Table of Contents Abstract I 摘要 IV Table of Contents VI Figures IX Schemes XVIII Tables XVIII Chapter 1 Synthesis of Paeonol Derivatives and Exploration of Their Inhibitory Activity against Hepatitis B Virus 1 1-1 Literature Review 2 1-1-1 Paeonol, previous works on structural modifications and the bioactivity of derivatives 2 1-1-2 Hepatitis B virus infection and chronic inflammation 6 1-1-3 Research Incentive 11 1-2 Material and Methods 12 1-2-1 Chemistry part 12 1-2-1-1 Chemicals and characterization methods 12 1-2-1-2 Synthetic procedures (see synthetic scheme 1-3-1-1 in section 1-3-1) 13 1-2-2 Biology part 22 1-2-2-1 Cell culture and reagents 22 1-2-2-2 Cell viability assay 22 1-2-2-3 Determination of HBsAg and HBeAg antigen levels 23 1-2-2-4 Real-time PCR analysis of HBV DNA level during treatment 23 1-2-2-5 Statistical analysis and quantification of data 23 1-2-2-6 Giemsa staining and flow cytometry 24 1-3 Results and Discussions 25 1-3-1 Synthesis 25 1-3-2 Cytotoxic effect of the compounds on HepG2 2.2.15 hepatoma cells 26 1-3-3 Antiviral effect of compounds 4a-4h and 5a-5e on HBV viral antigen expression in HepG2 2.2.15 cell culture medium 30 1-3-4 Antiviral effect of compounds 4a-4h and 5a-5e on HBV DNA replication in HepG2 2.2.15 cells 33 1-3-5 Inhibitory effect of compound 4a on HBV viral gene expression 34 1-4 Conclusions and Perspectives 36 1-5 References 37 Chapter 2 Development and Expansion of Unprecedented Desulfitative Substitution Reactions on Poly(2-chloro-3butylthiothiophene) with Five-Membered Heteroaromatic Terminal Alkynes 40 2-1 Literature Review 41 2-1-1 The synthetic methodology of polythiophenes 41 2-1-2 The introduction of -C≡CR as side chains 44 2-1-3 Sonogashira coupling 47 2-1-4 Thioorganics as electrophiles in some palladium catalyzed reactions 49 2-1-5 Research Incentive 54 2-2 Material and Methods 55 2-2-1 Chemicals (listed in alphabetical order) 55 2-2-2 Characterization methods 56 2-2-3 Synthetic procedures 59 2-2-3-1 Preparation of the five-membered heteroaryl terminal alkynes 59 2-2-3-2 Preparation of PBTT-Cl 64 2-3 Results and Discussion 67 2-3-1 Pilot experiments 67 2-3-2 Optimization study 72 2-3-3 Infrared spectroscopy analysis 119 2-3-4 X-ray photoelectron spectroscopy analysis 126 2-4 Conclusions and Perspectives 131 2-5 References 132 Appendix I 135 Appendix II 167 Figures Fig. 1-1-1 1 The structure of Paeonol 2 Fig. 1-1-1 2 The derivatives of Paeonol embodying (A) Schiff-base, (B) heterocyclic alkoxy and (C) sulfonate ester functional groups 3 Fig. 1-1-1 3 The structures of (A) thiazole, (B) 2-aminothiazole, (C) Pramipexole and (D) 2-(2-aminothiazol-4-yl)-5-methoxyphenol 4 Fig. 1-1-1 4 The sulfonyl conjugates of 2-(2-aminothiazol-4-yl)-5-methoxyphenol 5 Fig. 1-1-1 5 The sulfonate ester Paeonol derivatives functionalized with aminothiazole 5 Fig. 1-1-2 1 The mechanisms of HBV virus associated hepatocarcinogenesis. The predominant carcinogenic mechanism of HBV associated HCC is through the process of liver cirrhosis, but direct oncogenic effects of HBV may also contribute. 6 Fig. 1-1-2 2 Schematic representation of mechanisms of HBV replication and inhibition 8 Fig. 1-1-2 3 Inflammation resulting from chronic viral infection contributes to cancer development. Chronic activation of inflammatory signaling can be mediated directly by the virus or indirectly as a result of viral propagation. Direct activation of inflammation is generally mediated by viral proteins capable of activating inflammatory signaling cascades and/or host inflammatory cytokines. HBV and HCV especially, induce significant indirect inflammation as a result of viral replication induced ROS, lipid accumulation and immune recognition. A considerable amount of cell death and resulting hepatocyte regeneration ultimately leads to cancer development. 10 Fig. 1-3-2 1 The structure of Sorafenib 29 Fig. 1-3-3 1 Effects of compounds 4a-4h anf 5a-5e on HBV viral (A) HBsAg and (B) HBeAg secretion. HepG2 2.2.15 cells were treated with compounds 4a-4h and 5a-5e at three concentrations (0.75, 1.5 and 3.0 μM) or DMSO 0.1% (v/v) (control) for 72 h. The cultural medium of each treatment was collected for viral HBsAg and HBeAg EIA analysis. The data was expressed as the mean and the standard deviation of the mean (n = 3, *P < 0.05 vs. untreated cells). 31 Fig. 1-3-3 2 Effect of 4a on cellular morphology and DNA content analysis of HepG2 2.2.15 cells. HepG2 2.2.15 cells were exposed to three noncytotoxic concentrations (0.75, 1.5 and 3.0 M) of 4a. After 72 h of culture, cells were fixed and stained with Giemsa's solution to identify the morphology of HepG2 2.2.15 cells. (A) Based on cellular morphology, inhibition of HepG2 2.2.15 cell growth was not observed in cells treated with 8a. (B) Cell cycle distribution analysis of HepG2 2.2.15 treated with 8a was performed on flow cytometry. 32 Fig. 1-3-4 1 Effects of compounds 4a–4h and 5a-5e on HBV DNA replication in HepG2 2.2.15 cells. Cells were treated at three concentrations (0.75, 1.5 and 3.0 M) or with DMSO 0.1% (v/v) as the control for 72 h, and the culture medium of was collected for viral DNA extraction. The measurement of viral DNA was performed on real-time PCR analysis. The data was expressed as the mean and the standard deviation of the mean (n = 3) (* P < 0.05 vs. untreated cells). 33 Fig. 1-3-4 2 The SI values of compound 2, 4a-4h, 5a-5e and 3TC of inhibition against HBV DNA replication 34 Fig. 1-3-5 1 Inhibitory effect of compound 4a on HBV RNA expression in HepG2 2.2.15 cells. (A) HepG2 2.2.15 cells were treated at three non-cytotoxic concentrations (0.75, 1.5 and 3.0 M) for 72 h and total cellular RNA was extracted and subjected to northern blot analysis. (B) The intensity of each RNA band was quantitated with a densitometer and the relative amount was normalized with GAPDH loading control. The data shown was representative of three replicate experiments (* P < 0.05 vs. untreated cells). 35 Fig. 2-1-1 1 The mechanism of electropolymerization of five-membered heterocycles (X = S)2 41 Fig. 2-1-1 2 The proposed mechanism for oxidative polymerization with the use of FeCl37 42 Fig. 2-1-1 3 The proposed mechanism for the nickel-initiated cross-coupling polymerization8b 43 Fig. 2-1-1 4 The plausible mechanism for a chain-growth cationic polymerization induced by Brønsted acids6 44 Fig. 2-1-2 1 Packing model of HH-P3 (-C≡CR-alkyl)Th in solid state10 45 Fig. 2-1-2 2 Structures of PPA (poly(phenylacetylene)s) and polymers P1-P3 45 Fig. 2-1-3 1 The reaction mechanism proposed by Sonogashira et al.13 47 Fig. 2-1-3 2 The Sonogashira reaction. (a) General representation of Pd/Cu catalyzed and Cu-free Sonogashira reaction. (b) Textbook mechanism for the Pd/Cu catalyzed Sonogashira cross-coupling reaction that is synergistically catalyzed by Pd and Cu. (c) Textbook mechanism for Cu-free Sonogashira reaction. (d) The mechanistic proposal for Cu-free Sonogashira reaction. OA oxidative addition, TM transmetallation, RE reductive elimination (cis–trans isomerization steps are omitted for clarity)15 48 Fig. 2-1-4 1 The organosulfur electrophiles utilized in transition metal catalyzed C-C bond-forming reactions when the nucleophiles are Grignard reagents 49 Fig. 2-1-4 2 Metal-catalyzed cross-coupling with tetramethylenesulfonium salts21 50 Fig. 2-1-4 3 Nickel-catalyzed cross-coupling reactions of thioglycolic acids with organozinc reagents (a) and the transmetallation proceeded from an external zinc reagent to nickel, rather than from the internal organozinc R group bound to the thioglycolate (b)22 50 Fig. 2-1-4 4 Copper carboxylate mediated thiol ester-boronic acid cross-coupling23,24 51 Fig. 2-1-4 5 Pepitidyl ketones from peptidyl thiol esters and boronic acids25 51 Fig. 2-1-4 6 Palladium catalyzed carbon-sulfur bond methathesis26 51 Fig. 2-1-4 7 Desulfitative Sonogashira-type cross-coupling of heteroaryl thioethers with terminal alkynes27 52 Fig. 2-1-4 8 Cooperative catalysis of copper for the Sonogashira-type desulfitative reaction on cyclic thionocarbamates (oxazolinethiones and oxazolidinethiones) and the proposed mechanism.28 53 Fig. 2-1-4 9 Palladium-catalyzed Sonogashira-type cross-coupling of sulfonyl chlorides and terminal alkynes and its proposed mechanism19 53 Fig. 2-3-1 1 The 1H NMR spectra of 2-ethynyl-N-methylpyrrole (top; the peak from -CH3 (4.27 ppm) on CH3NO2 was used to estimate the concentration of product solution), PBTT-Cl (middle), and product after the 24-hour reaction with 10 mol% Pd(PPh3)4 and 10 mol% CuI as catalysts, TEA as base and solvent and toluene as solvent under 80 ℃ (bottom). 67 Fig. 2-3-1 2 The MALDI-TOF mass spectrum of product after the coupling between 2-ethynyl-N-methylpyrrole and PBTT-Cl under the reaction condition illustrated in Fig. 2-3-1-1. The nomenclature shown will be explained later in section 2-3-2. 68 Fig. 2-3-1 3 The 1H NMR spectra of 2-ethynylfuran (top; the peak from -CH3 (4.27 ppm) on CH3NO2 was used to estimate the concentration of product solution.), PBTT-Cl (middle), and product afforded after the 24-hour reaction with 10 mol% Pd(PPh3)4 and 10 mol% CuI as catalysts, TEA as base and solvent and toluene as solvent under 80 ℃ (bottom). The increase and decrease in the integral value were indicated with filled arrows. 69 Fig. 2-3-1 4 The MALDI-TOF mass spectrum of product after the coupling between 2-ethynylfuran and PBTT-Cl under the reaction condition illustrated in Fig. 2-3-1-3. The nomenclature shown will be explained in section 2-3-2. 70 Fig. 2-3-2 1 The 1H NMR spectra of PBTT-Cl (top) and products afforded after catalysis with 10 mol% Pd(PPh3)2Cl2 (middle) and 10 mol% Ni(PPh3)2Cl2 (bottom). In both conditions were THF employed as solvent, TEA as base/solvent under reflux for 12 hours (see the details in entry 1 and 2 in Table 2-3-2-1). 75 Fig. 2-3-2 2 The structures of molecules 7-1, 7-Cl-1 and 7’-1 appearing in the MALDI-TOF mass spectrum shown in Fig. 2-3-2-3. The mechanism of laser induced C-Cl bond cleavage is shown in Fig. 2-3-2-4. 77 Fig. 2-3-2 3 The MALDI-TOF mass spectra of the product in entry 1 with Pd(PPh3)2Cl2 as catalyst (Table 2-3-2-1) 78 Fig. 2-3-2 4 The laser induced C-Cl bond cleavage for the generation of molecule 7-1from 7-Cl-1 in Figure 2-3-2-2 (a) 79 Fig. 2-3-2 5 The plausible mechanism of the formation of R-1275 and R-1445 upon laser irradiation 79 Fig. 2-3-2 6 The enlargements of peaks corresponding to molecules 7 (top), 7-1 (middle), 7-Cl and 7-2 (bottom) in the MALDI-TOF mass spectrum of product in entry 1 (Table 2-3-2-1), and the predicted isotope patterns for the molecules above 80 Fig. 2-3-2 7 The enlargements of peaks corresponding to molecules 8 (top), 8-1 (middle), 8-Cl and 8-2 (bottom) in the MALDI-TOF mass spectrum of product in entry 1 (Table 2-3-2-1), and the predicted isotope patterns for the molecules above 83 Fig. 2-3-2 8 The MALDI-TOF mass spectra of product after the reaction with 10 mol% Ni(PPh3)2Cl2 as catalyst, 3 mL TEA as base and 3 mL THF as solvent under reflux for 12 hours (entry 2, Table 2-3-2-1) 86 Fig. 2-3-2 9 The enlargements and predicted patterns of peaks corresponding to molecules 7-Cl-3 (upper) and 8-Cl-3 (lower) in the MALDI-TOF mass spectra shown in Fig. 2-3-2-8 87 Fig. 2-3-2 10 The mechanisms of formation and predicted isotope patterns for rearranged molecules R-1259, R-1307, R-1347 and R-1429 in the MALDI-TOF mass spectra shown in Fig. 2-3-2-8 88 Fig. 2-3-2 11 The MALDI-TOF mass spectra of product after the reaction with 10 mol% Pd(PPh3)2Cl2 as catalyst, 3 equiv TEA as base and 10 mL THF as solvent under reflux for 3 hours (entry 3, Table 2-3-2-1) 90 Fig. 2-3-2 12 The MALDI-TOF mass spectra of product after the reaction with 10 mol% Pd(PPh3)2Cl2 as catalyst, 3 equiv DBU as base and 10 mL THF as solvent under reflux for 3 hours (entry 4, Table 2-3-2-1) 90 Fig. 2-3-2 13 The MALDI-TOF mass spectra of product after the reaction with 10 mol% Pd(PPh3)2Cl2 as catalyst, 3 equiv DBN as base and 10 mL THF as solvent under reflux for 3 hours (entry 5, Table 2-3-2-1) 90 Fig. 2-3-2 14 The correlation between max in UV spectroscopy and substitution degree of products from entry 3 to 8 (Table 2-3-2-1) 91 Fig. 2-3-2 15 The MALDI-TOF mass spectra of experiments in entry 7 (top) and entry 8 (bottom) with 3equiv DBU and DBN as bases respectively 92 Fig. 2-3-2 16 The full MALDI-TOF mass spectrum (A) and that of products having seven (B) and eight (C) repeat units from the experiment in which 10 mol% Pd(PPh3)2Cl2 was used as catalyst, 3 equiv DBN as base and 15 mL DMF as solvent under 80 ℃ for 24 hours (entry 14, Table 2-3-2-1) 94 Fig. 2-3-2 17 The enlargements of peaks corresponding to molecules 7 (top), 7-1 (middle) and 7-2 (bottom) in the MALDI-TOF mass spectrum of product in entry 14 (Table 2-3-2-1), and the predicted isotope patterns for the molecules listed above 95 Fig. 2-3-2 18 The MALDI-TOF mass spectra of products from 6-hour (upper two) and 24-hour (lower two) experiments where DMF was employed as solvent, 10 mol% Pd(PPh3)2Cl2 as catalyst and 3 equiv DBN as base under 80 ℃. A bit higher ratio of one side chain substituted product to its non-substituted starting molecule (the molecules 7-1 to 7, 7’-1 to 7’, 8-1 to 8 and 8’-1 to 8’) was observed in 24-hour trial. 106 Fig. 2-3-2 19 The MALDI-TOF spectra of experiments in entry 15 (top) and 16 (bottom). It can be seen clearly that the ratio of molecule 7-1 to 7 became higher with the aid of 5 mol% CuI. The same was true for the ratio of 7’-1 to 7’. 107 Fig. 2-3-2 20 The MALDI-TOF mass spectra of the experiments in entry 21without CuI co-catalyst (top, Table 2-3-2-1) and entry22 with 5 mol% CuI co-catalyst (bottom, Table 2-3-2-1). Both reactions were performed in DMF where 10 mol% Pd(PPh3)2Cl2 was used, DBN as base under 60 ℃ for 15 hours. 109 Fig. 2-3-2 21 The 1H NMR spectra of PBTT-Cl (top) and the product P-NMePy (bottom) 111 Fig. 2-3-2 22 The MALDI-TOF spectrum of the product P-NMePy. The enlargements of peaks, speculated molecules for the peaks, and theoretical isotope patterns for the molecules can be referred to Fig. 2-3-2-23 111 Fig. 2-3-2 23 The enlargements of peaks corresponding to molecules 7 (top), 7-N1 (middle) and 7-N2 (bottom) in the MALDI-TOF mass spectrum of product PNMePy (Table 2-3-2-2), and the predicted isotope patterns for the molecules listed above 112 Fig. 2-3-2 24 The MALDI-TOF mass result of product P-Furan 115 Fig. 2-3-2 25 The putative routes for deriving the molecules with M+1 value of 1209 from molecule 6’-2 (A) and 6-1 (B), and with M+1 value of 1211 (C) 116 Fig. 2-3-2 26 The enlargements together with structural assumptions and theoretical isotope patterns corresponding to peaks presented in the mass spectrum of product P-Furan 117 Fig. 2-3-2 27 The full (upper left), enlarged (upper right) MALDI-TOF mass spectra of product P-Se, and the predicted isotope pattern for molecule 7-Se1 119 Fig. 2-3-3 1 The IR spectrum of PBTT-Cl 120 Fig. 2-3-3 2 The IR spectra of PBTT-Cl and product afforded after the cross-coupling between PBTT-Cl and 2-ethynylthiophene with substitution degree of 32 % (entry 16; Table 2-3-2-1) 121 Fig. 2-3-3 3 The IR spectra of PBTT-Cl and PNMePy (substitution degree: 6 %) 122 Fig. 2-3-3 4 The IR spectra of PBTT-Cl and PFuran (substitution degree: 13 %) 122 Fig. 2-3-3 5 The IR spectra of PBTT-Cl and PSe (substitution degree: 10 %) 123 Fig. 2-3-4 1 The survey spectrum of Product_entry 16 (upper; the signals of silicon and oxygen atoms were from SiO2 wafer), and the chemical state spectra of S2p of PBTT-Cl (lower left) and the Product_entry 16 (lower right) 127 Fig. 2-3-4 2 The survey (upper; the signals of silicon and oxygen atoms were from SiO2 wafer) and curve-fitted chemical state spectra of S2p (lower left) and N1s (lower right) of PNMePy 128 Fig. 2-3-4 3 The survey (upper; the signals of silicon and oxygen atoms were from SiO2 wafer) and curve-fitted chemical state spectra of S2p (lower left) and O1s (lower right) of PFuran 129 Fig. 2-3-4 4 The survey (upper; the signals of silicon and oxygen atoms were from SiO2 wafer) and curve-fitted chemical state spectra of S2p (lower left) and Se3d (lower right) of PSe 130 Schemes Scheme 1-3-1 1 The synthetic route for preparation of amide 4a-4h, imide 5a-5c and urea 6a-6g derivatives. 25 Scheme 2-1-2 1 The desulfitative Sonogashira-type cross-coupling reaction on PBTT-Cl 46 Scheme 2-1-3 1 The plausible mechanism for the reduction of PdⅡ to Pd0 with TEA 48 Scheme 2-1-5 1 Desulfitative substitution reactions on PBTT-Cl with 2-ethynyl-N-methylpyrrole, 2-ethynylfuranm 2-ethynylthiophene and 2-ethynyl selenophene 54 Scheme 2-3-2 1 The calculation of substitution degree with 1H NMR analysis was assessed by assuming the amount of repeat units without substitution as X, and that of repeat units undergoing substitution as Y. 76 Scheme 2-3-2 2 The proposed mechanism for the laser induced formation of molecules R-1255, R-1275, R-1293 and R-1445 presented in Fig. 2-3-2-16 (B) and (C) 104 Tables Table 1-3-2 1 Cytotoxic effect of compounds 8a–9e on HepG2 2.2.15 cells, inhibition potential of HBV viral antigen and DNA replication after 72 h treatmenta 27 Table 1-3-2 2 The cytotoxicity (TC50; M) of compounds 6a-6g on HepG2 2.2.15, two human liver cancer cell lines HepG2 and Huh7, and human lung cancer cell lines A549, H23, H838, H2087 and H226 28 Table 2-1-2 1 Polymerization conditions and results for P1-P3a, 12 45 Table 2-3-2 1 The optimization study of reaction condition with 2-ethynylthiophene 72 Table 2-3-2 2 The exploration of reaction with three other substrates 110 Table 2-3-3 1 The summary of IR absorption information of PBTT-Cl, Product_entry 16, PNMePy, PFuran and PSe 124 Table 2-3-4 1 The substitution degree calculated from XPS and 1H NMR analyses of PBTT-Cl and the other four representative samples 130