156-14F1. I. Taxonomy, production, isolation, physico-chemical properties and biological activities[J]. J Antibiot,1991,44(6):573-578.
[69]Chung MC, Lee HJ, Chun HK, et al. Bestatin analogue from Streptomyces neyaga-waensis SL-387[J]. Biosci Biotechnol Biochem,1996,60(5):898-900.
[70]王俊丽.L-异谷氨酰胺类氨肽酶N抑制剂的设计合成及初步活性评价[D].山东大学,2004.
[71]王亚洲.L-异谷氨酰胺类氨肽酶N抑制剂的设计合成及初步活性研究[D].山东大学,2005.
[72]陈茂营.L-赖氨酸类氨肽酶N抑制剂的设计、合成与初步活性研究[D].山东大学,2007.
[73]张丽,徐文方,张杰.吡咯烷类明胶酶抑制剂的合成及初步活性[J].中国药物化学杂志,2007,17(2)：78-84.
[74]李乾斌.环酰亚胺类肽金属蛋白酶抑制剂的设计、合成及其抗肿瘤活性研究[D].山东大学,2008.
[75]Shenvi AB, alpha-Aminoboronic acid derivatives:effective inhibitors of aminopeptidases[J]. Biochem,1986,25 (6):1286-1291.
[76]Giannousis PP, Bartlett PA, Phosphorus amino acid analogues as inhibitors of leucine amino-peptidase[J]. J Med Chem,1987,30 (9):1603-1609.
[77]Chen H, Noble F, Roques BP, et al. Long lasting antinociceptive properties of enkephalin degrading enzyme (NEP and APN) inhibitor prodrugs[J]. J Med Chem,2001,44 (21):3523-3530.
[78]Lejczak B, Kafarski P, Zygmunt J. Inhibition of aminopeptidases by aminophosphonates[J]. Biochem,1989,28 (8):3549-3555.
[79]Joseph MD,Computational approaches to structure-based ligand design[J]. Pharmacol Ther, 1999,84(2):179-191.
[80]Robertus J, Structures-based drug design ten years on[J]. Nat Struct Biol,1994,1 (6):352-354.
[81]Ito K, Nakajima Y, Onohara Y, et al. Crystal structure of aminopeptidase N (proteobacteria alanyl aminopeptidase) from Escherichia coli and conforma-tional change of methionine 260 involved in substrate recognition[J]. J Biol Chem,2006,281 (44):33664-33676.
[82]Mina-Osorio P,The moonlighting enzyme CD13:old and new functions to target[J]. Trends Mol Med,2008,14(8):361-371
[83]Kramer RZ, Bella J, Mayville P, et al. Sequence dependent conformational variations of collagen triple-helical structure[J]. Nat Struct Biol,1999,6(5):454-457.
[84]汪多仁,陈体庆.L-苹果酸的开发与应用[J].饮料工业,2004,1(7)：21-26.
[85]王锐清.L-苹果酸的生产及应用研究进展[J].化工时刊,2002,16(5)：1-9.
[86]Morris GM, Goodsell DS, Halliday RS, et at. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function [J]. J Com Chem,1998, 199(14):1639-1662.
[87]Moriguchi I, Hirono S, Liu Q, et al. Simple Method of Calculating Octanol/Water Partition Coefficient[J]. Chem. Pharm. Bull,1992,40(1):127-130.
[88]杨二冰,李正名.药物分子设计中的Lipinski规则[J].化学通报,2006,69(1)：16-19.
[89]Kubinyi H, Drug research:myths, hype and reality[J]. Nature Reviews Drug Dicovery,2003, 2 (8):665-668.
[90]Lipinski CA, Chris Lipinski discusses life and chemistry after the Rule of Five[J]. Drug Discovery Today,2003,8(1):12-16.
[91]郑浩,董慧民,丁呈华等,2-氨基-1,3,4-噻二唑的合成新方法[J].河南师范大学学报：自然科学版.2006,5(2)：68-70.
[92]孟歌,韩紫岩,牛保华等.2-氨基-5-苯基噻二唑及其衍生物的合与表征[J].开封医专学报.1999,18(1)：44-46.
[93]吕艳阳,张玉霞.2,5-二(邻羟苯基)-1,3,4-噻二唑与钴的显色反应的研究[J].分析实验室,2006,25(4)：17-19.
[94]Henrot, S.; Larcheveque, M.; Petit, Y. Amino-acids as chiral synthons-preparation of enantiomerically pure (R) and (S) malic-acids and its application to the synthesis of 3-hydroxy 4-butanlide[J]. Synthetic Commun,1986,16 (2),183-190.
[95]刘全忠,龚流柱,蒋耀忠ZnCl2-KBH4还原苹果酸和天冬氨酸的研究[J].有机化学,2004,24(6)：637-640.
[96]Golovlyova SM, Moskvichev YA, Alov EM, et al. Synthesis of Novel Five-membered Nitrogen-containing Heterocyclic Compounds from Derivatives of Arylsulfonyl and Arylthioacetic and Propionic Acids[J]. Chemistry of Heterocyclic Compounds,2001,37 (9): 1102-1106.
[97]Spillane, WJ.; Kelly, LM.; Feeney, BG., et al. Synthesis of heterosulfamates.Search for structure-taste relationships[J]. Arkivoc,2003(vii):297-309.
[98]Li, Z.; Yu, J.-Y.; Yang, J.-Y, et al. Polymer-supported dichlorophosphate:a recoverable new reagent for synthesis of 2-amino-1,3,4-thiadiazoles[J]. J Chem Res,2005,36(43):341-343..
[99]Drag M, Grembecka J, Pawelczak M, et al. alpha-Aminoalkylphosphonates as a tool in experimental optimisation of Pl side chain shape of potential inhibitors in Sl pocket of leucine and neutral aminopeptidases[J]. Eur J Med Chem,2005,40 (8):764-77'1.
[100]Slater TF, Sawyer B, Strauli U,Studies on succinate-tetrazolium reductase system. Ⅲ. Points of coupling of four different tetrazolium salts[J]. Biochem. Biophys. Acta.,1963,77 (11):383-393.
[101]Berridge, MV.; Tan, AS. Characterization of cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT):subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction[J]. Arch. Biochem. Biophys,1993,303(2):474-482.
[102]Cell Proliferation Kit I (MTT). Roche Mol. Biochem.,1993,3:1-4. (http://www.roche-applied-science.com/pack-insert/1465007a.pdf)
[103]Vlachy, N; Touraud, D; Heilmann, J, et al. Determining the cytotoxicity of catanionic surfactant mixtures on HeLa cells[J]. Colloid Surface B,2009,70(2):278-280.
[104]吴舟锋,焦宛,王炳生.肿瘤化疗药物敏感性试验[J].国外医学外科学分册,1998,25(3)：153-155.
[105]郭杨,王增林,任金荣.MTT法在肿瘤化疗药物敏感性试验中的应用[J].华北煤炭医学院学报,1999,1(2)：99-101.
[106]Zhang ZH B, Jia L, Feng YJ, et al. Overexpression of follicle-stimulating hormone rece-ptor facilitates the development of ovarian epithelial cancer [J]. Cancer Lett,2009, 278(1):56-64.
[107]Gonzalez MA, Correa RJ, Agudelo L, et al. Synthesis and biological evaluation of abietic acid derivatives[J]. Eur J Med Chem,2009,44(6):2468-2472.
[108]Chandregowda V, Kush A, Reddy GC, Synthesis of benzamide derivatives of anacardic acid and their cytotoxic activity[J]. Eur J Med Chem,2009,44(16):2711-2719.
[109]Liu ZH, Zeng S,Cytotoxicity of ginkgolic acid in Hep-2 cells and primary rat hepat-ocytes[J]. Toxicol Lett,2009,187(3):131-136.
[110]Benkli K, Tunali Y, Canturk Z, et al. Cytotoxic and genotoxic effects of [Ru(phi)3]2+eval-uated by Ames/Salmonella and MTT methods[J]. Eur J Med Chem,2009,44(18):2601-2605.
[111]Liu F, Wang JJ, You ZY, et al. Radiosensitivity of prostate cancer cells is enhanced by EGFR inhibitor C225[J]. Urol oncol,28(1):59-66.
[112]Chang CJ, Hsu CC, Yung MC, et al. Enhanced radiosensitivity and radiation induced apoptosis in glioma CD133-positive cells by knockdown of SirT1 expression[J]. Biochem Bioph Res Co,2009,380(2):236-242.
[113]Zhou JJ, Yue XF, Han JX, et al. Improved MTT Assay for Activity of Antitumor Agents [J]. Chinese Journal of Pharm,1993,24(10):455-457.
[114]MTT Cell Proliferation Assay Instructions. ATCC,2001,1-6. http://www.atcc.org/ portals/1/pdf/30-1010k.Pdf
[2]Rawlings ND, Barrett AJ. Evolutionary families of peptidases[J]. Biochem J,1993,290 (Pt 1): 205-218.
[3]Hooper NM. Families of zinc metalloproteases[J]. FEBS Letters,1994,354 (1):1-6.
[4]Bauvois B, Dauzonne D. Aminopeptidase N/CD13 (EC 3.4.11.2) inhibitors:chemistry, biolo-gical evaluations, and therapeutic prospects[J]. Med Res Rev,2006,26 (1):88-130.
[5]Mizutani S, Shibata K, Kikkawa F, et al. Essential role of placental leucine aminopeptidase in gynecologic malignancy[J]. Expert Opin Ther Targets,2007,11 (4):453-461.
[6]Saiki I, Fujii H, Yoneda J, et al. Role of aminopeptidase N (CD13) in tumor-cell invasion and extracellular matrix degradation[J]. Int J Cancer,1993,54 (1):137-143.
[7]Menrad A, Speicher D, Wacker J, et al. Biochemical and functional characterization of amino-peptidase N expressed by human melanoma cells[J]. Cancer Res,1993,53(6):1450-1455.
[8]陈茂营,徐文方.蛋白酶体与蛋白酶体抑制剂[J].中国药物化学杂志,2007,17(4)：249-253
[9]Pauly RP, Demuth HU, Rosche F, et al. Improved glucose tolerance in rats treated with the dipeptidyl peptidase IV (CD26) inhibitor Ile-thiazolidide[J]. Metabolism,1999,48 (3):385-389.
[10]Rawlings ND, Barrett AJ. MEROPS:the peptidase database[J]. Nucleic Acids Res,1999,27 (1):325-331
[11]Xu W, Li Q. Progress in the development of aminopeptidase N (APN/CD13) inhibitors[J]. Curr Med Chem Anticancer Agents,2005,5 (3):281-301.
[12]Sjostrom H, Noren O, Olsen J. Structure and function of aminopeptidase N[J]. Adv Exp Med Biol,2000,477(8):25-34.
[13]Beate F, Marco A, Karin,et al. Estracellular cysteines define ectopeptidase (APN,CD13) esp-ression and function[J]. Free Radic. Biol.Med,2002,32(7):584-595.
[14]Ito K, Nakajima Y, Onohara Y, et al. Crystal structure of aminopeptidase N (proteobacteria alanyl aminopeptidase) from Escherichia coli and conformational change of methionine 260 involved in substrate recognition[J]. J Biol Chem,2006,281 (44):33664-33676.
[15]Jiang, M, Bond, JS, Families of metalloendopeptidases and their relationships[J]. FEBS Lert, 1992,312(2-3):110-114.
[16]Riemann D, Kehlen A, and Langner J. CD13-Not just a marker in leukemia typing[J]. Imm-unol Today,1999,20 (2):83-88.
[17]Teruki S, Keuji T, Kaoyoko H, et al. CD 13/Aminopeptidase N-Induced lymphocyte involvement in inflamed jionts of patients with rheumatoid arthritis[J]. Arthritis Rheum, 2002,46 (9):2330-2338.
[18]Emre S, Levent M, Senturk, et al. Expression of aminopeptidase N in human endom trium and regulation of its activity by estrogen[J]. Fertil Steril,2001,75 (6):1172-1176.
[19]Santos O A, Botbol V,Bestatin as experimental tool in mammals[J]. Curr Drug Metab,2001, 2(1):67-85.
[20]Santos AN, Langner J, Herrmann M, et al. Aminopeptidase N/CD13 is directly linked to sig-nal transduction pathways in monocytes[J]. Cell Immunol,2000,201 (1):22-32.
[21]Takeo H, Masahiro K, Kuniki K, et al. Enhancement of sensitivity by bestatin of acute promyelocytic leukemia NB4cell to all-trans retinoic acid[J]. Leukemia Res,2002,26 (12): 1097-1103.
[22]Lochter A. Bissell MJ. An odyssey from breast to bone:muftistep control ofmammary meta-stases and osteolysis by matrix metalloproteinases[J]. APMIS,1999,107(1):128-136.
[23]Folkman J. Seminars in Medicine of the Beth Israel Hospital, Boston. Clinical applications of research on angiogenesis[J]. N Engl J Med 1995,333(26):1757-1763.
[24]Martin DC, Fowlkes JL, Babic B, et al. Insulin-like growth factor Ⅱ signaling in neoplastic proliferation is blocked by transgenic expression of the metalloproteinase inhibitor TIMP-1 [J]. J Cell Biol,1999,146 (4):881-892.
[25]David E, Wentworth, Kathryn VH, Molecular determinants of species specificity in the coro-navirus receptor aminopeptidase N(CD13):influence of N-linked glycosylation[J]. J Virol, 2001,75 (20):9741-9752.
[26]Hanahan D, Folkman J,Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis[J]. Cell,1996,86 (3):353-364.
[27]R Pasqualini, E.Koivunen, R.Kain, et al. Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis[J]. Cancer Res,2000,60(12):722-727.
[28]Shim JS, Kim JH, Cho HY, et al. Irreversible inhibition of CD13/aminopeptidase N by the antiangiogentic agent curcumin[J]. Chem Biol,2003,10 (8):695-704.
[29]Alexander NS, Juergen L, Manfred H, et al. Aminopeptadase N/CD13 is directly linked to signal transduction pathways in monocytes[J]. Cellular Immunology,2000,201:22-32.
[30]Zetter BR, Angiogensis and tumor metastasis[J]. Annu Rev Med,1998,49(3):407-424.
[31]Shripad V, Bhagwat, Nenad P, et al. The angiogenic regulator CD13/APN is a transcriptional target of Ras signaling pathways in endothelial morphogenesis[J]. Blood,2003,101(5): 1818-1826.
[32]Ikeda N, NakajimaY, Tokuhara T, et al. Clinical significance of aminopeptidase N/CD13 exp-ression in human pancreatic carcinoma[J]. Clin Cancer Res,2003,9(4):1503-1508.
[33]Bhagwat SV, Lahdenranta J, Giordano R, et al. CD13/APN is a activated by angiogenic signals and is essential for capillary tube formation[J]. Blood,2001,97(3):652-659.
[34]Hiroki H, Arimichi T, Michiyuki K, Aminopeptidase N is involved in cell motility angio-gensis:Its clinical significance in human colon cancer[J]. Gastroenterology,2002,122(2): 376-386.
[35]Krakoff IH, Magill GB, Effects of 2-ethylamino-1,3,4 thiadiazole HCI on uric acid produc- tion in man[J]. Proc Soc Exp Biol Med,1956,91 (3):470-472.
[36]Elson PJ, Kvols LK, Vogl SE, et al. Phase Ⅱ trials of 5 day vinblastine infusion (NSC 49842), L-alanosine (NSC 153353), acivicin (NSC 163501), and aminothiadiazole (NSC 4728) in patients with recurrent or metastatic renal cell carcinoma[J]. Invest New Drugs,1988,6 (2): 97-103.
[37]Locker GY, Kilton L, Khandekar JD, et al. High-dose aminothiadiazole in advanced color-ectal cancer. An Illinois Cancer Center phase Ⅱ trial[J]. Invest New Drugs,1994,12 (4):299-301.
[38]Asbury RF, Wilson J, Blessing JA, et al. Aminothiadiazole (NSC 4728) in patients with advanced ovarian carcinoma. A phase Ⅱ study of the Gynecologic Oncology Group[J]. Am J Clin Oncol,1986,9 (4):334-336.
[39]Asbury RF, Blessing JA, McGuire WP, et al. Aminothiadiazole (NSC 4728) in patients with advanced carcinoma of the endometrium. A phase Ⅱ study of the Gynecologic Oncology group[J]. Am J Clin Oncol,1990,13(1):39-41.
[40]李少华,李刚,黄惠明,等.1,3,4-噻二唑衍生物的合成及初步活性测定[J].南昌大学学报,2009,33(3)：261-263.
[41]刘玉婷,晏会新,尹大伟,等.2,5-二取代-1,3,4-噻二唑类化合物的合成机应用研究[J].精细化工中间体,2009,39(01)：8-12.
[42]宋宝安,陈才俊,杨松,等.2-取代硫醚-5-(3,4,5-三甲氧基苯基)-1,3,4-噻二唑类化合物的合成、结构与体外抗癌活性[J].化学学报,2005,63(18)：1720-1726.
[43]杨锐生,胡国强,谢松强,等.2-(5-取代-1,3,4-噁二唑-2-亚甲硫)-5-吡啶-3-基-1,3,4-噻二唑的合成与抗癌活性[J].中国药学杂志,2008,43(5)：388-390.
[44]郑开波,何俊,张杰.含1,3,4-噻二唑的-5-氟尿嘧啶衍生物的合成及其抗肿瘤活性[J].华西药学杂志,2008,23(5)：528-530.
[45]蒋庆琳,吴林艳,吴勇.N1-(芳)烷酰氧亚甲基-5-氟尿嘧啶的合成及其初步抗肿瘤活性的研究[J].华西药学杂志,2005,20(1)：12-14.
[46]石德清,陈琦,李中华.含氨基酸希夫碱的5-氟尿嘧啶衍生物的合成及其抗肿瘤活性[J].有机化学,2005,25(5)：549-553.
[47]Masao Tada, Antineoplastic Agents.The preparation of 5-fluorouracil-l-acetic acid derivat ives[J].Bull Chem Soc Japan,1975,48 (11):3427-3428.
[48]Rzeski W, Matysiak J, Kandefer-Szerszeh M.Anticancer,neuroprotective activities and com-putation studies of 2-amino-1,3,4-thiadiazole based compound [J]. Bioorg Med Chem,2007, 15(9):3201-3207.
[49]Barve V, Ahmed F, Adsule S, et al.Synthesis, molecular charaterization, and biological acti-vity of chromen-4-one in human cancer cell[J].J Med Chem,2006,49(13):3800-3808.
[50]胡国强,毋晓魁,王新,等.氟喹诺酮C3杂环取代衍生物的合成及抗肿瘤活性研究(Ⅰ)：环丙沙星噻二唑希夫碱[J].药学学报,2008,43(11)：1112-1115.
[51]张耀洲,王海燕,王新,等.噻二唑硫醚双腙的合成及抗肿瘤活性[J].化学试剂,2009,31(9)：682-684.
[52]Umezawa H, Aoyagi T, Suda H, et al. Bestatin, an inhibitor of aminopeptidase B, produced by actinomycetes[J]. J Antibiot (Tokyo),1976,29 (1):97-99.
[53]Pulido Cejudo G, Conway B, Proulx P, et al. Bestatin-mediated inhibition of leucine amino-peptidase may hinder HIV infection[J]. Antiviral Res,1997,36 (3):167-177.
[54]Mathe G. Bestatin, An aminopeptidase inhibitor with a multi-pharmacological function[J]. Biomed Pharmacother,1991,45 (2-3):49-54.
[55]Ota K, Kurita S, Yamada K, et al. Immunotherapy with bestatin for acute non-lymphocytic leukemia in adults[J]. Cancer Immunol Immunother,1986,23 (1):5-10.
[56]Ino K, Bierman PJ, Varney ML, et al. Monocyte activation by an oral immuno-modulator (bestatin) in lymphoma patients following autologous bone marrow transplantation[J]. Cancer Immunol Immunother,1996,43 (4):206-212.
[57]Aoyagi T, Yoshida S, Nakamura Y, et al. Probestin, a new inhibitor of aminopeptidase M, produced by Streptomyces azureus MH663-2F6. I. Taxonomy, production, isolation, phy-sico chemical properties and biological activities[J]. J Antibiot,1990,43(2):143-148.
[58]Repic Lampret B, Kidric J, Kralj B, et al. Lapstatin, a new aminopeptidase inhibitor prod-uced by Streptomyces rimosus, inhibits autogenous aminopeptidases[J]. Arch Microbiol, 1999,171(6):397-404.
[59]Nagai M, Kojima F, Naganawa H, et al. Phebestin, a new inhibitor of aminopeptidase N, produced by Streptomyces sp. MJ716-m3[J]. J Antibiot,1997,50(1):82-84
[60]Aoyagi T, Tobe H, Kojima F, et al. Amastatin, an inhibitor of aminopeptidase A, produced by actinomycetes[J]. J Antibiot,1978,31(6):636-638.
[61]Rich DH, Moon BJ, Harbeson S Inhibition of aminopeptidases by amastatin and bestatin derivatives. Effect of inhibitor structure on slow-binding processes [J]. J Med Chem,1984, 27(4):417-422.
[62]Umezawa H, Aoyagi T, Tanaka T, et al. Production of actinonin, an inhibitor of amino-peptidase M, by actinomycetes[J]. J Antibiot,1985,38(11):1629-1630.
[63]Sui Z., Salto R, Li J, et al. Inhibition of the HIV-1 and HIV-2 proteases by curcumin and curcumin boron complexes[J]. Bioorg Med Chem,1993,1(6):415-420.
[64]Shim JS, Kim JH, Cho HY, et al. Irreversible inhibition of APN/aminopeptidase N by the antiangiogenic agent curcumin[J]. Chem Biol,2003,10(8):695-704.
[65]Egan ME, Pearson M, Weiner SA, et al. Curcumin, a major constituent of turmeric, corrects cystic fibrosis defects[J]. Science,2004,304(5670):600-602.
[66]Shin JS, Lee HS, Shin J, et al. Psammaplin A, a marine natural product, inhibits amino-peptidase N and suppresses angiogenesis in vitro[J]. Cancer Lett,2004,203(2):163-169.
[67]Melzig MF, Bormann H, Betulinic acid inhibits aminopeptidase N activity[J]. Planta Med, 1998,64(7):655-657.
[68]Aoyagi T, Yoshida S, Matsuda N, et al. Leuhistin, a new inhibitor of aminopeptidase M, produced by Bacillus laterosporus BMI156-14F1. I. Taxonomy, production, isolation, physico-chemical properties and biological activities[J]. J Antibiot,1991,44(6):573-578.
[69]Chung MC, Lee HJ, Chun HK, et al. Bestatin analogue from Streptomyces neyaga-waensis SL-387[J]. Biosci Biotechnol Biochem,1996,60(5):898-900.
[70]王俊丽.L-异谷氨酰胺类氨肽酶N抑制剂的设计合成及初步活性评价[D].山东大学,2004.
[71]王亚洲.L-异谷氨酰胺类氨肽酶N抑制剂的设计合成及初步活性研究[D].山东大学,2005.
[72]陈茂营.L-赖氨酸类氨肽酶N抑制剂的设计、合成与初步活性研究[D].山东大学,2007.
[73]张丽,徐文方,张杰.吡咯烷类明胶酶抑制剂的合成及初步活性[J].中国药物化学杂志,2007,17(2)：78-84.
[74]李乾斌.环酰亚胺类肽金属蛋白酶抑制剂的设计、合成及其抗肿瘤活性研究[D].山东大学,2008.
[75]Shenvi AB, alpha-Aminoboronic acid derivatives:effective inhibitors of aminopeptidases[J]. Biochem,1986,25 (6):1286-1291.
[76]Giannousis PP, Bartlett PA, Phosphorus amino acid analogues as inhibitors of leucine amino-peptidase[J]. J Med Chem,1987,30 (9):1603-1609.
[77]Chen H, Noble F, Roques BP, et al. Long lasting antinociceptive properties of enkephalin degrading enzyme (NEP and APN) inhibitor prodrugs[J]. J Med Chem,2001,44 (21):3523-3530.
[78]Lejczak B, Kafarski P, Zygmunt J. Inhibition of aminopeptidases by aminophosphonates[J]. Biochem,1989,28 (8):3549-3555.
[79]Joseph MD,Computational approaches to structure-based ligand design[J]. Pharmacol Ther, 1999,84(2):179-191.
[80]Robertus J, Structures-based drug design ten years on[J]. Nat Struct Biol,1994,1 (6):352-354.
[81]Ito K, Nakajima Y, Onohara Y, et al. Crystal structure of aminopeptidase N (proteobacteria alanyl aminopeptidase) from Escherichia coli and conforma-tional change of methionine 260 involved in substrate recognition[J]. J Biol Chem,2006,281 (44):33664-33676.
[82]Mina-Osorio P,The moonlighting enzyme CD13:old and new functions to target[J]. Trends Mol Med,2008,14(8):361-371
[83]Kramer RZ, Bella J, Mayville P, et al. Sequence dependent conformational variations of collagen triple-helical structure[J]. Nat Struct Biol,1999,6(5):454-457.
[84]汪多仁,陈体庆.L-苹果酸的开发与应用[J].饮料工业,2004,1(7)：21-26.
[85]王锐清.L-苹果酸的生产及应用研究进展[J].化工时刊,2002,16(5)：1-9.
[86]Morris GM, Goodsell DS, Halliday RS, et at. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function [J]. J Com Chem,1998, 199(14):1639-1662.
[87]Moriguchi I, Hirono S, Liu Q, et al. Simple Method of Calculating Octanol/Water Partition Coefficient[J]. Chem. Pharm. Bull,1992,40(1):127-130.
[88]杨二冰,李正名.药物分子设计中的Lipinski规则[J].化学通报,2006,69(1)：16-19.
[89]Kubinyi H, Drug research:myths, hype and reality[J]. Nature Reviews Drug Dicovery,2003, 2 (8):665-668.
[90]Lipinski CA, Chris Lipinski discusses life and chemistry after the Rule of Five[J]. Drug Discovery Today,2003,8(1):12-16.
[91]郑浩,董慧民,丁呈华等,2-氨基-1,3,4-噻二唑的合成新方法[J].河南师范大学学报：自然科学版.2006,5(2)：68-70.
[92]孟歌,韩紫岩,牛保华等.2-氨基-5-苯基噻二唑及其衍生物的合与表征[J].开封医专学报.1999,18(1)：44-46.
[93]吕艳阳,张玉霞.2,5-二(邻羟苯基)-1,3,4-噻二唑与钴的显色反应的研究[J].分析实验室,2006,25(4)：17-19.
[94]Henrot, S.; Larcheveque, M.; Petit, Y. Amino-acids as chiral synthons-preparation of enantiomerically pure (R) and (S) malic-acids and its application to the synthesis of 3-hydroxy 4-butanlide[J]. Synthetic Commun,1986,16 (2),183-190.
[95]刘全忠,龚流柱,蒋耀忠ZnCl2-KBH4还原苹果酸和天冬氨酸的研究[J].有机化学,2004,24(6)：637-640.
[96]Golovlyova SM, Moskvichev YA, Alov EM, et al. Synthesis of Novel Five-membered Nitrogen-containing Heterocyclic Compounds from Derivatives of Arylsulfonyl and Arylthioacetic and Propionic Acids[J]. Chemistry of Heterocyclic Compounds,2001,37 (9): 1102-1106.
[97]Spillane, WJ.; Kelly, LM.; Feeney, BG., et al. Synthesis of heterosulfamates.Search for structure-taste relationships[J]. Arkivoc,2003(vii):297-309.
[98]Li, Z.; Yu, J.-Y.; Yang, J.-Y, et al. Polymer-supported dichlorophosphate:a recoverable new reagent for synthesis of 2-amino-1,3,4-thiadiazoles[J]. J Chem Res,2005,36(43):341-343..
[99]Drag M, Grembecka J, Pawelczak M, et al. alpha-Aminoalkylphosphonates as a tool in experimental optimisation of Pl side chain shape of potential inhibitors in Sl pocket of leucine and neutral aminopeptidases[J]. Eur J Med Chem,2005,40 (8):764-77'1.
[100]Slater TF, Sawyer B, Strauli U,Studies on succinate-tetrazolium reductase system. Ⅲ. Points of coupling of four different tetrazolium salts[J]. Biochem. Biophys. Acta.,1963,77 (11):383-393.
[101]Berridge, MV.; Tan, AS. Characterization of cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT):subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction[J]. Arch. Biochem. Biophys,1993,303(2):474-482.
[102]Cell Proliferation Kit I (MTT). Roche Mol. Biochem.,1993,3:1-4. (http://www.roche-applied-science.com/pack-insert/1465007a.pdf)
[103]Vlachy, N; Touraud, D; Heilmann, J, et al. Determining the cytotoxicity of catanionic surfactant mixtures on HeLa cells[J]. Colloid Surface B,2009,70(2):278-280.
[104]吴舟锋,焦宛,王炳生.肿瘤化疗药物敏感性试验[J].国外医学外科学分册,1998,25(3)：153-155.
[105]郭杨,王增林,任金荣.MTT法在肿瘤化疗药物敏感性试验中的应用[J].华北煤炭医学院学报,1999,1(2)：99-101.
[106]Zhang ZH B, Jia L, Feng YJ, et al. Overexpression of follicle-stimulating hormone rece-ptor facilitates the development of ovarian epithelial cancer [J]. Cancer Lett,2009, 278(1):56-64.
[107]Gonzalez MA, Correa RJ, Agudelo L, et al. Synthesis and biological evaluation of abietic acid derivatives[J]. Eur J Med Chem,2009,44(6):2468-2472.
[108]Chandregowda V, Kush A, Reddy GC, Synthesis of benzamide derivatives of anacardic acid and their cytotoxic activity[J]. Eur J Med Chem,2009,44(16):2711-2719.
[109]Liu ZH, Zeng S,Cytotoxicity of ginkgolic acid in Hep-2 cells and primary rat hepat-ocytes[J]. Toxicol Lett,2009,187(3):131-136.
[110]Benkli K, Tunali Y, Canturk Z, et al. Cytotoxic and genotoxic effects of [Ru(phi)3]2+eval-uated by Ames/Salmonella and MTT methods[J]. Eur J Med Chem,2009,44(18):2601-2605.
[111]Liu F, Wang JJ, You ZY, et al. Radiosensitivity of prostate cancer cells is enhanced by EGFR inhibitor C225[J]. Urol oncol,28(1):59-66.
[112]Chang CJ, Hsu CC, Yung MC, et al. Enhanced radiosensitivity and radiation induced apoptosis in glioma CD133-positive cells by knockdown of SirT1 expression[J]. Biochem Bioph Res Co,2009,380(2):236-242.
[113]Zhou JJ, Yue XF, Han JX, et al. Improved MTT Assay for Activity of Antitumor Agents [J]. Chinese Journal of Pharm,1993,24(10):455-457.
[114]MTT Cell Proliferation Assay Instructions. ATCC,2001,1-6. http://www.atcc.org/ portals/1/pdf/30-1010k.Pdf