Table of Contents Chapter One: Introduction. 1 1-1. Multi-Function of Nitric Oxide 1 1-2. Physical / Chemical Properties of NO and Nitrosyl Metal Complexes. 2 1-3. Nitric Oxide Biosynthesis. 5 1-4. The Role of Nitric Oxide in Tumor. 8 1-5. Storage and Transportation of NO in Biology. 10 1-6. Nitrosyl Iron Complexes Containing Cysteamine Ligand. 18 1-7. Study on the RREs Formation Pathways via Reaction of Fe(CO)2(NO)2 and Thiols: the Synthetic Route for Synthesizing Neutral Mixed-Thiolate Containing DNICs/RRE/rRREs. 22 Chapter Two: Experimental Section. 25 General Procedures: 25 Preparation of [(μ-CO)(μ-L)Fe2(NO)4] (L = -S(CH2)2NH3 (1-NH3), L = S(CH2)2OH2 (1-OH2)). 26 Preparation of [(NO)2Fe(μ-S(CH2)2NH2)(μ-S(CH2)2NH3)Fe(NO)2] (2). 27 Reaction of Complex 2 and O2. 29 Preparation of [(S(CH2)2NH2)(S(CH2)2NH3)Fe(NO)2] (4). 29 Preparation of [(S(CH2)2OH)(S(CH2)2NH3)Fe(NO)2] (5). 30 Reaction of Complex 5 and Half Equiv of [HS(CH2)]2 (5-1). 31 Preparation of the Neutral DNIC [(SC6H5)(S(CH2)2NH3)Fe(NO)2] (6). 32 Reaction of Complex 2 and Diphenyl Disulfide. 32 Preparation of [(SC2H3(CO2)(NH3))(S(CH2)2NH3)Fe(NO)2] (7). 33 Preparation of the Neutral Mixed-Thiolate rRRE [(μ-L)(μ-S(CH2)2NH3)Fe2(NO)4] (L= -S(CH2)2OH (8), L= -S(C5H6) (9)). 34 Oxidation of Complex 9 Forming the Neutral Asymmetric [{Fe(NO)2}9]2 RRE [(μ-SC6H5)(μ-S(CH2)2NH2)Fe2(NO)4] (10). 35 Preparation of {Fe(NO)2}10 DNIC [PPN][(CO)(SCN)Fe(NO)2] (11). 36 Preparation of {Fe(NO)2}9 DNIC [PPN][(SCN)2Fe(NO)2] (12). 36 EPR Spectroscopy. 37 Magnetic Measurements. 39 XAS Measurements. 39 X-ray Crystallography. 40 Chapter Three: Results and Discussion 51 3-1. Preparation of the Neutral [{Fe(NO)2}10]2 Mixed-CO-Thiolate-Bridging RREs [(μ-CO)(μ-SR)Fe2(NO)4] (R = -(CH2)2NH3 (1-NH3); R = -(CH2)2OH2 (1-OH2)). 51 3-2. Conversion of the Neutral [{Fe(NO)2}10]2 Mixed-CO-Thiolate-Bridged RRE 1-NH3 to Neutral {Fe(NO)2}9-{Fe(NO)2}10 Mixed-Valence and Mixed-Thiolate Containing rRRE [(μ-S(CH2)2NH2)(μ-S(CH2)2NH3)Fe2(NO)4] (2) . 58 3-3. Synthesis of the Neutral [{Fe(NO)2}9]2 Thiolate-Containing RRE [(μ-S(CH2)2NH2)Fe (NO)2]2 (3) via Oxidation of Complex 2 by O2. 64 3-4. Synthesis of the Neutral {Fe(NO)2}9 Mixed-Thiolate-Containing DNICs [(S(R)(S(CH2)2NH3)Fe(NO)2] (R = -(CH2)2NH2 (4); -(CH2)2OH (5); -C6H5 (6)). 67 3-5. The Chelate Effect of 1.2-Ethanedithiol for Neutral DNIC 5 and RRE 3. 73 3-6. Reactivity Study of the Reduced-Form RRE (rRRE) 2. 76 3-7. Synthesis of Water-Soluble Neutral Cysteine-Containing DNIC [(SCys)(S(CH2)2NH3)Fe(NO)2] (7). 78 3-8. Preparation of the Mixed-Thiolate-Containing Reduced-Form RREs (rRREs): [(μ-SR)(μ-S(CH2)2NH3)Fe2(NO)4] (R = -(CH2)2OH (8); R = -C6H5 (9)). 82 3-9. The Half Maximal Inhibitory Concentration (IC50) for the Water Soluble Complex 5 toward Cancer Cell Lines: Non-Small Cell Lung Cancer (NSCLC: CRL-5889 and CRL-5866), Prostate Cancer Cell (PC3) and Breast Cancer Cell (SKBR3). 86 3-10. Preparation of {Fe(NO)2}10 DNIC [PPN] [(CO)(SCN)Fe(NO)2] (11). 93 3-11. Conclusion 100 Chapter Four: References 103 List of Figures Figure 1-1-1. Mulitifaceted biological effects of nitric oxide. 1 Figure 1-2-1. Molecular orbitals diagram of nitric oxide. 2 Figure 1-3-1. Overall reaction catalysed and cofactors of NOS. 6 Figure 1-3-2. Important functions of the different NOS isoforms. 7 Figure 1-4-1. The concentration of NO has been suggested to be responsible for cell proliferation and death. 9 Figure 1-4-2. Mechanisms and influences of NO in tumour cells. 10 Figure 1-5-1. The structures of CysNO and GSNO. 11 Figure 1-5-2. The Synthesis mechanism of the DNDGIC-bound GST P1-1(left) and crystal structure (right). 13 Figure 1-5-3. The anionic, neutral and cationic forms of {Fe(NO)2}9 DNICs and [{Fe(NO)2}9-{Fe(NO)2}9] RREs. 16 Figure 1-5-4. The anionic and dianionic form of RREs and the neutral form DNICs with {Fe(NO)2}10 motif. 17 Figure 1-5-5. The single crystal of aionic RRE [Fe(μ-StBu)(NO)2]2-. 18 Figure 1-6-1. The structure (left) and single crystal of Fe(LH)(NO)2 with tetradentate N2S2 ligand (L= N,N’-dimethyl-N,N’-bis(2-mercaptoethyl)-l,3-propanediamine) (right). 20 Figure 1-6-2. X-ray structures of the dications [(S(CH2)2NH3)Fe(NO)2]22+ showing the atom labeling scheme and 50% probability thermal ellipsoids (left), and the packing construction in aqueous solution (right). 21 Figure 3-1-1. IR spectrum of complex [(μ-CO)(μ-S(CH2)2OH2)Fe2(NO)4] (1-OH2) with stretching frequencies at 1689 s, 1700 s (νNO), 1843 s (νCO) cm-1 in THF solution. The IR stretching frequencies 1749 s, 1775 s, 1809 w (νNO) cm-1 assigning as RRE [(μ-S(CH2)2OH)2Fe2(NO)4] was produced via oxidation of complex 1-OH2. 52 Figure 3-1-2. IR stretching frequencies 1693 s, 1707 s (νNO), and 1851 m (νCO) cm-1 (THF) shows the formation of complex 1-NH3. 53 Figure 3-1-3. 1H NMR (500 MHz; d8-THF) of complex 1-NH3: δ 2.47 (s, 1H (-SH)), 2.60 (t, 2H (-CH2)), 3.09 (t, 2H (-CH2)), 7.31 (s, 3H (-NH3)) ppm. 18-crown-6-ether (δ 3.67 ppm). H grease (br s, δ 1.29 ppm, m, δ 0.89 ppm). 54 Figure 3-1-4. ORTEP drawing and labeling scheme of complex 1-NH3 with thermal ellipsoids drawn at 30% probability. Selected bond lengths (Å) and angles (deg). 55 Figure 3-1-5. ORTEP drawing and labeling scheme of complex 1-OH2 with thermal ellipsoids drawn at 30% probability. Selected bond lengths (Å) and angles (deg). 55 Figure 3-2-1. FT-IR spectrum of complex [(μ-SC2H4NH2)(μ-SC2H4NH3)Fe2(NO)4] (2) with νNO stretching frequencies at 1658 s, 1678 s cm-1 (THF). 59 Figure 3-2-2. 2H NMR (500 MHz; THF) of DHO (δ 2.56 ppm) obtained from reaction of complex 1-NH3, d-cysteamine (DS(CH2)2NH2) and O2 in a 1 : 1 : 0.25 molar ratio in THF. The THF solvent containing DHO was trapped by liquid nitrogen under vacuum. The trapped DHO in THF was identified by 2H NMR spectroscopy [(2H NMR; THF): δ 2.56 (DHO) vs δ 5.32 (CD2Cl2)]. 60 Figure 3-2-3. Time-tracing UV-Vis spectrum was detected per minute that showed the reaction of 10-4 M cysteamine was added into one equiv of complex 1-NH3 (10-4 M) and quartered equiv of O2 under N2 condition at 298K. 61 Figure 3-2-4. EPR spectra of complex 2 in THF (a) at 298 K (gav = 1.998), and (b) at 77 K (g⊥ = 2.011, g∥ = 1.968). 62 Figure 3-2-5. The µeff vs T plot of complex 2 under 0.5 Tesla applied field. 62 Figure 3-2-6. ORTEP drawing and labeling scheme of complex 2 with thermal ellipsoids drawn at 30% probability. Selected bond lengths (Å) and angles (deg). 63 Figure 3-3-1. IR stretching frequencies 1749 s, 1774 s, 1808 w cm-1 (THF) shows the formation of complex 3. 65 Figure 3-3-2. UV-Vis spectrum of complex 3 in THF [nm, λmax (M-1 cm-1, ε)]: 307 (19070), 358 (11880). 66 Figure 3-3-3. 1H NMR spectrum (400 MHz; d8-THF) of complex 3: δ 1.12 (br), 1.77 (br), 3.38 (br) ppm (-CH2CH2NH2). 66 Figure 3-4-1. FT-IR spectra of (a) neutral mixed-thiolate containing DNIC [(S(CH2)2NH2)(S(CH2)2NH3)Fe(NO)2] (4) with νNO stretching frequencies at 1685 s, 1731 s cm-1 (THF), (b) [(SC2H4OH)(SC2H4NH3)Fe(NO)2] (5) with νNO stretching frequencies at 1688 s, 1733 s cm-1 (THF), and (c) [(SC6H5)(S(CH2)2NH3)Fe(NO)2] (6) with νNO stretching frequencies at 1693 s, 1739 s cm-1 (THF). 68 Figure 3-4-2. EPR spectra indicated the principal pattern for complex 4 in MeOH: (a) gav = 2.029 at 298 K (left); g1 = 2.039, g2 = 2.029 and g3 = 2.014 at 77 K in MeOH, (b) complex 5 in THF: gav = 2.029 at 298 K (left); g1 = 2.038, g2 = 2.027 and g3 = 2.016 at 77 K (right) and (c) complex 6 in THF: gav = 2.028 at 298 K (left); g1 = 2.034, g2 = 2.026 and g3 = 2.019 at 77 K (right). 70 Figure 3-4-3. ORTEP drawing and labeling scheme of complex 4 with thermal ellipsoids drawn at 30% probability. Selected bond lengths (Å) and angles (deg). 71 Figure 3-4-4. ORTEP drawing and labeling scheme of complex 5 with thermal ellipsoids drawn at 30% probability. Selected bond lengths (Å) and angles (deg). 71 Figure 3-4-5. ORTEP drawing and labeling scheme of complex 6 with thermal ellipsoids drawn at 30% probability. Selected bond lengths (Å) and angles (deg). 72 Figure 3-5-1. The IR spectra of (a) complex 5-1 displayed the stretching frequencies at 1699 cm-1 (THF); (b) the reaction of RRE 3 and 1.2-ethanedithiol showed the stretching frequencies at 1681 s, 1721 s cm-1 (THF). 74 Figure 3-5-2. ORTEP drawing and labeling scheme of complex 5 with thermal ellipsoids drawn at 30% probability. Selected bond lengths (Å) and angles (deg). 75 Figure 3-5-3. The UV-Vis spectrum of complex 5-1 in THF shows absorbed bands at 312 (7865), 366 (3410), 587 (415) M-1 cm-1 (ε). 76 Figure 3-6-1. The normalized absorbing bands of IR spectrum indicates the reaction of complex 2 and diphenyl disulfide producing complex 6 accompanied by the formation of complex 3 (red line: complex 3; black line: complex 6; Blue line: complexes 3 and 6). 77 Figure 3-7-1. Aqueous IR spectrum (1719 s, 1770 s (νNO) cm-1 (D2O)) of complex 7. 79 Figure 3-7-2. Aqueous UV-Vis spectrum of complex 7 with absorption band at 378 (5240) M-1 cm-1 (ε). 80 Figure 3-7-3. EPR spectra of complex 7 in H2O (a) at 298 K (gav = 2.03, AN(NO) = 2.36 G, AH(Cys) = 1.15 G and AH(cysteamine) = 1.45 G), and (b) at 77 K (g1 = 2.038, g2 = 2.030 and g3 = 2.021). 81 Figure 3-8-1. The normalized IR stretching frequencies 1658 s, 1679 s and 1665 s, 1685 s νNO cm-1 (THF) indicated the formation of complex 8 (black line) and complex 9 (red line) respectively. 83 Figure 3-8-2. (a) The EPR spectra of complex 8 revealed gav = 1.998 at 298 K (left) and g1 = 2.011, g2 = 2.000 and g3 = 1.970 at 77 K (right) in THF; (b) complex 9 revealed gav = 1.999 at 298 K (left) and g1 = 2.010, g2 = 1.999 and g3 = 1.972 at 77 K (right) in THF. 84 Figure 3-8-3. Fe K-edge spectra of (a) complex 8 shows the pre-edge energy at 7113.3 eV and (b) complex 9 shows the pre-edge energy at 7113.4 eV. 85 Figure 3-8-4. The IR spectrum of RRE 10 indicated that the stretching frequencies 1754 s, 1780 s, 1812 w cm-1 (red line) in THF compared with [(NO)2Fe(μ-SPh)]2 (IR νNO: 1757 s, 1784 s, 1814 vw cm-1 (THF) (black line) and complex 3 (IR νNO: 1749 s, 1774 s, 1808 vw cm-1 (THF) (blue line)). 86 Figure 3-9-1. Organic nitrate (GNT) and the RSNO compounds (SANP and GSNO). 87 Figure 3-9-2. Percent viability of cell growth vs concentration of Lu-2 on prostate cancer cell PC-3. 89 Figure 3-9-3. Percent viability of cell growth vs. concentration of Lu-2 on breast cancer cell SKBR-3. 89 Figure 3-9-4. Percent viability of cell growth vs. concentration of Lu-2 on Lung cancer cell CRL5866. 90 Figure 3-9-5. Percent viability of cell growth vs. concentration of Lu-2 on Lung cancer cell CRL5889. 90 Figure 3-9-6. PC-3 cell proliferation as a function of DNIC concentration. Cells were incubated with different doses of DNIC ligands and were assayed at 24 hour using the MTT reagent. 91 Figure 3-9-7. SKBR-3 cell proliferation as a function of DNIC concentration. Cells were incubated with different doses of DNIC ligands and were assayed at 24 hour using the MTT reagent. 92 Figure 3-9-8. CRL5866 cell proliferation as a function of DNIC concentration. Cells were incubated with different doses of DNIC ligands and were assayed at 24 hour using the MTT reagent. 92 Figure 3-9-9. CRL5889 cell proliferation as a function of DNIC concentration. Cells were incubated with different doses of DNIC ligands and were assayed at 24 hour using the MTT reagent. 93 Figure 3-10-1. The IR spectrum of complex 11 displays the stretching frequencies at 1735 s, 1688 s (υNO), 1986 s (υCO) and 2098 s (υSCN) cm-1 (THF). 94 Figure 3-10-2. The UV-Vis spectrum of complex 11 in THF shows absorption bands at 312 (7865), 366 (3410), 587 (415) M-1 cm-1 (ε). 94 Figure 3-10-3. ORTEP drawing and labeling scheme of complex 11 with thermal ellipsoids drawn at 30% probability. Selected bond lengths (Å) and angles (deg). 95 Figure 3-10-4. The IR stretching frequencies 1718 s, 1786 s (υNO), 2054 s, 2076 s (υSCN) cm-1 (THF) indicated the formation of complex 12. 97 Figure 3-10-5. ORTEP drawing and labeling scheme of complex 12 with thermal ellipsoids drawn at 30% probability. Selected bond lengths (Å) and angles (deg). 98 Figure 3-10-6. EPR spectra of complex 12 in THF (a) at 298 K (gav = 2.032), and (b) at 77 K (g1 = 2.038, g2 = 2.027 and g3 = 2.016). 99 Figure 3-10-7. The UV-Vis spectrum of complex 12 in THF shows absorbed bands at 257 (10280), 400 (3675), 709 (270) M-1 cm-1 (ε). 99 List of Tables Table 1-2-1. Physical properties of nitrogen monoxides. 3 Table 1-5-1. List of the reported protein-bound DNICs based on EPR spectra. 14 Table 2-1. Crystal data and structure refinement for complex 1-NH3. 42 Table 2-2. Crystal data and structure refinement for complex 1-OH2. 43 Table 2-3. Crystal data and structure refinement for complex 2. 44 Table 2-4. Crystal data and structure refinement for complex 4. 45 Table 2-5. Crystal data and structure refinement for complex 5. 46 Table 2-6. Crystal data and structure refinement for complex 5-1. 47 Table 2-7. Crystal data and structure refinement for complex 6. 48 Table 2-8. Crystal data and structure refinement for complex 11. 49 Table 2-9. Crystal data and structure refinement for complex 12. 50 Table 3-1-1. Selected bond lengths (Å) of the [{Fe(NO)2}9]2 RRE, reduced-form {Fe(NO)2}9-{Fe(NO)2}10 RREs and di-reduced-form [{Fe(NO)2}10]2 RREs. 58