以NOS为靶点的吡咯烷衍生物的设计、合成及初步抑制活性研究
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摘要
第一部分NO和NOS研究进展
NO是一种由一氧化氮合酶(NOS)氧化L-精氨酸(L-Arg)产生的生物第二信使。高浓度时，NO是一种防御肿瘤细胞和病原体的细胞毒素；低浓度时，作为一种信号在各种生理活动中起作用，包括血流的控制、神经传导、学习和记忆。NO的过度产生涉及到炎症、神经退化和血管性疾病，例如中风、阿耳茨海默氏病、败血性休克、炎性关节炎、结肠炎和糖尿病。病理条件下，和疾病相关的NO的产生发生改变，这使一氧化氮合酶成为一个吸引人的药物开发靶点。
一氧化氮合酶(NOS)主要有两类：结构型和诱导型。结构型一氧化氮合酶需要Ca~(2+)／钙调蛋白，进一步分为：神经亚型和内皮亚型。内皮亚型主要分布在血管内皮组织，产生低浓度的NO能降低血压和阻止血小板聚集。神经型一氧化氮合酶产生的NO作为神经递质控制神经传导。诱导亚型分布在激活的巨噬细胞和其它类型细胞，产生的NO在宿主防御反应中发挥重要作用。在生理条件下，诱导型一氧化氮合酶不存在哺乳动物的细胞里。许多种细胞包括巨噬细胞和平滑肌细胞被促炎症因子如内毒素(细菌脂多糖，LPS)，肿瘤坏死因子-α(TNF-α)和干扰素-γ(IFN-γ)诱导产生诱导型一氧化氮合酶。一氧化氮合酶涉及各种循环休克和炎症的发病机理。诱导型一氧化氮合酶过度产生的NO病理生理学的重要性表明抑制iNOS具有潜在的治疗价值。
一氧化氮合酶(NOS)抑制剂分为氨基酸衍生物抑制剂和非氨基酸类抑制剂。迄今报道的多数NOS抑制剂包含脒、胍和异硫脲等功能基。在这些化合物中，氨基酸类化合物对iNOS具有好的活性和选择性。
第二部分目标化合物的设计
亚氨基吡咯烷衍生物对hiNOS(与heNOS相比较)显示了较好的活性和选择性。构象严格限制的含吡咯烷的类肽显示了较好的活性和选择性。因此，吡咯烷环可被用作潜在的先导骨架用以开发新的选择性的NOS抑制剂。用构象限制的吡咯烷环作为骨架开发选择性NOS抑制剂。我们对吡咯烷骨架进行了如下修饰：(ⅰ)6-(3-硝基胍)正己酸被连接到吡咯烷的4-位以模拟L-NNA结构；(ⅱ)保留吡咯烷1-位自由仲氨基或引入Boc基团；(ⅲ)用具有不同取代基的苯胺和氨基酸甲酯连接到吡咯烷2-位的羧基上。参考鼠的iNOS(PDB ID 1r35)的三维结构，柔性对接被用来模拟目标化合物与靶酶对接。
第三部分目标化合物的合成
所有的目标化合物从反式羟脯氨酸经过10步或11步反应制得。从反式羟脯氨酸经过酯化，Boc保护，甲烷磺酰化，叠氮钠的S_N 2亲核取代反应，氢化还原得到中间体6，S-甲基异硫脲硫酸盐和6-氨基正己酸反应得到6-胍基正己酸，6-胍基正己酸在发烟硝酸和发烟硫酸中硝化得到中间体9，9和6反应得到(2S，4S)-1-叔丁基2-甲基4-[6-(3-硝基胍)正己酰胺]吡咯烷-1，2-二羧酸酯，脱掉甲酯后生成相应羧酸中间体11。11、氯甲酸异丁酯和N-甲基吗啡啉生成混合酸酐，然后氨解得到A和C系列目标化合物。A和C脱掉Boc就得到B和D系列目标化合物。所有化合物都未见报道，目标化合物用红外、核磁共振氢谱和电喷雾质谱进行了结构确证。
第四部分目标化合物的活性评价
对70个目标化合物的iNOS抑制活性进行了评价，初步的测试表明多数化合物都有抑制iNOS的活性。16个化合物A1、A3、A11、A18、A19、A21、A26、A27、B1、B5、B14、C1、C2、C3、C6、C11显示了较好的iNOS抑制活性并且优于阳性对照药L-NNA。化合物C2显示了最好的抑制活性(IC_(50)=0.24μM)。这些化合物将来可被用作研究新的iNOS抑制剂的先导物。
初步活性评价也表明：A系列抑制活性比B系列活性好：C系列抑制活性比D系列好。含苯胺和环己胺的目标化合物都显示了较好的iNOS抑制活性。目标化合物抑制nNOS和eNOS的活性评价正在进行。
结论，我们设计、合成了一系列的新的类肽吡咯烷衍生物，并对这些化合物进行了初步抑制活性评价。结果表明，一些化合物显示了比L-NNA更好的iNOS抑制活性。
Part 1 Advances in study of NO and NOS inhibitors
Nitric oxide(NO), an important biological second messenger, is produced by the oxidation of Z-arginine by nitric oxide synthases(NOS). Nitric oxide functions at high concentrations as a defensive cytotoxin against tumor cells and pathogens; and at low concentrations as a signal in many physiological processes including blood flow regulation, neurotransmission, learning and memory. Overproduction of NO has also been implicated in the pathogenesis of a number of inflammatory, neurodegenerative, and vascular diseases such as stroke Alzheimer's disease, septic shock, inflammatory arthritis, colitis and diabetes. The various pathophysiological conditions associated with alterations in the body's NO homeostasis make NOS an attractive target for drug development.
There are two major classes of NOS: constitutive and inducible. The constitutive enzymes require Ca~(2+)/calmodulin for activation and are further divided into neuronal and endothelial isoforms. The endothelial isoform is found predominantly in the vascular endothelium and generates low concentrations of nitric oxide which lowers blood pressure and inhibits platelet aggregation. Nitric oxide generated by the neuronal enzyme appears to function as a neurotransmitter regulating neuronal transmission. The inducible isoform is found in activated macrophages as well as many other cell types and produces nitric oxide which plays an important role in host defense. iNOS, not existed in mammal cells in the condition of physiology, can be induced in a variety of cells including macrophages and smooth muscle cells by pro-inflammatory agents such as endotoxin (bacterial lipopolysaccharide, LPS), tumor necrosis factor-α(TNF-α) and interferon-γ(IFN-γ), which has been implicated in the pathogenesis of various forms of circulatory shock and inflammation. The pathophysiological importance of over-production of NO by iNOS suggests that iNOS inhibitors have potential therapeutic use.
NOS inhibitors can be divided into two types, amino-acid derivative inhibitors and non-amino acid-based inhibitors, by structural features. Most inhibitors of NOS thus far reported contain amidine, guanidine and isothiourea function groups. Among those, compounds of amino acids have been reported to exhibit a good activity and selectivity to inhibit NOS.
Part 2 Design of targeted compounds
It was reported that 2-iminopyrrolidines had been shown to be potent and selective inhibitors of the human inducible nitric oxide synthase (hiNOS) isoform versus the human endothelial nitric oxide synthase (heNOS). Conformationally restricted pyrrolidine-containing peptidomimetics showed better potent and selective inhibitory activities against iNOS. Therefore, the pyrrolidine ring could be used as potential lead scaffold to develop new selective iNOS inhibitors.
We use conformationally restricted pyrrolidine ring as a scaffold to develop selective pyrrolidine peptidomimetics inhibitors of the isoforms of NOS. This scaffold is optimized with following chemical modification: (i) 6-(3-nitroguanidino)hexanoic acid fragment was linked to 4-position of pyrrolidine so as to mimic the structure of L-NNA; (ii) keep the free secondary amine or introduce Boc in 1-position of pyrrolidine, respectively; (iii) using different substituted aromatic ring and amino acids methyl ester connected to the nitrogen atoms of amide derived from carboxylic group of proline. According to the 3D structure of murine iNOS (PDB ID 1r35), FlexX Docking was used for modeling the target compounds with the enzyme.
Part 3 Synthesis and discussion of targeted compounds
All target compounds were prepared in 10 or 11 steps from trans-4-hydroxy-L-proline. Briefly the intermediate 6 was prepared starting from commercially available trans-4-hydroxy-L-proline, followed by the sequential reactions of esterification, N-Boc protection, mesylation, S_N 2 displacement with sodium azide, and hydrogenation over 5% Pd/CaCO_3. The sulfate salt of 2-methyl-2-thiopseudourea, reacted with 6-aminocaproic acid to give intermediate 8 and then converted to 9 by nitration in fuming nitric acid and 50% fuming sulfuric acid. Acylation of compound 6 with 9 afforded the key intermediate 10. The series A and C were obtained through the reaction sequence including saponification, condensation from intermediate 10, and series B and D were obtained through N-Boc deprotection from A and C. All the targeted compounds have not been reported by now, and the structures of new compounds are identified by IR, ~1H-NMR and ESI-MS.
Part 4 Study on the inhibitory activityies against iNOS
The inhibitory effects on iNOS of 70 target compounds were evaluated in this thesis. The preliminary test results showed that most of compounds have ability to inhibit iNOS. Sixteen compounds, A1、A3、A11、A18、A19、A21、A26、A27、B1、B5、B14、C1、C2、C3、C6、C11 displayed good ability to inhibit iNOS and superior to positive control drug L-NNA. Compound C2 showed the best inhibitory activity (IC_(50)=0.24μM). These compounds could be used as lead compounds for exploring novel iNOS inhibitors in the future.
The preliminary test results also showed that series A displayed better inhibitory activities than series B, and series C than D, respectively. Aniline-containing and cyclohexylamine-containing compounds displayed better inhibitory activities.
In conclusion, a series of novel pyrrolidine peptidomimetics derivatives have been designed and synthesized. The preliminary inhibitory activities of target compounds were evaluated in this thesis. Some of the compounds displayed better inhibitory activities against iNOS than L-NNA.
NO是一种由一氧化氮合酶(NOS)氧化L-精氨酸(L-Arg)产生的生物第二信使。高浓度时，NO是一种防御肿瘤细胞和病原体的细胞毒素；低浓度时，作为一种信号在各种生理活动中起作用，包括血流的控制、神经传导、学习和记忆。NO的过度产生涉及到炎症、神经退化和血管性疾病，例如中风、阿耳茨海默氏病、败血性休克、炎性关节炎、结肠炎和糖尿病。病理条件下，和疾病相关的NO的产生发生改变，这使一氧化氮合酶成为一个吸引人的药物开发靶点。
一氧化氮合酶(NOS)主要有两类：结构型和诱导型。结构型一氧化氮合酶需要Ca~(2+)／钙调蛋白，进一步分为：神经亚型和内皮亚型。内皮亚型主要分布在血管内皮组织，产生低浓度的NO能降低血压和阻止血小板聚集。神经型一氧化氮合酶产生的NO作为神经递质控制神经传导。诱导亚型分布在激活的巨噬细胞和其它类型细胞，产生的NO在宿主防御反应中发挥重要作用。在生理条件下，诱导型一氧化氮合酶不存在哺乳动物的细胞里。许多种细胞包括巨噬细胞和平滑肌细胞被促炎症因子如内毒素(细菌脂多糖，LPS)，肿瘤坏死因子-α(TNF-α)和干扰素-γ(IFN-γ)诱导产生诱导型一氧化氮合酶。一氧化氮合酶涉及各种循环休克和炎症的发病机理。诱导型一氧化氮合酶过度产生的NO病理生理学的重要性表明抑制iNOS具有潜在的治疗价值。
一氧化氮合酶(NOS)抑制剂分为氨基酸衍生物抑制剂和非氨基酸类抑制剂。迄今报道的多数NOS抑制剂包含脒、胍和异硫脲等功能基。在这些化合物中，氨基酸类化合物对iNOS具有好的活性和选择性。
第二部分目标化合物的设计
亚氨基吡咯烷衍生物对hiNOS(与heNOS相比较)显示了较好的活性和选择性。构象严格限制的含吡咯烷的类肽显示了较好的活性和选择性。因此，吡咯烷环可被用作潜在的先导骨架用以开发新的选择性的NOS抑制剂。用构象限制的吡咯烷环作为骨架开发选择性NOS抑制剂。我们对吡咯烷骨架进行了如下修饰：(ⅰ)6-(3-硝基胍)正己酸被连接到吡咯烷的4-位以模拟L-NNA结构；(ⅱ)保留吡咯烷1-位自由仲氨基或引入Boc基团；(ⅲ)用具有不同取代基的苯胺和氨基酸甲酯连接到吡咯烷2-位的羧基上。参考鼠的iNOS(PDB ID 1r35)的三维结构，柔性对接被用来模拟目标化合物与靶酶对接。
第三部分目标化合物的合成
所有的目标化合物从反式羟脯氨酸经过10步或11步反应制得。从反式羟脯氨酸经过酯化，Boc保护，甲烷磺酰化，叠氮钠的S_N 2亲核取代反应，氢化还原得到中间体6，S-甲基异硫脲硫酸盐和6-氨基正己酸反应得到6-胍基正己酸，6-胍基正己酸在发烟硝酸和发烟硫酸中硝化得到中间体9，9和6反应得到(2S，4S)-1-叔丁基2-甲基4-[6-(3-硝基胍)正己酰胺]吡咯烷-1，2-二羧酸酯，脱掉甲酯后生成相应羧酸中间体11。11、氯甲酸异丁酯和N-甲基吗啡啉生成混合酸酐，然后氨解得到A和C系列目标化合物。A和C脱掉Boc就得到B和D系列目标化合物。所有化合物都未见报道，目标化合物用红外、核磁共振氢谱和电喷雾质谱进行了结构确证。
第四部分目标化合物的活性评价
对70个目标化合物的iNOS抑制活性进行了评价，初步的测试表明多数化合物都有抑制iNOS的活性。16个化合物A1、A3、A11、A18、A19、A21、A26、A27、B1、B5、B14、C1、C2、C3、C6、C11显示了较好的iNOS抑制活性并且优于阳性对照药L-NNA。化合物C2显示了最好的抑制活性(IC_(50)=0.24μM)。这些化合物将来可被用作研究新的iNOS抑制剂的先导物。
初步活性评价也表明：A系列抑制活性比B系列活性好：C系列抑制活性比D系列好。含苯胺和环己胺的目标化合物都显示了较好的iNOS抑制活性。目标化合物抑制nNOS和eNOS的活性评价正在进行。
结论，我们设计、合成了一系列的新的类肽吡咯烷衍生物，并对这些化合物进行了初步抑制活性评价。结果表明，一些化合物显示了比L-NNA更好的iNOS抑制活性。
Part 1 Advances in study of NO and NOS inhibitors
Nitric oxide(NO), an important biological second messenger, is produced by the oxidation of Z-arginine by nitric oxide synthases(NOS). Nitric oxide functions at high concentrations as a defensive cytotoxin against tumor cells and pathogens; and at low concentrations as a signal in many physiological processes including blood flow regulation, neurotransmission, learning and memory. Overproduction of NO has also been implicated in the pathogenesis of a number of inflammatory, neurodegenerative, and vascular diseases such as stroke Alzheimer's disease, septic shock, inflammatory arthritis, colitis and diabetes. The various pathophysiological conditions associated with alterations in the body's NO homeostasis make NOS an attractive target for drug development.
There are two major classes of NOS: constitutive and inducible. The constitutive enzymes require Ca~(2+)/calmodulin for activation and are further divided into neuronal and endothelial isoforms. The endothelial isoform is found predominantly in the vascular endothelium and generates low concentrations of nitric oxide which lowers blood pressure and inhibits platelet aggregation. Nitric oxide generated by the neuronal enzyme appears to function as a neurotransmitter regulating neuronal transmission. The inducible isoform is found in activated macrophages as well as many other cell types and produces nitric oxide which plays an important role in host defense. iNOS, not existed in mammal cells in the condition of physiology, can be induced in a variety of cells including macrophages and smooth muscle cells by pro-inflammatory agents such as endotoxin (bacterial lipopolysaccharide, LPS), tumor necrosis factor-α(TNF-α) and interferon-γ(IFN-γ), which has been implicated in the pathogenesis of various forms of circulatory shock and inflammation. The pathophysiological importance of over-production of NO by iNOS suggests that iNOS inhibitors have potential therapeutic use.
NOS inhibitors can be divided into two types, amino-acid derivative inhibitors and non-amino acid-based inhibitors, by structural features. Most inhibitors of NOS thus far reported contain amidine, guanidine and isothiourea function groups. Among those, compounds of amino acids have been reported to exhibit a good activity and selectivity to inhibit NOS.
Part 2 Design of targeted compounds
It was reported that 2-iminopyrrolidines had been shown to be potent and selective inhibitors of the human inducible nitric oxide synthase (hiNOS) isoform versus the human endothelial nitric oxide synthase (heNOS). Conformationally restricted pyrrolidine-containing peptidomimetics showed better potent and selective inhibitory activities against iNOS. Therefore, the pyrrolidine ring could be used as potential lead scaffold to develop new selective iNOS inhibitors.
We use conformationally restricted pyrrolidine ring as a scaffold to develop selective pyrrolidine peptidomimetics inhibitors of the isoforms of NOS. This scaffold is optimized with following chemical modification: (i) 6-(3-nitroguanidino)hexanoic acid fragment was linked to 4-position of pyrrolidine so as to mimic the structure of L-NNA; (ii) keep the free secondary amine or introduce Boc in 1-position of pyrrolidine, respectively; (iii) using different substituted aromatic ring and amino acids methyl ester connected to the nitrogen atoms of amide derived from carboxylic group of proline. According to the 3D structure of murine iNOS (PDB ID 1r35), FlexX Docking was used for modeling the target compounds with the enzyme.
Part 3 Synthesis and discussion of targeted compounds
All target compounds were prepared in 10 or 11 steps from trans-4-hydroxy-L-proline. Briefly the intermediate 6 was prepared starting from commercially available trans-4-hydroxy-L-proline, followed by the sequential reactions of esterification, N-Boc protection, mesylation, S_N 2 displacement with sodium azide, and hydrogenation over 5% Pd/CaCO_3. The sulfate salt of 2-methyl-2-thiopseudourea, reacted with 6-aminocaproic acid to give intermediate 8 and then converted to 9 by nitration in fuming nitric acid and 50% fuming sulfuric acid. Acylation of compound 6 with 9 afforded the key intermediate 10. The series A and C were obtained through the reaction sequence including saponification, condensation from intermediate 10, and series B and D were obtained through N-Boc deprotection from A and C. All the targeted compounds have not been reported by now, and the structures of new compounds are identified by IR, ~1H-NMR and ESI-MS.
Part 4 Study on the inhibitory activityies against iNOS
The inhibitory effects on iNOS of 70 target compounds were evaluated in this thesis. The preliminary test results showed that most of compounds have ability to inhibit iNOS. Sixteen compounds, A1、A3、A11、A18、A19、A21、A26、A27、B1、B5、B14、C1、C2、C3、C6、C11 displayed good ability to inhibit iNOS and superior to positive control drug L-NNA. Compound C2 showed the best inhibitory activity (IC_(50)=0.24μM). These compounds could be used as lead compounds for exploring novel iNOS inhibitors in the future.
The preliminary test results also showed that series A displayed better inhibitory activities than series B, and series C than D, respectively. Aniline-containing and cyclohexylamine-containing compounds displayed better inhibitory activities.
In conclusion, a series of novel pyrrolidine peptidomimetics derivatives have been designed and synthesized. The preliminary inhibitory activities of target compounds were evaluated in this thesis. Some of the compounds displayed better inhibitory activities against iNOS than L-NNA.
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