Design, Synthesis And Preliminary Activity Assay Of Pyrrolidine Derivatives Targeted At NOS

Part 1 Advances in study of NO and NOS inhibitorsNitric 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 Ca²⁺/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 compoundsIt 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 compoundsAll 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/CaCO₃. 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 iNOSThe 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 (IC₅₀=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.