Organometallic scaffolds as protein tyrosine phosphatase 1b inhibitor

ORGANOMETALLIC SCAFFOLDS AS PROTEIN TYROSINE PHOSPHATASE 1B INHIBITOR ONG JUN XIANG (B.Sc.(Hons.), NUS A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2012 I Declaration Thesis Declaration The work in this thesis is the original work of Ong Jun Xiang, performed independently under the supervision of Dr Ang Wee Han, (in the laboratory S15-05-01), Chemistry Department, National University of Singapore, between 1/8/2010 and 24/8/2012. Ong Jun Xiang Name Signature 23/8/2012 Date I Acknowledgements Acknowledgements I would like to express my greatest gratitude to my supervisor Dr Ang Wee Han for his guidance, valuable advice and patience throughout my course of research. I am really fortunate to be his student as he taught me many things within and outside the realm of Chemistry. I would also like to express my heartfelt thanks to Dr Yap Chun Wei for his input on molecular docking studies. My sincere thanks to the technical staff at X-ray diffraction (Prof. Koh, Ms Tan and Ms Hong), Nuclear Magnetic Resonance (Mdm. Han), Mass spectrometry and Elemental Analysis laboratories for their technical support. I would like to thank my group members Chee Fei, Daniel, Mun juinn, Diego and Tian Quan for their assistance and helpful discussions. I am grateful to NUS for my research scholarship. Lastly, I am very thankful to my family members and friends for their love and support. II Table of Contents Table of Contents Summary ....................................................................................................................................... V List of Tables .............................................................................................................................. VII List of Figures ........................................................................................................................... VIII List of Schemes ............................................................................................................................ IX List of Abbreviations ................................................................................................................... X 1. Introduction ............................................................................................................................... 1 1.1. Protein Tyrosine Phosphatase 1B as drug target ................................................................. 1 1.2. Organic inhibitors of Protein Tyrosine Phosphatases ......................................................... 4 1.3. Metal complexes as inhibitors of Protein Tyrosine Phosphatases ...................................... 6 2. Design concept of metallo-inhibitors ....................................................................................... 9 2.1. Design of Ruthenium-based inhibitor ................................................................................. 9 2.2. Design of Gold-based inhibitor ......................................................................................... 10 3. Synthesis of organoruthenium and organogold PTP inhibitors ......................................... 12 3.1. Synthesis of ligands ........................................................................................................... 12 3.2. Synthesis of organoruthenium inhibitors .......................................................................... 15 3.3. Synthesis of organogold inhibitors .................................................................................... 21 4. Biological evaluation of organoruthenium PTP inhibitors ................................................. 24 4.1. Aqueous stability of organoruthenium PTP inhibitors ...................................................... 24 III Table of Contents 4.2. Inhibition of PTP-1B and TC-PTP by organoruthenium PTP inhibitors .......................... 25 4.3. Determination of inhibition constants of 4a towards PTP-1B and TC-PTP ..................... 30 5. Conclusion ............................................................................................................................... 32 6. Experimental Section .............................................................................................................. 33 6.1. Preparation of organoruthenium complexes...................................................................... 35 6.2. Preparation of organogold complexes ............................................................................... 44 Appendix ...................................................................................................................................... 50 References .................................................................................................................................... 67 IV Summary Summary This thesis describes the synthesis and evaluation of two novel classes of organoruthenium and organogold complexes as inhibitors of protein tyrosine phosphatases (PTPs). The findings of the research are presented in four chapters. Chapter 1 gives an introduction to protein tyrosine phosphatase 1B (PTP-1B) and its potential as a therapeutic drug target. The major challenge in the attempt to design inhibitors of PTPs arose from their highly conserved enzymatic active-site across the PTP family. Selectivity against closely related PTPs could be low due to the structural homologies. This is particularly problematic for PTP-1B since it is 80% homologous with T-cell protein tyrosine phosphatase (TC-PTP) in their catalytic domains. So far, the organic and metallo-inhibitors synthesized to date have yet to achieve high selectivity towards PTP-1B against TC-PTP. Chapter 2 describes the concepts behind the design of the organoruthenium and organogold complexes respectively. Since PTPs possess an active-site which is highly conserved across the PTP family, targeting the active-site to achieve selectivity among the various PTPs will not be a feasible approach. We reasoned that the proximal space to the PTP active-site must be important for the molecular recognition of their protein substrate. By capitalizing on the potential difference in the structural environment proximal to the active-site, we hope to develop metallo-inhibitors that are selective towards specific PTPs. To do so, we would rationally build molecules which can simultaneously bind to the active-site as well as being able to interact with the chemical space proximal to the active-site of the PTPs. Chapter 3 describes the synthesis of the ligands and their respective organoruthenium and organogold complexes. The reaction of imidazole/benzimidazole difluoromethylphosphonate esters, 1 and 3, and imidazole/benzimidazole difluoromethylphosphonic acids, 2 and 4, with V Summary [(η6-arene)RuCl2]2 where arene = cymene or 1,3,5-triisopropyl-benzene, yielded mononuclear Ru(II) complexes 1a-b/3a-b and 2a-b/4a-b respectively. The molecular structures of complexes 1b, 3b, 4a and 4b were also determined by X-ray crystallography. Based on a different approach, ethynyl difluoromethylphosphonate ester 9 was reacted with [Au(PR3)Cl] where R = phenyl in the presence of base to yield mononuclear alkynyltriphenylphosphinegold(I) complex 9a. Chapter 4 describes the biological studies of the organoruthenium complexes as inhibitors of PTP-1B and TC-PTP. The organoruthenium complexes containing phosphonic acid groups were found to inhibit PTP-1B at low micromolar level with 7-10 fold selectivity towards PTP-1B over TC-PTP whereas the inhibitors containing phosphonate ester groups were found to be inefficacious. Steady-state kinetics experiments have shown that the complexes competitively bind to the enzymes at their active-sites. In addition, molecular docking studies have also shown that the organoruthenium complexes bind better than their parent ligands in PTP-1B due to additional hydrophobic interactions with Phe182. Overall, these results suggest that this novel class of organoruthenium complexes may be promising therapeutic agents to target PTP-1B. The manuscript entitled “Rational Design of Selective Organoruthenium Inhibitors of Protein Tyrosine Phosphatase 1B” have been submitted for publication. VI List of Tables List of Tables Table 3.1. Comparison of bond distances [Å] and angles [°] of 1b and 4a. 21 Table 4.1. Initial screening of inhibitors against PTP-1B and TC-PTP and IC50 of selected compounds. 30 VII List of Figures List of Figures Figure 1.1. Role of PTP-1B in insulin and leptin signaling pathways. 2 Figure 1.2. Overlap of protein crystal structures of TC-PTP and PTP1B. 3 Figure 1.3. Mimetics of phosphotyrosine. 4 Figure 1.4. Selected PTP-1B inhibitors of difluoromethylphosphonic acid class of inhibitors. 5 Figure 1.5. Mimicking protein kinase inhibitor staurosporine with octahedral metal complexes. 7 Figure 2.1. Approach to designing ruthenium-based PTP inhibitors. 9 Figure 2.2. Approach to designing gold-based PTP inhibitors. 10 Figure 3.1. Molecular representations of 1b and 3b. 20 Figure 3.2. Molecular representations of 2a, 4a and 4b. 20 Figure 4.1. UV-Vis stability studies of complex 4a. 25 Figure 4.2. Inhibition of PTP-1B and TC-PTP at inhibitor concentration of 100 µM. 27 Figure 4.3. Dose-response curves of inhibition of compounds towards PTP-1B and TCPTP. 28 Figure 4.4. Molecular docking of ligand 4 and complex 4a in PTP-1B. 29 Figure 4.5. Steady-state kinetic studies of complex 4a towards PTP-1B and TC-PTP. 31 VIII List of Schemes List of Schemes Scheme 3.1. Synthetic route for the synthesis of ligands 1, 2, 3 and 4. 13 Scheme 3.2. Synthetic route for the synthesis of ligand 9. 15 Scheme 3.3. Synthetic route for the synthesis of ruthenium(II)-arene complexes. 17 Scheme 3.4. Synthetic route for the synthesis of gold(I) complexes. 23 IX List of Abbreviations List of Abbreviations d doublet (NMR) J Coupling constant DMF Dimethylformamide DMSO Dimethylsulfoxide δ NMR chemical shift ESI Electronspray Ionization h Hour m multiplet (NMR) min Minute MS Mass Spectrometry m/z mass to charge ratio MeOH Methanol NMR Nuclear Magnetic Resonance pTyr Phosphotyrosine RT Room temperature s singlet (NMR) t triplet (NMR) THF Tetrahydrofuran UV-Vis Ultraviolet-Visible X Chapter 1 Chapter 1. Introduction Sedentary lifestyle and lack of physical exercises, which are common in many developed and developing countries, have been the major contributors to obesity in both the adult and child populations.[1-3] Obesity is strongly linked to Type 2 diabetes mellitus (TD2M) which is a chronic disease where the body loses its sensitivity to the blood glucose-regulating hormone insulin. People with diabetes have elevated blood glucose level and over time, their eyes, kidney, nerves and blood vessels will be damaged. Consequently, they incur long-term health problems leading to kidney failure, heart disease and stroke. World Health Organization reported in 2004 that an estimated of 3.4 million people died from consequences of high blood sugar, and that diabetes-linked death will double between 2005 and 2030. To address these problems, there has been an intensified search for new therapeutic treatments for T2DM and obesity. 1.1. Protein Tyrosine Phosphatase 1B as drug target Protein tyrosine phosphatases (PTPs) belong to a large family of 107 enzymes that play a vital role in the regulation of various signaling transduction pathways in mammalian systems.[4, 5] PTP enzymes catalyze the dephosphorylation from phosphorylated tyrosine residues and in conjunction with protein tyrosine kinases (PTKs), they are responsible for managing the levels of phosphorylation within the cells.[6, 7] Studies have shown that the dysregulation of PTP can lead to several pathological conditions including diabetes, obesity, cancer and autoimmune disorders.[8-10] Amongst the members of the PTP family, PTP-1B is a key negative regulator of the insulin and leptin signaling pathways associated with obesity and diabetes. PTP-1B is responsible for dephosphorylation of activated insulin receptor (IR) or insulin receptor substrates (IRS) in insulin signaling[11, 12] and dephosphorylates JAK2, which is downstream of the ObR 1 Chapter 1 receptor in the leptin signaling pathway[13, 14]. Cell cultures and gene coding studies have shown that aberrant expression of PTP-1B can contribute to obesity and diabetes.[15-17] In addition, PTP1B knockout mice experiments showed that PTP-1B deficiency lead to increased sensitivity towards insulin and resistance to diet-induced obesity, suggesting that inhibition of PTP-1B could address obesity and insulin resistance.[18, 19] These pioneering studies have validated the notion that inhibition of PTP-1B could serve to address issues of obesity and diabetes, and there have been growing interest in the development of PTP-1B inhibitors as potential therapeutic agents.[20-22] Figure 1.1. Role of PTP-1B in insulin and leptin signaling pathways. One major challenge in the attempt to design inhibitors of PTPs arose from their highly conserved enzymatic active site across the PTP family.[4] Within the conserved PTP catalytic domain, a unique signature sequence motif, CX5R, which is invariant among all PTPs, can be found. This motif contains residues 214-221 which bind to the phosphate group of pTyr and the cysteine 215 (Cys215) residue which is responsible for catalyzing the dephosphorylation of pTyr. When pTyr residues enter the catalytic site, the mobile WPD loop of the enzyme 2 Chapter 1 transforms into a closed conformation and stabilizes the substrate within the active site. This brings the pTyr substrates into close proximity to Cys215 which is in a position to undergo a nucleophilic attack on the substrate phosphorous atom. The activity of PTPs is regulated by Cys215 through a deactivation/activation pathway mediated by signaling molecules, hydrogen peroxide and glutathione, in the body.[23-26] The oxidation (deactivation) of the thiol of Cys215 leads to the formation of a sulfenyl-amide intermediate rendering it ineffective in catalyzing the dephosphorylation of pTyr. The subsequent reduction (deactivation) of the sulfenyl-amide intermediate restores the active thiol. Figure 1.2. Overlap of protein crystal structures of TC-PTP and PTP1B. Selectivity against closely related PTPs could be low due to the structural homologies. This is particularly problematic for PTP-1B since it is 80% homologous with TC-PTP in their catalytic domains.[27] TC-PTP is widely distributed throughout the body and is responsible for modulating the immune functions of the body.[28] The consequences of using a non-selective PTP-1B inhibitor that also inhibits TC-PTP can lead to severe side-effects and recent studies have shown that TC-PTP deficient mice die within 3-5 weeks of age.[29, 30] Developing PTP-1B inhibitors with high selectivity towards PTP-1B, as opposed to TC-PTP, remain a daunting task. 3 Chapter 1 1.2. Organic inhibitors of Protein Tyrosine Phosphatases The organic inhibitors that target the active site of PTPs can be classified into several classes based on the type of functional groups that binds to the catalytic site. One of the most potent classes of organic inhibitors is the difluoromethylphosphonic acids and they have been the core of many inhibitor designs. The main strategy behind the design of this class of active-site inhibitors is to build molecules that contain units that mimic pTyr. The fact that it is almost structurally identical to pTyr is the key reason why inhibitors incorporating difluoromethylphosphonic acids moieties are able to exhibit high inhibitory activities. These units are non-hydrolysable and compete for the active-site. In this way, it prevents the overdephosphorylation of pTyr on protein substrates. Figure 1.3. Mimetics of pTyr. A) pTyr; B) Methylenephosphonic acid; C) Difluoromethylphosphonic acid. The structure of difluoromethylphosphonic acid, a non-hydrolyzable mimetic of pTyr, is shown in Figure 1.3. The difluoromethylphosphonic acid has been shown to be a 1000-fold more superior than its non-fluorinated derivative.[31] The increased binding affinity of difluoromethylphosphonic acid to the active-site compared to methylenephosphonic acid has 4 Chapter 1 been attributed to interactions between the fluorine atoms and residues in the active site.[32] In addition, the geometry brought about by the CF2 group makes the Ph−CF2−PO3H2 angle resemble that of Ph−O−PO3H2 observed in pTyr. This allowed for a better fit to the PTP active site which would otherwise not be observed for methylenephosphonic acid or even the monofluorinated derivative.[33] Some selected inhibitors of the difluoromethylphosphonic acid class of PTP-1B inhibitors are shown in Figure 1.4. Compound I, which is the most potent and selective PTP-1B inhibitor identified to date (Ki = 2.4 nM), exhibits a 1000- to 10000-fold selectivity against a panel of other PTPs, but only 10-fold against the structurally similar TC-PTP.[34] Figure 1.4. Selected PTP-1B inhibitors of difluoromethylphosphonic acid class of inhibitors. Some other classes of active-site inhibitors include the 2-carbomethoxybenzoic acids[35] and the 2-oxalylaminobenzoic acids[36]. The strategy adopted in designing these inhibitors was the same, which was to design pTyr mimetics to bind to the catalytic pocket. However, as these compounds were often negatively charged owing to the high polar nature of the active-site, they exhibited low cellular penetration levels. This was often the drawback associated with such 5 Chapter 1 active-site inhibitors. An interesting class of inhibitors was the allosteric inhibitors which bind to a novel site located ~20Å away from the catalytic pocket.[37] This site is amenable to binding small molecules, considerably less polar and not well-conserved among PTPs, thus affording an opportunity to circumvent the problems associated with active-site inhibitors. The binding of an allosteric inhibitor to the allosteric site prevents the closure of catalytic WPD loop (Trp179, Pro180 and Asp181) at the active-site. This in turn rendered the catalytic site inactive as the WPD loop needed to close over the active-site in order to facilitate the cleavage of pTyr substrates.[38] Interestingly, it was found that the allosteric site also differed from the corresponding region in the closely related TC-PTP at the central position 280 (cysteine instead of phenylalanine). Hence, this difference at this central position between PTP-1B and TC-PTP can potentially be exploited to develop selective inhibitors. 1.3. Metal complexes as inhibitors of Protein Tyrosine Phosphatases Although the field of synthesizing organic inhibitors of PTPs has been extensively studied, the use of metal complexes to target PTPs remains largely unexplored. So far, several metallocomplexes have been investigated as potential PTP inhibitors. A series of vanadium and copper complexes containing Schiff base ligands were found to be very potent PTP inhibitors but low selectivity was observed between PTP-1B and TC-PTP.[39-44] These Schiff-based vanadium and copper complexes exhibited at most a 2-fold and 3-4 fold selectivity towards PTP-1B over TCPTP respectively, although several higher magnitudes of selectivity were observed against other PTPs such as SHP-1, SHP-2 and PTP-MEG2. Recently, a library of gold-phosphine and goldcarbene complexes was screened and several found to exhibit good PTP-1B inhibitory activity with modest levels of selectivity.[45, 46] However, these gold complexes were only selective 6 Chapter 1 towards lymphoid tyrosine phosphatase (LYP) and protein tyrosine phosphatase PEST (PTPPEST). Their mechanism were not known but given the affinity of these metal centres, especially Au, towards S-containing cysteine residues within the enzyme active-site, it was possible that the metal centre was directly binding to the S-atom which could account for the poor selectivity between the two enzyme homologues. Indeed, some reports have shown that inhibition of PTPs was attributed to binding of the metal centres in these organo-metallic complexes to the sulphur atom of the Cys215 at the active-site.[47, 48] The coordination of the metal center to Cys215 led to the retardation of the redox pathway which was critical in modulating the activity of PTPs. The search for a selective metal-based PTP inhibitor remained elusive. Earlier on, Meggers et al had shown that highly selective active-site inhibitors of PTKs can be prepared using a known kinase inhibitor, staurosporine, as a template. By replacing the glycoside motif with an organoruthenium fragment, improved inhibitory profiles against specific PTKs were achieved, representing some of the most efficacious PTK inhibitors reported. In this manner, the octahedral ruthenium framework provided the scaffold upon which ligands could be structurally organized and as a basis for structure-activity studies.[49-59] Figure 1.5. Mimicking protein kinase inhibitor staurosporine with octahedral metal complexes. 7 Chapter 1 We were interested in applying these principles in the design of PTP inhibitors using metal-ligand interactions as a structural element. PTPs presented a different challenge since the active-site was small, designed to bind a pTyr motif, as compared to PTKs which had a large cleft capable of accommodating a bulky ATP substrate. We reasoned that the proximal space to the PTP active site must be important for the molecular recognition of their protein substrate. By capitalizing on the potential difference in the structural environment proximal to the active site, we hope to develop metallo-inhibitors that are selective towards specific PTPs. 8 Chapter 2 Chapter 2. Design concept of metallo-inhibitors metallo 2.1. Design of Ruthenium-based Ruthenium inhibitor Figure 2.1. Approach to designing ruthenium ruthenium-based PTP inhibitors. As the active-site site is highly conserved across the PTP fa family, targeting the active-site active to achieve selectivity among the various PTPs would not be a feasible approach. As such, our approach is to target the structural environment proximal to the active-site site of the various PTPs to achieve selectivity. We hypothesize tthat hat there is potential difference in the structural st environment proximal to the substrate binding site of various PTPs which can be exploited to obtain ain selectivity. Our strategy was to build molecules which can simultaneously multaneously bind to the active-site as welll as being able to interact with the chemical space proximal to the active-site of the PTPs. Figure 2.1 depicted the approach to designing ruthenium-based based inhibitors of PTPs. The design of the organoruthenium ruthenium inhibitors consist of two fragments joined toge together by a bi-dentate linker. One portion of the molecule consisted of the phenyl yl difluoromethylphosphonic acid moiety which was a well-studied studied non-hydrolyzable mimetic of pTyr that bind to the active-site of PTPs.[33] The other portion tion of the molecule will consist of a 3-D D globular fragment which had the 9 Chapter 2 potential to interact with amino-acid residues surrounding the substrate binding site. The ruthenium metal center in the design solely serves a structural role to organize the ligands in three-dimensional space thereby creating molecules with unique and well-defined 3-D globular shapes, owing to its ability to form octahedral coordination geometry. With the ligands organize in a three dimensional fashion, interactions with amino-acid residues can be maximized leading to better binding to the PTPs. This was otherwise not easily achievable in purely organic molecules as the carbon center is only limited to a coordination number of four. By capitalizing on the potential difference in structural environment proximal to the active site of PTP-1B and TC-PTP, we hope to achieve selectivity between the two PTPs arising from contrasting interactions with the globular ruthenium fragment. In addition, with the established coordination chemistry of ruthenium metal, structurally diverse complexes can be assembled with relative ease as compared to organic molecules whose synthesis often involved multiple synthetic steps which is an arduous process. 2.2. Design of Gold-based inhibitor peripheral structural space PTP active site Figure 2.2. Approach to designing gold-based inhibitors. 10 Chapter 2 The strategy adopted to designing gold-based inhibitors towards PTPs is somewhat similar to the approach previously described for the design of the ruthenium-based inhibitors. This novel class of gold(I) complexes will consist of the phenyl difluoromethylphosphonic acid moiety and a 3-D globular fragment which was provided by a bulky phosphine ligand. The acetylene group in conjunction with the gold metal center served to link these two fragments together as depicted in Figure 2.2. The gold metal center in the design solely served a structural role and was not expected to interact with the enzymes. Similarly, this class of gold(I) inhibitors was expected to bind at the active-site of PTPs with the globular phosphine ligand at the exterior, poised to interact with amino-acid residues surrounding the substrate binding site. By capitalizing on the potential difference in structural environment proximal to the active-site of the various PTPs, we hope to achieve selectivity between the various PTPs arising from contrasting interactions with the globular phosphine ligand. This approach was attractive as it allowed an easy access to a library of structurally diverse compounds due to the commercial availability of a wide range of phosphine ligands. In addition, the typically strong gold-phosphorous and gold-carbon bonds will give rise to the formation of stable gold(I) complexes which was critical in biological evaluation. 11 Chapter 3 Chapter 3. Synthesis of organoruthenium and organogold PTP inhibitors 3.1. Synthesis of ligands The ligands 1, 2, 3 and 4 were synthesized as shown in Scheme 3.1 from p-tolualdehyde. p-Tolualdehyde was first treated with diethylphosphite in the presence of a catalytic amount of base to give the hydroxyl-phosphonate ester P1. Subsequent oxidation of P1 with Dess-Martin periodinane (DMP) afforded the keto-phosphonate ester P2. Treatment of compound P2 with diethylaminosulfur-trifluoride (DAST) resulted in the replacement of the ketone functional group by geminal fluorine atoms to give the difluoromethylphosphonate ester P3. It has been reported that P3 can be synthesized via Shibuya coupling using 4-iodotoulene and diethyl(bromodifluoromethyl)phosphonate with Zn dust and CuBr as the coupling agents,[60, 61] but after repeated unsuccessful attempts, the described three-step approach was adopted. Bromination was first carried out on P3 with N-bromosuccinimide (NBS) in the presence of azobisisobutyronitrile (AIBN) as the catalyst. The bromination reaction resulted in the formation of both mono-brominated P4 as the major product as well as di-brominated side-product which could not be effectively separated using flash-column chromatography. However, only P4 reacted with the 2-(2-pyridyl)imidazole and 2-(2-pyridyl)benzimidazole to afford the desired ligands 1 and 3 respectively in good yields, while the di-brominated side-product remained unreacted and can be removed using flash-column chromatography. Treatment of 1 and 3 with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and iodotrimethylsilane (TMSI) followed by MeOH resulted in the hydrolysis of the phosphonate ester groups to give ligands 2 and 4 respectively as the phosphonic acids. 12 Chapter 3 Scheme 3.1. Synthetic route for the synthesis of ligands 1, 2, 3 and 4. The ligand 9 was synthesized as shown in Scheme 3.2 from p-iodo benzaldehyde. Initial attempts to obtain compound 9 through the Stille coupling reaction of ethynyltributylstannane with compounds P7a or P7b, which were synthesized from p-triflate benzaldehyde and p-bromo benzaldehyde respectively through the hydroxyl- and keto- phosphonate ester intermediates P5ab and P6a-b, did not work well. Although several previous reports have shown that coupling reaction of ethynyltributylstannane with P7a or P7b was possible,[62-64] it was observed that there was no reaction with the triflate diethylphosphonate ester P7a after extended hours of reaction at 13 Chapter 3 elevated temperatures with a variety of polar solvents such as acetonitrile, 1,4-dioxane, THF, DMF, etc. On the other hand, coupling reaction with the bromo diethylphosphonate ester P7b resulted in a mixture of starting material and product which could not be readily separated using flash-column chromatography. Since iodo-substrates are known to be more activated towards palladium-catalyzed coupling reactions compared to the corresponding triflate- and bromosubstrates, iodo-diethylphosphonate ester P7c was chosen as the coupling reagent for the Stille coupling reaction. p-iodo benzaldehyde was first treated with diethylphosphite in the presence of a catalytic amount of base to give the hydroxyl-phosphonate ester P5c. Subsequent oxidation of P5c with DMP afforded the keto-phosphonate ester P6c. Treatment of compound P6c with DAST resulted in the replacement of the ketone functional group by geminal fluorine atoms to give the difluoromethylphosphonate ester P7c. Again, attempts were carried out to synthesize the difluoromethylphosphonate ester compounds P7a-c by the Shibuya coupling but they were unsuccessful after repeated attempts. Finally, compound P7c was subjected to the Stille coupling reaction with ethynyltributylstannane in DMF with PdCl2(PPh3)2 (5 mol%) as the catalyst to afford ligand 9 in moderate yield. Alternatively, ligand 9 can also be synthesized through the direct coupling reaction of P7c with trimethylsilylacetylene (TMSA) under palladium-catalyzed conditions to yield the trimethylsilyl (TMS) protected alkyne P8. De-protection of the compound P8 under basic conditions afforded compound 9 in relatively higher yield. 14 Chapter 3 Scheme 3.2. Synthetic route for the synthesis of ligand 9. 3.2. Synthesis of organoruthenium inhibitors Reaction of two equivalents of imidazole-diethylphosphonate ester 1 with [(η6-arene)RuCl2]2 where arene = cymene or 1,3,5-triisopropyl-benzene (TIPB), yielded mononuclear Ru(II) complexes 1a and 1b whereas treatment of imidazole-phosphonic acid 2 yielded complexes 2a and 2b as shown in Scheme 3.3. The pyridyl-imidazole ligand cleaved the dinuclear ruthenium precursor as well as displacing a chlorido ligand by forming a stable 5- 15 Chapter 3 membered chelate with the metal centre. Increased steric encumbrance afforded by the larger TIPB ligand did not adversely affected the formation of the desired products. Notably, 1a-b were obtained as monocationic complexes with hexafluorophosphate as the counter-anion after anion exchange with NH4PF6. Anion exchange was necessary to improve the yields and purity of the product. On the other hand, complexes 2a-b were isolated directly from the reaction mixture without anion exchange as the chloride salts. Under similar reaction conditions, benzimidazolediethylphosphonate ester 3 and benzimidazole-phosphonic acid 4 gave similar organoruthenium complexes 3a-b and 4a-b in good yields. All the complexes were isolated as yellow or orangeyellow solids. Complexes with the diethylphosphonate ester group 1a-b and 3a-b were soluble in common organic solvents namely dichloromethane, chloroform, MeOH, DMSO and acetone. In contrast, 2a-b and 4a-b are only soluble in polar solvents MeOH and DMSO presumably due to the highly polar phosphonic acid groups. All of the synthesized ruthenium complexes were soluble in water to concentrations exceeding 1 mM which is important for subsequent biological investigations. 16 Chapter 3 R R N N N N N F N F F O P HO OH P EtO OEt 2 : R = imidazole 4 : R = benzimidazole 1 : R = imidazole 3 : R = benzimidazole R' Cl R' Cl Ru Cl NH4PF6 Ru Cl Cl Ru Cl R' Ru Cl Cl R' R' Cl R' PF6 Ru F O R Cl N N Cl Ru R N N N N F F F O EtO P OEt 1a : R = imidazole, R' = cymene 1b : R = imidazole, R' = 1,3,5-iPr-benzene 3a : R = benzimidazole, R' = cymene 3b : R = benzimidazole, R' = 1,3,5-iPr-benzene F O HO P OH 2a : R = imidazole, R' = cymene 2b : R = imidazole, R' = 1,3,5-iPr-benzene 4a : R = benzimidazole, R' = cymene 4b : R = benzimidazole, R' = 1,3,5-iPr-benzene Scheme 3.3. Synthetic route for the synthesis of ruthenium(II)-arene complexes. The compounds were analyzed by 1H, 31P{1H}, 19F{1H}-NMR, ESI-MS and RP-HPLC. A distinct feature of the ruthenium-arene complexes is the presence of resonances at 5-6 ppm due the aryl-CH protons of the facially-bound arene ligand. The resonances were shifted upfield from the aromatic region, indicating a more shielded environment in the presence for the metal centre. In addition, the arene protons of the cymene ligand of complexes 1a-4a could be observed as four sets of doublets, compared to two sets of doublets of the precursor, indicating the 17 Chapter 3 desymmetrization of the arene ligand upon coordination of the imidazole or benzimidazole ligands. Likewise, the six methyl protons on the isopropyl group were observed as two sets of doublets, as opposed to a set of doublet in the precursor. This indicated that the ligands were bound at more than one coordination site around the metal centre. Similar observations were made for complexes 1b-4b with the TIPB ligand. A set of triplet and a set of doublet were observed in the 31 P{1H}- and 19 F{1H}-NMR respectively for all the complexes due to 2JPF coupling. All the complexes were observed as M+ parent molecular ions in the ESI-MS and confirmed with MS/MS fragmentation analysis. However, organic CHN elemental analyses did not yield results consistent with the molecular formula of the desired compounds despite using crystalline and highly-pure samples. In comparison, Ru content analysis on the samples by ICPOES was within error limits. We hypothesized that the discrepancy was due to the presence of the –CF2– group which could yield interfering HF on combustion, giving rise to inaccurate results. In order to ascertain purity, RP-HPLC were carried out and the newly synthesized compounds were found to be >95% pure. We investigated whether it was possible to hydrolyze the phosphonate ester groups in these organoruthenium complexes directly as facile entry to 2a-b and 4a-b. The direct hydrolysis of phosphonate ester groups has been reported for several octahedral polyaromatic ruthenium complexes[65] but not ruthenium-arene compounds which are more reactive and susceptible to ligand displacement. Treatment of 1a directly with TMSI followed by MeOH yielded quantitatively an unknown species with a similar 1H NMR profile to 2a. The 1H NMR spectrum of the unknown complex revealed the disappearance of the ethyl peaks of the phosphonate ester group indicating hydrolysis, while other peaks remained unchanged suggesting that organometallic scaffold remained intact. Closer inspection by ESI-MS analysis however 18 Chapter 3 suggested that the chlorido ligand coordinated to the Ru(II) centre had been displaced by an iodido ligand, with a single peak observed at m/z 728 corresponding to [(ŋ6-cymene)RuI(2)]+. In contrast, 2a is observed by ESI-MS as the parental molecular ion at m/z 636. The source of iodido ligand was presumably excess TMSI reagent which underwent halide exchange reaction at the Ru(II) centre after hydrolysis. Although direct hydrolysis of phosphonate groups was technically feasible, further steps would be required to convert the iodido ligand back to the chlorido ligand and the method was thus abandoned. Single crystals of 1b, 2a and 3b suitable for X-ray diffraction studies were grown by layer diffusion of diethyl ether into methanolic solutions of the complexes whereas single crystals of 4a and 4b was grown by slow evaporation of methanolic and aqueous solutions, respectively. To the best of our knowledge, these complexes are the first metallo-complexes reported which contain the difluoromethylphosphonate ester and difluoromethylphosphonic acid functional groups respectively. The structures of complexes 1b, 2a, 3b, 4a and 4b with atomic numbering are depicted in Figure 3.1 and Figure 3.2 respectively and selected X-ray crystallographic data are shown in Table A1 in the appendix. Selected bond lengths and angles of complexes 1b and 4a are also shown in Table 3.1. The high R1 value observed for complex 3b was due to poor quality of the crystal. Some problems were also encountered when solving the crystal structures of complexes 2a and 4b. As the solvent molecules and chlorido ligand in 2a and 4b respectively were highly disordered, their identities cannot be determined unambiguously. Given the poor Xray diffraction data, analysis of the bond parameters of 2a, 3b and 4b was not carried out. 19 Chapter 3 1b 3b Figure 3.1. Molecular representations of 1b and 3b; atoms are represented as spheres of arbitrary radii; [PF6]- anion was excluded for clarity. 4a 2a 4b 20 Chapter 3 Figure 3.2. Molecular representations of 2a, 4a and 4b; atoms are represented as spheres of arbitrary radii. The bond lengths and angles observed in these complexes are typical values of piano-stool ruthenium-arene complexes. The pyridyl-imidazole and pyridyl-benzimidazole rings in 1b and 4a were essentially in the planar conformation. The solid state structure of 4a showed that the complex crystallised as a zwitterionic structure with deprotonated monobasic phosphonate group. The negative charge on the deprotonated phosphonate group was delocalized between O2-P1-O3 with similar O2-P1 and O3-P1 bond lengths of 1.481(3) Å and 1.512(3) Å as observed in 4a. The distances were consistent with a bond order of 1 to 2. In comparison, the protonated O1-P1 bond was significantly longer at 1.558(3). Table 3.1. Comparison of bond distances [Å] and angles [°] of 1b and 4a. Complex Ru1-N1 [Å] Ru1-N2 [Å] Ru1-Cl1 [Å] average Ru-Carene [Å] N2-Ru1-N1 [°] N2-Ru1-Cl1 [°] N1-Ru1-Cl1 [°] P1-O1 [Å] P1-O2 [Å] P1-O3 [Å] 1b 2.099(2) 2.080(2) 2.3861(7) 2.185 − 2.239 76.32(8) 85.44(6) 83.78(6) 1.443(2) 1.506(4) 1.635(4) 4a 2.103 (3) 2.078 (3) 2.4063 (12) 2.163 − 2.289 75.68(13) 86.12(10) 84.66(10) 1.558(3) 1.481(3) 1.512(3) 3.3. Synthesis of organogold inhibitors The reaction of one equivalent of ligand 9 with [Au(PR3)Cl] (where R = phenyl) precursor in the presence of an equimolar of base yielded the mononuclear complex 9a as shown in Scheme 3.4. Compound 9 was first treated with base to deprotonate the acidic acetylene proton to form the negatively charged ligand, followed by reaction with Au(I) precursor to afford the neutral mononuclear complex 9a by displacement of the chlorido ligand from the labile Au-Cl bond. The complex was isolated as pale-yellow solid and was soluble in common organic 21 Chapter 3 solvents namely dichloromethane, chloroform, MeOH and DMSO. However, complex 9a was only moderately soluble in water presumably due to the complex being in the neutral charge. Compound 9a was analyzed by 1H, 31 P{1H}, 19 F{1H}-NMR and ESI-MS. The acetylene proton of ligand 9 which was at a chemical shift of 3.16 ppm was no longer observed in complex 9a after complexation indicated the coordination of the alkyne to the gold metal center. Ligand 9 is most likely to coordinate to the gold metal center in a σ fashion. In addition, a set of singlet and a set of triplet were observed in the 31P{1H}-NMR due to the presence of two phosphorous atoms in complex 9a each coming from the triphenylphosphine ligand and ligand 9 respectively. The singlet resonance of triphenylphosphine at around 40 ppm is typical of that found for other alkynylphosphinegold(I) complexes.[66, 67] The set of triplet observed in the together with another set of doublet observed in the 19 31 P{1H}-NMR F{1H}-NMR were due to 2JP-F coupling. The formation of complex 9a was also supported by ESI-MS in which 9a showed as its M+ parent molecular ion in the ESI-MS mass spectrum, although the spectrum was dominated by signal corresponding to [Au(PR3)2]+ (100%) fragment, as has previously been reported for other alkynyl(phosphine)gold(I) complexes.[68, 69] With the synthesis of the alkynyltriphenylphosphinegold(I) complex 9a established, a library of compounds can be generated by reaction of ligand 9 with various Au(I) precursors of the form [Au(PR3)Cl] (where R = methyl, ethyl, cyclohexyl, etc.) following the general reaction procedure. Work is also on-going to synthesize the phosphonic acid derivative of ligand 9 and its corresponding gold(I) phosphine complexes. This novel class of gold(I)-based inhibitors will subsequently be evaluated for their inhibition towards PTPs, in particular PTP-1B and TC-PTP. 22 Chapter 3 Scheme 3.4. Synthetic route for the synthesis of gold(I) complexes. 23 Chapter 4 Chapter 4. Biological evaluation of organoruthenium PTP inhibitors 4.1. Aqueous stability of organoruthenium PTP inhibitors The aqueous stabilities of 3a and 4a were investigated using UV-Vis spectroscopy over a 24 h period. There were no significant shifts in their UV-Vis spectra in water over the 24 h period indicating good aqueous stability. In the presence of 1 mM glutathione (GSH), an endogenous intracellular thiol-containing tripeptide, however, there was a significant blue shift in both spectra of both compounds suggesting that organoruthenium complexes could potentially react with these nucleophiles. Such reactions have been reported via a myriad of different reaction pathways resulting in both mononuclear and dinuclear species. In the presence of 200 mM NaCl however, this reactivity was suppressed suggesting that the aquation of the Ru-Cl bond was important for their reactivity and not via the degradation of the imidazole linker or phosphonate group. Therefore under physiological conditions at high chloride concentrations, the organoruthenium PTP inhibitors can be expected to maintain their high stability even in the presence of nucleophiles. Upon cell entry, where the chloride levels are lower, reaction with intracellular nucleophiles may occur. 24 0.5 0.7 Absorbance Absorbance Chapter 4 0.7 0 220 270 320 370 420 24h 0h 0 0 220 270 320 370 Wavelength (nm) 420 220 270 320 370 420 Wavelength (nm) Figure 4.1. UV-Vis aqueous stability studies of complex 4a. (Left) UV-Vis spectrum of complex 4a in H2O. (Right) UV-Vis spectrum of complex 4a in 1mM GSH. (Inset) UV-Vis spectrum of complex 4a in 1mM GSH + 200mM NaCl. 4.2. Inhibition of PTP-1B and TC-PTP by organoruthenium PTP inhibitors The organoruthenium inhibitors are expected to bind to the PTP active-sites through their phenyl difluoromethylphosphonic acid moiety, which is a good non-hydrolyzable mimetic of pTyr.[33] The bulky organoruthenium scaffold can then be utilized to achieve selectivity amongst the PTPs by exploiting on the potential difference in structural environment peripheral to the substrate binding site. Several metal complexes have been previously been reported to be strong inhibitors of PTP enzymes, presumably via direct covalent binding to the Cys215 at the activesite. However, such an approach would render the metallo-inhibitor to be poorly selective across broad spectrum of PTPs since the enzymatic active-site would be highly conserved and homologous amongst with PTP family. The compounds were subjected to an initial screen at a concentration of 100 µM against PTP-1B and TC-PTP using the para-nitrophenylphosphate (pNPP) assay. Phosphatases catalyze the hydrolysis of the phosphate group on the pNPP substrate to yield para-nitrophenol which can 25 Chapter 4 be quantitated by UV-Vis spectroscopy at A405. Briefly, the enzymes were treated with the inhibitors for 30 min and pNPP substrate was added, incubated for a further 30 min before levels of para-nitrophenol were determined. ICP-OES was carried out to determine the amount of ruthenium metal content in the stock solutions so as to determine the actual concentrations of the prepared solutions and to account for weighing error and purity of the compounds. In addition, the same stock of organoruthenium solution was applied against two different enzymes to minimize errors arising to sample preparation. Based on the initial screen as shown in Figure 4.2, compounds containing the diethylphosphonate ester ligands, namely 1a-b and 3a-b, were ineffective regardless of the nature of their Ru-arene fragment or linker groups. Indeed only compounds containing the phosphonic acid moiety, namely 2a-b and 4a-b, were efficacious. This was expected since the ester groups would increase the steric encumbrance around activesite targeting moiety and preventing effective binding. In addition, it rendered the phosphonate group strongly hydrophobic and lowers the affinity towards the hydrophilic enzyme pocket. Because the inhibitor contains a second-row transition metal with known affinity towards soft nucleophiles such cysteine, we investigated whether the Ru-arene fragment can itself directly inhibit enzymatic activity. Therefore, [(ŋ6-cymene)Ru(pyridylimidazole)Cl]PF6 5 and [(ŋ6-cymene)Ru(pyridylbenzimidazole)Cl]PF6 6 which model the organoruthenium imidazole and organoruthenium benzimidazole fragments respectively and themselves do not contain phenyl difluoromethylphosphonic acid moiety were prepared. These model compounds were themselves inactive indicating that the phenyl difluoromethylphosphonic acid groups were important and that the organoruthenium imidazole/benzimidazole groups were not solely responsible for inhibitory activity observed in 2a-b and 4a-b. 26 Chapter 4 Inhibition of PTPs at 100 µM concentration 120 100 % Activity 80 60 PTP-1B TC-PTP 40 20 0 1a 1b 2 2a 2b 3a 3b 4 Compounds 4a 4b 5 6 Figure 4.2. Inhibition of PTP-1B and TC-PTP at inhibitor concentration of 100 µM. Comparing between the organoruthenium inhibitors and their parent ligands, it was immediately obvious that at high concentrations, the parent ligand was equally efficacious against both PTP-1B and TC-PTP. When the organoruthenium moiety was incorporated, selectivity against PTP-1B was drastically improved with the organoruthenium inhibitors being 7-11 fold more efficacious against PTP-1B. This observation prompted a more detailed investigation examining a dose-response of the organoruthenium inhibitors against both PTP enzymes. The IC50 values, which depicted the concentration at which the enzymatic activity is reduced to 50% level vs untreated controls, was shown in Table 4.1. As with the initial screen, the inhibitors were some 7-10 fold effective against PTP-1B than TC-PTP. In general, with the incorporation of the ruthenium scaffold, the inhibition of PTP-1B is enhanced with respect to the parent ligands. This enhanced effect could be due to favorable interactions of the ruthenium 27 Chapter 4 scaffold in the inhibitors with amino-acid residues surrounding the active site in PTP-1B leading to better binding of the complexes in PTP-1B. Figure 4.3. Dose-response curves of inhibition of compounds towards PTP-1B (bold) and TC-PTP (dotted). Molecular docking studies of PTP-1B against 4a with ligand 4 as comparison were done by our collaborator, Dr Yap Chun Wei from the Department of Pharmacy. Results from the docking study, as shown in Figure 4.4, indicated that the cymene ligand in 4a was able to interact with Phe182. This additional interaction increased its binding energy to -10.84 kcal/mol, which was stronger than the -9.90 kcal/mol seen in the parent ligand 4. On the other hand, the observed diminishing effect in the potency of the inhibitors compared to their parent ligands towards TC- 28 Chapter 4 PTP is postulated to be attributed to steric effects whereby by the bulkiness of the ruthenium-arene ruthenium moiety impeded the effective binding of the complexes at the active-site off the enzyme. Figure 4.4. Conformation of parent ligand 4 in PTP-1B substrate binding site (-9.90 9.90 kcal/mol) (left); conformation of 4a in PTP-1B 1B substrate binding site (-10.84 ( kcal/mol) (right). The hydrophobic regions around of the phosphatase substrate binding site are highlighted in yellow. From these results, we can see that with the presence of the ruthenium scaffold in the structures, the complexes are able to inhibit PTP-1B PTP with increased potency while the inhibition inhibi towards TC-PTP PTP was diminished diminished. As such, selectivity towards PTP-1B 1B is achie achieved which is otherwise not prominent in the parent ligands. Interestingly, the imidazole analogues are more potent in the inhibition of TC TC-PTP PTP as compared to the benzimidazole counterparts. We postulated that the he steric effect which the imidazole analogues exhibit is less pronounced and this enables the compounds to bind relatively better in TC-PTP TC PTP thereby giving lower IC50 values. However, this decrease in steric bulk has ha no effect on PTP-1B 1B as both the imidazole and 29 Chapter 4 benzimidazole families of inhibitors bind equally well in PTP-1B. In essence, we can see that with the presence of the ruthenium scaffold, the parent ligand 2 which was initially selective towards TC-PTP, was eventually tuned to be more selective towards PTP-1B in complexes 2a-b. Overall, it can be concluded that these four ruthenium-arene complexes, 2a-b and 4a-b, are able to selectivity inhibit PTP-1B over TC-PTP with IC50 values in the low micro-molar range. Table 4.1. Initial screening of inhibitors against PTP-1B and TC-PTP and IC50 of selected compounds. Compounds Screeninga PTP-1B TC-PTP 99.3 ± 1.7 99.7 ± 0.7 1a 99.6 ± 0.7 100.2 ± 0.8 1b 43.4 ± 1.8 34.8 ± 1.0 2 7.8 ± 0.3 45.1 ± 1.2 2a 7.3 ± 0.7 44.0 ± 1.6 2b 99.1 ± 1.9 99.4 ± 1.6 3a 99.7 ± 0.6 100.4 ± 0.6 3b 42.8 ± 1.1 42.1 ± 2.5 4 6.7 ± 0.7 57.4 ± 1.2 4a 7.0 ± 0.3 56.5 ± 0.6 4b 99.5 ± 0.9 99.4 ± 1.2 5 100.6 ± 1.3 100.1 ± 0.9 6 a Screening was performed at inhibitor concentration of 100 µM. IC50 PTP-1B 72.0 ± 1.1 20.9 ± 1.0 18.7 ± 1.0 68.6 ± 1.1 14.2 ± 1.0 16.6 ± 1.0 - TC-PTP 45.1 ± 1.0 73.4 ± 1.0 69.1 ± 1.1 72.4 ± 1.0 112.1 ± 1.1 108.7 ± 1.1 - 4.3. Determination of inhibition constants of 4a towards PTP-1B and TC-PTP Concerning whether the observed difference in inhibition effects of the complexes arose due to different binding mechanisms of the complexes towards PTP-1B and TC-PTP, detailed kinetic studies were performed to elucidate the binding modes of the complexes towards the enzymes. The steady state kinetic experiments were conducted with six different concentrations of pNPP and five different concentrations of 4a with each of the enzymes. Both LineweaverBurk plots (Figure 4.5) intersected at the same point on the 1/v axis indicating competitive mode of inhibition and implying that 4a bound to both PTP-1B and TC-PTP at their substrate binding sites. The inhibition constant (Ki) of complex 4a was determined to be 7.3 µM and 80.1 µM for 30 Chapter 4 PTP-1B and TC-PTP respectively, consistent with the IC50 values. Taken together, these data indicated that the inhibition of the organoruthenium complexes of PTP-1B and TC-PTP could be attributed to the different structural environment of the PTPs proximal to the substrate binding site resulting in different binding affinities and inhibitory efficacies. PTP-1B 0.6 PTP-1B 0.15 50 µM 0.4 0.1 25 µM 1/v K 12.5 µM 0.2 6.3 µM 0 µM 0 0 -1 Ki = 7.3 µM 0.05 0 1 1.2 2 3 1/[pNPP] (mM -1) 4 5 -10 10 30 50 Inhibitor (µM) 0.3 TC-PTP TC-PTP 150 µM 125 µM 100 µM 0.8 50 µM 1/v 0 µM 0.4 K Ki = 80.1 µM 0.1 0 0 -1 0.2 0 1 2 1/[pNPP] 3 (mM -1) 4 5 -100 0 100 200 Inhibitor (µM) Figure 4.5. Steady-state kinetic studies of complex 4a towards PTP-1B and TC-PTP. (Left-Top/Bottom) Lineweaver-Burk plot of 1/v (min µM-1) vs the reciprocal of pNPP concentration (mM-1) at five fixed concentrations of complex 4a for the inhibition towards PTP-1B and TC-PTP. (Right-Top/Bottom) Ki determination for complex 4a inhibiting PTP-1B and TC-PTP. The value of -Ki was determined from the x-intercept of a plot of the slope of the line from the double-reciprocal plot as a function of inhibitor concentration (µM). 31 Chapter 5 Chapter 5. Conclusion In summary, several ruthenium(II)-arene complexes were rationally designed and synthesized to be selective inhibitors of PTP enzymes. Only inhibitors containing phosphonic acid groups were efficacious and they inhibit PTP-1B at low micromolar level with 7-10 fold selectivity towards PTP-1B over TC-PTP. Addition of the organoruthenium-arene fragments improved inhibitory activities against PTP-1B while activities against TC-PTP were diminished, leading to pronounced selectivities not observed in the parent ligands. Alone, the organoruthenium-imidazole/benzimidazole fragments were not phosphatase inhibitors indicating that they are not solely responsible for the improved efficacies. Steady-state kinetics and molecular docking indicated that the complexes competitively bind to the enzymes at their active sites. These results showed that molecular recognition of PTPs can be achieved by targeting both the substrate site and its periphery using organometallic principles of assembly and structural diversity. As a continuation of this work, future research will be focus on the synthesis of the gold-based inhibitors and their activities towards inhibition of PTPs. 32 Chapter 6 Chapter 6. Experimental Section Materials and methods: All reagents were purchased from commercial vendors and used without further purification. Solvents were dried and distilled by standard procedures, and reactions were performed under nitrogen using standard Schlenk techniques. PTP-1B and TCPTP were purchased from Sino-Biological. 2-(2-pyridyl)imidazole,[70] p-triflate-benzaldehyde,[71] p-iodo-benzaldehyde,[72] [(ŋ6-cymene)RuCl2]2,[73] [(ŋ6-TIPB)RuCl2]2,[74] and [Au(PPh)3Cl],[75] were synthesized according to literature methods. 1H, 31 P and 19 F NMR was recorded on a Bruker Avance 300 MHz or 400 MHz model. Chemical shifts are reported in parts per million relative to residual solvent peaks. Electrospray ionization mass spectra (ESI-MS) were obtained on a Thermo Finnigan MAT ESI-MS system. UV-Vis spectra were recorded on a Shimadzu UV1800 UV-vis spectrophotometer. Determination of Ru levels was carried out by CMMAC (National University of Singapore) on a Optima ICP-OES (Perkin Elmer). Purity of ruthenium(II) compounds were conducted using analytical HPLC on a Shimadzu Prominence using a Shimpack VP-ODS C18 (5 µM, 120 Å, 250 x 4.60 mm i.d) column at r.t at a flow rate of 1.0 ml/min with 254 nm UV detection. The gradient eluent conditions were as follows: 20-80% B over 20 min where solvent A is H2O and solvent B is MeCN + 0.1% TFA. PTP inhibition assays and enzymatic kinetic studies: PTP inhibition assays and enzymatic kinetic studies were carried out in accordance with a reported procedure, using p-nitrophenyl phosphate (pNPP) as the substrate.[76] The inhibition assays were performed in buffer (20 mM MOPS, 200 mM NaCl, pH 7.2, 100 µL) on a 96-well plate. Stock solutions of the inhibitors were prepared in ultrapure water and serially diluted to concentrations of 0.03-1 mM. The ruthenium concentrations of these stock solutions were determined using ICP-OES. Inhibitors (0.03-1 mM, 33 Chapter 6 10 µL) were incubated with enzymes (150 nM) in buffer (20 mM MOPS, 200 mM NaCl, pH 7.2, 75 µL) at r.t. for 30 min. The reaction was initiated by the addition of pNPP (20 mM MOPS, 10 µL) and incubated for further 30 min before being terminated using the stop buffer (1 M NaOH, 5 µL). A405 was recorded using a microplate reader (Tecan) and IC50 values were obtained by fitting the concentration-dependent inhibition curves using Prism (GraphPad). The experiments were carried out in triplicates. Solutions of the compounds were freshly prepared before each inhibition assays and ruthenium levels determined using ICP-OES where applicable. Kinetic analysis was performed according to the following rate equation.[77] ‫=ݒ‬ ܸ௠௔௫ ሾܵሿ ሾ‫ ܫ‬ሿ ‫ܭ‬௠ ൬1 + ൰ + ሾܵሿ ‫ܭ‬௜ where v = initial velocity, Vmax is the maximum initial velocity, Km is the Michaelis constant for the substrate, [S] and [I] for concentrations of substrate and inhibitor, and Ki is the inhibition constant, derived from the slope of the Lineweaver-Burk plots. The initial hydrolysis rates were measured at different substrate and inhibitor concentrations. The reciprocal of the reaction rate was plotted as a function of the reciprocal of the substrate concentration for each concentration of the inhibitor. The Ki values measured at the various inhibitor concentrations were plotted against concentration of the inhibitor to calculate the inhibition constants. X-ray Diffraction Studies: X-ray data were collected with a Bruker AXS SMART APEX diffractometer using Mo-Kα radiation at 223(2) K with the SMART suite of Programs.[78] Data were processed and corrected for Lorentz and polarization effects using SAINT software,[79] and for absorption effects using the SADABS software[80]. Structural solution and refinement were 34 Chapter 6 then carried out using the SHELXTL suite of programs.[81] The structure was solved by Direct Methods. Non-hydrogen atoms were located using difference maps and were given anisotropic displacement parameters in the final refinement. All H atoms were put at calculated positions using the riding model. 6.1. Preparation of organoruthenium complexes Synthesis of hydroxyl-phosphonate ester (P1). p-Tolualdehyde (2.34 mL, 20 mmol) was dissolved in THF (10 mL) and diethyl phosphite (3.09 mL, 24 mmol) was added dropwise. Tetramethylguanidine (0.25 mL, 2 mmol) was added dropwise and the reaction mixture was stirred at r.t. for 1 h. The reaction mixture was diluted with ethyl acetate (15 mL) and washed two times with 1 M HCl (10 mL). The organic portion was dried over MgSO4 and the solvent was removed to give the product as white solid. Yield: 4.65 g (90%). 1H-NMR (300 MHz, CDCl3): δ 7.35 (2H, d), 7.15 (2H, d), 4.97 (1H, d), 4.05 (4H, m), 2.34 (3H, s), 1.24 (6H, t); 31P NMR (121 MHz, CDCl3): δ 22.3 (s). Synthesis of difluoromethylphosphonate ester (P3). P1 (516.5 mg, 2 mmol) was dissolved in dichloromethane (10 mL) and cooled to 0°C. DMP (1.27 g, 3 mmol) was added to the solution and the reaction mixture was stirred at r.t. for 2 h. The reaction mixture was filtered and to the filtrate was added a solution containing 0.5 M Na2S2O3 (15 mL) and 1 M NaHCO3 (15 mL) and stirred for 30 min. The solution was then extracted three times with ethyl acetate (20 mL) and the organic portion was dried over MgSO4. The solvent was removed to give crude P2 as yellow oil. P2 was re-dissolved in dry dichloromethane (8 mL) and cooled to 0°C. DAST (0.53 ml, 4 mmol) was then added drop-wise under nitrogen atmosphere. The reaction mixture was allowed to 35 Chapter 6 warm to r.t. and stirred for 12 h. Thereafter, the mixture was diluted with dichloromethane (10 mL) and ice-cold 1 M NaHCO3 (20 mL) and stirred for an additional 30 min. The reaction mixture was extracted three times with dichloromethane (15 mL) and the organic portion was dried over MgSO4. The solvent was removed to give the crude compound as dark-brown oil and purified by flash column chromatography (2:3 v/v ethyl acetate:hexane, Rf = 0.7) to give the product as yellow oil. Final yield: 306.1 mg (55%). P2: 1H-NMR (300 MHz, CDCl3): δ 8.17 (2H, d), 7.30 (2H, d), 4.26 (4H, m), 2.39 (3H, s), 1.37 (6H, t); 31P NMR (121 MHz, CDCl3): δ 0.3835 (s). P3: 1H-NMR (300 MHz, CDCl3): δ 7.50 (2H, d), 7.25 (2H, d), 4.20 (4H, m), 2.39 (3H, s), 1.31 (6H, t); 31 P NMR (121 MHz, CDCl3): δ 7.16 (t, 2JP-F = 118.5 Hz); 19 F NMR (282 MHz, CDCl3): δ -31.9 (d, 2JP-F = 118.5 Hz). Synthesis of bromobenzyl difluoromethylphosphonate ester (P4). P3 (322.7 mg, 1.16 mmol), NBS (231.4 mg, 1.30 mmol) and AIBN (9.5 mg, 58 µmol) were dissolved in CCl4 (10 mL) and the reaction mixture was refluxed for 3 h. After the reaction, the reaction mixture was diluted with CCl4 (10 mL) and washed once with water (15 mL), 1 M NaHCO3 (15 mL) and brine (15 mL). The organic portion was dried over MgSO4 to give the crude product as pale yellow oil. 1H NMR analysis of the crude material indicated a mixture of starting material, mono-brominated P4 and the di-brominated side-product in a ratio of 16:67:17%. The crude product was purified by flash column chromatography (1:2 v/v ethyl acetate:hexane, Rf = 0.8) to isolate monobrominated compound P4 and di-brominated side-product as mixture of colourless oils. The mixture was used without further purification for the following step. P4: Yield: 220.2 mg (53%). 1 H NMR (300 MHz, CDCl3): δ 7.60 (2H, d), 7.48 (2H, d), 4.49 (2H, s), 4.18 (4H, m), 1.23 (6H, 36 Chapter 6 t); 31P NMR (121 MHz, CDCl3): δ 6.75 (t, 2JP-F = 115.5 Hz); 19 F NMR (282 MHz, CDCl3): δ - 32.5 (d, 2JP-F = 115.5 Hz). Synthesis of imidazole-diethylphosphonate ester (1). The crude mixture (0.177 g containing 67% of mono-brominated compound P4) and 2-(2-pyridyl)-imidazole (47.7 mg, 0.331 mmol) were dissolved in DMF (4 mL) and potassium tert-butoxide (44.6 mg, 0.40 mmol) in butanol (1 mL) was added drop-wise to the reaction mixture with stirring. The reaction mixture was stirred at r.t. for 12 h. After the reaction, the solvent was removed and the crude compound was purified by column chromatography (4:1 v/v ethyl acetate:hexane, Rf = 0.35) to give product as viscous colourless oil. Yield: 66.9 mg (48%). 1H NMR (300 MHz, CDCl3): 8.48 (1H, d); 8.20 (1H, d); 7.74 (1H, td); 7.53 (2H, d); 7.17-7.23 (4H, m); 7.00 (1H, d); 5.98 (2H, s); 4.15 (4H, m); 1.27 (6H, t); 31P NMR (121 MHz, CDCl3): δ 6.80 (t, 2JP-F = 117.0 Hz); 19F NMR (282 MHz, CDCl3): δ -32.4 (d, 2JP-F = 117.0 Hz). ESI (+ve mode): m/z 422.1 (M+H+), 444.1 (M+Na+). Synthesis of imidazole-phosphonic acid (2). Ligand 1 (18 mg, 43 µmol) was dissolved in dichloromethane (3 mL) and N,O-bis(trimethylsilyl) trifluoroacetamide (57 µL, 0.214 mmol) was added to the solution. After stirring for 15 min, iodotrimethylsilane (30 µL, 0.214 mmol) was added to the reaction mixture. After stirring for an additional 2 h, the solvent was removed and the residue was co-evaporated three times with dichloromethane (4 mL). The resulting residue was re-dissolved in dichloromethane (2 mL) followed by addition of MeOH (10 mL) and stirred at r.t. overnight. After the reaction, solvent was removed and the residue was washed three times with water (4 mL). The residue was then suspended in water (2 mL) and 1 M NH4OH (1mL) was added drop-wise till the residue dissolved followed by stirring for 0.5 h. The solvent was 37 Chapter 6 removed and the residue was washed once with dichloromethane (3 mL) and twice with diethyl ether (3 mL) to give the product as white solid. Yield: 11.0 mg (70%). 1H NMR (400 MHz, D2O): δ 8.58 (1H, d); 7.88 (1H, td); 7.67 (1H, d); 7.46 (1H, s); 7.43 (2H, d); 7.36 (1H, s); 7.18 (1H, s); 7.03 (2H, d); 5.57 (2H, s); 31 P NMR (162 MHz, D2O): δ 4.98 (t, 2JP-F = 93.0 Hz); 19 F NMR (376 MHz, D2O): δ -106.2 (d, 2JP-F = 93.0 Hz). ESI (+ve mode): m/z 366.1 (M+H+); (-ve mode): m/z 364.2 (M-H-). Purity (HPLC): 95.8% (based on chromatogram at 254 nm). Synthesis of benzimidazole-diethylphosphonate ester (3). The crude mixture (0.242 g containing 67% of mono-brominated compound P4) and 2-(2-pyridyl)-benzimidazole (88.1 mg, 0.451 mmol) were dissolved in DMF (4 mL) and potassium tert-butoxide (60.7 mg, 0.542 mmol) in butanol (1 mL) was added drop-wise to the reaction mixture with stirring. The reaction mixture was stirred at r.t. for 12 h. After the reaction, the solvent was removed and the crude compound was purified by column chromatography (1:1 v/v ethyl acetate:hexane, Rf = 0.5) to give product as viscous yellow oil. Yield: 131.8 mg (62%). 1H NMR (400 MHz, CDCl3): 8.60 (1H, d); 8.44 (1H, dd); 7.81-7.88 (2H, m); 7.50 (2H, d); 7.28-7.33 (4H, m); 7.25 (2H, d); 6.23 (2H, s); 4.12 (4H, m); 1.26 (6H, t); 31P NMR (162 MHz, CDCl3): δ 6.21 (t, 2JP-F = 115.8 Hz); 19F NMR (376 MHz, CDCl3): δ -108.3 (d, 2JP-F = 115.8 Hz). ESI (+ve mode): m/z 472.4 (M+H+), 494.4 (M+Na+). Synthesis of benzimidazole-phosphonic acid (4). Ligand 2 (30 mg, 63.6 µmol) was dissolved in dichloromethane (3 mL) and N,O-bis(trimethylsilyl)trifluoroacetamide (85 µL, 0.318 mmol) was added to the solution. After stirring for 15 min, iodotrimethylsilane (46 µL, 0.318 mmol) was added to the reaction mixture. After stirring for an additional 2 h, the solvent was removed 38 Chapter 6 and the residue was co-evaporated three times with dichloromethane (4 mL). The resulting residue was re-dissolved in dichloromethane (2 mL) followed by addition of MeOH (10 mL) and stirred at r.t. overnight. After the reaction, solvent was removed and the residue was washed three times with water (4 ml). The residue was then suspended in water (2 mL) and 1 M NH4OH (1mL) was added drop-wise till the residue dissolved followed by stirring for 0.5 h. The solvent was removed and the residue was washed once with dichloromethane (3 mL) and twice with diethyl ether (3 mL) to give the product as colourless solid. Yield: 18.2 mg (69%). 1H NMR (400 MHz, D2O): δ 8.66 (1H, d); 7.89-8.00 (2H, m); 7.78 (1H, d); 7.55 (2H, d); 7.35-7.40 (4H, m); 6.99 (2H, d); 5.85 (2H, s); 31P NMR (162 MHz, D2O): δ 5.05 (t, 2JP-F = 92.6 Hz); 19F NMR (376 MHz, D2O): δ -106.1 (d, 2JP-F = 92.6 Hz). ESI (+ve mode): m/z 416.2 (M+H+); (-ve mode): m/z 414.3 (M-H-). Purity (HPLC): 98.5% (based on chromatogram at 254 nm). Synthesis of [(ŋ6-cymene)Ru(1)Cl]PF6 (1a). The ligand 1 (9.3 mg, 22.1 µmol) was dissolved in MeOH (10 mL) and [(ŋ6-cymene)RuCl2]2 (6.7 mg, 11.0 µmol) was added. The reaction mixture was stirred at r.t. for 4 h. After the reaction, solid NH4PF6 (3.6 mg, 22.1 µmol) was added to the reaction mixture and stirred for an additional 0.5 h. The solvent was removed and the residue redissolved in small amount of dichloromethane (2 mL), filtered through celite and diethyl ether (8 mL) was added to give an orange-yellow precipitate. The precipitate was collected, washed twice with diethyl ether (6 mL) and vacuum dried. Yield: 17.6 mg (95%). 1H NMR (300 MHz, CDCl3): δ 9.24 (1H, d); 7.85 (1H, t); 7.66-7.69 (2H, m), 7.59 (2H, d); 7.42 (1H, t); 7.17-7.20 (3H, m); 5.85 (1H, d); 5.78(1H, d); 5.67(3H, s); 5.60(1H, d); 4.16 (4H, m); 2.79 (1H, m); 2.19 (3H, s); 1.28 (6H, t); 1.19 (3H, d); 1.17 (3H, d); 31P NMR (121 MHz, CDCl3): δ 6.21 (t, 2JP-F = 114.4 Hz); -143.7 (m, PF6-); 19F NMR (282 MHz, CDCl3): δ -32.7 (d, 2JP-F = 114.4 Hz); 3.81 (d, 39 Chapter 6 PF6-). ESI (+ve mode): m/z 692.1 (M+, 100%). Purity (HPLC): 98.1% (based on chromatogram at 254 nm). Synthesis of [(ŋ6-TIPB)Ru(1)Cl]PF6 (1b). Complex 1b was obtained from ligand 1 (11.2 mg, 26.6 µmol) and [(ŋ6-TIPB)RuCl2]2 (10.0 mg, 13.3 µmol) using the same procedure as used for 1a. Yield: 24.0 mg (99%). 1H NMR (300 MHz, CDCl3): δ 9.14 (1H, d); 7.85 (1H, t); 7.68 (1H, d); 7.56-7.58 (3H, m); 7.48 (1H, t); 7.26 (1H, s), 7.16 (2H, d); 5.70 (1H, d); 5.63 (1H, d); 5.55 (3H, s); 4.17 (4H, m); 2.93 (3H, m); 1.31 (9H, d); 1.25 (9H, d); 31P NMR (121 MHz, CDCl3): δ 6.24 (t, 2JP-F = 115.5 Hz); -143.7 (m, PF6-); 19 F NMR (282 MHz, CDCl3): δ -32.7 (d, 2JP-F = 115.5 Hz); 3.45 (d, PF6-). ESI (+ve mode): m/z 762.3 (M+, 100%). Purity (HPLC): 99.2% (based on chromatogram at 254 nm). Synthesis of [(ŋ6-cymene)Ru(3)Cl]PF6 (3a). Complex 3a was obtained from ligand 3 (13.0 mg, 27.6 µmol) and [(ŋ6-cymene)RuCl2]2 (8.4 mg, 13.7 µmol) using the same procedure as used for 1a. Yield: 24.3mg (99%). 1H NMR (300 MHz, CDCl3): δ 9.16 (1H, d); 7.86-7.94 (3H, m); 7.497.67 (6H, m); 7.11(2H, d); 6.03-6.10 (3H, m); 5.87-5.93 (2H, m); 5.80 (1H, d); 4.15 (4H, m); 2.63 (1H, m); 2.61 (3H, s); 1.27 (6H, t); 1.04 (6H, d); 31P NMR (121 MHz, CDCl3): δ 6.23 (t, 2JPF = 114.4 Hz); -143.7 (m, PF6-); 19F NMR (282 MHz, CDCl3): δ -32.6 (d, 2JP-F = 114.4 Hz); 3.84 (d, PF6-). ESI (+ve mode): m/z 742.1 (M+, 100%). Purity (HPLC): 99.6% (based on chromatogram at 254 nm). Synthesis of [(ŋ6-TIPB)Ru(3)Cl]PF6 (3b). Complex 3b was obtained from ligand 3 (12.5 mg, 26.5 µmol) and [(ŋ6-TIPB)RuCl2]2 (9.9 mg, 13.2 µmol) using the same procedure as used for 1a. 40 Chapter 6 Yield: 24.5 mg (97%). 1H NMR (300 MHz, CDCl3): 9.16 (1H, d); 8.00 (1H, d); 7.89 (2H, d); 7.52-7.65 (6H, m); 7.10 (2H, d); 6.17 (1H, d); 5.87 (1H, d); 5.65 (3H, s); 4.14 (4H, m); 2.95 (3H, m); 1.26 (9H, d); 1.22 (9H, d); 31P NMR (121 MHz, CDCl3): δ 6.26 (t, 2JP-F = 114.4 Hz); -143.9 (m, PF6-); 19 F NMR (282 MHz, CDCl3): δ -32.6 (d, 2JP-F = 114.4 Hz); 3.42 (d, PF6-). ESI (+ve mode): m/z 812.2 (M+, 100%). Purity (HPLC): 98.6% (based on chromatogram at 254 nm). Synthesis of [(ŋ6-cymene)Ru(2)Cl]Cl (2a). [(ŋ6-cymene)RuCl2]2 (8.5 mg, 14.0 µmol) was dissolved in MeOH (10 mL) and ligand 2 (10.2 mg, 28.0 µmol) dissolved in water (1 mL) was added. The reaction mixture was stirred at r.t. overnight. After the reaction, the solvent was reduced to a small volume (2 mL) and diethyl ether (8 mL) was added to give a yellow precipitate. The yellow precipitate was collected, washed once with dichloromethane (4mL), once with ethyl acetate (4mL), once with diethyl ether (4mL) and vacuum dried. Yield: 16.9 mg (90%). 1H NMR (400 MHz, MeOD): 9.37 (1H, d), 7.94-7.98 (2H, m); 7.83 (1H, d); 7.65 (1H, s); 7.57 (2H, d); 7.50 (1H, t); 7.06 (2H, d); 6.06 (1H, d); 5.94 (1H, d); 5.77-5.81 (3H, m); 5.70 (1H, d); 2.59 (1H, m); 2.18 (3H, s); 1.03 (3H, d); 0.98 (3H, d); 31P NMR (162 MHz, MeOD): δ 2.96 (t, 2JP-F = 98.1 Hz); 19F NMR (376 MHz, MeOD): δ -109.1 (d, 2JP-F = 98.1 Hz). ESI (+ve mode): m/z 636.1 (M+, 100%). Purity (HPLC): 95.6% (based on chromatogram at 254 nm). Synthesis of [(ŋ6-TIPB)Ru(2)Cl]Cl (2b). Complex 2b was obtained from ligand 2 (10.2 mg, 28.0 µmol) and [(ŋ6-TIPB)RuCl2]2 (10.5 mg, 14.0 µmol) using the same procedure as used for 2a. Yield: 18.3 mg (88%). 1H NMR (400 MHz, MeOD): 9.15 (1H, d), 7.72-7.76 (2H, m); 7.61 (1H, d); 7.43 (1H, s); 7.35 (2H, d); 7.28 (1H, t); 6.84 (2H, d); 6.06 (1H, d); 5.93 (1H, d); 5.81 (3H, s); 2.87 (3H, m); 1.10 (9H, d); 1.07 (9H, d); 31P NMR (162 MHz, MeOD): δ 3.05 (t, 2JP-F = 41 Chapter 6 99.1 Hz); 19F NMR (376 MHz, MeOD): δ -109.0 (d, 2JP-F = 99.1 Hz). ESI (+ve mode): m/z 706.1 (M+, 100%). Purity (HPLC): 95.5% (based on chromatogram at 254 nm). Synthesis of [(ŋ6-cymene)Ru(4)Cl]Cl (4a). Complex 4a was obtained from ligand 4 (12.6 mg, 30.3 µmol) and [(ŋ6-cymene)RuCl2]2 (9.2 mg, 15.1 µmol) using the same procedure as used for 2a. Yield: 20.4 mg (93%). 1H NMR (400 MHz, MeOD): 9.54 (1H, d); 8.08-8.16 (3H, m); 7.84 (1H, d), 7.62-7.70 (3H, m); 7.53 (2H, d); 7.02 (2H, d); 6.34 (1H, d); 6.08-6.15 (3H, m); 6.04 (1H, d); 5.95 (1H, d); 2.43 (1H, m); 2.27 (3H, s); 0.90 (3H, d); 0.88 (3H, d); 31 P NMR (162 MHz, MeOD): δ 3.08 (t, 2JP-F = 98.1 Hz); 19F NMR (376 MHz, MeOD): δ -109.2 (d, 2JP-F = 98.1 Hz). ESI (+ve mode): m/z 686.1 (M+, 100%). Purity (HPLC): 97.3% (based on chromatogram at 254 nm). Synthesis of [(ŋ6-TIPB)Ru(4)Cl]Cl (4b). Complex 4b was obtained from ligand 4 (12.5 mg, 30.0 µmol) and [(ŋ6-TIPB)RuCl2]2 (11.3 mg, 15.0 µmol) using the same procedure as used for 2a. Yield: 21.4 mg (90%). 1H NMR (400 MHz, MeOD): 9.32 (1H, d); 8.09-8.20 (3H, m); 7.637.85 (4H, m); 7.56 (2H, d); 7.07 (2H, d); 6.16 (1H, d); 6.03 (1H, d); 5.91 (3H, s); 2.97 (3H, m); 1.20 (9H, d); 1.17 (9H, d); 31 P NMR (162MHz, MeOD): δ 3.07 (t, 2JP-F = 99.5 Hz); 19 F NMR (376 MHz, MeOD): δ -109.2 (d, 2JP-F = 99.5 Hz). ESI (+ve mode): m/z 756.2 (M+, 100%). Purity (HPLC): 98.4% (based on chromatogram at 254 nm). Synthesis of [(ŋ6-cymene)Ru(pyridylimidazole)Cl]PF6 (5). 2-(2-pyridyl)-imidazole (7.26 mg, 50.0 µmol) was dissolved in MeOH (10 mL) and [(ŋ6-cymene)RuCl2]2 (15.3 mg, 25.0 µmol) was added. The reaction mixture was stirred at room temperature for 4 h. After the reaction, solid 42 Chapter 6 NH4PF6 (8.2 mg, 50.0 µmol) was added to the reaction mixture and stirred for an additional 0.5 h. After the reaction, the solvent was reduced to a small amount (2 mL), filtered through celite and diethyl ether (8 mL) was added to give an orange-yellow precipitate. The precipitate was collected, washed twice with diethyl ether (6 mL) and vacuum dried. Yield: 26.9 mg (96%). 1H NMR (400 MHz, MeOD): δ 9.33 (1H, d); 8.08 (1H, td); 7.97 (1H, d), 7.83 (1H, d), 7.53-7.57 (2H, m); 6.03 (1H, d); 5.94 (1H, d); 5.79 (1H, d); 5.70 (1H, d); 2.62 (1H, m); 2.15 (3H, s); 1.04 (3H, d); 0.99 (3H, d); P NMR (162 MHz, MeOD): δ -144.5 (m, PF6-); 31 19 F NMR (376 MHz, MeOD): δ -74.5 (d, PF6-). Syntheis of [(ŋ6-cymene)Ru(pyridylbenzimidazole)Cl]PF6 (6). Complex 6 was obtained from 2-(2-pyridyl)-benzimidazole (9.8 mg, 50.0 µmol) and [(ŋ6-cymene)RuCl2]2 (15.3 mg, 25.0 µmol) using the same procedure as used for 5. Yield: 28.7 mg (94%). 1H NMR (400 MHz, MeOD): δ 9.48 (1H, d); 8.18-8.23 (2H, m); 8.00-8.02 (1H, m); 7.67-7.71 (2H, m); 7.51-7.58 (2H, m); 6.27 (1H, d); 6.13 (1H, d); 5.98 (1H, d); 5.95 (1H, d); 2.47 (1H, m); 2.20 (3H, s); 0.92 (3H, d); 0.91 (3H, d); 31P NMR (162 MHz, MeOD): δ -144.5 (m, PF6-); 19F NMR (376 MHz, MeOD): δ -74.5 (d, PF6-). Synthesis of [(ŋ6-cymene)RuI(2)]PF6. Complex 1a (8.4 mg, 10.0 µmol) was dissolved in dichloromethane (5 mL) and iodotrimethylsilane (11 µL, 75.0 µmol) was added and stirred at r.t. overnight. The solvent was then evaporated and to the residue was added MeOH (10 mL). The reaction mixture was stirred at r.t. for 24 h. After the reaction, the solvent was reduced to ca. 2 mL and diethyl ether (8 mL) was added to give a yellow precipitate. The yellow precipitate was collected, washed once with dichloromethane (6 mL), once with ethyl acetate (6mL), twice with 43 Chapter 6 diethyl ether (6 mL) and vacuum dried. Yield: 7.1 mg (81%). 1H NMR (300 MHz, MeOD): δ 9.40 (1H, d); 7.99 (1H, s); 7.96 (1H, s); 7.89 (1H, d); 7.74 (1H, s); 7.64 (2H, d); 7.51 (1H, t); 7.13 (2H, d); 6.03(1H, d); 5.94 (1H, d); 5.88 (2H, s); 5.84 (1H, d); 5.79 (1H, d); 2.87 (1H, m); 2.47 (3H, s); 1.15 (3H, d); 1.11(3H, d); 31P NMR (121 MHz, MeOD): δ 3.63 (t, 2JP-F = 99.0 Hz); -144.0 (m, PF6-); 19 F NMR (282 MHz, MeOD): δ -33.3 (d, 2JP-F = 99.0 Hz); 1.09 (d, PF6-). ESI (+ve mode): m/z 728.0 (M+, 100%). 6.2. Preparation of organogold complexes Synthesis of triflate-hydroxyl-phosphonate ester (P5a). p-triflate-benzaldehyde (0.508 g, 2 mmol) was dissolved in THF (6 mL) and diethyl phosphite (0.283 mL, 2.2 mmol) was added dropwise. Tetramethylguanidine (0.025 mL, 0.2 mmol) was added dropwise and the reaction mixture was stirred at r.t. for 1 h. The reaction mixture was diluted with ethyl acetate (10 mL) and washed two times with 1 M HCl (10 mL). The organic portion was dried over MgSO4 and the solvent was removed to give the product as white solid. Yield: 0.690 g (88%). 1H-NMR (300 MHz, CDCl3): δ 7.58 (2H, d), 7.28 (2H, d), 5.07 (1H, d), 4.09 (4H, m), 1.25 (6H, t); 31P NMR (121 MHz, CDCl3): δ 21.0 (s); 19F NMR (282 MHz, CDCl3): δ 3.16 (s). Synthesis of bromo-hydroxyl-phosphonate ester (P5b). p-bromo-benzaldehyde (0.370 g, 2 mmol) was dissolved in THF (6 mL) and diethyl phosphite (0.283 mL, 2.2 mmol) was added dropwise. Tetramethylguanidine (0.025 mL, 0.2 mmol) was added dropwise and the reaction mixture was stirred at r.t. for 1 h. The reaction mixture was diluted with ethyl acetate (10 mL) and washed two times with 1 M HCl (10 mL). The organic portion was dried over MgSO4 and the solvent was removed to give the product as white solid. Yield: 0.595 g (92%). 1H-NMR (300 44 Chapter 6 MHz, CDCl3): δ 7.49 (2H, d), 7.35 (2H, d), 4.98 (1H, d), 4.08 (4H, m), 1.25 (6H, t); 31P NMR (121 MHz, CDCl3): δ 21.2 (s). Synthesis of iodo-hydroxyl-phosphonate ester (P5c). p-iodo-benzaldehyde (0.464 g, 2 mmol) was dissolved in THF (6 mL) and diethyl phosphite (0.283 mL, 2.2 mmol) was added dropwise. Tetramethylguanidine (0.025 mL, 0.2 mmol) was added dropwise and the reaction mixture was stirred at r.t. for 1 h. The reaction mixture was diluted with ethyl acetate (10 mL) and washed two times with 1 M HCl (10 mL). The organic portion was dried over MgSO4 and the solvent was removed to give the product as pale pink solid. Yield: 0.637 g (86%). 1H-NMR (300 MHz, CDCl3): δ 7.71 (2H, d), 7.23 (2H, d), 4.97 (1H, d), 4.06 (4H, m), 1.26 (6H, t); 31 P NMR (121 MHz, CDCl3): δ 21.2 (s). Synthesis of triflate-difluoromethylphosphonate ester (P7a). P5a (0.785 g, 2 mmol) was dissolved in dichloromethane (10 mL) and cooled to 0°C. DMP (1.27 g, 3 mmol) was added to the solution and the reaction mixture was stirred at r.t. for 2 h. The reaction mixture was filtered and to the filtrate was added a solution containing 0.5 M Na2S2O3 (15 mL) and 1 M NaHCO3 (15 mL) and stirred for 30 min. The solution was then extracted three times with ethyl acetate (20 mL) and the organic portion was dried over MgSO4. The solvent was removed to give crude P6a as yellow oil. P6a was re-dissolved in dry dichloromethane (8 mL) and cooled to 0°C. DAST (0.53 mL, 4 mmol) was then added drop-wise under nitrogen atmosphere. The reaction mixture was allowed to warm to r.t. and stirred for 12 h. Thereafter, the mixture was diluted with dichloromethane (10 mL) and ice-cold 1 M NaHCO3 (20 mL) and stirred for an additional 30 min. The reaction mixture was extracted three times with dichloromethane (15 mL) and the 45 Chapter 6 organic portion was dried over MgSO4. The solvent was removed to give the crude compound as dark-brown oil and purified by flash column chromatography (1:2 v/v ethyl acetate:hexane, Rf = 0.7) to give the product as yellow oil. Final yield: 0.429 g (52%). P6a: 1H-NMR (300 MHz, CDCl3): δ 8.40 (2H, d), 7.43 (2H, d), 4.29 (4H, m), 1.39 (6H, t); 31P NMR (121 MHz, CDCl3): δ -1.63 (s); 19 F NMR (282 MHz, CDCl3): δ 3.28 (s). P7a: 1H-NMR (300 MHz, CDCl3): δ 7.73 (2H, d), 7.39 (2H, d), 4.20 (4H, m), 1.32 (6H, t); 31P NMR (121 MHz, CDCl3): δ 6.18 (t, 2JP-F = 113.4 Hz); 19 F NMR (282 MHz, CDCl3): δ 3.24 (s), δ -32.7 (d, 2JP-F = 113.4 Hz). ESI (+ve mode): m/z 412.8 (M+). Synthesis of bromo-difluoromethylphosphonate ester (P7b). P5b (0.646 g, 2 mmol) was dissolved in dichloromethane (10 mL) and cooled to 0°C. DMP (1.27 g, 3 mmol) was added to the solution and the reaction mixture was stirred at r.t. for 2 h. The reaction mixture was filtered and to the filtrate was added a solution containing 0.5 M Na2S2O3 (15 mL) and 1 M NaHCO3 (15 mL) and stirred for 30 min. The solution was then extracted three times with ethyl acetate (20 mL) and the organic portion was dried over MgSO4. The solvent was removed to give crude P6b as yellow oil. P6b was re-dissolved in dry dichloromethane (8 mL) and cooled to 0°C. DAST (0.53 mL, 4 mmol) was then added drop-wise under nitrogen atmosphere. The reaction mixture was allowed to warm to r.t. and stirred for 12 h. Thereafter, the mixture was diluted with dichloromethane (10 mL) and ice-cold 1 M NaHCO3 (20 mL) and stirred for an additional 30 min. The reaction mixture was extracted three times with dichloromethane (15 mL) and the organic portion was dried over MgSO4. The solvent was removed to give the crude compound as dark-brown oil and purified by flash column chromatography (1:2 v/v ethyl acetate:hexane, Rf = 0.7) to give the product as yellow oil. Final yield: 0.384 g (56%). P6b: 1H-NMR (300 MHz, 46 Chapter 6 CDCl3): δ 8.14 (2H, d), 7.66 (2H, d), 4.27 (4H, m), 1.38 (6H, t); 31P NMR (121 MHz, CDCl3): δ -1.13 (s). P7b: 1H-NMR (300 MHz, CDCl3): δ 7.60 (2H, d), 7.49 (2H, d), 4.18 (4H, m), 1.32 (6H, t); 31P NMR (121 MHz, CDCl3): δ 6.43 (t, 2JP-F = 114.4 Hz); 19F NMR (282 MHz, CDCl3): δ -32.8 (d, 2JP-F = 114.4 Hz). ESI (+ve mode): m/z 342.8 (M+). Synthesis of iodo-difluoromethylphosphonate ester (P7c). P5c (0.740 g, 2 mmol) was dissolved in dichloromethane (10 mL) and cooled to 0°C. DMP (1.27 g, 3 mmol) was added to the solution and the reaction mixture was stirred at r.t. for 2 h. The reaction mixture was filtered and to the filtrate was added a solution containing 0.5 M Na2S2O3 (15 mL) and 1 M NaHCO3 (15 mL) and stirred for 30 min. The solution was then extracted three times with ethyl acetate (20 mL) and the organic portion was dried over MgSO4. The solvent was removed to give crude P6c as yellow oil. P6c was re-dissolved in dry dichloromethane (8 mL) and cooled to 0°C. DAST (0.53 mL, 4 mmol) was then added drop-wise under nitrogen atmosphere. The reaction mixture was allowed to warm to r.t. and stirred for 12 h. Thereafter, the mixture was diluted with dichloromethane (10 mL) and ice-cold 1 M NaHCO3 (20 mL) and stirred for an additional 30 min. The reaction mixture was extracted three times with dichloromethane (15 mL) and the organic portion was dried over MgSO4. The solvent was removed to give the crude compound as dark-brown oil and purified by flash column chromatography (1:2 v/v ethyl acetate:hexane, Rf = 0.7) to give the product as yellow oil. Final yield: 0.421 g (54%). P6c: 1H-NMR (300 MHz, CDCl3): δ 7.98 (2H, d), 7.90 (2H, d), 4.27 (4H, m), 1.38 (6H, t); 31P NMR (121 MHz, CDCl3): δ -1.13 (s). P7c: 1H-NMR (300 MHz, CDCl3): δ 7.81 (2H, d), 7.34 (2H, d), 4.19 (4H, m), 1.32 (6H, t); 31P NMR (121 MHz, CDCl3): δ 6.41 (t, 2JP-F = 115.5 Hz); 19F NMR (282 MHz, CDCl3): δ -33.0 (d, 2JP-F = 115.5 Hz). ESI (+ve mode): m/z 390.8 (M+). 47 Chapter 6 Synthesis of ethynyl difluoromethylphosphonate ester (9). Method 1: To a solution of 7c (52.8 mg, 0.135 mmol) in DMF (2mL) in a sealed tube, trimethylsilylacetylene (28 µL, 0.2 mmol), triethylamine (98 µL, 0.7 mmol) and PdCl2(PPh3)2 (4.8 mg, 5 mol %) were added. The reaction mixture was purged with N2 gas for 10 min and then stirred at 90 oC for 1 h. After cooling, the reaction mixture was diluted with diethyl ether (10 mL) and filtered through a pad of celite. The filtrate was washed once with brine (10 mL) and the organic portion was dried over MgSO4. The solvent was removed to give the crude compound as dark-orange oil and purified by flash column chromatography (1:2 v/v ethyl acetate:hexane, Rf = 0.75) to give P8 as yellow oil. P8 was re-dissolved in methanol (2mL) and K2CO3 (10 equivalent excess) was added. The reaction mixture was then stirred at r.t. for 2 h. After the reaction, the reaction mixture was filtered and concentrated in vacuo. The residue was purified by flash column chromatography (1:2 v/v ethyl acetate:hexane, Rf = 0.65) to give the product as pale yellow oil. Final yield: 28.1 mg (72%). Method 2: To a solution of 7c (52.8 mg, 0.135 mmol) in DMF (2 mL) in a sealed tube, ethynyltributylstannane (41 µL, 0.142 mmol) and PdCl2(PPh3)2 (4.8 mg, 5 mol %) were added. The reaction mixture was purged with N2 gas for 10 min and then stirred at 60 oC for 1 h. After cooling, the reaction mixture was diluted with water (8 mL) and diethyl ether (8mL), and filtered through a pad of celite. The filtrate was extracted three times with diethyl ether (10mL) and the organic portion was dried over MgSO4. The solvent was removed to give the crude compound as dark-brown oil and purified by flash column chromatography (1:2 v/v ethyl acetate:hexane, Rf = 0.65) to give the product as pale yellow oil. Yield: 23.3 mg (60%). P8: 1HNMR (300 MHz, CDCl3): δ 7.54 (4H, s), 4.17 (4H, m), 1.30 (6H, t), 0.26 (9H, s); 31P NMR (121 MHz, CDCl3): δ 6.68 (t, 2JP-F = 115.5 Hz); 19F NMR (282 MHz, CDCl3): δ -32.9 (d, 2JP-F = 115.5 Hz). ESI (+ve mode): m/z 360.8 (M+). 9: 1H-NMR (300 MHz, CDCl3): δ 7.57 (4H, s), 4.18 (4H, 48 Chapter 6 m), 3.16 (1H, s), 1.32 (6H, t); 31 P NMR (121 MHz, CDCl3): δ 6.60 (t, 2JP-F = 114.4 Hz); 19 F NMR (282 MHz, CDCl3): δ -33.0 (d, 2JP-F = 114.4 Hz). ESI (+ve mode): m/z 288.8 (M+). Synthesis of [Au(PPh3)(9)] (9a). Ligand 9 (6.8 mg, 23.6 µmol) was dissolved in methanol (4 mL) and potassium tert-butoxide (2.9 mg, 26.0 µmol) was added. The reaction mixture was allowed to stir at r.t. for 0.5 h. [Au(PPh3)Cl] (11.7 mg, 23.6 µmol) dissolved in dichloromethane (2 mL) was then added and the resulting mixture was stirred at r.t. overnight. After the reaction, the solvent was removed and the residue re-dissolved in small amount of dichloromethane (2 mL) and diethyl ether (8 mL) was added to give a pale-yellow precipitate. The precipitate was collected, washed twice with diethyl ether (6 mL) and vacuum dried. Yield: 14.9 mg (85 %). 1HNMR (300 MHz, CDCl3): δ 7.43-7.59 (19H, m), 4.14 (4H, m), 1.28 (6H, t); 31P NMR (121 MHz, CDCl3): δ 7.01 (t, 2JP-F = 116.5 Hz), δ 42.8 (s, PPh3); 2 19 F NMR (282 MHz, CDCl3): δ -32.5 (d, JP-F = 116.5 Hz). ESI (+ve mode): m/z 721.2 ([Au(PPh3)2]+, 100%), 746.5 (M+, 30%). 49 Appendix Appendix Table A1. Selected X-ray crystallographic data for 1b, 2a, 3b, 4a and 4b. Complex Formula Formula weight Temperature [K] Wavelength [Å] Crystal size [mm3] Crystal system Space group a [Å] b [Å] c [Å] α [deg] β [deg] γ [deg] V [Å3] Z Dc [Mg/m3] µ [mm-1] θ range [deg] no. of unique data max., min. transmn final R indices [I>2σ(I)] R indices (all data) 4a·(CH3OH)4 4b·(H2O)8 2a·(CH3OH)1.5·(H2O) 3b·(CH3OH) 1b·(C2H5)2O C27.5H33ClF2N3O5.5PRu C40H52ClF8N3O4P2Ru C34H45ClF2N3O7PRu C34H45ClF2N3O7PRu C39H56ClF8N3O4P2Ru 989.31 813.22 813.22 981.33 699.06 100(2) 100(2) 100(2) 293(2) 100(2) 0.71073 0.71073 0.71073 0.71073 0.71073 0.30 x 0.04 x 0.03 0.36 x 0.26 x 0.10 0.36 x 0.26 x 0.10 0.36 x 0.20 x 0.10 0.31 x 0.30 x 0.15 Monoclinic Triclinic Triclinic Triclinic Monoclinic P2 /c P ī Pī Pī P21/n 1 9.991(2) 10.653(3) 10.653(3) 9.8539(4) 12.6459(17) 17.720(4) 12.504(3) 12.504(3) 13.3013(6) 19.272(3) 24.098(6) 14.449(4) 14.449(4) 18.0943(8) 12.8815(17) 90 101.457(4) 101.457(4) 110.4200(10) 90 91.018(5) 101.362(4) 101.362(4) 93.6680(10) 104.048(3) 90 92.915(4) 92.915(4) 96.1320(10) 90 4265.4(17) 1841.3(8) 1841.3(8) 2196.78(16) 3045.5(7) 4 2 2 2 4 1.541 1.467 1.467 1.484 1.525 0.584 0.601 0.601 0.566 0.710 2.04 to 25.00 1.67 to 27.50 1.67 to 27.50 1.66 to 27.50 2.11 to 25.00 24522 23737 23737 28789 17591 0.9827 and 0.8442 0.9423 and 0.8126 0.9423 and 0.8126 0.4305 and 0.3930 0.9010 and 0.8099 R1 = 0.1022 R1 = 0.0653 R1 = 0.0653 R1 = 0.0437 R1 = 0.0782 wR2 = 0.2548 wR2 = 0.1432 wR2 = 0.1432 wR2 = 0.0989 wR2 = 0.1913 R1 = 0.1852 R1 = 0.0860 R1 = 0.0860 R1 = 0.0524 R1 = 0.0891 wR2 = 0.2938 wR2 = 0.1524 wR2 = 0.1524 wR2 = 0.1030 wR2 = 0.1981 goodness-of-fit on F2 1.024 1.040 1.040 1.042 1.093 peak/hole [e Å-3] 2.322 and -0.873 1.767 and -1.120 1.767 and -1.120 0.716 and -0.543 1.729 and -0.719 Remarks CCDC 897052 Ambiguous solvent High R1 value CCDC 897053 Ambiguous Cl1 R = Σ||Fo| - |Fc||/Σ|Fo |, wR2 = {Σ[w(Fo2 - Fc2)2]/Σ[w(Fo2)2]}1/2. Goodness-of-fit (GOF) = {Σ[w(Fo2 - Fc2)2]/(n - p)}1/2, where n is the number of data and p is the number of parameters refined 50 Appendix H2 H6 H7 H4 H3 H8 5.6192 5.5989 5.6745 5.7945 5.7742 5.8646 5.8444 7.2600 7.2019 7.1970 7.1663 7.4446 7.4233 7.4019 H2O * CDCl3 * H13 H14 H16 H12 H11 H15 7.8 7.6 7.4 5.9 7.2 5.8 H19 H18 H1 – H4 + H15 8.0 7.6 7.2 6.8 6.4 6.0 H20 5.6 5.2 4.8 4.4 1.2172 3.9373 H6 1.0737 2.8607 1.1288 0.9890 3.0022 1.1484 2.0862 1.9853 1.0000 H9 8.4 H7 + H8 ether * dcm * 8.8 5.5 H21 H10 – H14 + H16 – H19 9.2 5.6 H21 H20 9.6 5.7 (ppm) (ppm) 1.1104 H10 H5 H17 4.0 3.6 3.2 2.8 3.2867 H9 ether * 2.4 6.0950 H5 2.0 1.6 5.9985 H1 7.6944 7.6901 7.6643 7.6002 7.5734 7.8802 7.8544 7.8314 (a) 1.2 0.8 (ppm) 51 -32.5219 8 7 6 -32.9272 (c) 5.2742 7.1520 (b) 6.2131 Appendix 5 (ppm) -32.0 -33.0 -34.0 (ppm) 20 0 -20 -40 -60 -80 -100 -120 -140 -160 10 0 -10 (ppm) -20 -30 -40 -50 -60 -70 -80 -90 -100 -110 (ppm) (d) 98.2% Figure A1. Characterisation and analysis of 1a: (a) 1H NMR, (b) 31P{1H} NMR, (c) 19F{1H} NMR, (d) HPLC chromatogram. 52 Appendix H2 H1 CDCl3 * N H9 H10 N N H7 H8 H12 H11 H15 H13 5.5518 5.6017 5.6773 5.6597 5.7326 H1 H2O * F F O H14 O P H17 O 1b 7.8 7.6 7.4 5.80 7.2 5.70 5.60 (ppm) (ppm) H16 H6 – H10 + H12 – H15 H16 9.6 9.2 8.8 8.4 8.0 7.6 7.2 6.8 6.4 6.0 5.6 5.2 4.8 4.4 4.0 3.6 3.2 24.143 3.0414 3.9228 2.7087 2.2107 1.9608 1.0857 2.9377 H5 H2 ether * H11 1.0824 H6 Ru 1.0756 H5 H3 – H4 + H17 PF6 1.0000 Cl H4 7.2605 7.2595 7.1718 7.1450 H3 7.6939 7.6676 7.5843 7.5580 7.4989 7.4759 7.4550 7.8769 7.8500 7.8243 (a) 2.8 2.4 2.0 1.6 1.2 0.8 (ppm) 53 9 8 7 6 5 -32.5183 4 3 -32.9272 (c) 5.2986 7.1886 (b) 6.2375 Appendix 2 (ppm) -31.0 -32.0 -33.0 -34.0 -35.0 (ppm) 40 20 0 -20 -40 -60 (ppm) -80 -100 -120 -140 -160 10 0 -10 -20 -30 -40 -50 -60 -70 -80 -90 -100 -110 (ppm) (d) 99.2% Figure A2. Characterisation and analysis of 1b: (a) 1H NMR, (b) 31P{1H} NMR, (c) 19F{1H} NMR, (d) HPLC chromatogram. 54 Appendix H2O H1 H2 H4 H3 N N H12 H10 H11 5.8070 5.7917 5.7744 5.7656 5.7107 5.6954 5.9434 5.9284 6.0733 6.0579 7.0734 7.0539 7.6473 7.5770 7.5573 7.5127 7.4958 7.4795 H13 N H14 H16 H15 H19 2a H17 F F H18 O HO P OH 8.0 7.8 7.6 7.4 7.2 6.1 6.0 (ppm) 5.9 5.8 H5 5.7 (ppm) H7 + H8 H10 – H14 + H16 – H19 H1 – H4 + H15 9.2 8.8 8.4 8.0 7.6 7.2 6.8 6.4 6.0 5.6 4.8 4.4 4.0 3.6 3.2 2.8 2.4 3.0309 0.9815 5.2 2.0 1.6 1.2 3.0170 ether * H6 1.1523 2.9847 1.0369 1.0175 2.0168 1.0080 2.0395 1.0403 1.0000 9.6 ether * dcm * 3.0081 H9 1.0519 H9 * Cl H8 2.0437 Cl Ru MeOD * H7 H6 H5 7.8439 7.8238 7.9788 7.9587 7.9436 (a) 0.8 (ppm) 55 8 6 4 2 -109.0133 (c) -109.2739 2.7058 (b) 3.2976 3.9136 Appendix 0 (ppm) -109.0 (ppm) 80 60 40 20 0 -20 -40 -60 -80 -65 -75 -85 -95 -105 -115 -125 -135 -145 -155 -165 (ppm) (ppm) (d) 95.6% Figure A3. Characterisation and analysis of 2a: (a) 1H NMR, (b) 31P{1H} NMR, (c) 19F{1H} NMR, (d) HPLC chromatogram. 56 Appendix 5.7925 MeOD * 5.8527 5.8345 5.8951 * 5.9198 7.1235 7.1043 8.0276 8.0107 8.0078 7.9109 7.8909 7.8310 7.8272 7.7331 7.7297 (a) 7.6390 7.6199 7.6048 7.6026 7.5870 7.5716 7.5691 H2O H3 H3 + H4 H2 H1 H4 Cl H1 Ru H9 N N H7 H8 H12 H11 H15 H13 F F H14 O HO P OH 8.0 7.8 7.6 7.4 7.2 6.00 5.95 (ppm) 5.90 5.85 5.80 5.75 (ppm) 2b H6 – H10 + H12 – H15 H2 9.6 9.2 8.8 8.4 8.0 7.2 6.8 6.4 6.0 5.6 5.2 4.8 4.4 4.0 3.6 3.2 9.4210 2.9268 2.1614 2.9185 1.9837 2.9857 1.0502 1.0304 7.6 ether * ether * 2.8 2.4 2.0 1.6 9.2677 H11 H5 1.0382 H6 H10 N 1.0000 H5 1.0611 Cl 1.2 0.8 (ppm) 57 6 5 4 3 (c) 2 1 -109.4729 -109.2087 2.5005 (b) 3.1044 3.7203 Appendix 0 (ppm) -108 -110 (ppm) 60 40 20 0 -20 -40 -60 -80 -100 -75 -85 -95 (ppm) -105 -115 -125 -135 -145 -155 -165 (ppm) (d) 95.5% Figure A4. Characterisation and analysis of 2b: (a) 1H NMR, (b) 31P{1H} NMR, (c) 19F{1H} NMR, (d) HPLC chromatogram. 58 H21 H23 9.0 8.0 8.0 H22 7.0 7.6 (ppm) 6.0 7.2 H9 5.0 (ppm) 6.2 4.0 5.9309 5.9150 5.8953 5.8706 5.8093 5.7901 6.0958 6.0695 6.0520 6.0339 ether * H5 H20 6.0 (ppm) 5.8 3.0 2.0 6.2240 H17 5.6035 H11 H16 H18 H 19 3.0981 H9 1.0917 H12 H8 H13 H 14 3.8123 7.8615 7.6747 7.6681 7.6539 7.6473 7.6276 7.6199 7.6062 7.5799 7.5575 7.5252 7.5038 7.4945 7.2600 7.1187 7.0918 (a) 3.0363 2.0922 1.0458 H10 H3 2.0084 H4 1.2967 4.7429 H5 H2 2.9830 H1 1.0000 Appendix ether * H7 H6 CDCl3 * H15 H2O * H7 + H8 H23 H10 – H16 + H18 – H21 H1 – H4 + H17 H22 H6 1.0 59 10 8 6 (ppm) 4 -32.4270 -32.8323 (c) 5.2864 7.1764 (b) 6.2253 Appendix 2 -30 20 0 -20 -40 -60 (ppm) -80 -100 -120 -140 -160 -180 0 -20 (ppm) -40 -32 (ppm) -60 -34 -80 -100 (d) 99.6% Figure A5. Characterisation and analysis of 3a: (a) 1H NMR, (b) 31P{1H} NMR, (c) 19F{1H} NMR, (d) HPLC chromatogram. 60 Appendix (a) H9 H 10 H7 5.6466 5.8383 5.8997 H12 H14 H 15 H5 H6 H2O * H1 CDCl3 * H11 6.1429 H4 6.2048 H2 7.1159 7.0885 H1 8.0111 7.9865 7.8977 7.8840 7.6479 7.6413 7.6265 7.6199 7.6013 7.5936 7.5706 7.5520 7.5482 7.5197 H3 7.2600 H3 – H4 + H19 H8 H 13 H17 H16 H19 H18 8.0 7.8 7.6 7.4 7.2 6.2 7.0 6.0 5.8 5.6 (ppm) (ppm) H6 – H12 + H14 – H17 H18 9.2 8.8 8.4 8.0 7.6 7.2 6.8 6.4 6.0 5.6 5.2 4.8 4.4 2.9958 4.0077 3.0203 1.0716 1.0363 2.0728 6.0544 1.9899 1.0209 1.0000 9.6 ether * H13 4.0 3.6 3.2 23.918 H5 H2 2.8 2.4 2.0 1.6 1.2 0.8 (ppm) 61 9 8 7 6 5 4 3 -32.8140 -32.4087 (c) 5.3230 7.2130 (b) 6.2619 Appendix 2 -31.0 (ppm) -32.0 -33.0 -34.0 (ppm) 20 0 -20 -40 -60 (ppm) -80 -100 -120 -140 -160 -180 10 0 -10 -20 -30 -40 -50 -60 -70 -80 -90 -100 -110 (ppm) (d) 98.6% Figure A6. Characterisation and analysis of 3b: (a) 1H NMR, (b) 31P{1H} NMR, (c) 19F{1H} NMR, (d) HPLC chromatogram. 62 Appendix H3 N N H16 H18 N H11 H12 H17 H21 H19 F H20 O HO P OH 8.2 8.0 7.8 7.6 7.4 6.40 7.2 6.30 6.20 6.10 6.0261 6.0111 6.00 (ppm) (ppm) H7 + H8 ether * H10 – H16 + H18 – H21 H1 – H4 + H17 H9 9.6 ether * H5 F 4a 10.0 6.1108 6.0970 6.2334 6.2171 6.2018 6.1883 6.4153 6.4000 7.0943 7.0742 H15 dcm * 9.2 8.8 8.4 8.0 7.6 7.2 6.8 6.4 6.0 1.0281 0.9988 1.1863 2.9138 1.0058 1.9546 2.0834 3.1400 H6 1.0332 H10 14 2.9964 H9 Ru 1.0000 Cl H8 H13 H Cl 5.6 5.2 4.8 4.4 4.0 3.6 3.2 2.8 2.4 5.8732 H4 H7 H6 3.0797 H2 7.9119 7.8927 7.7638 7.7613 7.7428 7.7262 7.7221 7.7002 7.6823 7.6108 7.5904 8.1700 8.1505 8.1301 8.1270 H1 H5 MeOD * H2O * 6.1475 (a) 2.0 1.6 1.2 0.8 0.4 (ppm) 63 7 6 5 4 -109.0676 -109.3281 2.4764 (b) 3.0802 3.6962 Appendix (c) 3 2 1 0 -1 (ppm) -108 -110 (ppm) 40 20 0 -20 -40 -60 (ppm) -80 -100 -120 -140 -160 -100 (ppm) -120 -140 -160 (d) 97.4% Figure A7. Characterisation and analysis of 4a: (a) 1H NMR, (b) 31P{1H} NMR, (c) 19F{1H} NMR, (d) HPLC chromatogram. 64 10.0 H3 9.6 H13 H17 9.2 H15 F H16 O HO P OH 8.8 8.4 8.2 8.0 7.6 8.0 7.2 7.8 6.8 7.6 H5 6.4 7.4 6.0 5.6 7.2 5.2 6.30 4.8 4.4 4.0 6.1889 6.2344 8.1537 8.1348 8.1176 8.1145 7.8978 7.8918 7.8796 7.8755 7.7588 7.7560 7.7413 7.7372 7.7312 7.7262 7.7231 7.7168 7.7134 7.7039 7.6989 7.6936 7.6839 7.6807 7.6660 7.6629 7.6221 7.6017 7.1263 7.1059 H2O * 6.20 (ppm) 3.6 6.10 H6 – H12 + H14 – H17 3.2 5.9628 6.0597 6.1049 ether * 18.077 H8 H12 H14 3.0203 H7 H9 H 10 2.8965 H6 N 1.0069 N 1.0483 N 1.9678 H5 H4 2.0864 (a) 3.1735 Ru 1.0063 Cl H2 1.0344 H1 2.0442 1.0000 Appendix 6.00 2.8 ether * MeOD * H3 + H4 Cl H1 H11 F (ppm) 5.90 4b H2 H13 (ppm) 2.4 2.0 1.6 1.2 0.8 65 7 6 5 4 3 2 1 0 -1 -109.0893 (c) -109.3535 2.4522 (b) 3.0682 3.6841 Appendix -2 (ppm) -109.5 (ppm) 40 20 0 -20 -40 -60 (ppm) -80 -100 -120 -140 -100 (ppm) -120 -140 -160 -180 (d) 98.4% Figure A8. Characterisation and analysis of 4b: (a) 1H NMR, (b) 31P{1H} NMR, (c) 19F{1H} NMR, (d) HPLC chromatogram. 66 References References [1] P. Zimmet, K. G. M. M. Alberti, J. Shaw, Nature 2001, 414(6865), 782-787. [2] R. Sinha, G. Fisch, B. Teague, W. V. Tamborlane, B. Banyas, K. Allen, M. Savoye, V. Rieger, S. Taksali, G. Barbetta, R. S. Sherwin, S. Caprio, New Engl. J. Med. 2002, 346(11), 802-810. [3] A. P. Rocchini, New Engl. J. Med. 2002, 346(11), 854-855. [4] S. Zhang, Z.-Y. Zhang, Drug Discov. Today 2007, 12(9–10), 373-381. [5] A. Alonso, J. Sasin, N. Bottini, I. Friedberg, I. Friedberg, A. Osterman, A. Godzik, T. Hunter, J. Dixon, T. Mustelin, Cell 2004, 117(6), 699-711. [6] A. Salmeen, J. N. Andersen, M. P. Myers, N. K. Tonks, D. Barford, Mol. Cell 2000, 6(6), 1401-1412. [7] N. K. Tonks, Nat. Rev. Mol. Cell. Biol. 2006, 7(11), 833-846. [8] Z.-Y. Zhang, Curr. Opin. Chem. Biol. 2001, 5(4), 416-423. [9] J. N. Andersen, P. G. Jansen, S. M. Echwald, O. H. Mortensen, T. Fukada, R. Del Vecchio, N. K. Tonks, N. P. H. MØller, FASEB J. 2004, 18(1), 8-30. [10] S. Arena, S. Benvenuti, A. Bardelli, Cell Mol. Life Sci. 2005, 62(18), 2092-2099. [11] D. Bandyopadhyay, A. Kusari, K. A. Kenner, F. Liu, J. Chernoff, T. A. Gustafson, J. Kusari, J. Biol. Chem. 1997, 272(3), 1639-1645. [12] S. Dadke, J. Kusari, J. Chernoff, J. Biol. Chem. 2000, 275(31), 23642-23647. [13] J. M. Zabolotny, K. K. Bence-Hanulec, A. Stricker-Krongrad, F. Haj, Y. Wang, Y. Minokoshi, Y.-B. Kim, J. K. Elmquist, L. A. Tartaglia, B. B. Kahn, B. G. Neel, Dev. Cell 2002, 2(4), 489-495. 67 References [14] A. Cheng, N. Uetani, P. D. Simoncic, V. P. Chaubey, A. Lee-Loy, C. J. McGlade, B. P. Kennedy, M. L. Tremblay, Dev. Cell 2002, 2(4), 497-503. [15] R. Di Paola, L. Frittitta, G. Miscio, M. Bozzali, R. Baratta, M. Centra, D. Spampinato, M. G. Santagati, T. Ercolino, C. Cisternino, T. Soccio, S. Mastroianno, V. Tassi, P. Almgren, A. Pizzuti, R. Vigneri, V. Trischitta, Am. J. Hum. Genet. 2002, 70(3), 806-812. [16] S. M. Echwald, H. Bach, H. Vestergaard, B. Richelsen, K. Kristensen, T. Drivsholm, K. Borch-Johnsen, T. Hansen, O. Pedersen, Diabetes 2002, 51(1), 1-6. [17] A. Mok, H. Cao, B. Zinman, A. J. G. Hanley, S. B. Harris, B. P. Kennedy, R. A. Hegele, J. Clin. Endocrinol. Metab. 2002, 87(2), 724-727. [18] M. Elchebly, P. Payette, E. Michaliszyn, W. Cromlish, S. Collins, A. L. Loy, D. Normandin, A. Cheng, J. Himms-Hagen, C.-C. Chan, C. Ramachandran, M. J. Gresser, M. L. Tremblay, B. P. Kennedy, Science 1999, 283(5407), 1544-1548. [19] L. D. Klaman, O. Boss, O. D. Peroni, J. K. Kim, J. L. Martino, J. M. Zabolotny, N. Moghal, M. Lubkin, Y.-B. Kim, A. H. Sharpe, A. Stricker-Krongrad, G. I. Shulman, B. G. Neel, B. B. Kahn, Mol. Cell. Biol. 2000, 20(15), 5479-5489. [20] M. A. T. Blaskovich, Curr. Med. Chem. 2009, 16(17), 2095-2176. [21] L. M. Scott, H. R. Lawrence, S. M. Sebti, N. J. Lawrence, J. Wu, Curr. Pharm. Des. 2010, 16(16), 1843-1862. [22] P. Heneberg, Curr. Med. Chem. 2009, 16(6), 706-733. [23] A. Salmeen, J. N. Andersen, M. P. Myers, T.-C. Meng, J. A. Hinks, N. K. Tonks, D. Barford, Nature 2003, 423(6941), 769-773. [24] B. K. Sarma, G. Mugesh, J. Am. Chem. Soc. 2007, 129(28), 8872-8881. 68 References [25] S. Sivaramakrishnan, K. Keerthi, K. S. Gates, J. Am. Chem. Soc. 2005, 127(31), 1083010831. [26] R. L. M. van Montfort, M. Congreve, D. Tisi, R. Carr, H. Jhoti, Nature 2003, 423(6941), 773-777. [27] S. Lee, Q. Wang, Med. Res. Rev. 2007, 27(4), 553-573. [28] K. M. Doody, A. Bourdeau, M. L. Tremblay, Immunol. Rev. 2009, 228(1), 325-341. [29] K. E. You-Ten, E. S. Muise, A. Itié, E. Michaliszyn, J. Wagner, S. Jothy, W. S. Lapp, M. L. Tremblay, J. Exp. Med. 1997, 186(5), 683-693. [30] T. Tiganis, B. E. Kemp, N. K. Tonks, J. Biol. Chem. 1999, 274(39), 27768-27775. [31] T. R. Burke, H. K. Kole, P. P. Roller, Biochem. Biophys. Res. Commun. 1994, 204(1), 129-134. [32] T. R. Burke, B. Ye, X. Yan, S. Wang, Z. Jia, L. Chen, Z.-Y. Zhang, D. Barford, Biochemistry 1996, 35(50), 15989-15996. [33] L. Chen, L. Wu, A. Otaka, M. S. Smyth, P. P. Roller, T. R. Burke, J. Denhertog, Z. Y. Zhang, Biochem. Biophys. Res. Commun. 1995, 216(3), 976-984. [34] K. Shen, Y.-F. Keng, L. Wu, X.-L. Guo, D. S. Lawrence, Z.-Y. Zhang, J. Biol. Chem. 2001, 276(50), 47311-47319. [35] J. E. Bleasdale, D. Ogg, B. J. Palazuk, C. S. Jacob, M. L. Swanson, X.-Y. Wang, D. P. Thompson, R. A. Conradi, W. R. Mathews, A. L. Laborde, C. W. Stuchly, A. Heijbel, K. Bergdahl, C. A. Bannow, C. W. Smith, C. Svensson, C. Liljebris, H. J. Schostarez, P. D. May, F. C. Stevens, S. D. Larsen, Biochemistry 2001, 40(19), 5642-5654. [36] H. S. Andersen, L. F. Iversen, C. B. Jeppesen, S. Branner, K. Norris, H. B. Rasmussen, K. B. Møller, N. P. H. Møller, J. Biol. Chem. 2000, 275(10), 7101-7108. 69 References [37] C. Wiesmann, K. J. Barr, J. Kung, J. Zhu, D. A. Erlanson, W. Shen, B. J. Fahr, M. Zhong, L. Taylor, M. Randal, R. S. McDowell, S. K. Hansen, Nat. Struct. Mol. Biol. 2004, 11(8), 730-737. [38] T. O. Johnson, J. Ermolieff, M. R. Jirousek, Nat. Rev. Drug. Discov. 2002, 1(9), 696-709. [39] C. Yuan, L. Lu, X. Gao, Y. Wu, M. Guo, Y. Li, X. Fu, M. Zhu, J. Biol. Inorg. Chem. 2009, 14(6), 841-851. [40] C. Yuan, L. Lu, Y. Wu, Z. Liu, M. Guo, S. Xing, X. Fu, M. Zhu, J. Inorg. Biochem. 2010, 104(9), 978-986. [41] L. Lu, J. Yue, C. Yuan, M. Zhu, H. Han, Z. Liu, M. Guo, J. Inorg. Biochem. 2011, 105(10), 1323-1328. [42] Q. Wang, L. Lu, C. Yuan, K. Pei, Z. Liu, M. Guo, M. Zhu, Chem. Commun. 2010, 46(20), 3547-3549. [43] L. Ma, L. Lu, M. Zhu, Q. Wang, Y. Li, S. Xing, X. Fu, Z. Gao, Y. Dong, Dalton Trans. 2011, 40(24), 6532-6540. [44] Q. Wang, M. Zhu, L. Lu, C. Yuan, S. Xing, X. Fu, Dalton Trans. 2011, 40(48), 1292612934. [45] D. Krishnamurthy, M. R. Karver, E. Fiorillo, V. Orrú, S. M. Stanford, N. Bottini, A. M. Barrios, J. Med. Chem. 2008, 51(15), 4790-4795. [46] M. R. Karver, D. Krishnamurthy, R. A. Kulkarni, N. Bottini, A. M. Barrios, J. Med. Chem. 2009, 52(21), 6912-6918. [47] L. Piovan, L. Wu, Z.-Y. Zhang, L. H. Andrade, Org. Biomol. Chem. 2011, 9(5), 13471351. 70 References [48] Y. Han, Q. Luo, X. Hao, X. Li, F. Wang, W. Hu, K. Wu, S. Lu, P. J. Sadler, Dalton Trans. 2011, 40(43), 11519-11529. [49] S. Blanck, T. Cruchter, A. Vultur, R. Riedel, K. Harms, M. Herlyn, E. Meggers, Organometallics 2011, 30(17), 4598-4606. [50] J. Spencer, J. Amin, P. Coxhead, J. McGeehan, C. J. Richards, G. J. Tizzard, S. J. Coles, J. P. Bingham, J. A. Hartley, L. Feng, E. Meggers, M. Guille, Organometallics 2011, 30(11), 3177-3181. [51] J. Maksimoska, L. Feng, K. Harms, C. Yi, J. Kissil, R. Marmorstein, E. Meggers, J. Am. Chem. Soc. 2008, 130(47), 15764-15765. [52] E. Meggers, G. E. Atilla-Gokcumen, H. Bregman, J. Maksimoska, S. P. Mulcahy, N. Pagano, D. S. Williams, Synlett 2007, 8, 1177-1189 [53] N. Pagano, J. Maksimoska, H. Bregman, D. S. Williams, R. D. Webster, F. Xue, E. Meggers, Org. Biomol. Chem. 2007, 5(8), 1218-1227. [54] S. P. Mulcahy, E. Meggers, Top. Organometallic Chem. 2010, 32, 141-153. [55] E. Meggers, Chem. Commun. 2009(9), 1001-1010. [56] H. Bregman, P. J. Carroll, E. Meggers, J. Am. Chem. Soc. 2005, 128(3), 877-884. [57] L. Feng, Y. Geisselbrecht, S. Blanck, A. Wilbuer, G. E. Atilla-Gokcumen, P. Filippakopoulos, K. Kräling, M. A. Celik, K. Harms, J. Maksimoska, R. Marmorstein, G. Frenking, S. Knapp, L.-O. Essen, E. Meggers, J. Am. Chem. Soc. 2011, 133(15), 59765986. [58] N. Pagano, E. Y. Wong, T. Breiding, H. Liu, A. Wilbuer, H. Bregman, Q. Shen, S. L. Diamond, E. Meggers, J. Org. Chem. 2009, 74(23), 8997-9009. 71 References [59] G. E. Atilla-Gokcumen, N. Pagano, C. Streu, J. Maksimoska, P. Filippakopoulos, S. Knapp, E. Meggers, ChemBioChem 2008, 9(18), 2933-2936. [60] T. Yokomatsu, T. Murano, K. Suemune, S. Shibuya, Tetrahedron 1997, 53(3), 815-822. [61] T. Yokomatsu, T. Murano, I. Umesue, S. Soeda, H. Shimeno, S. Shibuya, Bioorg. Med. Chem. Lett. 1999, 9(4), 529-532. [62] G. Kennedy, A. D. Perboni, Tetrahedron Lett. 1996, 37(42), 7611-7614. [63] G. S. Cockerill, H. J. Easterfield, J. M. Percy, Tetrahedron Lett. 1999, 40(13), 26012604. [64] G. S. Cockerill, H. J. Easterfield, J. M. Percy, S. Pintat, J. Chem. Soc., Perkin Trans. 1 2000(16), 2591-2599. [65] M. Montalti, S. Wadhwa, W. Y. Kim, R. A. Kipp, R. H. Schmehl, Inorg. Chem. 1999, 39(1), 76-84. [66] J. Vicente, M. a. T. Chicote, M. M. Alvarez-Falcón, M. a.-D. Abrisqueta, F. J. Hernández, P. G. Jones, Inorg. Chim. Acta 2003, 347(0), 67-74. [67] J. Vicente, M.-T. Chicote, M.-D. Abrisqueta, P. G. Jones, Organometallics 1997, 16(26), 5628-5636. [68] G. Hogarth, M. M. Álvarez-Falcón, Inorg. Chim. Acta 2005, 358(5), 1386-1392. [69] M. I. Bruce, M. J. Liddell, J. Organometallic Chem. 1992, 427(2), 263-274. [70] G. Stupka, L. Gremaud, A. F. Williams, Helv. Chim. Acta 2005, 88(3), 487-495. [71] F. Y. Kwong, C. W. Lai, Y. Tian, K. S. Chan, Tetrahedron Lett. 2000, 41(52), 1028510289. 72 References [72] P. Fesser, C. Iacovita, C. Wäckerlin, S. Vijayaraghavan, N. Ballav, K. Howes, J.-P. Gisselbrecht, M. Crobu, C. Boudon, M. Stöhr, T. A. Jung, F. Diederich, Chem. Eur. J. 2011, 17(19), 5246-5250. [73] M. A. Bennett, T.-N. Huang, T. W. Matheson, A. K. Smith, Inorg. Synth. 1982, 21, 7478. [74] J. W. Hull, W. L. Gladfelter, Organometallics 1984, 3(4), 605-613. [75] C. Nieto-Oberhuber, S. López, A. M. Echavarren, J. Am. Chem. Soc. 2005, 127(17), 6178-6179. [76] J. Montalibet, K. I. Skorey, B. P. Kennedy, Methods 2005, 35(1), 2-8. [77] B. Ember, T. Kamenecka, P. LoGrasso, Biochemistry 2008, 47(10), 3076-3084. [78] Bruker AXS Inc., Madison, WI, SMART ver. 5.628, 2001. [79] Bruker AXS Inc., Madison, WI, SAINT+ ver. 6.22a, 2001. [80] Sheldrick G. W., University of Gottingen, Gottingen, Germany, SADABS ver. 2.10, 2001. [81] Bruker AXS Inc., Madison, WI, SHELXTL ver. 6.14, 2000. 73 [...]... using a non-selective PTP -1B inhibitor that also inhibits TC-PTP can lead to severe side-effects and recent studies have shown that TC-PTP deficient mice die within 3-5 weeks of age.[29, 30] Developing PTP -1B inhibitors with high selectivity towards PTP -1B, as opposed to TC-PTP, remain a daunting task 3 Chapter 1 1.2 Organic inhibitors of Protein Tyrosine Phosphatases The organic inhibitors that target... problems, there has been an intensified search for new therapeutic treatments for T2DM and obesity 1.1 Protein Tyrosine Phosphatase 1B as drug target Protein tyrosine phosphatases (PTPs) belong to a large family of 107 enzymes that play a vital role in the regulation of various signaling transduction pathways in mammalian systems.[4, 5] PTP enzymes catalyze the dephosphorylation from phosphorylated tyrosine. .. between PTP -1B and TC-PTP can potentially be exploited to develop selective inhibitors 1.3 Metal complexes as inhibitors of Protein Tyrosine Phosphatases Although the field of synthesizing organic inhibitors of PTPs has been extensively studied, the use of metal complexes to target PTPs remains largely unexplored So far, several metallocomplexes have been investigated as potential PTP inhibitors A... and PTP-MEG2 Recently, a library of gold-phosphine and goldcarbene complexes was screened and several found to exhibit good PTP -1B inhibitory activity with modest levels of selectivity.[45, 46] However, these gold complexes were only selective 6 Chapter 1 towards lymphoid tyrosine phosphatase (LYP) and protein tyrosine phosphatase PEST (PTPPEST) Their mechanism were not known but given the affinity of... Selected PTP -1B inhibitors of difluoromethylphosphonic acid class of inhibitors Some other classes of active-site inhibitors include the 2-carbomethoxybenzoic acids[35] and the 2-oxalylaminobenzoic acids[36] The strategy adopted in designing these inhibitors was the same, which was to design pTyr mimetics to bind to the catalytic pocket However, as these compounds were often negatively charged owing to... levels This was often the drawback associated with such 5 Chapter 1 active-site inhibitors An interesting class of inhibitors was the allosteric inhibitors which bind to a novel site located ~20Å away from the catalytic pocket.[37] This site is amenable to binding small molecules, considerably less polar and not well-conserved among PTPs, thus affording an opportunity to circumvent the problems associated... that target the active site of PTPs can be classified into several classes based on the type of functional groups that binds to the catalytic site One of the most potent classes of organic inhibitors is the difluoromethylphosphonic acids and they have been the core of many inhibitor designs The main strategy behind the design of this class of active-site inhibitors is to build molecules that contain... derivative.[33] Some selected inhibitors of the difluoromethylphosphonic acid class of PTP -1B inhibitors are shown in Figure 1.4 Compound I, which is the most potent and selective PTP -1B inhibitor identified to date (Ki = 2.4 nM), exhibits a 1000- to 10000-fold selectivity against a panel of other PTPs, but only 10-fold against the structurally similar TC-PTP.[34] Figure 1.4 Selected PTP -1B inhibitors of difluoromethylphosphonic... redox pathway which was critical in modulating the activity of PTPs The search for a selective metal-based PTP inhibitor remained elusive Earlier on, Meggers et al had shown that highly selective active-site inhibitors of PTKs can be prepared using a known kinase inhibitor, staurosporine, as a template By replacing the glycoside motif with an organoruthenium fragment, improved inhibitory profiles against... 2.2 Approach to designing gold-based inhibitors 10 Chapter 2 The strategy adopted to designing gold-based inhibitors towards PTPs is somewhat similar to the approach previously described for the design of the ruthenium-based inhibitors This novel class of gold(I) complexes will consist of the phenyl difluoromethylphosphonic acid moiety and a 3-D globular fragment which was provided by a bulky phosphine ... 1.1 Protein Tyrosine Phosphatase 1B as drug target 1.2 Organic inhibitors of Protein Tyrosine Phosphatases 1.3 Metal complexes as inhibitors of Protein Tyrosine Phosphatases... problems, there has been an intensified search for new therapeutic treatments for T2DM and obesity 1.1 Protein Tyrosine Phosphatase 1B as drug target Protein tyrosine phosphatases (PTPs) belong... towards PTP -1B, as opposed to TC-PTP, remain a daunting task Chapter 1.2 Organic inhibitors of Protein Tyrosine Phosphatases The organic inhibitors that target the active site of PTPs can be classified