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The attempted synthesis of a thiazolidinedione-containing ligand for vanadyl complexation : investigating… Mitchell, Devin Paul 2000

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THE ATTEMPTED SYNTHESIS OF A THIAZOLIDINEDIONE-CONTAINING LIGAND FOR VANADYL COMPLEXATION: INVESTIGATING POTENTIAL SYNERGISTIC INSULIN MIMICS by Devin Paul Mitchell B.Sc. (Hons.), McGill University, Canada, 1997 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November 1999 © Devin Paul Mitchell, 1999 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my writ ten permission. Department of cC-Af/H/i//v The University of British Columbia Vancouver, Canada Date hjei/ f is/11 DE-6 (2/88) ABSTRACT Thiazolidinedione-containing compounds are a relatively recent class of oral hypoglycemic agents used today to treat type II diabetes mellitus. Vanadium is well known to be an effective agent for lowering blood-glucose levels. In this study, synthesis of the ligand precursors 5-[4-(3-hydroxypropoxy)benzyl]-2,4-thiazolidinedione (HPBT), 5-[4-(3-bromopropoxy)benzyl]-2,4-thiazolidinedione (BPBT), and 5-[4-(3-iodopropoxy)benzyl]-2,4-thiazolidinedione (IPBT) were accomplished at high purity in gram scale quantities. These were fully characterized by the methods mentioned below. Several synthetic pathways were examined, with varying success; 5-[4-(3-p-toluenesulfonylpropoxy)benzyl]-2,4-thiazolidinedione (TsPBT), N-BOC-5-[4-(3-hydroxypropoxy)benzyl]-2,4-thiazolidinedione (BocHPBT), N-BOC-5-[4-(3-iodopropoxy)benzyl]-2,4-thiazolidinedione (BocIPBT), and 5-[4-(3-sulfonylimidazolepropoxy)benzyl]-2,4-thiazolidinedione (ImPBT) were compounds on those pathways. 5-[4-(3-Acetyl-6-hexoxy-2-one)benzyl]-2,4-thiazolidinedione (APBT), was synthesized, but not successfully purified. The vanadium coordination complex, bis-(5-[4-(3-acetyl-6-hexoxy-2-one)benzyl]-2,4-thiazolidinedionato) oxovanadium VO(APBT)2) gave encouraging preliminary results, e.g., synthesis of the complex directly from IPBT and vanadyl acetylacetonate; however further purification of the ligand would be required to determine stability constants. The ligand precursors were characterized by [ H NMR spectroscopy, mass 13 spectrometry, elemental analyses, infrared spectroscopy and, in certain instances, C ii NMR spectroscopy and two dimensional NMR spectroscopy for HPBT and IPBT. BocHPBT was characterized as a pure compound with ! H NMR spectroscopy. TsPBT, BocIPBT and ImPBT were not purified enough for complete characterization. LSIMS characterization of the thiazolidinedione oxovanadium(IV) complex, VO(APBT)2, indicated that the synthetic pathway so far elucidated could ultimately be refined to yield this compound in greater purity and quantity. iii TABLE OF CONTENTS page Abstract ii Table of Contents iv List of Figures vi List of Tables viii List of Abbreviations ix Acknowledgements xiii Chapter 1 Introduction xiii 1.1 Diabetes Mellitus 1 1.2 Vanadium Background. 2 1.3 Insulin-Mimetic Effects of Vanadium 3 1.4 Thiazolidinediones 5 1.5 Research Focus 8 Chapter 2 Experimental 10 2.1 Materials 10 2.2 Instrumentation 11 2.3 Procedures 11 2.4 Synthesis: Reactions Accomplished 12 2.5 Synthesis: Reactions Attempted. 19 Chapter 3 Results and Discussion 27 3.1 Design and Syntheses 27 iv 3.2 Characterization 38 Chapter 4 Conclusions and Future Directions 49 4.1 Conclusions 49 4.2 Future Directions 50 References 53 v LIST OF FIGURES page Figure 1.1 Bis(maltolato)oxovanadium(IV) (BMOV) 4 Figure 1.2 Chemical structures of various thiazolidinediones 5 Figure 1.3 5-[4-(3-Acetyl-6-hexoxy-2-one)benzyl]-2,4-thiazolidinedione (APBT) and its target vanadyl complex (VO(APBT)2) 9 Figure 2.1 Schematic diagram of a dry flash column 12 Figure 3.1 Synthesis of 4-(3-hydroxypropoxy)benzaldehyde 27 Figure 3.2 Overall scheme for the reactions described in this thesis 28 Figure 3.3 Synthesis of 5-([4-(3-hydroxypropoxy)phenyl]methylene)-2,4-thiazolidinedione (oxidized HPBT) 29 Figure 3.4 Synthesis of 5-[4-(3-hydroxypropoxy)benzyl]-2,4-thiazolidinedione (HPBT) 30 Figure 3.5 Synthesis of 5-[4-(3-bromopropoxy)benzyl]-2,4-thiazolidinedione (BPBT).. 31 Figure 3.6 Synthesis of 5-[4-(3-iodopropoxy)benzyl]-2,4-thiazolidinedione (IPBT) 32 Figure 3.7 Synthesis of A^-BOC-5-[4-(3-hydroxypropoxy)benzyl]-2,4-thiazolidinedione (BocHPBT) 32 Figure 3.8 Synthesis of A^A '^-sulfuryldiimidazole 33 Figure 3.9 Synthesis of 5-[4-(3-acetyl-6-hexoxy-2-one)benzyl]-2,4-thiazolidinedione (APBT) 34 Figure 3.10 Synthesis of 5-[4-(3-/7-toluenesulfonylpropoxy)benzyl]-2,4-thiazolidinedione (TsPBT) 35 vi Figure 3.11 Synthesis of A^-BOC-5-[4-(3-iodopropoxy)benzyl]-2,4-thiazolidinedione (BocIPBT) 36 Figure 3.12 Synthesis of N-BOC-2,4-thiazolidinedione 36 Figure 3.13 Synthesis of 5-[4-(3-sulfonylimidazolepropoxy)benzyl-2,4-thiazolidinedione (ImPBT) 37 Figure 3.14 Synthesis of bis-(5-[4-(3-acetyl-6-hexoxy-2-one)benzyl]-2,4-thiazolidinedionato) oxovanadium (VO(APBT)2) 38 Figure 3.15 Selected FTIR spectra comparing various ligand precursors 40 Figure 3.16 Regions of *H NMR (200 MHz) spectra of various precursor compounds .. 42 Figure 3.17 ! H-NMR spectra (200 MHz) of BocHPBT 43 Figure 3.18 1 3 C NMR (125 MHz) spectrum of HPBT with assignments 45 Figure 3.19 1 3 C NMR (75 MHz) spectrum of IPBT with assignments 45 Figure 3.20 HMQC spectrum of HPBT 46 Figure 3.21 HMQC spectrum of IPBT 47 Figure 3.22 VO(APBT) 2 mass spectrum (LSIMS) 48 Figure 4.1 Synthesis of APBT using BOC protected IPBT 50 Figure 4.2 Synthesis of APBT using ImPBT 51 Figure 4.3 Synthesis of the desired complex (VO(APBT)2) from APBT and VOS0 4 .... 51 vii LIST OF TABLES page Table 2.1 Variations in conditions for acetylacetone reaction 20 Table 3.1 Characteristic IR absorptions (cm"1) of ligand precursors 39 Table 3.2 Characteristic IR absorptions (cm"1) of N'-sulfuryldiimidazole 40 viii LIST OF ABBREVIATIONS Abbreviation Meaning acac acetylacetone AIDS Acquired Immune Deficiency Syndrome Anal. Analysis APBT 5 - [4-(3 -acetyl-6-hexoxy-2-one)benzyl] -2,4-thiazolidinedione APT attached proton test ATPase adenosine triphosphatase BMOV bis(maltolato)oxovanadium(IV) BOC butyloxycarbonyl BocHPBT N-BOC-5-[4-(3-hydroxypropoxy)benzyl]-2,4-thiazolidinedione BocIPBT N-BOC-5- [4-(3 -iodopropoxy)benzyl] -2,4-thiazolidinedione (Boc)20 di-terr-butyl dicarbonate BPBT 5 - [4-(3 -bromopropoxy)benzyl] -2,4-thiazolidinedione Calc. Calculated °C degrees Celsius CDCI3 deuterated chloroform C D 3 O D deuterated methanol cm centimeter cm"1 wave number ix d doublet dd doublet of doublets 5 chemical shift (ppm, NMR) A heat DMAP 4-(dimethylamino)pyridine DMF N,N-dimethylformamide EI electron-impact ionization eq equivalents EtOH ethanol FDA Food and Drug Administration (U.S.) FTIR fourier transform infrared g gram GI gastrointestinal GLUT-4 insulin-activated transporter protein HMQC heteronuclear multiple quantum coherence HPBT 5-[4-(3-hydroxypropoxy)benzyl]-2,4-thiazolidinedione HPLC high performance liquid chromatography hr hour IDDM insulin-dependent diabetes mellitus ImPBT 5-(3-sulfonylimidazolepropoxy)benzyl)-2,4-thiazolidinedione IPBT 5-[4-(3-iodopropoxy)benzyl]-2,4-thiazolidinedione IR infrared x LD50 dose of a substance at which 50% of subjects die LSIMS liquid secondary ion mass spectrometry m multiplet M moles/liter [M] + positively charged parent mass MHz megahertz mL milliliter mmol millimole mol mole MS mass spectrometry m/z mass-to-charge ratio NIDDM non-insulin-dependent diabetes mellitus NMR nuclear magnetic resonance v stretching vibration (IR) oxidized HPBT 5-([4-(3-hydroxypropoxy)phenyl]methylene)-2,4-thiazolidinedione % percent p para pH negative log of the concentration of H3<_)+ pKa negative log of the deprotonation constant PPAR peroxisome proliferator activated receptor PPARy peroxisome proliferator activated receptor gamma PPI13 triphenylphosphine xi PPb.3«Br2 dibromotriphenylphosphine ppm parts per million PTPase protein tyrosine phosphatase RXR retinoid X receptor s singlet STZ streptozotocin t triplet temp temperature tert tertiary THF tetrahydrofuran TLC thin layer chromatography Ts j!?-toluenesulfonate TsPBT 5-[4-(3-p-toluenesulfonylpropoxy)benzyl]-2,4-thiazolidinedione tt triplet of triplets UV-vis ultraviolet-visible VOL2-2H vanadyl with two deprotonated ligands attached VO(acac)L-H vanadyl with one deprotonated ligand and one acetylacetonate ligand attached VO(APBT) 2 bis-(5- [4-(3 -acetyl-6-hexoxy-2-one)benzyl] -2,4-thiazolidinedionato)oxovanadium(IV) xii A C K N O W L E D G M E N T S The first person to whom I owe thanks is without a doubt Dr. Chris Orvig, who, was patient and understanding. His guidance and encouragement was invaluable and immensely appreciated. The past and present Orvig group members helped make this research project very memorable. Peter especially took me under his wing at the beginning and led me into my project. I felt that the group dynamics were very positive and that there was always somebody who would be willing to help. Plenty of non-Orvig group chemistry people helped in myriad ways and I would like to list them all by name but there are just too many. I would especially like to thank my family and Karycia for their loving support. My friends in organic chemistry were life-savers when it came time to find new directions of research. Thanks. Prof. R. V. Stick was extremely helpful and I appreciate the time he took out of his schedule to help lend his experience to my research. I would also like to thank Mr. Peter Borda for completing the elemental analyses and the UBC Chemistry support staff for their assistance in navigating the bureaucracy of UBC. The wonderful people who operated the instruments (MS and NMR) also deserve mention as their help made all the difference. And last but not least, I must thank UBC, the Department of Chemistry and the various companies and organizations who have contributed funding. xiii Chapter 1 Introduction 1.1 Diabetes Mellitus Diabetes mellitus, caused by an absolute or relative deficiency of insulin, is a metabolic disease in which carbohydrate utilization is diminished and that of lipid and protein enhanced.1 Chronic hyperglycaemia, ketoacidosis, glycosuria, water and electrolyte loss, and coma characterize the more severe cases of diabetes mellitus. Serious long-term complications may include eye, kidney, nerve and blood vessel damage, as well as decreased resistance to infection.2 Two main types of diabetes mellitus are recognized; Type I, insulin-dependent diabetes mellitus (IDDM) and Type II, non-insulin-dependent diabetes mellitus (NIDDM). In the United States, approximately 14 million people have diabetes mellitus, 10% of whom have IDDM; the remainder (90%) have NIDDM. IDDM patients require daily exogenous insulin replacement therapy to sustain life. NIDDM patients can be managed with therapies that supplement insulin or enhance insulin action at the cellular level. People with NIDDM are insulin resistant and produce insufficient insulin to prevent hyperglycaemia. Hyperinsulinemia, in which the body produces more insulin than normal, often arises due to the body's attempt to compensate for insulin resistance and is characteristic of NIDDM. 2 ' 3 The disease known as diabetes mellitus has been recorded as far back as 1550 BC when a diet was mentioned in an Egyptian papyrus that claimed "to drive away the passing of too much urine".2 However it was not until the discovery and subsequent synthesis of insulin that diabetics were able to live relatively normal lives. 1 Being a heterogeneous disease, NIDDM may not be explicable by a single physiological mechanism. Part of the problem is that there are both genetic and environmental contributions to the disease. Many different pieces of the puzzle have been discovered but, at this time, the mechanism is not completely known. Much of what is known has been obtained from studies of the effects of current treatments. From a genetic viewpoint, diabetes is clearly multifactorial.3 At the present time IDDM is treated with exogenous insulin. Treatment of NIDDM, because of the existence of insulin secretion, tends to rely on either increasing secretion or decreasing tissue resistance to the hormone. There are several main classes of pharmaceutical agents currently in use. These include biguanides, sulfonylureas, alpha-glucosidase inhibitors, insulin, and thiazolidinediones.4 Some vanadium compounds have been, or are in clinical trials (sodium vanadate, vanadyl sulfate, BMOV). 1.2 Vanadium Background Initially discovered in 1813 by the Spaniard del Rio, 5 and named Panchromium, vanadium was rediscovered in 1830 by Nils Sefstrom who named it after Vanadis, the Norse goddess of beauty, because of the various intense colours of its solutions. Like other transition metals, vanadium exists in many different oxidation states from -3 to +5. The oxidation state is highly dependent on the pH of the system. Vanadium is found almost exclusively as vanadate (VC»43",+5) or vanadyl (V02+,+4) in biological systems and exists in both forms in the plasma. Despite being a relatively common transition metal, with a concentration of -0.02% in the earth's crust,6 vanadium is an element found more rarely in mammals, 2 with humans having about 100 to 200 u.g in their bodies, mostly bound to transferrin, with only 1% remaining unbound intracellularly.7 Despite several studies suggesting that it is an essential element, at least for some species, its biochemical role in mammals is unknown. While generally not yet classified as an essential trace element, vanadium is essential for development and growth for many cells and organisms.8'11 Vanadium's high pharmacological activity makes it difficult to determine essentiality. The discovery of its insulin-mimetic behaviour arose from the investigation into its biological interactions. 1.3 Insulin-Mimetic Effects of Vanadium Vanadium was first demonstrated to have insulin-like properties in vitro in 1979,12 in skeletal muscle and adipose tissue, and in vivo in 1985,13 using streptozotocin (STZ)-treated insulin-deficient diabetic rats. The in vitro studies were accomplished using sodium orthovanadate (Na3V04), sodium metavanadate (NaVC>3) or vanadyl sulfate (VOSO4). The earliest in vivo study used sodium metavanadate. The important advantage of using vanadium as an anti-diabetic agent is that some of its compounds are orally active, unlike insulin, which is a protein and cannot pass through the digestive system intact. Vanadium demonstrates insulin-mimetic properties within many aspects of carbohydrate metabolism including glucose transport, glucose transporter translocation, glycolysis and glycolytic enzymes, glucose output, glucose oxidation, and glycogen synthesis.14'15 Lipid metabolic pathways and mitogenesis are also affected.16"19 Improved glucose transport in peripheral tissues is one major effect of vanadate and 3 could be attributed to an enhanced translocation of proteins responsible for glucose transport, specifically GLUT-4, to the plasma membrane.20 Vanadyl sulfate has a much lower LD50 (by 6 to 10 times)21 than sodium orthovanadate and exhibits anti-diabetic properties by either replacing or enhancing the effects of insulin in lowering blood-glucose levels. Treatment with oral vanadyl sulfate resulted in a sustained effect after withdrawal of treatment.21 Despite these successes, the poor absorption of inorganic vanadium from the gastrointestinal (GI) tract (and some GI toxicity) dictated that improvements were needed. Organic vanadium complexes have been synthesized to enhance absorption from the GI tract. One of the earliest of these complexes was bis(maltolato)oxovanadium(IV) (BMOV), (Figure 1.1) reported in 1992 by McNeill and Orvig. 2 2 This complex was shown to be more highly absorbed from the GI tract and to be 2-3 times more effective at lowering blood glucose levels than V O S O 4 . 2 3 ' 2 4 Figure 1.1 Bis(maltolato)oxovanadium(IV) (BMOV) Originally it was thought that a possible mechanism of insulin-mimetic action involved the inhibition of Na +, K+-ATPase by vanadate,25 but recently it has been proven that this effect, which requires a high intracellular concentration of V(V), is unrelated.26 This activity may be accounted for by the fact that vanadate, possessing the same charge and similar size to phosphate,27 can act as an analog of phosphate. 4 Some vanadium compounds, as phosphate analogues, are capable of inhibiting protein tyrosine phosphatases (PTPases),28-30 an inhibition which in turn stimulate protein tyrosine phosphorylation. This inhibition may be a key feature of the mechanism of activity for vanadium in exerting its insulin-like effects; however, evidence exists that suggests a post-receptor mechanism, possibly involving a non-receptor protein tyrosine kinase.31 This mechanism is supported by the finding that most intracellular vanadium is vanadyl,32 which, unlike vanadate, is not a strong PTPase inhibitor.33 1.4 Thiazolidinediones Thiazolidinediones are named for the five-membered heteronuclear ring common to all compounds of this class; Figure 1.2 depicts examples of thiazolidinediones. 0 Rosiglitazone (BRL49653) Figure 1.2 Chemical structures of various thiazolidinediones 5 Thiazolidinediones are a class of anti-diabetic agents which have recently become of interest; the first thiazolidinedione derivative, ciglitazone, was synthesized in 1982 3 4 and its anti-diabetic properties were discovered soon afterwards.35 Many different compounds containing the thiazolidinedione moiety were subsequently synthesized and tested, resulting in the discovery of several other active agents: pioglitazone, englitazone, troglitazone and rosiglitazone (BRL 49653). The majority of these compounds did not enter clinical development because of unacceptable side-effects, but troglitazone was approved for use by the FDA in January 1997. Rosiglitazone has now been filed for approval for clinical treatment of NIDDM. 3 6 Troglitazone is a combination of a hindered phenol, known for its anti-oxidative properties, and a thiazolidinedione. It was tested extensively in vitro, and in both animal and clinical trials,37 and was found to be effective at lowering not only plasma glucose levels, but also plasma levels of triglycerides. Preclinical studies indicated that troglitazone reduced insulin resistance and thus increased the glucose uptake in adipocytes; these effects were observed both in vitro and in vz'vo.37>38 The mechanisms for the anti-diabetic effects of thiazolidinediones are not completely known, but it is related to peroxisome proliferator-activated receptors (PPAR), a member of the nuclear hormone receptor super-family. The anti-diabetic effect arises from a complex multicomponent biological interaction. Thiazolidinedione-activated PPARy stimulates transcription of certain regulatory regions of genes involved in lipid metabolism and energy balance.39'40 Expression of PPARy induces adipocyte differentiation in the presence of thiazolidinedione.41"44 It is known that thiazolidinediones bind to PPARy with affinities in. the nanomolar range.45'46 In addition, 6 there is a strong correlation between the blood glucose lowering in vivo with both thiazolidinedione binding and activation of PPARy in vitro.47'48 Finally, it was noted that certain other ligands that bind to the retinoid X receptor (RXR) of the active component could also activate the heterodimer and result in antidiabetic activity in vivo.49 The exact tissue site of thiazolidinedione function is still unknown, as there remain several unsolved problems. Although troglitazone was generally well-tolerated, with up to 26% of patients experencing mild reversible side effects, a few subjects experienced severe liver dysfunction. Between January and October 1997, 800,000 patients with NIDDM used troglitazone; 147 cases (0.02%) of liver dysfunction were found in the US and Japan, six of which resulted in death.50 It was discovered, however, that those in Japan who later died continued treatment despite obvious evidence of liver dysfunction. This resulted in a change of prescribing information by the FDA and Japanese authorities, which dictated that treatment should be discontinued if laboratory or physical findings indicate hepatic damage. European countries responded either by removing troglitazone from the market (as in the United Kingdom) or by suspending clinical trials pending more information. Beside the side effects, the most significant obstacle remaining for troglitazone is the number of non-responders, which is approximately 30%. Rosiglitazone, a new thiazolidinedione-based pharmaceutical, may address these problems. Rosiglitazone has a stronger binding affinity for PPARy than troglitazone, suggesting an increased efficacy. Unlike troglitazone, rosiglitazone does not interact with cytochrome P450 and consequently does not interact with oral contraceptives, metformin, acarbose and other prescribed drugs. Most importantly, rosiglitazone has 7 shown no evidence of hepatotoxicity and has been tested at doses five times higher than the dose for troglitazone-induced hepatotoxicity.36 1.5 Research Focus Synergism is the interaction of two or more agents that results in an effect greater than the sum of the components. Pharmacological testing of certain drugs has been complicated by synergism. This development has resulted in the testing and use of so-called 'drug cocktails', that are more effective than the individual constituent drugs would be, such as those used in the treatment for AIDS. It is important, however, to note that often the difference between purely additive effects and synergistic effects is not differentiated. The ultimate goal of this research project was the synthesis of a vanadium complex that includes a thiazolidinedione moiety. It was hoped that the effect would be increased efficacy through either additive or synergistic effects. Either phenomenon would be possible because vanadium and thiazolidinediones act by different mechanisms, and the combination of the two could conceivably be effective at lower dosages of each. In a complex the ratio of vanadium to thiazolidinedione moieties is fixed and possible difficulties could arise because their activities may differ significantly. This could result in incompatibility, as it may be necessary to raise the dosage of one component above a toxic level to derive a measure of activity from the other component. This difficulty has been observed in previous attempts at synergism between vanadium and several different biguanides.51 Vanadium complexes of biguanides were not effective synergistic insulin-mimics because the two different moieties were effective at dosages that differed by a factor of ten. 8 This project was divided into two different parts. The first was the synthesis of a ligand incorporating a thiazolidinedione moiety. Due to the strong affinity of acetylacetonate for vanadium, it was decided that the most useful target would be the adduct of an acetylacetone molecule and the thiazolidinedione group to form the potential ligand depicted in Figure 1.3, 5-[4-(3-acetyl-6-hexoxy-2-one)benzyl]-2,4-thiazolidinedione (APBT). Figure 1.3 5-[4-(3-Acetyl-6-hexoxy-2-one)benzyl]-2,4-thiazolidinedione (APBT) and its target vanadyl complex (V0(APBT)2). This ligand would then be reacted with vanadyl sulfate to form the desired complex bis-(5-[4-(3-acetyl-6-hexoxy-2-one)benzyl]-2,4-thiazolidinedionato)oxovanadium(IV), also shown in Figure 1.3. 9 Chapter 2 Experimental 2.1 Materials All solvents and chemicals were reagent grade or higher, and were used without further purification, unless stated otherwise. Al l solvents were obtained from Fisher, with the exception of THF, which was obtained from E M Science. Dimethylglyoxime, pyridine, PPli3, PPh3»Br2, sulfuryl chloride, NaH, di-terr-butyl dicarbonate, 3-bromopropanol, DMAP, and vanadyl acetylacetonate were purchased from Aldrich; 4-hydroxybenzaldehyde and 2,4-thiazolidinedione were purchased from Avocado Chemicals; imidazole, anhydrous K2CO3, acetylacetone, anhydrous Na2SC>4, and tert-butanol were purchased from BDH and acetylacetone was purified according to a literature procedure. 5 2 Piperidine, NaBFL;, glacial acetic acid, iodine crystals, decolourizing carbon, and anhydrous MgSCM were purchased from Fisher; p-toluenesulfonyl chloride was purchased from Sigma and CoCi2»6H20 was purchased from Mallinckrodt Chemical Works. Sodium acetylacetonate was acquired from Dr. Zhiqiang Xu of our research group. Water was both distilled (Corning MP-1 Megapure Still) and deionized (Barnstead D8902 and D8904 cartridges) before use. Deuterated chloroform and methanol were purchased from Cambridge Isotope Laboratory. 10 2.2 Instrumentation ! H NMR (200 MHz), ] H COSY (200 MHz) and I 3 C NMR (50.3 MHz) spectra were recorded on a Bruker AC-200E spectrometer. C NMR (75 MHz) spectra were recorded on a Varian XL-300 spectrometer. ! H - 1 3 C HMQC spectra and 1 3 C NMR (125 MHz) spectra were recorded on a Bruker AVA-500 (AMX upgraded) spectrometer. Infrared spectra were obtained on a Galaxy Series 5000 FTIR spectrophotometer using KBr discs over the range 4000-400 cm"1. Mass spectra (positive ion detection mode) were obtained using either a Kratos Concept IIH32Q (Cs+ liquid secondary ion mass spectrometry, LSIMS) or Kratos MS 50 (electron-impact ionization, EI) instrument. HPLC chromatograms were obtained on a Symmetry 5um C18 column on a Waters Deltaprep 600 semi-prep instrument using a Waters Model 996 photodiode array detector and a 2410 refractive index detector. Elemental analyses of C, H, and N were performed by Mr. Peter Borda in the UBC Chemistry Department on a Carlo Erba instrument. 2.3 Procedures 2.3.1 Dry Flash Column Chromatography Using a large glass frit filter (coarse grade) filled to within 2 cm of the top with silica gel, crude product can be placed on top and solvent run through the filter. The eluent was pulled through the silica by means of a vacuum filter flask connected to an aspirator. The primary advantage of a dry flash column over traditional flash column 11 chromatography is speed. A dry flash column can be completed much more quickly than a regular column; however, this speed comes at the price of separation. Because of the short column length, the separation is poorer than for a traditional column and is thus limited to those cases in which only one compound migrates. Figure 2.1 Schematic diagram of a dry flash column 2.4 Synthesis: Reactions Accomplished 2.4.1 4-(3-Hydroxypropoxy)benzaldehyde 4-Hydroxybenzaldehyde (5.00 g, 41.0 mmol) was placed in a 500 mL round bottom flask with potassium hydroxide (2.2 g, 39 mmol) and dissolved in 150 mL ethanol. To this, 5 mL doubly distilled water was added to promote solubility of the base, and the reaction mixture was stirred until the base had completely dissolved. The 12 solution was clear yellow in colour. 3-Bromopropanol (3.80 mL, 42.0 mmol) was added via syringe to the stirring solution. The reaction mixture was then refluxed overnight for 18 hours with stirring. At this point, a white precipitate was visible on the sides of the flask. The reaction mixture was removed from heat and allowed to cool to room temperature. The precipitate was removed by vacuum filtration; the pungent filtrate was concentrated in vacuo leaving a yellow oil. The oil was diluted with 40 mL 1 M KOH and 60 mL methylene chloride. The organic layer was separated and the aqueous layer extracted with methylene chloride (2 X 30 mL). The combined organic fractions were dried over Na2SC>4, filtered and concentrated in vacuo. This yielded 6.07 g (82%) of a yellow oil. This was used in the next reaction without further purification. 'H NMR (CDC13): 5 2.01 (tt, 2H), 3.79 (t, 2H), 4.13 (t, 2H), 6.93 (d, 2H), 7.74 (d, 2H), 9.78 (s, 1H). 2.4.2 5-([4-(3-Hydroxypropoxy)phenyI]methylene)-2,4-thiazolidinedione (oxidized HPBT) Crude 4-(3-hydroxypropoxy)benzaldehyde (6.07 g, 0.034 mmol) was dissolved in 100 mL distilled ethanol. 2,4-Thiazolidinedione (3.70 g, 0.032 mmol) was added to this solution. Piperidine (3.1 mL, 0.03 mol) was added by syringe to the stirred solution. Upon the addition of piperidine the reaction mixture darkened immediately to a dark yellow. The reaction mixture was then refluxed with stirring for 24 hours. The resulting mixture had a strong unpleasant scent. The solvent was removed in vacuo leaving a yellow oil. The oil was adsorbed onto silica by first dissolving in acetone, adding silica 13 and removing the solvent in vacuo. This procedure was repeated until all of the oil had been adsorbed onto silica. The silica/product mixture was then ground using a mortar and pestle to a fine powder. A dry flash column was then performed using 9:1 chloroform/methanol as the eluent and five 250 mL fractions were collected. The fractions were left to evaporate over five days. A pale yellow precipitate remained which was collected by vacuum filtration. The filtrates from all the fractions were collected together and evaporated. The precipitate was washed with the eluent before placing in vials. The vials were placed on the vacuum line overnight and weighed. The combined yield was 3.08 g (35%). Anal. Calc (found) for C13H13NO4S: C, 55.90 (55.61); H, 4.69 (4.78); N , 5.01 (4.80). MS (EI): m/z 279, ([M]+, [C13H13NO4S] 4 ). ! H NMR (CDCI3): 5 2.00 (tt, 2H), 3.74 (t, 2H), 4.15 (t, 2H), 7.05 (d, 2H), 7.50 (d, 2H), 7.73 (s, IH). 2.4.3 5-[4-(3-Hydroxypropoxy)benzyl]-2,4-thiazolidinedione (HPBT) Dimethylglyoxime (0.082 g, 0.710 mmol) and CoCi2«6H20 (0.040 g, 0.160 mmol) were placed in a 1 L Erlenmeyer flask and dissolved in 250 mL distilled water. To this solution was added 10 drops aqueous potassium hydroxide (3 M). Upon addition of the potassium hydroxide the solution went from colourless to a dark orange brown. The flask was cooled to 0°C using an ice bath and NaBH 4 (2.3 g, 61 mmol) was added. 5-([4-(3-Hydroxypropoxy)phenyl]methylene)-2,4-thiazolidinedione (3.08 g, 11.0 mmol) was added slowly to the reaction mixture with 14 stirring. The mixture immediately became turbid. Shortly afterwards the mixture changed colour to an opaque purple. White bubbles evolved, forming a foam layer on the top of the mixture. A white precipitate began to form after about 8 minutes and the ice bath was removed. The flask opening was covered and left to stir overnight. After 16 hours, the reaction mixture was a translucent yellowish colour with a fine black precipitate. The reaction mixture was acidified using glacial acetic acid to pH 6. Although the mixture became a milky pale yellow colour with a visible off-white precipitate at pH 7, the acidification was continued to pH 6 to confirm that all of the product had been reprotonated. The contents of the flask were placed in a separatory funnel and extracted with chloroform (10 x 100 mL) until the precipitate had disappeared leaving a clear yellow aqueous layer. The collected extracts were combined and concentrated in vacuo. The organic phases were then dried with MgSC>4 and filtered. The dried extract was boiled with decolourizing carbon for five minutes and filtered. The solvent was removed from the pale yellow solution in vacuo, leaving an off-white precipitate. The precipitate was dried overnight on the vacuum line to give 3.02 g product (97%). Anal. Calc (found) for Ci 3 Hi 5 N0 4 S: C, 55.50 (55.43); H, 5.37 (5.41); N , 4.98 (4.88). MS (EI): m/z 281 ([M]+, [Ci 3Hi 5N0 4S] +). lH NMR (CD3OD): 1.96 (tt, 2H), 3.21 (m, 2H), 3.72 (t, 2H), 4.05 (t, 2H), 4.68 (dd, 1H), 6.86 (d, 2H), 7.15 (d, 2H). 1 3 C NMR (CD3OD, 125 MHz): 33.3, 38.4, 54.8, 59.6, 65.7, 116, 130, 132, 160, 173, 177. 15 2.4.4 5- [4-(3-Bromopropoxy)benzyl] -2,4-thiazolidinedione (BPBT)5 3 In a round bottom flask, 5-[4-(3-hydroxypropoxy)benzyl]-2,4-thiazolidinedione (1.97 g, 7.01 mmol) was dissolved in 100 mL distilled acetonitrile. Pyridine (0.60 mL, 7.4 mmol) was added via syringe to the stirring yellow mixture. After cooling the reaction mixture to 0°C in an ice bath PPri3»Br2 (4.01 g, 9.51 mmol) was slowly added to the stirring mixture. The reaction mixture was allowed to return to room temperature and left to stir overnight. As the reaction mixture heated up all of the solid dissolved. Additional PPli3»Br2 (0.968 g, 2.29 mmol) was added and the mixture was stirred for 4 hours. The solvent was removed in vacuo leaving a yellow oil, from which a precipitate eventually formed. An attempt was made to dissolve the precipitate in 1:1 diethyl ether/hexanes but a gummy solid formed. The solvent was removed in vacuo, and the solid was recovered and placed on top of a silica column (11 cm x 6 cm). The column was eluted with 1:1 diethyl ether/hexanes and followed by TLC. The fractions were combined and the solvent removed in vacuo. As the column chromatography was unsuccessful, the products were recovered from the column by dissolving in acetonitrile, filtering and removing the solvent, leaving an orange oil. The oil was adsorbed onto silica as previously described and a dry flash column was performed, using 1:1 diethyl ether/hexanes as eluent. Two 500 mL fractions were collected. The solvent was then removed in vacuo and the resulting white precipitate was placed on the vacuum line to dry. The yield of the reaction was 0.765 g (32 %). This reaction was only performed once and was not optimized. 16 Anal. Calc (found) for C 1 3H 1 4BrN03S: C, 45.36 (45.44); H, 4.10 (4.25); N , 4.07 (3.90). MS (EI): m/z 345 , ([M]+, [Ci 3Hi 4BrN0 3S] +). ! H NMR (CDC13): 2.29 (tt, 2H), 3.26 (m, 2H), 3.58 (t, 2H), 4.07 (t, 2H), 4.49 (dd, IH), 6.84 (d, 2H), 7.13 (d, 2H). 2.4.5 5-[4-(3-Iodopropoxy)benzyl]-2,4-thiazolidinedione (IPBT)53 Imidazole (1.17 g, 17.1 mmol), 5-[4-(3-hydroxypropoxy)benzyl]-2,4-thiazolidinedione (3.04 g, 10.8 mmol) and PPh3 (4.33 g, 16.5 mmol) were placed in a round bottom flask with 150 mL distilled toluene. Al l of the reactants did not dissolve- a clear yellow solution with a visible precipitate was the result. Iodine crystals (4.44 g, 17.5 mmol) were added to the reaction mixture, and the reaction mixture turned an opaque orange with red and white sludge stuck on the sides. Most of the solid was removed from the sides before heating the flask to 80°C in an oil bath. The heat was maintained while stirring constantly for 4 hours under an argon atmosphere. After 6 hours the solvent was removed in vacuo leaving an orange waxy precipitate. The precipitate was dissolved in acetonitrile and adsorbed onto silica. A dry flash column was performed, using 1:1 ether/hexanes as the eluent. Fractions were collected (10 x 250 mL) and concentrated. These fractions were allowed to evaporate over a week to collect 2.68 g (63%) of product. Anal. Calc (found) for Ci 3 Hi 4 IN0 3 S: C, 39.91 (40.19); H, 3.61 (3.60); N , 3.58 (3.49). 17 MS (EI): m/z 391 ([M]+, [Ci3H I 4rN03S]+). [ H NMR (CDC13): 2.26 (tt, 2H), 3.27 (m, 2H), 3.36 (t, 2H), 4.01 (t, 2H), 4.50 (dd, 1H), 6.84 (d, 2H), 7.14 (d, 2H). 1 3 C NMR (CDCI3, 75 MHz): 2.50, 32.9, 37.7, 53.7, 67.2, 115, 128, 130, 158, 168, 170, 174. 2.4.6 AyV'-sulfuiyldiimidazole54 Imidazole (10.81 g, 150.0 mmol) was dissolved in 250 mL dry THF and cooled to 0°C using an ice bath with stirring. To this solution, sulfuryl chloride (5.04 g, 37.3 mmol) was added slowly via syringe. The ice bath was removed and the reaction mixture was allowed, while stirring, to warm to room temperature over one hour. The opaque yellow mixture was concentrated to approximately 50 mL, whereupon white crystals fell out of the yellow solution. The crystals were vacuum filtered, left to dry on the vacuum filtration apparatus for 1 hour and then weighed. The mass of the product was 1.49 g Anal. Calc (found) for CeHeN^S: C, 36.36 (36.36); H, 3.05 (3.06); N , 28.27 (28.10). MS (EI): m/z 198 ([M]+,[C6H6N402S]+). 'H NMR (CDC13): 7.16 (d, 2H), 7.30 (t, 2H), 8.03 (s, 1H). o (20.5%). 18 2.5 Synthesis: Reactions Attempted Many attempts were made to synthesize the desired product (see summary Table 2.1). Those listed here were not altogether successful. Many reactions were performed for the synthesis of APBT using essentially the same conditions so only one example is given and the conditions are listed in Table 2.1. 2.5.1 5-[4-(3-Acetyl-6-hexoxy-2-one)benzyl]-2,4-thiazolidinedione (APBT) 5 - [4-(3 -Iodopropoxy)benzyl] -2,4-thiazolidinedione (0.073 g, 0.197 mmol) was dissolved in 20 mL DMF and oven-dried K2CO3 (0.084 g, 0.609 mmol) was added to the mixture (it did not dissolve). Previously distilled acetylacetone (0.040 mL, 0.390 mmol) was added to the mixture via syringe. The mixture was heated to 80°C with stirring for 3 hours over which time the reaction was followed by TLC using 3:7 ethyl acetate/hexanes as the eluent. During the course of the reaction, the mixture changed colour, darkening from pale yellow to orange and finally to almost black by the end of the reaction. After 3 hours, the reaction mixture was removed from heat and the solvent removed under reduced pressure. The residue was dissolved in 25 mL distilled water and acidified to pH 4 using glacial acetic acid. A dark chocolate brown precipitate formed upon acidification. An additional 10 mL distilled water was added in an unsuccessful attempt to dissolve the brown precipitate. The aqueous mixture was extracted with diethyl ether (4 x 40 mL) and then CH2CI2 (2 x 60 mL). The four ether portions and the two CH2CI2 portions were combined separately, the solvent removed and 'H-NMR spectra taken of each to determine identity and purity. 19 Table 2.1 Variat ions in conditions for acetylacetone reaction. Starting Material Reagent Base Solvent Temperature Time BPBT 4 eq Acetylacetone 4 eq NaH Benzene Reflux 20 hr IPBT 1.5 eq Acetylacetone Acetone Reflux 20 hr IPBT 1.5 eq Sodium acetylacetonate CH 3CN Reflux Overnight IPBT 1.2 eq Sodium acetylacetonate 1.2 eq K 2 C 0 3 THF Reflux Overnight IPBT 1.2 eq Acetylacetone 1.4 eq K 2 C 0 3 DMF 80°C temp up to 130°C 1.5 hr IPBT 1.3 eq Acetylacetone 1.2 eq K 2 C 0 3 DMF 40°C, then 80°C 2.5 hr, 0.5hr IPBT 1.9 eq Acetylacetone 1.4 eq K 2 C 0 3 CH 3CN 80°C 2hr IPBT 2.7 eq Acetylacetone 1.3 eq K 2 C 0 3 THF Reflux 6hr IPBT 1.3 eq Acetylacetone 1.2 eq K 2 C 0 3 DMF 80°C 3hr IPBT 1.8 eq Acetylacetone 1.2 eq K 2 C 0 3 DMF 80°C, then 110°C 1 hr, 0.5 hr IPBT 2 eq Acetylacetone 3 eq K 2 C 0 3 DMF 80°C 3 hr IPBT 2eq Acetylacetone 3 eq K 2 C 0 3 DMF 80°C 1 hr IPBT 3 eq Acetylacetone 4 eq NaH DMF 80°C 1.5 hr IPBT 29.2 eq Acetylacetone 3 eq K 2 C 0 3 None 70°C 1.5 hr 20 2.5.2 5-[4-(3-/>-Toluenesulfonylpropoxy)benzyl]-2,4-thiazolidinedione (TsPBT) In a 500 mL round bottom flask 5-[4-(3-hydroxypropoxy)benzyl]-2,4-thiazolidinedione (0.273 g, 0.970 mmol) was dissolved in 50 mL pyridine and cooled to 0°C using an ice bath. Using a dropping funnel, p-toluenesulfonyl chloride (0.291 g, 1.53 mmol), dissolved in 80 mL pyridine, was added drop-wise over 30 minutes. The mixture was left to stir overnight to warm up to room temperature. The next day, the reaction mixture had the same appearance (a clear pale yellow solution). The mixture was placed in a separatory funnel with 100 mL CH2CI2 resulting in a miscible solution, which separated upon the addition of 100 mL HC1 (1 M). The organic phase was washed twice with 1 M HC1, dried with anhydrous Na2SC<4, filtered, and the solvent removed under reduced pressure leaving a yellow oil. Upon standing 3 days, a yellowish precipitate was visible. The precipitate was washed onto a glass filter frit with ether and then washed with chloroform until all of the precipitate was a uniform colour. The 'H-NMR spectrum revealed unfortunately that the precipitate was entirely starting material. 2.5.3 /V-BOC-5-[4-(3-hydroxypropoxy)benzyl]-2,4-thiazolidinedione (BocHPBT) Method A 5 5 21 5-[4-(3-Hydroxypropoxy)benzyl]-2,4-thiazolidinedione (0.849 g, 3.02 mmol) was dissolved in 3 mL NaOH (1 M) and 2.25 mL ter/-butanol in a 10 mL round bottom flask. To this solution, di-tert-butyl dicarbonate (0.640 g, 3.10 mmol) was added and the mixture stirred, heated to 30°C, and allowed to stir overnight covered with parafilm. After 15 hours, the mixture was removed from heat and the solvent concentrated under reduced pressure. The aqueous layer was extracted with ethyl acetate (3x15 mL). The fractions were combined and the solvent removed in vacuo leaving an oily orange-brown solid. Method B 5 6 After dissolving 5-[4-(3-hydroxypropoxy)benzyl]-2,4-thiazolidinedione (0.570 g, 2.03 mmol) in 100 mL distilled THF and flushing the solution with argon, di-tert-butyl dicarbonate (0.839 g, 4.07 mmol) was added. DMAP (0.252 g, 2.06 mmol) was added to the solution yielding a clear pale yellow solution. The reaction mixture was left overnight at room temperature while stirring under argon. The reaction was followed by TLC. After 22 hours the solvent was removed under reduced pressure and the residue was mixed with 2:1 hexanes/ethyl acetate and filtered. A white insoluble material remained in the original flask. The solvent was removed from the filtrant in vacuo leaving a yellow-orange oil, which was dissolved in CH2CI2 and added to the top of a dry flash column by means of a Pasteur pipet. A dry flash column was performed using 3:7 ethyl acetate/hexanes as the eluent. Six 50 mL fractions were collected and then the column was eluted with 100 mL CH2CI2. The solvent was removed from the fractions in vacuo and 'H-NMR spectra were performed to determine identity and purity. The sample 22 was not pure, and was passed through a silica column using 95:5 CHCls/methanol as the eluent. An oil resulted showing near purity for BocHPBT by *H NMR spectroscopy. 2.5.4 AMBOC-5-[4-(3-iodopropoxy)benzyl]-2,4-thiazolidinedione (BocIPBT) Method A 5 5 5-[4-(3-Iodopropoxy)benzyl]-2,4-thiazolidinedione (0.201 g, 0.514 mmol) was dissolved in 0.5 mL 1 M NaOH and 0.37 mL ter/-butanol. The solid did not completely dissolve so 0.5 mL more NaOH was added. To this mixture di-terr-butyl dicarbonate (0.112 g, 0.543 mmol) was added and the mixture stirred. The mixture was heated to 35°C and left stoppered overnight. After heating for 24 hours the reaction mixture was removed from heat and then placed under reduced pressure to remove the rerr-butanol. The solution was then extracted with CH2CI2 (3x15 mL) and the organic fractions combined. The solvent was removed in vacuo and a *H-NMR spectrum was taken to determine purity. The result showed that more than one product was present. Method B 5 6 5-[4-(3-Iodopropoxy)benzyl]-2,4-thiazolidinedione (0.399 g, 1.02 mmol) was dissolved in 50 mL distilled THF and flushed with argon. Di-tert-butyl dicarbonate (0.414 g, 2.01 mmol) and DMAP (0.128 g, 1.04 mmol) were consecutively added to the reaction mixture. The mixture was stirred while flushing with argon and heated to 30°C and then left overnight. After 24 hours the orange reaction mixture was removed from heat and a waxy yellow solid was observed on the sides of the round bottom flask. The 23 solvent was removed under vacuum leaving an orange oil. The oil was then triturated with 2:1 hexanes/ethyl acetate (2 x 45 mL) giving a yellow-orange, sticky, extremely viscous wax and a faintly coloured solution. The solution was decanted off each time, combined and evaporated in vacuo. The resulting oil was a complex product mixture, as shown in its 'H-NMR spectrum. 2.5.5 /V-BOC-2,4-thiazoIidinedione56 2,4-Thiazolidinedione (1.18 g, 10.1 mmol) was dissolved in 100 mL distilled THF and DMAP (1.22 g, 9.95 mmol) was subsequently added. To this solution di-ter/-butyl dicarbonate (4.01 g, 19.4 mmol) was added, transforming the solution, within a minute, from a clear yellow solution to a slushy mixture, to a clear red solution. The reaction mixture was stirred under argon and the reaction followed by TLC using 3:7 ethyl acetate/n-pentane as the eluent. After 18 hours, the solvent was removed in vacuo, leaving a red solid that resembled a plastic film; this was subsequently dissolved in 2:1 hexanes/ethyl acetate leaving behind insoluble DMAP as an orange precipitate. The mixture was vacuum filtered resulting in an orange precipitate and a red solution. The solvent was removed from the solution under reduced pressure. Purification of the residue was attempted, using a silica gel column, 3:7 ethyl acetate/hexanes as the eluent. This procedure, however, was not successful. 2.5.6 5-[4-(3-Sulfonylimidazolepropoxy)benzyl]-2,4-thiazolidinedione (ImPBT) 24 5-[4-(3-Hydroxypropoxy)benzyl]-2,4-thiazolidinedione (0.278 g, 0.990 mmol) was dissolved in 5mL DMF. The reaction flask was then cooled to 0°C in an ice bath with stirring under argon. NaH (0.082 g, 3.42 mmol) was then added directly and the reaction mixture stirred at room temperature for 30 minutes. This mixture was then cooled to -40°C using dry ice/CH3CN slush and N'-sulfuryldiimidazole (0.304 g, 1.57 mmol), previously dissolved in 3 mL DMF, was added to the reaction mixture. The vial was rinsed with 2 mL DMF and this was also added to the flask. The mixture was left stirring for 30 minutes before adding 0.30 mL methanol by syringe and then stirring for an additional 35 minutes. The reaction mixture, now a clear yellow solution, was poured into 60 mL cold H 2 0 and acidified to pH 5 with glacial acetic acid. The aqueous fraction was extracted with diethyl ether (4 x 60 mL). The organic extracts were combined, dried over MgS04 and filtered. The solvent was then removed, leaving a yellow oil. 2.5.7 Bis-(5- [4-(3-acetyl-6-hexoxy-2-one)benzyl] -2,4-thiazolidinedionato)oxovanadium (VO(APBT)2) In a 25 mL round bottom flask, 5-[4-(3-iodopropoxy)benzyl]-2,4-thiazolidinedione (0.815 g, 2.08 mmol), oven-dried K2CO3 (0.575 g, 4.16 mmol) and vanadyl acetylacetonate (0.257 g, 0.967 mmol) were dissolved (the K2CO3 did not dissolve completely) in 20 mL CH 3 CN. The reaction mixture was heated to 60°C while stirring under argon. The reaction was followed by TLC, using 3:7 ethyl acetate/hexanes as the eluent. After 1 hour with no change, the temperature was raised to 75°C. The 25 reaction vessel was removed from heat after 4 hours and a peach/brown precipitate with a green translucent solution was observed. Removal of the solvent in vacuo left a greenish brown precipitate/oil. Distilled water (20 mL) was added to the flask and the residue scraped from the sides. The mixture was vigourously shaken and allowed to settle before decanting off the opaque dark green solution, to leave a light pink precipitate. The solution was extracted with C H 2 C I 2 (2 x 20 mL), combined and dried with anhydrous MgSC>4, filtered and the solvent removed under reduced pressure. The light pink precipitate was dissolved in 20 mL C H 2 C I 2 and washed with distilled water (3 x 20 mL). To the washed organic layer, 20 mL C H 2 C I 2 was added before drying with anhydrous MgSCU. This was filtered and the solvent removed under reduced pressure. A grey residue remained. Anal. Calc (found) for C 3 6 H 4 0 N 2 O 1 1 S 2 V : C, 54.61 (52.87); H, 5.09 (4.51); N , 3.54 (3.82). MS (LSIMS): m/z 790 ([M-H]+, [CaerLjo^Oi^V]*). 26 Chapter 3 Results and Discussion 3.1 Design and Syntheses The first 3 steps of the synthesis of the target compound 5-[4-(3-acetyl-6-hexoxy-2-one)benzyl]-2,4-thiazolidinedione (APBT) proceeded smoothly, based on procedures established by Dr. Peter Buglyo.57 Despite ephemeral glimpses of the target, APBT was not purified. The synthesis was attempted through multiple sequences. The first attempt utilized bromide as the leaving group (BPBT). Other leaving groups were also tried, including iodide, /?-toluenesulfonate and A^-sulfurylimidazolate. One consideration in attempting to bind the acetylacetone to the alkyl chain is the pK a of various protons. The pK a of acetylacetone is ~9 5 8 whereas the pK a of 2,4-thiazolidinedione is 6.7.59 Consequently, the sequence involving protection of the thiazolidinedione N-H proton as a BOC group was examined. 3.1.1 Synthesis: Reactions Accomplished 4-(3-Hydroxypropoxy)benzaldehyde O H H O O C H O C H O Figure 3.1 Synthesis of 4-(3-hydroxypropoxy)benzaldehyde 27 Figure 3.2 Overall scheme for the reactions described in this thesis 28 This reaction is a substitution reaction, involving the use of KOH as base to deprotonate the phenol. The addition of water was necessary to dissolve the KOH, because it is insoluble in common organic solvents. After the reaction was complete, a white insoluble solid was visible. The precipitate was assumed to be KBr because it would have been formed as the side product of this reaction. The water condenser was used to prevent evaporation of solvent during reflux. The extraction was accomplished by first adding more KOH to deprotonate any remaining starting material. Thus, only the desired product and any remaining 3-bromopropanol would be extracted by CH2CI2. The oil was not purified before the next step because the 3-bromopropanol impurity would be unreactive under the reaction conditions. 5-([4-(3-Hydroxypropoxy)phenyl] methylene)-2,4-thiazolidinedione (oxidized HPBT) Figure 3.3 Synthesis of 5-([4-(3-hydroxypropoxy)phenyl]methylene)-2,4-thiazolidinedione (oxidized HPBT) As noted above, the starting material was crude, so the theoretical yield was calculated using the moles of 2,4-thiazolidinedione. This reaction proceeds through a Knpevenagel condensation reaction,60 which is catalyzed by an amine, in this case, 29 piperidine. The product mixture gives off an acrid smell that necessitates performing all solvent removal in the fume hood. The pure compound is, however, odourless. The product mixture was purified using a dry flash column. 5-[4-(3-Hydroxypropoxy)benzyl]-2,4-thiazolidinedione (HPBT) Figure 3.4 Synthesis of 5-[4-(3-hydroxypropoxy)benzyl]-2,4-thiazoIidinedione (HPBT) The reduction of the double bond occurs using sodium borohydride as the reducing agent and a cobalt complex, formed in situ from CoCl2«6H20 and dimethylglyoxime, as a catalyst. To form the complex the ligand first needs to be deprotonated, which was accomplished by addition of base to the solution. Because this reaction is exothermic, the solution was cooled in an ice bath before adding the NaBH4. The starting material was added slowly, in a controlled manner, to the reducing mixture in order to prevent foaming over, with substantial loss of product. The reaction was allowed to continue overnight (18 hours) to ensure completion. After the reaction was completed, a fine black precipitate was visible, which was assumed to be cobalt metal that had been reduced by NaBH4. The reaction mixture was then acidified to reprotonate 30 the thiazolidinedione nitrogen atom, resulting in the precipitation of an off-white solid that was then extracted from the aqueous mixture. Decolourizing with activated charcoal gave only a slight increase in purity. The reaction was performed on a 3-4 gram scale, as there was greater difficulty in controlling the rate and exothermicity of reactions of larger scale. 5-[4-(3-Bromopropoxy)benzyl]-2,4-thiazolidinedione(BPBT)53 Figure 3.5 Synthesis of 5-[4-(3-bromopropoxy)benzyl]-2,4-thiazolidinedione (BPBT) This reaction uses commercially purchased PPh3»Br2, actually a salt ([PPh3Br]Br). The bromotriphenylphosphonium attacks the activated alcohol to liberate the thermodynamically stable triphenylphosphine oxide when the carbon attached to the oxygen is attacked by the Br". Pyridine was used as a base that was not a strong nucleophile, to activate the alcohol. The purification performed was by dry flash column. 5- [4-(3-Iodopropoxy)benzyl] -2,4-thiazolidinedione (IPBT) 5 3 31 Figure 3.6 Synthesis of 5-[4-(3-iodopropoxy)benzyl]-2,4-thiazolidinedione (IPBT) This reaction is very similar to the previous one. It proceeds by first activating the h with triphenylphosphine using imidazole as the base. Imidazole performs the same function that pyridine did in the previous reaction. The reaction mixture was heated to 80°C for 4 to 6 hours, resulting in a successful reaction. This reaction was purified by using a dry flash column and resulted in an analytically pure, pale yellow, crystalline solid. ^-BOC-5-[4-(3-hydroxypropoxy)benzyl]-2,4-thiazolidinedione (BocHPBT) 5 6 Figure 3.7 Synthesis of iV-BOC-5-[4-(3-hydroxypropoxy)benzyl]-2,4-thiazolidinedione (BocHPBT) 32 This reaction was attempted by two different routes as explained in the experimental section. Initially the reaction was carried out in water, using NaOH and ter/-butanol, producing sodium tert-butoxide because the solvent mixture was suggested in a literature preparation.55 No reaction occurred, as indicated by ! H-NMR spectroscopy. Conceivably, di-ter/-butyl dicarbonate may have reacted with the primary alcohol instead of the imine, so the reaction was repeated with IPBT. It was subsequently discovered that the thiazolidinedione ring had a deactivating effect on the imine and that the di-ter/-butyl dicarbonate was reacting with the water present before it could react with the imine. Another reaction was attempted using DMAP as a catalyst and THF as solvent.56 This variation was successful. A^'-sulfuiyldiimidazole54 C - l - C + 3eq ™Q] THF CH~0 + o Figure 3.8 Synthesis of NJV -sulfuryldiimidazole This reaction was performed according to a literature procedure.54 The reaction was performed at 0°C because the reactivity of the sulfuryl chloride needs to be moderated. This reaction results in the loss of HC1 and thus is driven thermodynamically to product. Filtration was required to isolate the crystals, which were not recrystallized as 'H-NMR and elemental analyses indicated a high level of purity. 33 3.1.2 Synthesis: Attempted Reactions 5- [4-(3-Acetyl-6-hexoxy-2-one)benzyl] -2,4-thiazolidinedione (APBT) Figure 3.9 Synthesis of 5-[4-(3-acetyl-6-hexoxy-2-one)benzyl]-2,4-thiazolidinedione (APBT) The synthesis of this compound was attempted by many different methods, none of which ultimately produced a pure product, even after column purification attempts. The different reaction conditions are listed in Table 2.1, pg 20. This reaction was initially performed using acetylacetone and BPBT with sodium hydride as the base in benzene at reflux for 20 hours. TLC revealed a complex product mixture and ^ - N M R did not display any of the required peaks. Thus, the bromide was replaced by the more reactive iodide and the benzene was replaced by acetone, as noted previously.61 Acetylacetone and IPBT were refluxed in acetone for 20 hours, to no avail. The complex product mixture could not be separated or characterized. Possibly, the acetone peak at 2.1 ppm might interfere with the determination of whether the acetylacetone had bound to the alkyl chain, and also the temperature might be too low for the reaction to proceed. Therefore the reaction was next run in acetonitrile. Another change was made to ensure deprotonation of acetylacetone: IPBT was treated with what was initially thought to be the more reactive sodium acetylacetonate. This reaction was 34 tried both with and without the addition of base (K2CO3). Potassium carbonate was used as the base for almost all of the attempted reactions, in accordance with literature precedent.61 Sodium acetylacetonate was also reacted with IPBT in THF. Since acetylacetonate is potentially too stable, acetylacetone was used with DMF as the solvent, at 8 0 ° C . DMF was chosen as an appropriate solvent as it is a polar aprotic solvent, conducive of S N 2 reactions. The first attempt under these conditions resulted in a 'H-NMR spectrum that was encouraging, including a triplet that integrated to one proton at the position corresponding to the proton on the P carbon of acetylacetone. However despite multiple efforts, reproducibility was not achieved. When added to the difficulty in purification (the compound seemed to hydrolyze on the column back to HPBT), this result was discouraging. 5-[4-(3-p-Toluenesulfonylpropoxy)benzy 1]-2,4-thiazolidinedione (TsPBT) Figure 3.10 Synthesis of 5-[4-(3-p-toluenesulfonylpropoxy)benzyl]-2,4-thiazolidinedione (TsPBT) A different leaving group, />-toluenesulfonate, was tried to bring about the preceeding reaction, Figure 3 . 1 0 , with greater ease and reproducibility. Pyridine, a base commonly used for this reaction, was needed to deprotonate the alcohol, and was used 35 neat. Unfortunately, the purification was not straightforward and this reaction was abandoned in favour of other synthetic pathways. N-BOC-S- [4-(3-iodopropoxy)benzyl] -2,4-thiazolidinedione (BocIPBT) 5 6 Figure 3.11 Synthesis of A7-BOC-5-[4-(3-iodopropoxy)benzyl]-2,4-thiazolidinedione (BocIPBT) This reaction was performed with the same reaction conditions as for BocHPBT and was equally unsuccessful for the reaction using NaOH and rerr-butanol. The reaction using DMAP as the catalyst and THF as the solvent was completed but was not purified. 7V-BOC-2,4-thiazolidinedione56 Figure 3.12 Synthesis of N-BOC-2,4-thiazolidinedione This was a test reaction to confirm that the reaction worked before trying it with HPBT and IPBT. This reaction proceeded very quickly, changing colour within a minute from a clear yellow solution to a clear red solution; however purification was 36 challenging. A column was performed using an eluent (3:7 ethyl acetate/n-pentane) as determined by TLC. Nonetheless, this reaction was encouraging enough (TLC showed a spot less polar than starting material) to proceed. As a model case for protecting IPBT and HPBT, no further exploration was attempted. 5-[4-(3-Sulfonylimidazolepropoxy)benzyl]-2,4-thiazolidinedione (ImPBT) 6 2 HO' Figure 3.13 Synthesis of 5-[4-(3-sulfonylimidazolepropoxy)benzyl]-2,4-thiazolidinedione (ImPBT) Sodium hydride was used in this reaction to deprotonate the alcohol so that the previously synthesized A^A^-sulfuryldiimidazole would react with the anion. More than two equivalents of NaH were added because of the acidic thiazolidinedione proton. This reaction was adapted from a literature preparation that dealt with sugars and consequently may not have been suitable in this case.62 There may have been an interaction with the nitrogen on the thiazolidinedione ring. 37 Bis-(5-(3-acetyI-6-hexoxy-2-one)benzyl-2,4-thiazolidinedionato)oxovanadium(IV) ( V O ( A P B T ) 2 ) tf o V •N H VO(acac)2, K 2 C0 3 D M F Figure 3.14 Synthesis of bis-(5-[4-(3-acetyI-6-hexoxy-2-one)benzyl]-2,4-thiazolidinedionato)oxovanadium ( V 0 ( A P B T ) 2 ) Direct alkylation of vanadyl acetylacetonate was expected to succeed due to a relatively more acidic p proton which would thus render the complex more reactive than acetylacetone itself. Reaction conditions were similar to those for attempted ABPT formation (above). It was hoped that a small quantity of complex could be isolated, even if the reaction did not go to completion or if a complex mixture resulted. The product was a greyish residue that contained multiple compounds, none of which appeared to be the desired ligand, although LSIMS data suggested that the complex was formed. 3.2 Characterization 3.2.1 Infrared Spectroscopy IR spectral peaks are summarized in Table 3.1 and Table 3.2. Examples of the spectra are shown in Figure 3.15. 38 Table 3.1 Characteristic IR absorptions (cm1) of ligand precursors Assignment oxidized HPBT HPBT IPBT 2,4-thiazolidinedione v(C=0) (SN) 1729 1743 1753 1737 v(C=0) (NC) 1688 1693 1704 1663 v(COC) 1265 1245 1033 1246 1054 v(CH) 2956 2954 2924 2936 2948 v(OH) 3443 3450 3425 v(NH) 3135 3047 phenyl 3111 3144 3202 39 4000 3500 3000 2500 2000 1500 1000 500 Wavenumbers (cm1) Figure 3.15 Selected FTIR spectra comparing various ligand precursors Table 3.2 Characteristic IR absorptions (cm1) of AyV'-sulfuryldiimidazole Assignment Wave numbers absorption (cm"1) v(S=0) 1435 1200 40 3.2.2 N M R Spectroscopy 'H - NMR spectra were obtained for each of the synthesized compounds using either deuterated chloroform (CDCI3) or deuterated methanol (CD3OD) as the solvents. Al l compounds were dissolved in CDCI3 except HPBT which was dissolved in CD3OD. ! H-NMR spectra for the ligand precursors are shown in Figure 3.16 with diagrams indicating proton assignments The spectra for compounds containing the thiazolidinedione moiety are all similar due to only slight differences in their structures (oxidized HPBT, HPBT, BPBT, IPBT), thus they are discussed together. The two doublets at low field are due to the para substituted aromatic ring protons and the asymmetric nature of the substituents. The protons adjacent to the carbon attached to the oxygen (H<0 are more deshielded than the protons adjacent to the carbon attached to the carbon (Hc) and are thus further downfield. The doublet of doublets observed in the spectra for HPBT, BPBT and IPBT at -4.5 ppm are from the proton on the thiazolidinedione ring (Ha) that is split by Hb and Hb' ( J H H = 2.1 Hz), which are diastereotopic by virtue of being adjacent to a chiral centre. The multiplets observed at -3.2 ppm correspond to the diastereotopic protons which are coupled to H a . Oxidized HPBT has a different spectrum in this region because the olefin eliminates the chiral proton and reduces Hb to one proton which is highly deshielded, shifted to very low fields (7.73 ppm), and is not split. Hf is coupled to H e and H g , resulting in a triplet of triplets at -2 ppm (JHH = 6.1 Hz). Both H e and H g can be assigned as triplets because they are each coupled to the two 41 CDCU J g e g 3-bromopropanol impurity c d 7.5 7.0 iii][|f|Wipt 4.5 4.0 3.5 3.02.4 Che mi cal Shi ft (ppm) Figure 3.16 Regions of *H NMR (200 MHz) spectra of various precursor compounds 42 Chemical shift (ppm) Figure 3.17 *H-NMR spectra (200 MHz) of BocHPBT equivalent Hf protons (JHH = 6.1 Hz). The protons of the terminal carbon of the alkane chain (Hg) can be differentiated from H e by comparing the spectra of the various compounds with different substituents. The upfield triplet is due to the protons on the carbon attached to the ether oxygen, as shown by an invariant chemical shift. In addition, the ether oxygen will deshield the protons on the adjacent carbon more than either an alcohol or a halogen. For N'N-sulfuryldiimidazole, only three signals are visible because of the symmetry of the molecule. H c is a singlet because it is remote. The other two can be 43 differentiated by the fact that Hb is adjacent to the nitrogen attached to the sulfur and H c is adjacent to the double-bonded nitrogen. The 'H-NMR spectrum of BocHPBT (Figure 3.17) exhibits many of the characteristics of the HPBT 1 H-NMR spectrum. Although most of the peaks have not shifted significantly from the positions of the HPBT spectrum, the doublet of doublets associated with H a is not visible leading to the conclusion that the product has been deprotonated in that spot. This is a possibility because the resonance of the thiazolidinedione ring could stabilize a positive charge. The diagnostic peak at ~1.5 ppm arises from the tert-butyl group on the BOC group. It should have an integration of 9 protons. Because the sample is not entirely pure, there is more than one peak in that region, complicating the spectrum. The 1 3 C-NMR spectra were assigned by using 'H- I 3 C HMQC and APT spectroscopic techniques. They were only taken for HPBT (Figure 3.18) and IPBT (Figure 3.19). As would be expected, most of the peaks are in similar regions. Carbons 3 , 4 , 6 , 7 , 9 , 1 0 , and 11 were easily assigned by correlating the proton spectrum to the carbon spectrum using the HMQC pulse experiment. The HMQC spectra used to assign the carbons of the C-NMR spectra are shown in Figure 3.20 and Figure 3.21. Carbon 1 was assigned to the lowest field peak (HPBT: 177 ppm, IPBT: 174 ppm) due to the strong deshielding from the oxygen, nitrogen and the sulfur atoms. Carbon 2 had next lowest field (HPBT: 173 ppm, IPBT 170 ppm) as it was only slightly less deshielded. Carbons 5 and 8 , being members of an aromatic ring were also fairly deshielded and consequently were found at 130 ppm and 160 ppm respectively for HPBT and 128 ppm and 158 ppm respectively for IPBT. 44 CD 3OD 11 9 Figure 3.18 1 3 C NMR (125 MHz) spectrum of HPBT with assignments 7 6 i | 11 9 j Figure 3.19 1 3 C NMR (75 MHz) spectrum of IPBT with assignments 45 i i « » r 0 • -i i-IT rniTiTiTnTTT -ppm Figure 3.20 HMQC spectrum of HPBT 46 Figure 3.21 HMQC spectrum of IPBT 3.2.3 Mass Spectrometry and Elemental Analysis Electron-impact ionization (EI) mass spectral studies of the successful reactions showed strong parent peaks (M+) for all compounds that were purified. +LSIMS was used for analysis of the reaction to form VO(APBT)2, which had two diagnostic peaks (m/z = 790, 527) which implied success. +LSIMS characteristically generate [M+l]+ peaks for VOL2 compounds. The first peak (m/z = 790) is equivalent to [M-H]+ which 47 may be explained by the loss of two H and is probably due to the labile thiazolidinedione imine protons. Each ligand would lose one hydrogen giving the expected mass of the deprotonated complex (VOL2-2H). The second peak (m/z = 527) could be explained by the partially reacted species (VOL(acac)-H). The +LSIMS is shown in Figure 3.22. The elemental analyses were generally within 0.2% except for oxidized HPBT which was within 0.3%. VO(APBT)2 was clearly impure. VOL2-H 790 732 I '/'- 1053 I i l l , 838 888 M 6 I 1105 1192 800 900 . 1000 1100 1200 m/z Figure 3.22 VO(APBT)2 mass spectrum (LSIMS) 48 Chapter 4 Conclusions and Future Directions 4.1 Conclusions This research project was initially proposed to be a routine synthesis followed by complexation, physical studies and finally biological studies. This goal was not fully realized, however, due to unforeseen difficulties encountered during the synthesis. Although the synthesis was taken up to the last step, that of a halogenated ligand (BPBT, IPBT), in a relatively short period of time, the binding of the acetylacetone to the thiazolidinedione moiety did not proceed as smoothly. Part of the difficulty arose from the complexity of the molecule; despite being a relatively small molecule, it has a variety of different functional groups. Another difficulty is the tautomerization of acetylacetone. Initially it was thought that the reaction did not proceed because the deprotonation of the acetylacetone was difficult. Thus several unsuccessful reactions were attempted using sodium acetylacetonate. The anion may be more stable than the neutral species and thus the reaction may only work through acetylacetone. The pK a of 2,4-thiazolidinedione is 6.7 and the pKa of acetylacetone was ~9. The thiazolidinedione thus deprotonated before the acetylacetone, leading to competition and side products. Attempts made to protect the amine nitrogen through the use of BOC were met with difficulty due to the unusual stability of the thiazolidinedione N-H. The nitrogen is straddled by two ketones which deactivate the nitrogen, attenuating its reactivity. After a number of false starts, the reaction was believed to have succeeded, as indicated by the ! H NMR spectrum (Figure 3.17); however complete purification and characterization were not completed due to time constraints. 49 4.2 Future Directions 4.2.1 Completion of Synthesis of APBT The BOC-protected IPBT would be a more useful starting material to carry out a substitution reaction on the alkyl iodide. More BocIPBT should be synthesized and reacted with acetylacetone. It is hoped that the protection of the thiazolidinedione will be sufficient for eliminating competing side reactions centered on the thiazolidinedione nitrogen. Figure 4.1 Synthesis of APBT using BOC protected IPBT Another route that may work and merits exploration is a follow-up on the imidazolate ligand (ImPBT, Figure 4.2). This reaction is another variation of a nucleophilic attack using a different leaving group. Although the purification of the compound was not accomplished due to time constraints, it should be possible in the future. If this reaction does not work, it should also be attempted on the BOC protected version. 50 Figure 4.2 Synthesis of APBT using ImPBT 4.2.2 Synthesis of VO(APBT) 2 Most vanadium complexes can be formed by merely combining the ligand and metal together under basic conditions. The source of metal ion should be vanadyl sulphate because it is readily available and is used in the synthesis of B M O V . 2 2 Figure 4.3 Synthesis of the desired complex (VO(APBT) 2) from A P B T and V O S 0 4 Another direction that has merit is the reaction that was attempted near the end of the experimental project, the reaction between IPBT and vanadyl acetylacetonate (see Figure 3.14). 51 4.2.3 Determination of Stability Constants and Other Physical Constants Once the complex has been formed, its physical properties should be studied. Of most importance is the stability of the complex under physiological conditions. Potentiometry would be used to determine the stability constants of the bis complex and describe the solution chemistry over a broad pH range. The electrochemical properties of the complex are also valuable because oxidation and reduction reactions are important in biological systems. Complete characterization of these properties could be examined formally through cyclic voltammetry and through the complex's reaction chemistry. 4.2.4 Testing of Biological Efficacy The final step would be to test the effects of the complex on biological models of diabetes. This is important to determine the viability of the complex for clinical use. Collaborators in the Faculty of Pharmaceutical Science would perform these tests. They would test the toxicity, effectiveness and dose levels in a variety of animal (rodent) models. Biological models of diabetes mellitus are either chemically induced (STZ treated rats) or through breeding (BioBreeding Wistar rat, db/db and ob/ob mice and fa/fa Zucker rats). Knowledge of possible side effects would arise from these studies. 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