Sinomenine-based Synthesis And Evaluation Of Anti-inflammatory Molecules

Rheumatoid Arthritis (RA), with a prevalence of 0.5-1% of the population, is a chronic inflammation and autoimmune disease that dispalys the feature of inflammation of the synovial lining of joints, progressive demolish of articular cartilage and thickening of the synovial tissue, followed by the limitation of bones’ action. However, its cause and pathology is very complicated and still remains ambiguous. Sinomenine [(9α,13α,14α)-7,8-didehydro-4-hydroxy-3,7-dimethoxy-17-methyl-mophinan-6-one] is a nature alkaloid isolated from the stem of Sinomenine acutum (Thunb.) Rehd. et Wils and Sinomenine acutum (Thunb.) Rehd. Et Wils. var. cinereum Rehd. et Wils. It has been clinically used for the treatment of inflammation and RA in China, the whole effective rate of sinomenine could up to 85% clinically, also it was used auxiliarily in treatment of RA by methotrexate (MTX), however, the efficacy of sinomenine for the treatment of RA is quite weak. Additionally, certain adverse effects have also been reported. Therefore, to get derivatives with better activity based on sinomenine scaffold, a structure modification was conducted. In this thesis, in an effort to develop novel molecules with better bioactivity and understand the structure-activity relationships (SAR), a series of stereodimers of sinomenine analogues were synthesized and their inhibitory activity on NO production was assayed, and furthermore the suppressive effect on mRNA expression of iNOS of the compounds with potent activity was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Additionally, a novel series of bivalent ligands with sinomenine as pharmacophore, as well as monovalent ligands were designed and synthesized through different linkers with various lengths (2-10 atoms), the inhibitory activity on NO production and cytotoxicity of the ligands were firstly screened using RAW264.7 cells, then suppressive effects on IL-6 and TNF-α of the compounds with potent NO inhibitory activity were assayed in vitro, and finally the preliminary mechanism and anti-inflammatory effects in vivo of two compounds (3-11 and 3-14) were further investigated.1. Synthesis and biological evaluation of unique stereodimers of sinomenine analogues as potential inhibitors of NO productionWe have designed, synthesized and biologically evaluated a series of unique stereodimers of sinomenine analogues on the inhibitory effect of NO overproduction and mRNA expression of iNOS using LPS-induced murine RAW264.7 cells. Among the synthesized compounds, five compounds displayed a potent inhibitory activity and also exerted the activity via suppressing mRNA expression of iNOS in a dose-dependent manner. Reduction of ring-C (2) could be beneficial to increase of the activity. The present results unfolded that dimerization of sinomenine analogues with weak steric hindrance (S-dimers) showed a better inhibitory potency both on NO overproduction and mRNA expression of iNOS, while those with a strong steric hindrance (R-dimers) showed almost no benefit on the enhancement of the activity, some of (R)-dimers (2-6, 2-8) even promoted the secretion of NO.RT-PCR experiment was performed to assay the effect of five selected compounds on mRNA expression of iNOS. The RT-PCR results revealed that compounds 2-1,2,2-3,2-4 and 4 markedly inhibited the mRNA expression of iNOS in a dose-dependent manner, while the parent compound 1 only faintly restrained the expression at 100 μM. These results revealed that the sinomenine analogues might play crucial role in the pathway involved iNOS.Why the (R, S)-dimers only with different angle around C1-C1’ bond exhibited reversed activity clearly warrant further studies, and now the mechanism clarification and further expansion of the related compounds are under way in our laboratory.2. Design, Synthesis and Biological Evaluation of Sinomenine Bivalent Ligands as Novel Anti-inflammatory AgentsSinomenine bivalent ligands together with monovalent ligands that linked via ester and ether bonds were designed, prepared and evaluated. The linker included aliphatic esters, aliphatic ethers, aromatic ethers and glycol ethers. Bivalent ligands of other monomer including 7-Demethoxy-6-desoxo-7,8-dihydro sinomenine, N-demethyl sinomenine,6β-NH-Ts-7β-methoxy-7,8-dihydro sinomenine, and 6,7-Quinoxaline sinomenine, were also synthesized.Firstly, we evaluated the inhibitory activity on NO production of all the synthesized compounds using LPS-induced RAW264.7 cells. For the ester bivalent and monovalent ligands:(ⅰ) When the linker length is short (carbon number is 4 to 6 for bivalent ligands,4 to 5 for monovalent ligands), almost no impacts on both activity and cytotoxicity were observed; (ⅱ) When the linker length is middle (carbon number is 7 to 8 for bivalent ligands,6 to 7 for monovalent ligands), the cytotoxicity IC50 exceed the activity IC50; (ⅲ) Compounds with longer linkers showed good inhibitory profile on NO production without obvious cytotoxicity. The results above revealed that the linker length in the ester-linked bivalent and monovalent ligands did impact the bio-profiles of the compounds, and the longer linkers could ameliorate the NO inhibitory activity.For the ether linked ligands, however, an opposite effect was observed compared with those of ester linked ones. It was of great interest that the ether bivalent ligands with shorter linker, such as compound 3-14 possessed IC50 values of 15.2 μM in inhibition of NO production, while longer alkyl chains induced obvious cytotoxicity. The phenyl dimethyl could lead to a visible inhibition in cell viability. Glycol scaffolds served little effects in the structural modification, with neither visible activity nor obvious cytotoxicity.The novel bivalent ligand (3-14) and monovalent ligand (3-11) effectively inhibited NO production in LPS-induced RAW264.7 cells, and their activity was enhanced much more compared with that of parent compound 1, also 3-11 and 3-14 selectively inhibited IL-6 and TNF-α production, respectively. Furthermore, the mechanism studies on the modulation of signaling kinase and transcription factor activities revealed that 3-14 specifically inhibited the phosphorylation of NF-κB and the degradation of IκBa in the NF-κB signaling pathway; while 3-11 not only suppressed IκBα, NF-αB phosphorylations, but also ERK1/2, JNK, and p38 MAP kinase phosphorylations in the MAPK signaling pathway.In vivo assay demonstrated that 3-14 and 3-11 displayed the protective effects against systemic inflammatory toxicity by LPS at least via reduction of both serum levels of TNF-α and IL-6. Especially,3-14 significantly alleviated the LPS-induced mortality. These results suggested that bivalent ligands of sinomenine might have a great potential to be novel leads for anti-inflammatory drug discovery. Further syntheses of related compounds, detailed mechanism studies including identification of the protein targets of 3-14 and 3-11, and biactivity evaluation of other bivalent ligands of sinomenine derivatives are in process in our laboratory.