L-异谷氨酰胺类肽化合物的设计、合成及活性研究
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摘要
氨肽酶是一类广泛存在于动、植物细胞的肽链端解酶,哺乳类动物的氨肽酶,包括氨肽酶A、B、N等数种。氨肽酶N(APN,CD13)是一类膜型锌离子依赖性的外肽酶,在人各种组织和细胞(内皮细胞、上皮细胞、成纤维细胞和白细胞)中都有广泛表达。在体内该酶能调节活性肽反应,影响人免疫功能,同时APN在恶性肿瘤细胞的增殖、分化、侵袭以及血管生成过程中起重要作用。尚有研究表明APN是人冠状病毒TEGV和229E的受体。因此APN是研究抗肿瘤抗病毒药物的一个重要靶点。
基于酶活性位点的三维结构以及已知APN抑制剂与酶的结合模式,利用Chem 3D Ultra 7.0软件进行分析和优化,本文设计并合成了两个系列共十五个L—异谷氨酰胺类肽衍生物。本研究以L—谷氨酸为基本骨架,3,4-二氯苯甲酸形成酰氯后与之发生酰化反应,经环合反应得到关键中间体环状酸酐(化合物3),再经氨解反应合成目标化合物,其化学结构经过红外光谱、电喷雾质谱和核磁共振谱(氢谱、碳谱、碳氢异核多键相关谱)确证。
对所合成的化合物均进行了体外初步生物活性评价,体外抑酶试验中,选择两类酶(APN和MMP-2,9)分别进行了活性筛选。在抑制APN活性试验中,以L-亮氨酰—对硝基苯胺为底物,测定化合物对APN的抑制活性;试验结果表明,本文设计合成的大多数目标化合物均具有较好的抑制APN的活性。初步构效关系分析,含有短脂肪侧链和芳香环氨基侧链的化合物均具活性,后者活性相对较强;同时含杂环的氨基侧链亦具有较强的活性,含有异羟肟酸基的化合物表现出较好的抑制活性。在抑制MMP-2,9活性试验中,以琥珀酰明胶为底物,TNBSA为显色剂,测定化合物对明胶酶的抑制活性;结果表明所合成的化合物对明胶酶也有一定的抑制作用,但不及对APN的抑制活性强。
本文所设计合成的L—异谷氨酰胺类肽化合物,在体外对氨肽酶表现出较好的抑制活性;初步筛选出一个活性较强、结构全新的APN抑制剂,预计其在体内也应具有较好的抗肿瘤活性,为创新性抗肿瘤药物的研究开发提供了新的结构骨架和研究方向。
Aminopeptidases are a group of exopeptidase which are widely distributed throughout the animal and plant kingdoms.There are a number of mammalian aminopeptidases which include aminopeptidase A,aminopeptidase B and aminopeptidase N.Aminopeptidases N(APN,CD13),one of membrane-bound zinc-dependent exopeptidase,is widely expressed in a wide variety of human tissues and cell types(endothelial,epithelial,fibroblast,leukocyte).In vivo,this enzyme is able to modulate bioactive peptide responses and to influence immune functions,at same time APN has been also reported to play an important role in the cell proliferation,secretion,invasion and angiogenesis of malignant tumor cells.It is also reported that APN is a receptor for corona-viruses TEGV and 229E in humans.All these findings make this enzyme as an interesting target for possible anti-tumor drugs research.
Based on the 3D structures and binding models of some novel APN inhibitors in complex with APN,two series of L-iso-glutamine derivatives are designed with the aid of Chem3D Ultra 7.0 Program.All compounds are synthesized starting from the acylation reaction of 3,4-dichlorobenzoic acid with L-glutamine to form the basic scaffold,followed by the cyclic reaction in acetic anhydride and the key intermediate cyclic anhydride(compound 3)was obtained.The intermediate compound is condensed with different amino acids or primary amines,and produces the target compounds.The chemical structures of target compounds are identified by IR,ESI-MS and NMR(~1H-NMR,~(13)C-NMR and HMBC)spectra.
Preliminary bioactivity assays are carried out in vitro.Inhibitory activity of compounds against APN is measured with L-leucine p-nitroanilide as substrate.As a result,most of these newly synthesized compounds show good inhibitory activity on APN.Structure-activity relationships of fifteen tested compounds are elucidated and compounds containing aromatic heterocyclic substituents have higher activity than straight side chain;compounds para-substituted by electron withdrawing group such as chlorine and nitro group had weak inhibiting activity.To determine the selectivity of these inhibitors towards the targeted enzyme,inhibiting activity on gelatinase(MMP-2,9),which is one of endo-peptidases of Zn~(2+)metallopeptidases, are measured by using succinylated gelatin as substrate.Data of the comparative studies exhibits the selectivity towards APN is higher than towards MMP-2,9.All the data strongly support that compounds containing carbonyl and hydroxamates show higher inhibiting activity than others significantly,and support the hypothesis that compounds inhibit APN by chelating to the zinc ion of this enzyme.
In this thesis,APN inhibitors of L-iso-glutamate peptidomimetic derivatives have good inhibiting activity both on APN in vitro.A new and powerful APN inhibitor is found in preliminary screening and predicted to have good activity in vivo.
基于酶活性位点的三维结构以及已知APN抑制剂与酶的结合模式,利用Chem 3D Ultra 7.0软件进行分析和优化,本文设计并合成了两个系列共十五个L—异谷氨酰胺类肽衍生物。本研究以L—谷氨酸为基本骨架,3,4-二氯苯甲酸形成酰氯后与之发生酰化反应,经环合反应得到关键中间体环状酸酐(化合物3),再经氨解反应合成目标化合物,其化学结构经过红外光谱、电喷雾质谱和核磁共振谱(氢谱、碳谱、碳氢异核多键相关谱)确证。
对所合成的化合物均进行了体外初步生物活性评价,体外抑酶试验中,选择两类酶(APN和MMP-2,9)分别进行了活性筛选。在抑制APN活性试验中,以L-亮氨酰—对硝基苯胺为底物,测定化合物对APN的抑制活性;试验结果表明,本文设计合成的大多数目标化合物均具有较好的抑制APN的活性。初步构效关系分析,含有短脂肪侧链和芳香环氨基侧链的化合物均具活性,后者活性相对较强;同时含杂环的氨基侧链亦具有较强的活性,含有异羟肟酸基的化合物表现出较好的抑制活性。在抑制MMP-2,9活性试验中,以琥珀酰明胶为底物,TNBSA为显色剂,测定化合物对明胶酶的抑制活性;结果表明所合成的化合物对明胶酶也有一定的抑制作用,但不及对APN的抑制活性强。
本文所设计合成的L—异谷氨酰胺类肽化合物,在体外对氨肽酶表现出较好的抑制活性;初步筛选出一个活性较强、结构全新的APN抑制剂,预计其在体内也应具有较好的抗肿瘤活性,为创新性抗肿瘤药物的研究开发提供了新的结构骨架和研究方向。
Aminopeptidases are a group of exopeptidase which are widely distributed throughout the animal and plant kingdoms.There are a number of mammalian aminopeptidases which include aminopeptidase A,aminopeptidase B and aminopeptidase N.Aminopeptidases N(APN,CD13),one of membrane-bound zinc-dependent exopeptidase,is widely expressed in a wide variety of human tissues and cell types(endothelial,epithelial,fibroblast,leukocyte).In vivo,this enzyme is able to modulate bioactive peptide responses and to influence immune functions,at same time APN has been also reported to play an important role in the cell proliferation,secretion,invasion and angiogenesis of malignant tumor cells.It is also reported that APN is a receptor for corona-viruses TEGV and 229E in humans.All these findings make this enzyme as an interesting target for possible anti-tumor drugs research.
Based on the 3D structures and binding models of some novel APN inhibitors in complex with APN,two series of L-iso-glutamine derivatives are designed with the aid of Chem3D Ultra 7.0 Program.All compounds are synthesized starting from the acylation reaction of 3,4-dichlorobenzoic acid with L-glutamine to form the basic scaffold,followed by the cyclic reaction in acetic anhydride and the key intermediate cyclic anhydride(compound 3)was obtained.The intermediate compound is condensed with different amino acids or primary amines,and produces the target compounds.The chemical structures of target compounds are identified by IR,ESI-MS and NMR(~1H-NMR,~(13)C-NMR and HMBC)spectra.
Preliminary bioactivity assays are carried out in vitro.Inhibitory activity of compounds against APN is measured with L-leucine p-nitroanilide as substrate.As a result,most of these newly synthesized compounds show good inhibitory activity on APN.Structure-activity relationships of fifteen tested compounds are elucidated and compounds containing aromatic heterocyclic substituents have higher activity than straight side chain;compounds para-substituted by electron withdrawing group such as chlorine and nitro group had weak inhibiting activity.To determine the selectivity of these inhibitors towards the targeted enzyme,inhibiting activity on gelatinase(MMP-2,9),which is one of endo-peptidases of Zn~(2+)metallopeptidases, are measured by using succinylated gelatin as substrate.Data of the comparative studies exhibits the selectivity towards APN is higher than towards MMP-2,9.All the data strongly support that compounds containing carbonyl and hydroxamates show higher inhibiting activity than others significantly,and support the hypothesis that compounds inhibit APN by chelating to the zinc ion of this enzyme.
In this thesis,APN inhibitors of L-iso-glutamate peptidomimetic derivatives have good inhibiting activity both on APN in vitro.A new and powerful APN inhibitor is found in preliminary screening and predicted to have good activity in vivo.
引文
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