Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The design and synthesis of vanadyl-biguanide complexes as potential synergistic insulin mimics Woo, Lenny Chick Ying 1999

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata


831-ubc_1999-0109.pdf [ 2.66MB ]
JSON: 831-1.0059537.json
JSON-LD: 831-1.0059537-ld.json
RDF/XML (Pretty): 831-1.0059537-rdf.xml
RDF/JSON: 831-1.0059537-rdf.json
Turtle: 831-1.0059537-turtle.txt
N-Triples: 831-1.0059537-rdf-ntriples.txt
Original Record: 831-1.0059537-source.json
Full Text

Full Text

THE DESIGN AND SYNTHESIS OF VANADYL-BIGUANIDE COMPLEXES AS POTENTIAL SYNERGISTIC INSULIN MIMICS by Lenny Chick Ying Woo B.Sc. (Hons.), McGill University, Canada, 1996 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 December 1998 © Lenny Chick Ying Woo, 1998 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 written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT The coordination of biguanides, specifically biguanide, metformin, and phenformin, to oxovanadium(IV) was designed to function as a potentially synergistic approach towards insulin-mimetic compounds. Biguanides, most importantly metformin, are oral hypoglycemic agents used today to treat type II diabetes mellitus, and vanadium has well documented blood glucose-lowering properties in vivo. Highly coloured, air stable bis(biguanidato)oxovanadium(IV), [VO(big)2], bis(N', N'-dimethylbiguanidato)oxovanadium(IV), [VO(met)2], and bis(p-phenethylbiguan-idato)oxovanadium(IV), [VO(phenf)2], were prepared. Solvation with dimethylsulfoxide occurred with VO(big)2 and VO(met)2 to form new six-coordinate complexes. Tris(p-phenethylbiguanidato)chromium(III) was also synthesized as a possible bifunctional complex because chromium is thought to be responsible for maintaining glucose tolerance. The prepared ligand precursors and complexes were characterized by infrared spectroscopy, mass spectrometry, elemental analyses, magnetic susceptibility, and, where appropriate, 'H NMR spectroscopy. Due to the limited solubilities of these oxovanadium complexes, crystals suitable for X-ray diffraction studies were not obtained. Square pyramidal and octahedral geometries are predicted for the vanadyl-biguanide complexes and the chromium-phenformin complex, respectively. STZ-diabetic rat studies with VO(met)2, administered by intraperitoneal injection or oral gavage, resulted in ambiguous findings as summarized in Appendix A. The complex VO(met)2 did not demonstrate a synergistic mechanism of action in lowering ii blood plasma glucose at doses of 0.12 mmol kg"1 and 0.6 mmol kg"1; however, the complex was effective most likely from the vanadium contribution alone. i i i T A B L E O F C O N T E N T S page Abstract ii Table of Contents iv List of Figures vi List of Tables viii List of Abbreviations ix Acknowledgments xii Chapter 1. Introduction 1 1.1. Vanadium Background 1 1.2. Vanadium as an Insulin Mimic 3 1.3. Biguanides and Biguanide Complexes 6 1.4. Chromium in Nutrition 8 1.5. Research Focus - Syntheses of Bifunctional Complexes 9 Chapter 2. Experimental 12 2.1. Materials, Methods, and Syntheses 12 2.1.1. Materials 12 2.1.2. Syntheses ofLigand Precursors 12 2.1.3. Syntheses of Metal Complexes. 13 2.2. Characterization 16 2.2.1. Infrared Spectroscopy 17 2.2.2. Elemental Analysis 21 2.2.3. Mass Spectrometry 22 2.2.4. NMR Spectroscopy 23 2.2.5. Magnetic Susceptibility 24 Chapter 3. Results and Discussion : 25 iv 3.1. Introduction 25 3.2. Proligands 25 3.2.1. Synthesis 25 3.2.2. Characterization 26 3.2.3. Properties 27 3.3. Vanadium(IV) Complexes 30 3.3.1. Synthesis 30 3.3.2. Characterization 30 3.3.3. Properties 32 3.4. Chromium(III) Complexes 36 3.4.1. Synthesis 36 3.4.2. Characterization 37 3.4.3. Properties 37 3.5. Alternate Analogues 38 3.6. STZ-Diabetic Rat Studies 42 Chapter 4. Conclusions and Future Work 43 References 44 Appendix A. STZ-Diabetic Rat Studies with VO(met)2 50 V LIST OF FIGURES page Figure 1.2. Some designed vanadium coordination complexes that show insulin-mimetic properties in vivo 4 Figure 1.3. Structure of guanidine, biguanide, some substituted biguanides, and sulfonylurea 7 Figure 1.4. Structure of chromium(III) picolinate, [Cr(pic)3] 9 Figure 1.5.1. VO(bg)2 complexes prepared (bg = big, met, phenf) 10 Figure 1.5.2. Tris(P-phenethylbiguanidato)chromium(III), [Cr(phenf)3] ... 11 Figure 3.2.1. Preparation of biguanide following Rathke 26 Figure Resonance structures of biguanide 28 Figure Requisites for effective hypoglycemic activity in biguanides 28 Figure Predicted square pyramidal VO(big)2 with C 2 v symmetry .... 34 Figure Possible structures and symmetries of VO(met)2 and VO(phenf)2, point group assignments assume free rotation about C - N R ^ bond 34 Figure Possible structures of [VO(bg)2S] present in solution (S = dimethylsulfoxide) 35 Figure X-ray crystal structure of tris(biguanidato)chromium(III), [Cr(big)3] 38 Scheme 3.5.1. Attempted condensation reaction of maltol and biguanide to form a pyridinone 38 Scheme 3.5.2. Attempted condensation reaction of salicylaldehyde and biguanide to form a Schiff base, HsalHmet 40 Figure 3.5.1. Potential structure of bis(ligand)dioxovanadate(V) monoanion, [V02(bg)2]" 40 Figure 3.5.2. Potential structure of tris(ligand)vanadium(III) complexes, vi [V(bg)3] 41 Figure 3.5.3. Potential structure of protonated bis(biguanide)-oxovanadium(IV) cationic complexes 42 Figure A l Plasma glucose versus time - intraperitoneal injection study .. 54 Figure A2 Plasma glucose versus time - oral gavage study 55 Figure A3 Body weight versus time for (a) i.p. injection and (b) oral gavage 56 vii LIST OF TABLES page Table Characteristic IR absorptions for the biguanide ligand precursors 17 Table Characteristic IR absorptions for the vanadyl complexes 19 Table Characteristic IR absorptions for Cr(phenf)3 20 Table 2.2.2. Summary of elemental analyses of ligands, and vanadium and chromium complexes 21 Table EIMS data (m/z) for the ligand precursors and VO(met)2 22 Table +LSIMS data (m/z) for the vanadium complexes 22 Table +LSIMS data (m/z) for Cr(phenf)3 22 Table 2.2.4. 'H NMR chemical shifts (ppm) for the biguanide proligands 23 Table Diamagnetic susceptibility measurements of ligand precursors 24 Table Magnetic susceptibilty measurements (x g ) and calculated (spin only) magnetic moments (|aB) of the complexes 24 Table Acid dissociation constants for selected biguanides 29 Table A l Blood glucose concentrations over 72 hours for each treatment group - i.p. injection study 57 Table A2 Blood glucose concentrations over 72 hours for each treatment group - oral gavage study 59 Table A3 Animal weights over 72 hours for each treatment group -i.p. study 61 Table A4 Animal weights over 72 hours for each treatment group -gavage study 62 viii LIST OF ABBREVIATIONS Abbreviation Meaning A angstrom ATPase adenosine triphosphatase big" biguanidate anion BEOV bis(ethylmaltolato)oxovanadium(IV) BKOV bis(kojato)oxovanadium(IV) B M Bohr magneton BMOV bis(maltolato)oxovanadium(IV) BM02V bis(maltolato)dioxovanadate(V) anion °C degrees celsius cm"1 wave number Cr(phenf)3 tris(P-phenethylbiguanidato)chromium(III) 8 chemical shift (ppm, NMR); vibrational in-plane bending mode or deformation (IR) DMSO dimethylsulfoxide ED 5 0 effective dose to 50% of animals tested EI electron-impact ionization EIMS electron-impact ionization mass spectrometry EPR electron paramagnetic resonance FDA Food and Drug Administration (U.S.) g gram ix GI gastrointestinal GTF glucose tolerance factor 'H hydrogen Hbg generic biguanide FIbig biguanide H2bg+ generic protonated biguanide Hma maltol (3-hydroxy-2-methyl-4-pyrone) Hmet metformin (N1, N'-dimethylbiguanide) Hphenf phenformin (P-phenethylbiguanide) i.p. intraperitoneal IDDM insulin-dependent diabetes mellitus IR infrared K Kelvin kg kilogram L liter; ligand +LSIMS positive liquid secondary ion mass spectrometry m multiplet ma" maltolate anion met" N'.N'-dimethylbiguanidate anion mL millilitre mmol millimole mol mole x m/z mass-to-charge ratio v stretching vibration (IR) v a asymmetric stretching vibration (IR) v s symmetric stretching vibration (IR) NIDDM non-insulin-dependent diabetes mellitus NMR nuclear magnetic resonance pH negative log of the concentration of H 3 0 + phenf P-phenethylbiguanidate anion R alkyl or aryl substituent pr rocking s singlet STZ streptozotocin t time; triplet (NMR) T temperature UV-Vis ultraviolet-visible VO(big)2 bis(biguanidato)oxovanadium(IV) VO(met)2 bisfN1, N'-dimethylbiguanidato)oxovanadium(IV) VO(phenf)2 bis(phenethylbiguanidato)oxovanadium(IV) xi ACKNOWLEDGMENTS Firstly, I would like to thank Dr. Chris Orvig for being a patient, supportive, wise, encouraging, and constantly optimistic research supervisor. Next, I must thank the group, present and past members - Paul, Ashley, Nico, Grandpa Buglys, Leon, Marco, Ika, Kathie, Mike, Peter, Barry, Devin, Dave, and Jason. I still think our group is weird, but who am I to judge? Additional non-Orvig-group colleagues include Cerrie, Dan, Heather, Rob, and everyone else who made my experience at UBC very memorable. A special thank you goes to Violet Yuen and the McNeill group in the Faculty of Pharmaceutical Sciences at UBC for performing the animal experiments. This definitely completed the story of my thesis. I cannot forget to thank Mr. Peter Borda for performing the elemental analysis and the UBC Chemistry support staff for their skill. Finally, thanks to UBC and various agencies and companies for funding. Let's go Hoopsters! xii To my parents, sisters, brother, and niece for their enduring love and support. xi i i CHAPTER 1 INTRODUCTION 1.1. Vanadium Background Vanadium, discovered by Sefstrom (1831) and named for its multicoloured solutions, is widely distributed in the world, where it is essential to various organisms and present in trace quantities in mammals. It exists in oxidation states of -3 to +5; however, only V(III), V(IV), and V(V) are involved in physiological systems.1 Vanadium's biological importance was recognized in ascidians in the beginning of the 1900's. Insight came from Cantley and coworkers2 (1977) with their discovery that vanadium (as vanadate) inhibited Na+,K+-ATPase in vitro. Also, this element is present in the active site of haloperoxidases in sea algae and lichens3, and of nitrogenases in Azotobacter bacteria.4 The specific biological role of vanadium is indistinct, leading to the interest in its bioinorganic chemistry. Predominantly V(V) and V(IV) exist, as orthovanadate (HV0 4 2 7H 2 V0 4 ) and vanadyl (V0 2 +), respectively, in aerobic water at ambient pH and temperature. Hence, both the anionic and cationic forms can participate in biological processes. Vanadyl readily undergoes autoxidation to vanadate in the presence of oxygen. Vanadate (V043~) remarkably resembles phosphate (P043~) in size and geometry, and most importantly in function as they both inhibit, stimulate, and regulate phosphohydrolases and phosphotransferases.2'5'6 This is the likely explanation for vanadium's biological relevance, yet it is uncertain since the cyclic vanadate tetramer (V 4 0 ! 2 4 ) is also a potent inhibitor.7 1 Chapter 1 Simultaneously, vanadium can act as a competitor for phosphate, and as a transition metal ion which competes with other metal ions in coordination to biogenic molecules. In humans, vanadium is present in almost all cells at 0.1-1.0 uM 8 for an approximate body burden of 100 jug, and close to 90% of circulating vanadium is bound to proteins.910 Both oxidation states of vanadium exist in plasma. By anion transport, free vanadate passes into the cell, where it is then reduced to vanadyl by glutathione.1112 Less than 1% of intracellular vanadium is left unbound.13 Vanadate is also reduced to vanadyl in the blood plasma by ascorbate, catecholamines, and cysteine, yet due to oxygen tension, vanadate still exists in blood. Vanadyl is bound and transported by transferrin and albumin, while vanadate is transported by the former alone.13 Vanadium is targeted to iron-rich cells where it is mostly bound to ferritin. Accumulation occurs substantially in the liver and spleen, and moderately in the kidney, bone, and testis. The poorly understood entity - peroxovanadate(V) - is thought to be responsible for many biological actions of vanadium such as its insulin-mimetic action and haloperoxidase activity. The drawback of vanadium in therapeutic use is its toxicity, both chronic and acute, in humans. Taken in quantities of 10-50 |ig/day, vanadium is safe and adequate; however, there is a need to distinguish a fundamental biochemical function for vanadium in higher animals. The oxo cations V 0 2 + and V 0 2 + have an extensive chemistry and form numerous complex compounds. V 0 2 + can bind effectively to electronegative donor atoms such as fluorine, oxygen, chlorine, and nitrogen; complexes with the former two elements are particularly stable. The aqueous chemistry of V(V) and V(IV) species is characterized by complex equilibria and hydrolyses. For example, the speciation of vanadate is complicated 2 Chapter 1 by pH-dependent oligomer formation.14 Oligovanadates in biosystems, however, are important at toxic levels of vanadate only or within special cell compartments where vanadate accumulates. On the other hand, the vanadyl ion is the most stable oxocation of the first row transition metals and reacts to form cationic, anionic, and neutral square pyramidal complexes, with the oxo-0 in the axial position with approximately 1.6 A as the V=0 bond length. Vanadium(IV) is paramagnetic (3d1) which makes it a suitable EPR spin probe. At extremely low pH, VO(H 20) 5 2 + exists but increasing the pH above 2 results in vanadate formation from air oxidation. This can be slowed down with anionic chelating ligands incorporating hard donor atoms like oxygen and nitrogen. 1.2. Vanadium as an Insulin Mimic Diabetes mellitus is a syndrome resulting from a relative lack of metabolic action of insulin (or resistance to these actions from elevated concentrations of counter-insulin hormones, such as glucagon). People develop diabetes when the pancreas no longer makes enough insulin, or when the body is unable to utilize properly the insulin it makes; these are of two general types: type I (IDDM) and type II (NIDDM), respectively. Insulin, secreted by the pancreas, is a signalling hormone which regulates amino acid, fatty acid, and glucose uptake from the circulatory system for storage as proteins in muscle, as triglycerides in adipose tissue, and as glycogen in muscle and liver. Insulin is administered by subcutaneous injection to diabetic patients. Interest is high in developing oral drugs to replace insulin to provide increased ease and convenience of use, and because insulin is not orally active - it is a protein. 3 Chapter 1 CH 3 B K O V Figure 1.2. Some designed vanadium coordination complexes that show insulin-mimetic properties in vivo', bis(N-octylcysteineamide)oxovanadium(IV) [Naglivan], bis(maltolato)oxovanadium(rVO [BMOV], bis(ethylmaltolato)oxovanadium(IV) [BEOV], bis(maltolato)dioxovanadate(V) anion [BM02V], and bis(kojato)oxovanadium(IV) [BKOV] Since vanadium has been found to have insulin-like properties in biological systems, there is significant activity in studying the properties and potential applications of new synthetic vanadium compounds which could replace insulin in diabetes treatment.15'24 4 Chapter I Inorganic sodium metavanadate was first reported in 1985 by Heylinger et al. 1 6 to be an effective oral insulin mimic in vivo in STZ-diabetic rats. This led to studies with vanadyl sulfate, which was shown to be less toxic and longer lasting. Inorganic vanadate1718 and vanadyl1921 have been studied extensively; however they are poorly absorbed from the gastrointestinal tract22 and Gl difficulties have been reported with vanadyl and vanadate.23 Vanadium coordination complexes have shown increased potency over inorganic vanadium. Vanadium complexation with organic ligands may decrease toxicity by lowering the vanadium dose required for effectiveness and improved Gl absorption. Oxovanadium(IV) complexes have been found to be less toxic than vanadate(V) analogues. Vanadium compounds mimic insulin by lowering the average blood glucose levels of diabetic animals and also by reversing some or all of the symptoms in treated diabetic animals.24 Various vanadyl complexes have been proposed25'26 but lack stability in air and in water, which inhibits their use as oral drugs. In vivo testing with vanadium coordination complexes (Figure 1.2, p. 4), such as Naglivan25, BMOV 2 7 , BM02V 2 8 , BKOV 2 9 , BEOV, as well as some pervanadate17'30'31 complexes, has shown lower blood glucose values in STZ-diabetic rats. These vanadium compounds mimic insulin via oral administration. Naglivan was first studied in vivo by McNeill and co-workers25, but it lacked water solubility and adequate effectiveness. Next, the potent VO(ma)2 complex was reported by Orvig, McNeill, and co-workers in 199227 to have the favourable properties of water solubility, electrical neutrality, and low molecular weight, which led to high potency. Also known by its acronym as BMOV, this compound is effective, non-toxic, and two to three times more potent than vanadyl sulfate.32 Analogous to BMOV but more water soluble, 5 Chapter 1 BKOV was less effective than BMOV in lowering plasma glucose levels both chronically and acutely.28 Likewise, BM02V showed little effectiveness and decreased tolerability.28 In summary, both inorganic and organic vanadium compounds are effective in the treatment of hyperglycemia in diabetes mellitus and the prevention of secondary complications associated with this disease. Organically chelated vanadium complexes are effective insulin-mimetic agents at significantly lower doses, with reduced Gl side effects. 1.3. Biguanides and Biguanide Complexes Treatment of adult-onset diabetes is generally carried out by prescription of a diet followed by an antidiabetic medication. At present, pharmacological treatment of patients is mainly carried out with the use of sulphonylureas, which stimulate the release of insulin, helping to regulate fasting and postprandial glycemia. Because resistance to these drugs may increase and because their activity is limited to the pancreatic level, alternatives such as the biguanide class of oral drugs (Figure L3, p. 7) are also employed in the pharmacological treatment of diabetes. Since the early 1900's, biguanide and substituted biguanides containing the active guanidine moiety, have proven useful for the treatment of hyperglycemia33, malaria34, fileria35, and influenza36. Biguanides, as metformin, phenformin, and buformin, introduced in the 1950's37, are widely used for the treatment of non-insulin dependent diabetes as antihyperglycemic drugs. They have no activity on the pancreas but can significantly improve peripheral glucose management by impairing oxidative phosphorylation and gluconeogenesis, in addition to enhancing glycolysis. The suggested mechanism of hypoglycemic action of biguanides is increased glucose uptake into muscle, adipocytes, 6 Chapter 1 and lymphocytes, decreased hepatic gluconeogenesis, and decreased intestinal glucose absorption.38'39 Metformin's mechanism is linked to an improved peripheral sensitivity to insulin, through a stimulated tissue glucose uptake by a transporter linked system.39 Favourably, the biguanides do not cause hypoglycemia or hyperinsulinemia, and do not lead to weight gain - potential problems with sulfonylureas. N H H 2 N C N H 2 guanidine N H N H R i II II \ II H II N C N C N H 2 generic biguanide (Hbg) / (R, = R 2 = H;Hbig) K 2 N H N H H 3 C \ II II N C N C N H 2 metformin (Hmet) H 3 C < ^ - < C H 2 ) , f f \ 11 H 11 N C N C N H 2 phenformin (Hphenf) H N H N H CH 3(CH 2)3 | | | | N C N C N H 2 buformin / H O / \ II H H . . , . R, P J S N C N R 2 genenc sulfonylurea O O Figure 1.3. Structure of guanidine, biguanide, some substituted biguanides (shown as bases), and generic sulfonylurea Chapter 1 Bidentate biguanide is a strong base that can form brilliantly colourful, stable metal and non-metal chelate compounds with extensive n derealization40 by coordinating through two nitrogens with lone electron pairs. Such chelates undergo electrophilic substitution reactions due to their pseudo-aromatic nature.41 Vanadyl biguanide complexes were first reported by Banerjee and Ray in 195942a, and complex compounds of biguanides with various transition metals such as Cu(II), Ni(II), Co(II), Co(III), Cr(III), Mn(III), Mn(IV), V(IV), Re(V), Os(VI), Ag(III), Pd(II), and Zn were reviewed by Ray4 2 b in 1961, but with no synthetic detail, incomplete characterization, and no reaction chemistry. 1.4. Chromium in Nutrition Chromium was recognized as an essential trace metal in 1955.43 Proposed by Mertz44 (1959), the bioactive "chromium complex" glucose tolerance factor (GTF) was postulated to function as a cofactor for insulin potentiation; hence it is thought to have a role in glucose metabolism. GTF consists of Cr, 2 nicotinic acids, glycine, glutamic acid, and cysteine.45 Chromium supplementation studies in humans46 showed it to be effective in maintaining euglycemia, and it is also needed for normal carbohydrate and lipid metabolism in mammals via its role in potentiation of insulin action.46 Chromium deficiency in man and higher animals results in an impairment of intravenous glucose tolerance and symptoms like those associated with adult-onset diabetes (NIDDM) and cardiovascular disease. Cr(III) is a hard, d3 metal ion which undergoes facile hydrolysis and its complexes are substitutionally inert - factors which make it difficult to study the thermodynamic stability of Cr(III). With this knowledge, controversy arises as to whether chromium has 8 Chapter 1 any biological role at all. Nevertheless, improvement in glucose tolerance after chromium supplementation is well documented. This beneficial effect on glycemic control from chromium replacement is only observed for people who are chromium deficient; most people with diabetes are not chromium deficient and thus, chromium supplementation has no benefit. There is much research on chromium(III) picolinate47, Cr(pic)3, (Figure 1.4), a com-mercially available, non-toxic, readily absorb-able source of chromium. Chromium picolinate presumably enhances insulin action, builds lean muscle tissue, reduces body fat, and lowers cholesterol.48'49 Studies have also shown that Cr(pic)3 damages chromosomes in animals, Figure 1.4. Structure of chromium(III) picolinate, [Cr^ ic^] 5 0 posing a threat as a potential carcinogen. Employing the same idea as with vanadium, organic coordination complexes of chromium may prove to be more effective sources of so-called nutritional chromium. Already synthesized with a crystal structure is the complex Cr(big)35 1. Analogous preparations should readily yield Cr(met)3 and Cr(pheni)3. J.S. Research Focus - Syntheses of Bifunctional Complexes As discussed, new organic vanadium coordination complexes may prove to be useful as oral drugs in diabetes treatment. It has been clearly indicated that vanadyl and biguanides individually lower blood glucose; therefore the chelation of biguanides to a 9 ChapterJ vanadium metal centre (Figure 1.5.1) could potentially result in new synergistic compounds that boost effectiveness while simultaneously decreasing required dosage, and favourably reducing vanadium toxicity. The design of these complexes is novel, not to mention that biguanides are appealing ligands as they are proven antidiabetic agents in addition to being strong chelators. The preparation and characterization of oxovanadium(IV) combined with each of biguanide, metformin, and phenformin (VO(big)2, VO(met)2, and VO(phenf)2, respectively) are discussed here. Emphasis is placed on metformin (Glucophage, U.S. FDA approved) because it is a common drug used today to treat type II diabetes in monotherapy or combination treatment. Although phenformin was withdrawn from the market in 1977 due to serious lactic acidosis side effects, it was used in this investigation for comparison purposes as the three biguanides are structurally and chemically distinct. Furthermore, phenformin coordination to trivalent chromium is presented (Figure 1.5.2). As there is considerable research in chromium and its link to diabetes treatment, R 2 N Figure 1.5.1. VO(bg) 2 complexes prepared (bg = big, met, phenf) 10 Chapter 1 combination of this metal with biguanides is another design of potentially useful hypoglycemic bifunctional complexes. Figure 1.5.2. Tris(P-phenethylbiguanidato)chromium(III), [Cr(phenf)3] All the complexes were characterized by infrared spectroscopy, mass spectrometry, elemental analyses, and magnetic susceptibility. Attempts to synthesize modified derivatives are discussed briefly. Preliminary intraperitoneal and oral gavage STZ-diabetic rat studies with VO(met)2 (Appendix A, p. 50) were performed by the McNeill lab to observe any plasma glucose lowering, and consequently any anticipated synergy. The results from these experiments show that the complex is active in lowering plasma glucose, but synergy was not observed with the doses administered. 11 CHAPTER 2 EXPERIMENTAL 2.1. Materials, Methods, and Syntheses 2.1.1. Materials All solvents (Fisher, Aldrich) and chemicals were reagent grade and used without further purification unless otherwise specified: metformin (N^N'-dimethylbiguanide hydrochloride, Sigma), P-phenethylamine hydrochloride (Aldrich), dicyandiamide (Sigma), maltol (3-hydroxy-2-methyl-4-pyrone, Sigma), ethylmaltol (Pfizer), vanadyl sulfate trihydrate (Aldrich), ammonium metavanadate (Sigma), and sodium metavanadate (Sigma). Biguanide sulfate52 and phenformin hydrochloride373 were prepared following literature procedures. Water was deionized (Barnstead D8902 and D8904 cartridges) and distilled (Corning MP-1 Megapure Still) before use. The yields are for analytically pure compounds and calculations are based on vanadium. Al l complexation reactions were carried out under argon unless noted. Melting points were measured on a Mel-Temp apparatus and are uncorrected. 2.1.2. Syntheses of Ligand Precursors Biguanide sulfate52, C 2 H 7 N 5 H 2 S0 4 , (HbigH2S04). Dicyandiamide (5 g, 0.059 mol) and ammonium chloride (8 g, 0.150 mol) were separately ground to a fine state, mixed, and heated in an oil bath until a liquid melt was obtained. For 15 minutes at 160-165°C and with constant stirring, this melt was maintained. After cooling in air, the solid was broken up and dissolved in 30 mL boiling water. The resulting mixture was filtered and the 12 Chapter 2 precipitate (ammeline) was washed with 2 x 5 mL hot water. Ammoniacal copper(II) sulfate solution was added in excess to the filtrate, precipitating red copper biguanide sulfate. This was filtered, washed with water, and dissolved in 7 mL hot 10% sulfuric acid solution. Upon cooling in an ice bath, crude biguanide sulfate precipitated, was filtered out and dissolved in boiling water, and this solution was cooled in ice. The white product of biguanide sulfate dihydrate was filtered, washed with water, then ethanol, and dried at 110°C overnight. The anhydrous yield obtained was 1.12 g (9.5%); m.p. 231-232°C. Phenformin373, C 1 0 H 1 5 N 5 H C 1 , (HphenfHCl). p-phenethylamine hydrochloride (5 g, 0.0316 mol) and dicyandiamide (2.65 g, 0.0315 mol) were heated gradually with stirring in an oil bath up to 130°C. The mixture was further heated for an hour at 145-150°, initiating an exothermic reaction as the mixture fused. When cool, the product was recrystallized from isopropanol, filtered, and dried in vacuo overnight to obtain a yield of 4.21 g (55%); m.p. 170-172°C. 2.1.3. Syntheses of Complexes Bis(biguanidato)oxovanadium(rV), VO(big) 21.5H 2 0. Biguanide sulfate (0.46 g, 2.31 mmol) was dissolved in 5 mL hot water. VOS0 4 3H 2 0 (0.25 g, 1.15 mmol) in 3 mL H 2 0 was added to the biguanide solution to yield a clear blue solution. Dilute 2M NaOH (0.28g, 7 mmol) was added dropwise slowly, turning the solution gray, then light brown, and finally green (pH ~ 11). The mixture was stirred for 1 hour and the green precipitate was filtered out under vacuum, washed with water followed by ethyl ether, and dried overnight in vacuo. The yield was 0.25 g (81%). 13 Chapter 2 Bis^ N^dimethylbiguanidato^xovanadiumflV), VO(met)2 H 20. VOS0 4 3H 2 0 (1 g, 4.6 mmol) was dissolved in 5 mL water and added slowly to a solution of 2 equivalents metformin (1.52 g, 9.2 mmol) in 5 mL H 2 0. 2M NaOH (0.9 g, 23 mmol) was added dropwise to bring the pH to ~12. Initial addition of base resulted in brown hydroxide complex formation, however, upon complete addition, a light green solid eventually precipitated. The solution was stirred for 2-3 hours and the solid was collected by vacuum filtration, washed with cold water followed by ether, and dried overnight in vacuo. The yield was 0.9 g (56%). Bis(p-phenethylbiguanidato)oxovanadium(IV), VO(phenf)2 HzO. VOS0 4 .3H 20 (0.45 g, 2.07 mmol) was dissolved in ~3 mL water and added slowly to a solution of 2 equivalents phenformin (1 g, 4.14 mmol) in 5 mL H 2 0. Dilute NaOH (0.166 g, 4.14 mmol) in 3 mL H 2 0 was added dropwise to bring the pH to ~11. Initial addition of base resulted in light brown hydroxide formation; however, upon complete addition and after stirring for 1 hour, a light blue solid eventually precipitated. The solution was stirred for an additional hour and the solid was collected by vacuum filtration, washed with cold water followed by ether, and dried overnight in vacuo. The yield was 0.72 g (37%). Bis(biguanidato)oxovanadium(IV) dimethylsulfoxide, VO(big)2(DMSO). 1 mL of dimethylsulfoxide was added to green VO(big)2 (0.02 g, 0.075 mmol) with stirring. The solid turned dark green-purple and dissolved over a few days, leaving a purple solution. Before dissolution, however, the bluish-green solid was collected by vacuum filtration, washed with ethyl ether, and dried in vacuo overnight. The yield was 0.0134 g (52%). 14 Chapter 2 Bis(N1,N1-dimethylbiguanidato)oxovanadium(IV) dimethylsulfoxide, VO(met)2 (DMSO). 1 mL of dimethylsulfoxide was added to green VO(met)2 (0.02 g, 0.062 mmol) with stirring. The solid turned light purple and dissolved over a period of 2-3 days, resulting in a purple solution. Before dissolution, the purple solid was collected by vacuum filtration, washed with ethyl ether, and dried in vacuo overnight. The yield was 0.007 g (28%). Tris(phenethylbiguanidato)chromium(III), Cr(phenf)3. Phenformin (0.3 g, 1.24 mmol) was dissolved in 5 mL water. NaOH (0.1 g, 2.5 mmol) in 1 mL H 2 0 was added, raising the pH to 12. An aqueous blue solution of KCr(S0 4) 2 6H 20 (0.2 g, 0.401 mmol) in 2 mL H 2 0 was then added dropwise, immediately forming a sticky red precipitate. After stirring the mixture for 1 hour, this precipitate was filtered out and recrystallized from ethanol. The yield was 0.1 g(38%). SaUcylidene-metformin, HsalHmet. Metformin (3 g, 0.018 mol) was partially dissolved in 20 mL ethanol with gentle heating. A solution of salicylaldehyde (2.2 g, 0.018 mol) in 5 mL ethanol was added and the mixture was refluxed overnight. Dilute 2M NaOH was added dropwise to the reaction flask resulting in the formation of a precipitate and a yellow solution. After stirring and refluxing for 30 minutes, a very pale green-yellow solid was filtered out under vacuum, washed with ethanol, and dried in vacuo overnight. The yield was 3.54 g (84 %). 15 Chapter 2 2.2. Characterization All complexes were characterized by infrared (IR) spectroscopy, LSIMS or EIMS, elemental analysis, and magnetic susceptibilty. Where appropriate, ! H NMR was used for further characterization. Infrared spectra were recorded as KBr disks in the range 4000-400 cm"1 on a Galaxy Series 5000 FTIR spectrometer. Mass spectra (+ ion) were obtained with a Kratos MS 50 (electron-impact ionization, EI), or a Kratos Concept II H32Q (Cs+ liquid secondary ion mass spectrometry, LSIMS) instrument. Room temperature (293 K) magnetic susceptibilities were measured on a Johnson Matthey balance, using Hg[Co(NCS)4] as the susceptibility standard. Diamagnetic corrections were estimated by using Pascal's constants.53 Al l C, H, N analyses were performed by Mr. Peter Borda on a Carlo Erba instrument in this department. 'H NMR spectra of samples in c?6-DMSO were recorded on a Bruker AC-200E instrument at 200 MHz. 16 Chapter 2 2.2.1. Infrared Spectroscopy The relevant IR data are summarized in the following tables. A l l the bands reported are sharp and strong except where indicated. Table Characteristic IR absorptions (cm1) for the biguanide ligand precursors Assignment biguanide H 2 S 0 4 Metformin H C 1 phenformin H C 1 v a(NH 2) v s(NH 2) 5(NH 2) v(C=N) 3285 b 3083 b 1727 m 1691 1642 m 1630 m 3371 3320 3300 3169 b 1628 3409 3320 b 3166 1675 1630 1614 v(C=C) v a(NCN) v(CH 2) 1549 m 1530 m 1583 1568 1554 1594 1573 1538 1535 1493 m 1483 m v(CN) v(CH 3) v(CH 2) 1443 w 1478 1450 1418m 1471 m 1452 m 1299 1216 17 Chapter 2 pr(NH2) 1138 1080 m 1151 HOOvs 1064 m 1057 vs(NCN) 974 w 937 m 5(NCN) 673 m 737 m 664 m 656 m 640 m phenyl 762 w 700 w w = weak, m = medium, b = broad, v = very, s = strong 18 Chapter 2 Table Characteristic I R absorptions (cm1) for the vanadyl complexes Assignment VO(big)2 VO(met)2 VO(phenf)2 VO(met)2(DMSO) VO(big)2(DMSO) va(NH2) 3371 3327 3304 3495 m 3355 3325 3305 m 3356 3332 3294 b 3409 3373 3330 3306 v s(NH2) 3214 mb 3142 mb 3208 w 3201 mb 3208 3157 3139 8(NH2) 1658 m v(C=N) 1626 1620 1630 m 1630 v(C=C) 1571 va(NCN) 1564 vs 1513 vs 1502 vs 1548 1527 1516 1503 1520 1546 1516 1502 1564 1501 v(CN) 1462 1449 m 1470 1453 v(CH3) 1428 1402 m 1426 1404 v(CH2) 1327 m 1293 1247 w 1359 w 1296 m 1257 w 1316m 1291 m 1252 m 8(NH) 1303 1254 m 1297 1253 m Pr(NH2) 1119w 1051 m 1111 1055 m 1121 w v(SO) 1024 969 1019w 980 w v(V=0) 942 929 m 953 m 952 m 947 19 Chapter 2 phenyl 746 w 698 m 8(NCN) 712 w 718 w 706 m 713 m Table Characteristic IR absorptions (cm1) for Cr(phenf)3 Assignment Cr(phenf)3 va(NH2) 3379 v(C=C) 1589 va(NCN) 1514 1501 5(NH) 1412 v(CH2) 1356 1233 pr(NH2) 1147 phenyl 751 700 20 Chapter 2 2.2.2. Elemental Analyses Adequate elemental analyses were obtained for all biguanide ligand precursors and complexes (Table 2.2.2.). Vanadyl-biguanide complexes were not purified before analysis; however, all samples were dried in vacuo at 60°C for 24 hours. Table 2.2.2. Summary of elemental analyses of ligands, and vanadium and chromium complexes (Calculated [Found]) Compound Formula %C % H %N MetforminHCl C 4 H n N 5 H C l 29.00[29.23] 7.25[7.19] 42.30[42.32] Biguanidesulfate C 2 H 7 N 5 H 2 S0 4 12.06 [12.09] 4.55 [4.51] 35.16 [35.17] PhenforminHCl C 1 0H 1 5N 5HC1 49.69 [49.80] 6.67 [6.58] 28.97 [28.64] VO(met)2(H20) C 8 H 2 0 N 1 0 OVH 2 O 28.16 [28.44] 6.50 [6.59] 41.04 [41.26] VO(big) 2(H 20) 1 5 C 4H 1 2N 1 0OV1.5H 2O 16.33 [16.52] 5.14 [4.81] 47.61 [47.15] VO(phenf)2(H20) C 2 0 H 2 8 N 1 0 OVH 2 O 48.68 [48.64] 6.13 [5.80] 28.38 [26.59] VO(met)2(DMSO) C 8 H 2 0 N 1 0 OV2DMSO H 2 0 28.97 [29.42] 6.89 [6.70] 28.15 [27.90] VO(big)2(DMSO) C 4 H 1 2 N 1 0 OV2DMSO H 2 0 21.77 [21.27] 5.94 [6.35] 31.73 [32.87] Cr(phenf)3 C 3 0 H 4 2 CrN 1 5 C 2 H 5 OH 49.51 [50.09] 6.79 [6.21] 28.87 [28.06] HsalHmet C n H 1 5 N 5 OC 2 H 5 OH 55.91 [55.95] 7.53 [7.13] 25.09 [24.31] 21 Chapter 2 2.2.3. Mass Spectrometry Table EIMS data (m/z) for the prepared ligands and VO(met)2 H V O L V fy? HmetHCl ~- 130 HbigH 2S0 4 -— 102 HphenfHCl -— 205 VO(met)2 323 VO(met)2(DMSO) 323 VO(big)2(DMSO) 268 HsalHmetEtOH -— 234 Table +LSIMS data (m/z) for the vanadium complexes HVOL 2 + VO(met)2 130 VO(big)2 268 102 VO(phenf)2 476 206 VO(met)2(DMSO) -— 130 Table +LSIMS data (m/z) of Cr(phenf)3 _____ _ _ ___. _ _ Cr(phenf)3 665 460 256 206" 22 Chapter 2 2.2.4. NMR Spectroscopy B C NH NH R i R 2 H Hbig C H 3 Hmet C H 2 C H 2 C 6 H 5 Hphenf Table J H NMR chemical shifts (ppm) for the biguanide proligands Assignment (a) Biguanide (sulfate) (b) Metformin (hydrochloride) (c) Phenformin (hydrochloride) A 8.75 s,br 7.24 s 7.47 br B 6.99 s 6.81 s 6.95 s,br C 6.99 s 6.81 s 6.95 s,br D 6.84 s 6.81 s 6.95 s,br R. 6.84 s 2.94 s 7.47 br R2 6.84 s 2.94 s 7.29 m (5H) 2.78 t (2H) 3.37 m (2H) s = singlet, t = triplet, m = multiplet, br = broad •N N H A NH 2 D (a) H (b) C H 3 (c) H 23 Chapter 2 2.2.5. Magnetic Susceptibilty Table Diamagnetic susceptibility measurements of ligand precursors Ligand X G ( X 1 ° ? ) _ _ _ _ _ _ _ .1.989 HmetHCl -7.279 Hphenf.HCl -5.790 Table Magnetic susceptibilty measurements and calculated (spin only) magnetic moments (pa) of complexes Complex X g (x IO"6) MB (BM) VO(met)2(H20) 4.563 1.99 VO(big)2(1.5H20) 4.586 1.70 VO(phenf)2(H20) 2.856 1.94 VO(met)2(DMSO) 3.304 1.93 Cr(phenf)3 9.181 3.89 24 CHAPTER 3 RESULTS AND DISCUSSION 3.1. Introduction The design and synthesis of bifunctional complexes is interesting for biological applications as well as for exploring the coordination and reaction chemistry of transition metals such as vanadium(IV). The metals and ligands chosen in these experiments are particularly appealing as they individually play a role in glucose metabolism in vivo. Vanadyl sulfate and other vanadium(IV) compounds, biguanides, and various chromium(III) compounds may be effective agents for treating hyperglycemia in humans. The preparation and analysis of the ligand precursors will first be discussed, followed by complex synthesis and characterization, ligand variation attempts, and diabetic animal experiments. 3.2. Proligands 3.2.1. Synthesis In 1879 Rathke mixed thiourea and guanidine thiocyanate with phosphorous trichloride, forming cyanamide from thiourea; cyanamide then reacts with guanidine to give biguanide in low yield (Scheme 3.2.1).54 A more effective route is heating a mixture of dicyandiamide and ammonium chloride.52 Biguanide sulfate can be isolated in moderate yield and is used immediately in further reactions as the anhydrobase turns yellow on exposure to the atmosphere and slowly decomposes on heating or long standing in aqueous solution. Similarly, substituted biguanides can be prepared from dicyandiamide 25 Chapter 3 and the appropriate aryl- or alkylamine either by heating the reactants in aqueous solution in the presence of copper(II) sulfate or by fusing the amine hydrochloride with dicyandiamide. The latter method was employed to prepare phenformin in reasonable quantities from P-phenethylamine hydrochloride and dicyandiamide. SC(NH2)2 • NCNH2 + H2S NH NH NH H 2 N — c - - N + / A X H2N NH2 H 2N N NH 2 H Scheme 3.2.1. Preparation of biguanide following Rathke 5 4 3.2.2. Characterization Because biguanide sulfate and phenformin hydrochloride were prepared from literature procedures, and metformin was purchased, each of the compounds was easily confirmed by various characterization methods. Each of the three ligand precursors was given IR spectral frequency, mass spectrometric, and NMR spectroscopic assignments. These are found in Tables,, and 2.2.4 (pp. 17, 22, 23), respectively. Distinctive vibrations in the 3300-3400 cm"1 region are indicative of the N-H stretching mode (vN.H). The infrared spectrum of guanylurea hydrochloride56 was referenced to assign the vibrations of the biguanides. The electron impact ionization mass spectra show the protonated base as the parent mass (base peak). The broad peaks in the ! H NMR are characteristic of the fast exchanging hydrogens due to the numerous biguanide 26 Chapter 3 tautomerizations. This explains the choice of tV6-dimethylsulfoxide as the solvent rather than an alcohol. Table 2.2.2 (p. 21) also gives the experimental elemental analyses, which correlate well with the calculated values for each organic moiety. Diamagnetic susceptibilities were obtained (Table, p. 24) for later magnetic moment calculations of V(IV) in the prepared complexes. 3.2.3. Properties Biguanides are already identified to be strong chelating ligands that bind readily to a variety of metals and so coordination to vanadium was facile. The biguanides are very strong diacid bases characterized by strongly basic primary dissociation constants and considerably weaker secondary ones (See Table, p.29). Biguanide can be drawn in many resonance forms as illustrated in Figure There is a conjugated double bond system stabilized as an intramolecularly hydrogen-bonded six-membered ring.42b Hydrogen bonding giving the cyclic structures is supposed to influence the physiological properties of biguanides.37 Biguanides have a conjugated -C=N-C=N- chromophore and additional N atoms with lone pairs capable of forming a delocalized n electron system. Both of the structures of the basic (Hbg) and normal salt (H2bg+) forms possess a conjugated single and double bond system whereby biguanide is capable of complex formation with transition elements. The relationship of structure to hypoglycemic activity was observed from studying a series of N'-alkyl- and arylalkylbiguanides and pharamcologically testing them in the 27 Chapter 3 guinea pig.37 The data suggest that hypoglycemic activity is associated with selected biguanides in the form of an intramolecularly hydrogen-bonded cation (Figure NH NH H 2 N N N H 2 "NH NH HjN* N N H 2 H NH "NH A A, H,N N +NH2 H A "NH "NH H 2 N N T f f l 2 H + "NH NH A A H 2 N N N H 2 H + H,N NH "NH A A N+ H + "NH "NH H , N + A A N 4 H N H , Figure Resonance structures of biguanide H N NH H 2 N N H+ N R[ R? R! = C 4 ) C 5 alkyl, Ar(CH 2 ) n (n=l,2) Ar = phenyl, furyl, thienyl, pyridyl, chlorophenyl, methoxyphenyl R 2 = H , C H 3 Figure Requisites for effective hypoglycemic activity in biguanides37 28 Chapter 3 Biguanides generally behave as mono-acidic bases whose strengths may be directly compared by examination of their first dissociation constants. Experimental pK^ and pK 2 values for some selected biguanides55 are listed in Table There are no literature protonation constants for phenformin; however, the pKa's would be expected to be in the same range as, but slightly lower than, those given. The electron-withdrawing phenyl substituent would make phenformin a weaker base and the protonated base would be a stronger acid. The end amine group is stabilized by derealization of a lone pair of electrons into the 7 1 system of the biguanide and subsequently the ring, decreasing the electron density at the nitrogen. Biguanides are stronger bases than ammonia (pK^ = 9.2) and approach similar basicity values to alkylamines. Their ampholytic character allows them to form coordination complexes with an assortment of metal ions. Table Acid dissociation constants of selected biguanides1 (Ionic strength = 0.05 M , Temperature = 32°C) Compound pK, pK 2 Biguanide 11.50 2.93 N 1 ,N' -Dimethylbiguanide 11.53 2.73 Ethylbiguanide 11.47 3.08 Phenylbiguanide 10.76 2.13 K, = (BgH)(H+)/(BgH2+); K 2 = (BgH2+)(H+)/(BgH32+) Some substituted biguanides undergo rapid hydrolysis in acid solution. In aqueous solution of a biguanide, there is an equilibrium between biguanide and the hydrolysis products - guanylurea and ammonia.41 29 Chapter 3 3.3. Vanadium(IV) Complexes 3.3.1. Synthesis The oxovanadium(IV)-biguanide complexes were prepared by the addition of vanadyl sulfate to alkaline aqueous solutions of each biguanide, in a ratio of 1:2, with moderate yields. A slight excess of ligand precursor was used to drive the reaction to completion. The procedure required the slow addition of dilute NaOH to the reaction mixture, whereupon vanadyl hydroxide initially precipitated. Once the pH was increased to ~ 11, the bis(biguanidato)oxovanadium(IV) complex predominated and precipitated out of solution as light green, green, and light blue solids, characterized as VO(met)2, VO(big)2, and VO(phenf)2, respectively. 3.3.2. Characterization The infrared vibrations are listed in Table (p. 19) for each of the oxovanadium(IV) complexes. Characteristic stretching frequencies of the V=0 bond in oxovanadium(IV) complexes generally occur in the region of 930 to 1030 cm"1.57 The complexes VO(big)2, VO(met)2, and VO(phenf)2 have low v v = 0 , 942, 929, and 953, respectively, due to the presence of strong out-of-plane d_-p_ bonding. This considerable lowering of the V=0 stretch is not unexpected in view of the base strength and strong coordinating ability of the biguanides.58 The vanadyl multiple bond involves one strong a bond between the spa (2s, 2pJ oxygen hybrid orbital and the (4s+3dz2) vanadium hybrid orbital, and two K bonds between the oxygen 2px and 2py, and the vanadium 3d*- and 3dy_ orbitals. Assuming that the 5-coordinated bis(ligand)oxovanadium(IV) complexes have a square pyramidal geometry with V=0 in the axial position, the equatorial biguanide 30 Chapter 3 ligands will form four a bonds with the metal 4s, 4px, 4py, and 3dx2.y2 orbitals. The degree of oxide O p_-d_ donation depends on the preference of vanadium to accept electrons. This will be opposed by the presence of ligands which are primarily strong a donors. Such is the case with biguanides, their lone electron pairs on N considerably increase the metal d-orbital electron density. Therefore, the subsequent repulsive interaction lowers the V=0 bond order and consequently the V=0 stretching frequency.58 A sixth ligand, such as water, is weakly bonded in the axial position and perturbs the complex slightly. Upon the addition of DMSO, v v = 0 increases slightly. This is because of the increased electron density on V from the donating solvent coordinated trans to the axial V=0. This enhances the a and n donations from V to the oxo-O, subsequently raising the V=0 frequency. Typical N-H stretching vibrations were observed in each spectrum.58d The carbon, hydrogen, and nitrogen elemental analyses were experimentally determined for the ligands and complexes and are summarized in Table 2.2.2 (p. 21). The results are generally consistent with the calculated values. The low nitrogen values in some oxovanadium(IV) complexes may be due to the formation of vanadium nitride during incomplete combustion of the sample. This is a known occurrence in complexes with metal-nitrogen coordination; the biguanide ligands, in particular, are highly nitrogen rich, and each coordinate at two N sites. Even after drying in vacuo at 60°C for 24 hours, residual water could not be eliminated. The biguanide proligands and VO(met)2 were examined by electron impact ionization mass spectrometry (EIMS) while the remaining complexes were examined by positive ion detection liquid secondary ion mass spectrometry (+LSIMS). The distinctive 31 Chapter 3 fragment peaks support the suggested complex formulations. Complexes containing water or other solvent also show the HVOL 2 + peaks in the EI and +LSI mass spectra. Al l the oxovanadium(IV) complexes prepared are paramagnetic in the solid state and room temperature paramagnetic susceptibilities were obtained. The diamagnetic susceptibities of the ligand precursors were corrected using Pascal's contants53 to obtain the effective magnetic moments listed in Table (p. 24). With an electronic configuration of [Ar]3d', vanadium(IV) has one unpaired electron for which the spin-only formula predicts a magnetic moment of 1.73 BM. The experimental values are in the range of 1.70 - 2.00 for the vanadyl complexes. Literature values of 1.45-1.94 B M 4 2 a for some vanadyl-biguanide compounds are lower possibly from an exchange interaction leading to the formation of a metal-metal bond.42b The solution equilibria and stability data for the vanadyl-metformin system could not be determined because hydrolysis, forming VO(OH)2 presumably, predominates at approximately pH 4. An acid titration of a basic solution might remedy this problem but this was not attempted. UV-vis spectra were not obtained either as the complexes are not soluble enough in common organic solvents. 3.3.3. Properties All the complexes prepared are air stable, exhibiting a magnetic moment at room temperature in the solid state. The vanadyl complexes lack solubility in organic solvents such as alcohol, chloroform, methylene chloride, tetrahydrofuran, and acetone; hence they could not be recrystallized. In water, hydrolysis occurs and a brown solution results. In DMSO, each complex turns a distinct shade of purple and ultimately dissolves. VO(met)2 32 Chapter 3 immediately becomes a light purple solid upon the addition of small quantities of dimethylsulfoxide; this was characterized as VO(met)2(DMSO). VO(big)2 responds more slowly to the addition of solvent. After approximately 30 minutes, the solution turns purple and the solid a dark green-purple, this solid was identified as VO(big)2(DMSO). Finally, VO(phenf)2 is much more soluble and dissolves easily in DMSO to afford a purple solution; thus, no solid was isolated. The infrared spectra of the isolated solvated species show the sulfoxide stretching (v s = 0) in the 960 cm"' region, resulting from coordination. The solvent is most likely bonded through the O of DMSO to the metal centre as the v s = 0 stretching frequency is lower than that of free solvent (1055-1015 cm"1). Vibrations at 1025 and 1024 cm"1 for VO(big)2(DMSO) and VO(met)2(DMSO), respectively, are representative of residual free DMSO. Green VO(big)2 and VO(met)2 are recovered by the addition of water. From the DMSO solvated species, oxidation to V(V), a yellow solution, occurs when methanol is added, however, the species has not been isolated. The compounds are decomposed by acids. Biguanides combine with many elements of the transition series to give highly coloured chelate complexes. The ligands are monodeprotonated in all cases discussed, a favourable situation because a neutral complex is desirable for increased Gl absorption and lipophilicity in drug applications. When deprotonated, the bidentate biguanides act as hard Lewis bases which bind well with V 0 2 + , a hard Lewis acid. The resonating deprotonated ligand coordinates through the N donor atoms forming a planar, 6-membered, Ti-delocalized chelate ring with the metal, enhancing overall thermodynamic stability. Bis(ligand) metal complexes of neutral charge are obtained when deprotonated biguanide, metformin, and phenformin are bound to the vanadyl ion. 33 Chapter 3 Vanadium(IV) forms stable complexes with various ligands, most favourably with O and N coordination. Generally, bivalent metals combine with two molecules of biguanide to form 4-coordinated planar complexes. Vanadyl complexes typically have a square pyramidal geometry, which is postulated for the three prepared complexes with biguanides. Figure Predicted square pyramidal VO(big)2 with C 2 v symmetry R i Figure Possible structures and symmetries of VO(met)2 and VO(phenf)2, point group assignments assume free rotation about C-NR,R2 bond Deprotonation of the ring N is predicted, subsequently producing an increase of n conjugation along the central C-N-C system in the ligand, reducing the bond angle on the N to the theoretical value of 120° (evidenced by the crystal structure of Cr(big)351). This 34 Chapter 3 angle in typical protonated biguanide ligands ranges from 125.7° to 128.8°. As a consequence of the conjugation, the biguanide ligands are nearly planar.59 VO(big)2 would have C 2 v symmetry (Figure The other two complexes with substituents on the biguanide moiety would yield C 2 or C s symmetry, depending on the configuration of attachment onto the metal centre (Figure NH 2 (H 3C) 2N Figure Possible structures of [VO(bg)2S] present in solution (S = DMSO)' 35 Chapter 3 Preliminary electron paramagnetic resonance spectroscopic data for VO(big)2 and VO(met)2 show the characteristic eight-line pattern of V(IV). The unresolved, broad peaks, however, make it difficult to elucidate any hyperfine coupling information. Variable temperature studies may refine the spectra to give detailed, interpretable data. In all cases, the solvated DMSO species is monitored by the noticeable colour change to purple upon dissolution during sample preparation. Some structural positions where the solvent may coordinate are illustrated in Figure The predicted structure is the trans-ligand, trans-solvent species depicted as TI 6 0 ; however, a better understanding of the coordination environment would be derived from X-ray crystal analysis. No crystals of any of the complexes were obtained because of limited solubility. A structure similar to that of many 5-coordinate bis(bidentate ligand)oxovanadium(IV) complexes57 (e.g. BMOV) 6 1 is expected - a simple square pyramidal configuration with two biguanidato ligands in a trans arrangement around the base of the square pyramid and the V=0 unit axial. V=0 distances of approximately 1.52 - 1.68 A are common.62 3.4. Chromium(III) Complexes 3.4.1. Synthesis Following the known preparation of tris(biguanidato)chromium(III)51, Cr(phenf)3 was synthesized by combining a saturated chrome alum solution with alkaline aqueous phenformin in a ratio of 1:3. Slight excess of ligand precursor was used to complete the reaction. Cr(phenf)3 is red in colour, similar to literature analogues.42'5,5' Likewise, Cr(met)3 preparation was attempted. The addition of KCr(S0 4) 212H 20 to a basic solution 36 Chapter 3 of metformin resulted in a colour change to red; however, this could not be precipitated or crystallized, and the complex could not be isolated. 3.4.2. Characterization Characterization data for Cr(phenf)3 are listed throughout Chapter 2. The infrared spectrum shows the expected N-H stretching vibrations. The elemental analysis matches somewhat with the calculated C, H, and N values. The +LSIMS spectrum gives the distinctive peaks correlating to cationic tris-, bis-, and mono(P-phenethylbiguanidato)-chromium(III), in addition to the protonated ligand species, [H2phenf]+. The room temperature magnetic susceptibility was measured and the effective magnetic moment of Cr(III) was found to be 3.89, similar to the spin-only value of 3.87 BM, expected for a system with three unpaired electrons. 3.4.3. Properties Chromium(III) is the most stable and important oxidation state of this metal. Complexes of Cr(III) are almost exclusively six-coordinate with an octahedral arrangement, typical of trivalent metals which frequently combine with three molecules of ligand, as shown by the crystal structures of Cr(big)351 and Mn(big)363. Such complexes are kinetically inert, a trait which has caused much controversy regarding its biological role as an essential nutrient. Trivalent chromium is a hard Lewis acid, like vanadyl, and readily forms stable complexes with donor ligands. Competition occurs in water as hydroxide complexes form in aqueous solution. Neutral, red Cr(phenf)3 was prepared with the deprotonated (phenf)" ligand which contains a delocalized n electron system. An 37 Chapter 3 octahedral configuration is predicted for Cr(phenf)3, similar to that found in the X-ray structure of Cr(big)3 (Figure, consisting of planar chelate rings and D 3 symmetry. Figure X-ray crystal structure of tris(biguanidato)chromium(III), [Cr(big)3]5 1 3.5. Alternate Analogues Since the focus in this project was the potential synergy of coordinating biguanide to vanadium, alternate modes of attachment were investigated. Attempts were made to condense metformin and maltol (Scheme 3.5.1) to yield a pyridinone-type product. This might then coordinate more strongly to V 0 2 + as an [0,0] donor ligand at lower pH. o o N H N R, Scheme 3.5.1. Attempted condensation of maltol and biguanide to form a pyridinone 38 Chapter 3 The reaction chemistry and pharmacological properties of VO(ma)2 has already been comprehensively studied32,60'61,64"69 and deprotonated maltol has proven to be a formidable coordinating ligand for medicinal applications. Many synthetic variations were made in order to prepare the bis(pyridinone)oxovanadium(IV) complex. Pyridinone condensation, a one-pot synthesis of maltol, metformin, and vanadyl sulfate, and addition of metformin to VO(ma)2 were three unsuccessful approaches. It seems that VO(ma)2 is the favoured product even in the presence of excess metformin, which further confirms the chelating strength of the maltolate ligand. Another strategy tried was the coordination of a biguanide functionalized Schiff base to the metal centre. Following a similar procedure to that used in the preparation of copper-biguanide-salicylaldehde complexes70, biguanide and salicylaldehyde were refluxed in ethanol, vanadyl sulfate was added, and a green product was obtained. This did not characterize as the expected compound. The combination of VO(met)2 and salicylaldehyde yielded a light purple solid which is somewhat inconsistent with the calculated elemental analysis. The +LSIMS shows the presence of HsalHmet ligand and an unassignable peak at 503. Condensing metformin with salicylaldehyde to give the Schiff base type ligand (Scheme 3.5.2) was an alternate approach. Refluxing in ethanol yielded the desired product as characterized by elemental analysis and mass spectrometry. Nevertheless, combining it with an aqueous solution of vanadyl sulfate gave a dark green powder, whose composition could not be determined. The analyses of this powder showed it to contain the Schiff base moiety; however it did not match that of the bis(ligand)oxovanadium(IV) complex or a possible dimer. 39 Chapter 3 NH NH Scheme 3.5.2. Attempted condensation of salicylaldehyde and metformin to form a Schiff base, HsalHmet (R, = R2 = CH3) Vanadium(IV) readily autoxidizes to vanadium(V) in the presence of oxygen. Reactions with NaV0 3 or NH 4 V0 3 and each biguanide were attempted in hopes of synthesizing the cis-bis(ligand)dioxovanadate(V) anion, [V02(bg)2]" (Figure 3.5.1); however, the complex aqueous speciation of V(V) interfered. At low pH, the vanadate V 0 2 + ion exists. With the addition of ligand and increase of pH, oligomer formation occurs whereby the ligand presumably acts as a counter cation. NH 2 Figure 3.5.1. Potential structure of cis-bis(ligand)dioxovanadate(V) anion, [V02(bg)2] 40 Chapter 3 The synthesis of a tris(ligand)vanadium(III) complex was attempted by reducing vanadyl sulfate with sodium dithionite and then adding three equivalents of biguanide (Hbig, Hmet) ligand to form potential octahedral vanadium(III) complexes with N 6 coordination (Figure 3.5.2). Interest in these types of compounds was initially stimulated by the work of Marco Melchior71, another member of the Orvig group. His tris(ligand)vanadium(III) complexes appear to have some insulin-mimetic effects in STZ-diabetic rats.71 V(III) easily hydrolyzes and undergoes oxidation in air in this system; hence it is unstable and complex formation was not achieved. Figure 3.5.2. Potential structure of tris(ligand)vanadium(III) complexes, [V(bg)3] Another objective was to protonate the ligands in the corresponding vanadyl complexes. This was done by placing the solid in a closed beaker of HC1 vapour. Protonating each coordinated biguanide ligand would result in a cationic complex (Figure 3.5.3), as shown by Melchior's71 solid state protonation of VOL 2 to [H 2 VOL 2 ] 2 + via this simple technique (L = maltolate anion). This was attempted once, whereby there was a slight colour change of VO(big)2 or VO(met)2 to a blue colour; however both solids were very wet and could not be completely dried. The acids decomposed the prepared vanadyl complexes. 41 Chapter 3 I "I 2+ H 2 N N R 2 1 ^ =NH I I H N < ^ r ^ > N H - N N H , Figure 3.5.3. Potential structure of protonated bis(biguanide)oxovanadium(IV) cationic complexes, [VO(Hbg) 2] 2 + 3.6. STZ-Diabetic Rat Studies Preliminary STZ-diabetic rat experiments were performed to conclude this particular research on vanadium-biguanide synergy. These are initial investigations over a short time period with small treatment groups. Results from these studies give some information as to the effectiveness of VO(met)2 in lowering blood glucose and summarize the concept of bifunctionality with respect to vanadyl-biguanide complexes as potential insulin mimics. Streptozotocin (STZ) chemically induces a model of diabetes in rats. Intraperitoneal and oral gavage studies were carried out by Violet Yuen of the McNeill group, our collaborators in the Faculty of Pharmaceutical Sciences at UBC, using the synthesized complex VO(met)2. The experimental and results are summarized in Appendix A. 42 C H A P T E R 4 C O N C L U S I O N S A N D F U T U R E W O R K In conclusion, the design of the vanadium-biguanide complexes seemed promising as there is plenty of evidence for the glucose lowering properties of V(V) and V(IV), and biguanides are used today for the treatment of type II diabetes. From preliminary animal investigations with VO(met)2, it may be deduced that vanadium is much more effective than metformin in glucose metabolism. This is not surprising as metformin alone is taken up to a maximal daily human dosage of over 2 g in comparison to what would be |ag treatments of vanadium. When considering vanadium as a drug, high doses lead unfavourably to increased toxicity; thus increasing the VO(met)2 dose to observe potential synergy may not be favourable as high levels of vanadium may be toxic. Rather than complexing biguanides to vanadium, synergy may be invoked by combination treatment whereby a greater amount of biguanide can be administered with a lower dose of vanadium; this could ideally prove to be effective. Another suggestion for future work includes testing a chromium-biguanide complex in vivo to monitor any potential synergy. Again, similar results to those of VO(met)2 might be obtained, which could in turn suggest combination therapy. Modified derivatives such as the Schiff base and pyridinone ligands could prove to be effective by increasing the solubility of the complex upon coordination to vanadium. 43 REFERENCES 1. Butler, A.; Carrano, C.J. Coord. Chem. Rev. 1991,109, 61. 2. Cantley, L.C., Jr.; Josephson, L.; Warner, R.; Yanagisawa, M. ; Lechene, C ; Guidotti, G. Biol. Chem. 1977, 252, 7421. 3. (a) Vilter, H. Bot. 1983, 429, 451 (b) Vilter, H. Phytochemistry, 1984, 23, 1397. (c) Wever, R.; Drenn, B.E.; in Vanadium in Biological Systems: Vanadium Haloperoxidases (Chasteen, N.D., ed.); Kluwer Academic Publishers: Dordrecht, 1990; p.81. 4. (a) Robson, R.L.; Eady, R.R.; Richardson, T.J.; Miller, R.W.; Hawkins, M . ; Postgate, T.J. Nature, 1986, 322, 388. (b) Plat, H.; Krenn, B.E.; Wever, R. Biochem. J. 1987, 248, 277. 5. Josephson, L.; Cantley, L.C.Jr. Biochemistry, 1977,16, 4572. 6. Kobayashi, T.; Martensen, T.; Nath, J.; Flavin, M . Biochem. Biophys. Res. Commun. 1978, 80, 1313. 7. Crans, D.C.; Willging, E.M.; Butler, S.R. /. Am. Chem. Soc. 1990,112, 427. 8. Rehder, D. Biometals, 1992, 5, 3. 9. Nechay, B.R. Ann. Rev. Pharmacol. Toxicol. 1984, 24, 501. 10. Nechay, B.R.; Nanninga, L.B.; Nechay, P.S.E.; Post, R.L.; Grantham, J.J.; Macara, I.G.; Kubena, L.F.; Phillips, T.D.; Nielsen, F.H. Fed. Proc. 1986, 45, 123. 11. Macara, I.G. Trends Biochem. Sci. 1980, 5, 92. 12. Nielsen, F.H.; Utmus, E.O. in Vanadium in Biological Systems (Chasteen, N.D., ed.), 1990, p. 51. 44 References 13. Chasteen, N.D.; Grady, J.K.; Holloway, C.E. Inorg. Chem. 1986, 25, 2754. 14. Rehder, D. Angew. Chem., Int. Ed. Engl. 1991,103, 148. 15. (a) Shechter, Y.; Meyerovitch, J.; Farfel, Z.; Sack, J.; Bruck, R.; BarMeir, S.; Amir, S.; Degani, H.; Karlick, S.J.D. in Vanadium in Biological Systems (Chasteen, N.D., ed.), 1990, p. 129. (b) Shechter, Y. Diabetes, 1990, 39, 1. (c) Shechter, Y.; Shisheva, A. Endeavour, 1993,17, 27. (d) Brichard, S.M.; Lederer, J.; Henquin, J.-C. Diabetes Metab. 1991, 77,435. 16. Heyliger, C.E.; Tahiliani, A.G.; McNeill, J.H. Science, 1985, 227, \A1A. 17. Kadota, S.; Fantus, I.G.; Deragon, G.; Guyda, H.J.; Posner, B.L. J. Biol. Chem. 1987, 262, 8252. 18. Fantus, I.G.; Kadota, S.; Deragon, G.; Foster, B.; Posner, B.I. Biochemistry, 1989, 28, 8864 19. Pederson, R.A.; Ramanadham, S.; Buchan, A.M.J.; McNeill, J.H. Diabetes, 1989, 38, 1390. 20. Ramanadham, S.; Mongold, J.J.; Brownsey, R.W.; Cros, G.H.; McNeill, J.H. Am. J. Physiol. 1989, 257, H904. 21. Ramanadham, S.; Brownsey, R.W.; Cros, G.H.; Mongold, J.J.; McNeill, J.H. Metabolism, 1989, 38, 1022. 22. (a) Conklin, A.W.; Skinner, C.S.; Feltin, T.L.; Sanders, C L . Toxicol. Lett. 1982,11, 199. (b) Hansard, S.L. II; Ammerman, C.B.; Henry, P.R. Anim. Sci. 1982, 55, 350. 23. (a) Domingo, J.L.; Gomez, M. ; Llobet, J.M.; Corbella, J.; Keen, C L . Pharmacol. Toxicol. 1991, 68, 249. (b) Domingo, J.L.; Gomez, M. ; Llobet, J.M.; Corbella, J.; Keen, C L . Toxicology, 1991, 66, 279. 45 References 24. Orvig, C ; Thompson, K.H.; Battell, M. ; McNeill, J.H. Met. Ions Biol. Syst. 1 9 9 5 , 31, 575. 25. Cam, M . C ; Cros, G.H.; Serrano, J.J.; Lazaro, R.; McNeill, J.H. Diab. Res. Clin. Prac. 1 9 9 3 , 20, 111. 26. (a) Sakurai, H.; Tsuchiya, K.; Nukatsuka, M. ; Kawada, J.; Ishikawa, S.; Yoshida, H.; Komatsu, M . J. Clin. Biochem. Nutr. 1 9 9 0 , 8, 193. (b) Watanabe, H.; Nakai, M . ; Komazawa, K.; Sakurai, H. J. Med. Chem. 1 9 9 4 , 37, 876. 27. McNeill, J.H.; Yuen, V.G.; Hoveyda, H.R.; Orvig, C. J. Med. Chem. 1 9 9 2 , 35, 1489. 28. McNeill, J.H.; Yuen, V.G.; Dai, S.; Orvig, C. Mol. Cell. Biochem. 1995,153, 175. 29. Yuen, V.G.; Caravan, P.; Gelmini, L.; Glover, N. ; McNeill, J.H.; Setyawati, I.A.; Zhou, Y.; Orvig, C. J. Inorg. Biochem. 1 9 9 7 , 109. 30. Shisheva, A.; Shechter, Y. Endocrinology, 1993,133, 1562. 31. Posner, B.I.; Shaver, A.; Fantus, I.G. in New Diabetic Drugs (CJ. Bailey and P.R. Flatt, eds.), Smith Gordon, 1 9 9 0 , p. 107. 32. Yuen, V.G.; Orvig, C ; McNeill, J.H. Can. J. Physiol. Pharmacol. 1 9 9 5 , 73, 55. 33. (a) Bailey, C J . Int. Congr. Symp. Ser., R. Soc. Med. 1 9 8 5 , 79, 17. (b) Pradalier, A.; Casassus, H. Diabetes, 1 9 7 4 , 22, 203. 34. (a) Curd, F.H.S.; Rose, F.L. J. Chem. Soc. 1 9 4 6 , 729. (b) Bygbjerg, I.C Eur. J. Clin. Pharmacol. 1 9 8 5 , 28, 287. 35. Angelo, M. ; Ortwine, D.; Worth, D.; Werbel, L.M.; McCall, J.W. Med. Chem. 1 9 8 3 , 26, 1258. 36. Kashiwagi, S. Fukuoka Igaku Zasshi, 1 9 7 4 , 65, 157. 46 References 37. (a) Shapiro, S.L.; Parrino, V.A.; Freedman, L. J. Am. Chem. Soc. 1959, 81, 2220 (b) ibid. 1959, 81, 3725 (c) ibid. 1959, 81, 3728. 38. Klip, A.; Leiter, L.A. Diabetes Care, 1990,13(6), 696. 39. Sirtori, C.R.; Pasik, C. Pharmacol. Res. 1994, 30(3), 187. 40. Sen, D. J. Chem. Soc. (A), 1969, 2900. 41. (a) Saha, C ; Sen, D.; Guhu, S. J. Chem. Soc. Dalton Trans. 1975, 1701. (b) Sen, D.; Saha, C. J. Ind. Chem. Soc. 1977, LIV, 127. 42. (a) Banerjee, B.; Ray, P. Proceedings of the Symposium on the Chemistry of Coordination Complexes, Agra; Proc. Nat. Acad. Sci. 1959, Part III, 198. (b) Ray, P. Chem. Rev. 1961, 61, 313. 43. Mertz, W.; Schwarz, K. Arch. Biochem. Biophys. 1955, 58. 504. 44. Mertz, W.; Schwarz, K. Am. J. Physiol. 1959,196, 614. 45. Toepfer, E.W.; Mertz, W.; Polansky, M.M.; Roginski, E.E.; Wolf, W.R. J. Agric. Food Chem. 1977, 25, 162. 46. Glinsmann, W.H.; Mertz, W. Metabolism, 1966,15, 510. 47. (a) Evans, G.W.; Bowman, T.D. J. Inorg. Biochem. 1992, 46, 243. (b) Hasten, D.L.; Rome, E.P.; Franks, B.D.; Hegsted, M . Int. J. Sports Med. 1992, 2, 343. (c) Evans, G.W.; Pouchnik, D.J. J. Inorg. Biochem. 1993, 49, 177. 48. Press, R.; Geller, J.; Evans, G.W. West. J. Med. 1990,152, 41. 49. Evans, G.W. Int. J. Biosoc. Med. Res. 1989,11, 163. 50. Stearns, D.M.; Armstrong, W.H. Inorg. Chem. 1992, 31, 5178. 51. Coghi, L.; Nardelli, M. ; Pelizzi, G. Acta Cryst. 1976, B32, 842. 52. Karipides, D.; Fernelius, W.C. Inorg. Syn. 1963, VII, 56. 47 References 53. Mabbs, F.E.; Machin, D.J. Magnetism and Transition Metal Complexes, Chapman and Hall; London, 1961; p. 5. 54. Rathke, B. Ber. 1879,12,11 A: 55. Sarma, B.D. J. Ind. Chem. Soc. 1952, 29, 217. 56. Scoponi, M. ; Polo, E.; Bertolasi, V.; Carassiti, V.; Bertelli, G. J. Chem. Soc. Perkin Trans. 1991, 2, 1619. 57. Wilkinson, G.; Gillard, R.D.; McCleverty, J.A. eds.; Comprehensive Coordination Chemistry Volume 3: Pergamon Press: New York, 1987; p. 455. 58. (a) Selbin, J.; Holmes, H. Jr.; McGlynn, S.P. J. Inorg. Nucl. Chem. 1963, 25, 1359. (b) Dutta, R.L.; Lahiry, S. Ind. Chem. Soc. 1964, 41, 546. (c) Ray, M.M.; De, K.; Poddar, S.N. Ind. J. Chem. 1965, 3, 228. (d) Skabo, R.H.; Smith, P.W. Aust. J. Chem. 1969, 22, 659. 59. Schwarzenbach, G.; Scharzenbach, D. Ind. Chem. Soc. 1977, LIV, 23. 60. Hanson, G.R.; Sun, Y.; Orvig, C. Inorg. Chem. 1996, 35, 6507. 61. Caravan, P.; Gelmini, L.; Glover, N. ; Herring, F.G.; L i , H.; McNeill, J.H.; Rettig, S.J.; Setyawati, I.A.; Shuter, E.; Sun, Y.; Tracey, A.S.; Yuen, V.G.; Orvig, C. J. Am. Chem. Soc. 1995,117, 12759. 62. Caira, M.R.; Haigh, J.M.; Nassimbeni, L.R. J. Inorg. Nucl. Chem. 1972, 34, 111\. 63. Hart, R.O.C.; Bott, S.G.; Atwood, J.L.; Cooper, S.R. J. Chem. Soc. Chem. Commun. 1992, 894. 64. Dai, S.; Yuen. V.G.; Orvig, C ; McNeill, J.H. Pharmacol. Commun. 1993, 3, 311. 65. Yuen, V.G.; Orvig, C ; McNeill, J.H. Can. J. Physiol. Pharmacol. 1993, 71, 263. 48 References 66. Yuen, V.G.; Orvig, C ; Thompson, K.H.; McNeill, J.H. Can. J. Physiol. Pharmacol. 1993, 71, 270. 67. Yuen, V.G.; Pederson, R.A.; Dai, S.; Orvig, C ; McNeill, J.H. Can. J. Pharmacol. 1996, 74, 1001. 68. Sun, Y.; James, B.R.; Rettig, S.J.; Orvig, C. Inorg. Chem. 1996, 35, 1667. 69. Yuen, V.G.; Orvig, C ; McNeill, J.H. Am. J. Physiol. 1997, 272, E30. 70. Poddar, S.N.; Sarkar, A.K. /. Ind. Chem. Soc. 1968, 45(11), 979. 71. Melchior, M . Ph.D thesis to be submitted 1999. 49 APPENDIX A STZ-DIABETIC RAT STUDIES WITH VO(met)2 A.l. Experimental IP administration in STZ-Diabetic Rats - Initial Study of the Effect of VO(met)2. Nineteen male Wistar rats weighing 190-220 g were obtained from the Animal Care Unit at U.B.C. Animals were acclimatized for 7-14 days. With a single intravenous injection of streptozotocin (60 mg kg"1 in 0.9% NaCl, 1 mL kg"1) under light halothane anaesthesia they were made diabetic. On day 3 post-STZ, the diabetic state was confirmed via blood glucometer (Ames Glucometer and Glucostix) readings. Blood glucose levels > 13 mM were taken as diabetic. On day 7 post-STZ, the animals were randomly divided into 4 groups: diabetic (gum arabic, n=4), BEOV (n=5), metformin (n=5), and VO(met)2 (n=5). Al l drugs were administered as suspensions in 3% gum arabic. A control group received an equivalent volume of 3% gum arabic alone. Animals were not fasted prior to drug administration. 50 uL of blood were collected for glucose analysis immediately prior to drug administration and at 2, 4, 6, 8, 12, 16, 20, 24, 48, and 72 hours following drug administration. Blood was collected from the tail into heparinized capillary tubes and centrifuged at 10,000 g x 15 minutes. The plasma was analyzed immediately for glucose levels using Boehringer Mannheim kits (glucose oxidase method). Drugs were administered by intraperitoneal injection at a volume of 5 mL kg"1 at a dose of 0.12 mmol kg 1-Gavage of STZ-Diabetic Rats. Twenty-two male Wistar rats weighing 190-220 g were obtained from the Animal Care Unit at U.B.C. Animals were acclimatized for 7-14 50 Appendix A days. With a single intravenous injection of streptozotocin (60 mg kg"1 in 0.9% NaCl, 1 mL kg"1) under light halothane anaesthesia they were made diabetic. On day 3 post-STZ, the diabetic state was confirmed via blood glucometer (Ames Glucometer and Glucostix) readings. Blood glucose levels > 13 mM were taken as diabetic. On day 7 post-STZ, the animals were randomly divided into 4 groups: diabetic (gum arabic, n=5), BEOV (n=5), metformin (n=6), and VO(met)2 (n=6). All drugs were administered as suspensions in 3% gum arabic. A control group received an equivalent volume of 3% gum arabic alone. Animals were not fasted prior to drug administration. 50 uL of blood were collected for glucose analysis immediately prior to drug administration and at 2, 4, 6, 8, 12, 16, 20, 24, 48, and 72 hours following drug administration. Blood was collected from the tail into heparinized capillary tubes and centrifuged at 10,000 g x 15 minutes. The plasma was analyzed immediately for glucose levels using Boehringer Mannheim kits (glucose oxidase method). Drugs were administered by oral gavage at a volume of 5 mL kg"1 at a dose of 0.6 mmol kg"1. At all time points the animals were observed for any signs of toxicity (diarrhea, etc.) A.2. Results The i.p. experiment was performed with the objective of determining if VO(met)2 gave any plasma glucose lowering effects. At the dose used (0.12 mmol kg"1), there was no response to metformin alone, however, there was 40% response to both BEOV and VO(met)2. From the data and calculated standard error obtained (Figure A l , p. 54), there is statistically no difference between BEOV and VO(met)2 in lowering plasma glucose in 51 Appendix A STZ-diabetic rats. The dosage given is clearly much lower than the ED 5 0 for metformin. There was a marked percentage of incidence of diarrhea from responders given VO(met)2 compared to no diarrhea in responders given BEOV. This is most likely explained by the high pH (11.5) of the VO(met)2 solution administered. Unexpectedly, VO(met)2 dissolved in 3% gum arabic; as discussed earlier, this compound is virtually insoluble in common solvents. From this study, VO(met)2 was shown to be effective so the oral gavage study was undertaken. The gavage dose (0.6 mmol kg"1) gave 100% response in rats treated with VO(met)2. Again, there was no response from those given metformin because the dose was still too low to show any effects. Only 60% responded to BEOV; however the data for this treatment group were later deemed imprecise because 2 of the responders died. The data (Figure A2, p. 55) show that VO(met)2 considerably lowered plasma glucose levels at 24 hours; however, within the rest of the 72 hour study, the plasma glucose increased steadily back to initial levels (a similar jump to was seen with VOS0 4 treatment). Again, there were GI side effects with VO(met)2 administration. Blood glucose concentrations over 72 hours are listed in Tables A l and A2 (pp. 57, 59) for i.p. and gavage studies, respectively. Another observation was that there was no appreciable weight gain from VO(met)2 administration in both studies, as can be seen in Figure A3 (p. 56). Table A3 and A4 (pp. 61, 62) list the body weight measurements taken during the course of the experiments. In conclusion, it can be inferred that the vanadium component of the synergistic compound VO(met)2 is far more effective at the doses given than is metformin. The ED 5 0 of metformin is much greater than that of VOS0 4 or any of the vanadium compounds 52 Appendix A tested in vivo. Hence, in these preliminary rat studies, synergism of VO(met)2 was not observed. 5 3 Figure A l . Plasma glucose versus time - i.p. study 24.00 "I* 16.00 8 12 16 20 24 time (hours) 48 72 3% gum arabic; 0% R BEOV;40 % R metformin; 0% R VO(met)2; 40% R 54 Figure A 2 . Plasma glucose versus time - oral gavage study 8 12 16 20 24 time (hours) 48 72 3% gum arabic; 0% R BEOV; 60% R metformin; 0% R VO(met)2; 100%R 55 (8) jq§i3/U Xpoq IS S3 a s o u DC +-a s « o _ u es cu _ a cn 3 © _ CM U >• O m 0 O T3 « CU u O CJ CU CO O o 3 "©JD £ CM — £ CU _ es H CM 00 Tt Tt CN o CM o _ 43 CN So oo 43 NO 43 T t 43 tN m m OO ( os — * Tt — ^ Os NO NO rn so oo —H oo Tt o in NO NO m so NO m Tt ON oo oq in Os -_i -_i rn o —H tN tN tN tN tN tN ON Tt r - 00 NO tN Tt rn —< CN rn —H CN CN tN tN tN tN m r~ O NO Os NO rn m O oq c i —« CN ON d tN tN tN tN O oo oo Os tN vq Tf CM Tt rn rn -H rn tN tN tN tN tN CO oo o oo tN '—i Os 00 r -—H Tt tN CM tN <N tN tN tN fN IT) NO tN SO O 00 O in m l> O Tt rn CM tN tN tN tN tN tN O r- o o OS oq O Os Tt r --_i —< OS o d tN tN —H tN tN O tN tN 00 tN '—i Tt rn tN O OS Os CN d tN tN tN 00 Tt Tt o tN NO _ rn p —H Os d CN tN tN tN tN in o c-» tN NO oq lO oo tN —* —H tN oo —H tN tN tN tN m OS NO m CN OS r-; OO tN —< CN d tN tN tN tN tN r - H tN rn < < < < Tt d in Tt d d m NO _2 3 oo —; m -q-m S o o 2} tN oo un 2 I . tN . 2 ^ ^ o 43 tN "3- _ 00 tN II m d tN CN o tN 43 SO 43 CM 43 oo 43 s_ 43 43 tN VO O II o ^ > O W 05 Os ~ . OS iri p tN 00 in ° ^ m ^ oo ^ ° ^ C-N — H W f>| so tN rn CN 00 o —; p rn rn tN tN m o Os tr~-rn tN cs CN tN O in o tN —< m (N m _4. ^ > n in 3 CN —' rn i-; tN 1 — i N (N O O _ —< m vq in ^ tN tN in c^j* tN '—' tN tN SO 0O o fN (N ON O ON 00 r~ in - H (N o —< in —j sq CM sq Os t> 00 ON NO CN oo o o oo oq oq oq oq ON t-^  d d in —< —< CM tN —< -—i CN '—i NO NO OS oo in 00 O oo m m oo —< CM tN ON CM in NO tN O O rn - H r - i rn rn <—< CM CM CM CM CM m >o r - oo o pq P5 P5 CQ PQ CM o ON in ON CM O in o CM O m o CM CM 00 oo 00 in ^1 ^ in CM m 2 . m 2 cn-oo 0 0 ° ° . CM o m 2 o SO oo 2 © —i CM m d CM 00 CM o tN 43 SO 43 CM 43 oo 43 NO 43 43 CM m m NO NO ON CM CM ON oo ON OO OO — . ' NO CM in r-~ r~- in r~ ON in NO in in in CM i—i —H i-—: ——i ——i — NO —< r- —i os CM ON in oq — CM — O O CN CM CM fN CN o m o — TJ- oo NO in p — CM —< ON CM CM CM — i tN in rn CM NO CM ; oq ON m' CM CM CM —< CM CN in O ON ON ; —< oq ^t -t rn — CM CM CM CM CM CM CM t -H ON m o NO o oo NO m m CM CM m CM tN tN tN fN oo o m Tt Tt in CN CN CM r-^  rn in rn CM tN CM CM CM CM tN O Tt oo Tt rn Tt oo ON d - H - H CN Tt CN m ON NO d — CM 00 ON ON Tt - H in co oo ON ON OO oo m r-0O —* —H —< NO ^ tN — —• ON O ON 00 ON m m NO CM CN NO NO m CM — — CM CM CM O CM r - r~- Tt CN ON ON l> —< O ON CM —< CN d d tN CM CM CM fN o --i tN m Tt u u u u o Tt fN oo CM m Tt d ^ d CM ° •n 0 ^ in ^ d CM ° 0 . T ? ^ d CM ° 2 ^> 2 o m o NO CM 2 © m _ . <*2 m 2 © ^ Tt cl © OO in CM in Tt NO oo ON Os i n i n m ro ro —; o CN (N —< o CN CN T m NO ON CN CN ON OS J^" oo oo Os so ro CN —< — ( i n i n CN — i ^ ^ I/O 2 2 ^ o Os O —< CN CN NO O © CN CN t> o oo , — i NO o NO ro ON CN m I/O NO <n NO ro ro </0 NO CN •sr. NO ro 0 0 CN CN r ~ oo ro ro ro CN 0 0 CN CN 0 0 ON NO oo I/O l> ^H ro NO CN CN -3- ro OO ^r CN i n CN ro ON CN CN I/O CM NO O ro ro ON CN CN ro r -CN ro CN d CN CN o 0 0 ON o r-~ © d CN CN O CN NO d d (> CN CN NO CN uo KO o ON d d t> CN CN Os ro m •*t NO m CN CN d CN CN CN © < CN ro CN CN CN CN CN i n NO r ~ oo ON O Q Q Q Q ON od m ON NO NO NO CN ON o ON OO «n ON o ro r -9 S > w IS s «5 CU D C e s > O C I a 3 © u D C •+•> cu B +-CS CU ha — u CS CU CX) - I S O CN r~~ -. cu > o a CS - . ca CU U S3 o o CU O 3 "5c E SB _2 § 3 CU 05 H oo 4 3 •st CN O CN 4 3 CN < PH 4 3 NO 4 3 •st 4 3 CN 1 ca l l N O N O <n N O o N O '—' OO r- in N O CN •st" d CN O N N O <n oo O N o O N IT) N O m <n N O m ' " ' N O O N N O in N O —< m —< N O _ 4 co o CN CN CN CN CN CN t- r- 0 0 CN p N O in o C O CN CN C O CN CN CN CN CN CN CN «n 0 0 N O •st CN C O O oo O N N O O N O d d ,—< d CN CN CN CN CN CN o o N O O N •st 0 0 •st oo 0 0 C O •st O N CN C O CN CN C O CN CN CN CN CN CN CN N O m 0 0 O N 0 0 C O -ct p N O •st •st CN co C O co CN CO C O CN CN CN CN CN CN in N O O N in _, C O -—i OO in O N no CN CN CN CN CN CN CN CN CN CN CN CN O in 0 0 C O N O Tt-N O r- •st in OO o ,—< CN 1—4 d C O CN CN CN CN CN CN CN r- r- CO OO o in in oo C O O N d O N d d CN CN CN CN •st O N O N o CN <n O N O N CN '—1 <sf O N d _i O N 1—4 d CN CN i—1 CN CN CN O r- N O r- N O O N —< >n N O CN C O >n d d d d _i d CN CN CN CN CN CN O N CN N O in N O CN 7 * r-- O in CN CN CN CN CN CN CN CN CN CN CN C O m O < < < < < •st d •st d CN d CN 0 0 CN 0 0 •st • S t CN o CN 4 3 NO 4 3 CN 4 3 0 0 4 3 N O 4 3 4 3 CN i CO ON i in ! r-! c"> ! 1 CN 1 CN CN o <n ON NO o NO 1—4 •st CN CN CN CN CN CN > o w PQ NO 0 0 in t->n oo ON 1279 CN 1660 ON NO CN 1555 1008 CN CN C O N O N O C O N O o 0 0 N O CN C O N O OO in d CN : o £ CN C ^ « NO in c-—< CN —< C O ^ C O ON o in CN CN Tl" —< CN T T T T ON O CN O CN —< NO r- oo ON 2 P3 CQ CQ P3 fQ NO o in •sa-N O in cK * d d CN -—1 NO CN m O o NO ON oo d CN d d CN CN S3 ^ ° « N O 0 0 O N CO N O m CO CO 0 0 d 0 0 CN CN N O O N in N O O N oo d CN CN CN CO CN CN O CO CN T-4 CN CN CN in OO -d-oo in CN d i—4 CN CN CN CO o 0 0 O N O CN CN CN CN CN CN 0 0 T t co < oo CN oo CN o CN 4 3 NO C O - H 4 3 CN 4 3 oo 4 3 N O 4 3 4 3 CN cS "S s ON CN in C O o o CN C O ON oo ON OO NO d in oo in O N OO C O m ON NO in NO NO m ~ " O N CN C O SO 0 0 N O ON in 0 0 C O ON CN ON d CN d CN i—i CN CN CN CN O OO CN oo o O N CN t-; oo •st co CN C O C O CN CN CN CN CN CN CN NO NO •sl- "3- CN CN NO •st CN in d d d C O CN CN CN CN CN CN oo m NO C O o oo in —; o NO '—1 C O CN CN C O C O CN CN CN CN CN CN in CN ON oo oo in NO 0 0 CN O N —< C O CN CN Tf" C O C O CN CN CN CN CN CN C O NO o O N C O ON co in in C O ON ON C O CN CN ' 1 T""H CN CN CN t— NO CN ON <n NO CN ON o CN 0 0 O N C O d d CN CN CN CN —< CN OO C O m NO •st CN —J o d ON d _ i d CN CN CN CN CN NO 0 0 in C O f -C O O •sj- ON C O CN 0 0 ON ON d d CN CN CN o ON •sj" NO NO oo OO m f- NO 0 0 d OO ON O s d ON CN CN CN O N ON NO in in oo •sfr •st C O CN CN CN CN CN CN CN CN CN C O •st m NO — H ——H r—H — H 1—1 1— I O o o o u u O S 3 CN 43 ON oo ro ro d II +H 00 43 o •"3- NO 1/0 CN oo d II •«-» in 43 i f O o r -ro CN <n d I  •*-» NO NO 43 NO NO NO CN oo d I  oo 43 CN 43 OO 43 NO 43 43 CN O > m c-~ q in i> m CN ON 00 O ON oo CN o d in r-~ r-» NO O 00 —« NO ro NO in m NO q NO CN t> 1383. d ro CN ro m NO CN CN CN 00 NO OO CN o ON 00 IT T f d CN CN CN CN CN ON ON CN ro O ON m ON q ro CN CN CN CN in O q NO • * ON rr c-» CN m oo ro I> NO ro q ro ON NO o OO CN d 00 ON d oo ro CN in CN OO CN NO NO ON ON ro ro CN CN O CN NO q NO d CN CN in ro CN r-CN OO NO ON CN q t> 00 ON OO* 00 NO ON q ON oo ro CN 00 CN OO ON ON d CN ON r~ NO o q in r-; oo CN d CN CN ON d CN d CN so ON CN ON CN ON o •st-o ro m ON 00 NO o ON NO — i NO ON ro oo ro m d ON d —i d d d CN CN CN CN CN CN in r- CN 00 in CN CN in CN in oo o ON d CN CN CN CN f - H CN CN CN CN CN CN CN f - O O O N O - H C N O S H -H H O l N N > S Q Q Q Q Q Q !< £ Table A3. Animal weights over 72 hours for each treatment group - i.p. study ANIMAL WEIGHT (g) 3% gum arabic STZ t = 0 t= 12 h t = 24h t = 48h t = 72h A l 280 312 290 304 306 314 A2 286 318 312 316 308 310 A3 298 334 326 332 334 334 A4 300 312 308 302 316 320 AVG 291 319 309 314 316 320 SEM 5 5 7 7 6 5 . BEOV STZ t = 0 t= 12h t = 24h t = 48h t = 72 h B5 282 314 302 310 328 308 B6 300 348 330 338 326 348 B7 272 296 300 294 294 300 B8 310 320 320 322 346 326 B9 308 340 332 332 306 326 AVG 294 324 317 319 320 322 SEM 7 9 7 8 9 8 metformin STZ t = 0 t= 12 h t = 24h t = 48h t = 72h CIO 300 316 308 316 318 320 C l l 298 320 310 316 318 322 C12 290 312 308 310 314 318 C13 284 312 310 316 324 320 C14 270 300 294 296 296 300 AVG 288 312 306 311 314 316 SEM 5 3 3 4 5 4 VO(met)2 STZ t = 0 t= 12 h t = 24 h t = 48h t = 72 h D15 270 288 270 282 282 288 D16 278 338 314 328 332 334 D17 300 336 324 324 328 328 D18 296 326 312 314 316 316 D19 330 354 350 362 306 358 AVG 295 328 314 322 313 325 SEM 10 11 13 13 9 11 61 Table A4. Animal weights over 72 hours for each treatment group - gavage study ANIMAL WEIGHT (g) 3% gum arabic STZ t = 0 t= 12 h t = 24h t = 48h t = 72h A l 290 298 302 280 284 286 A2 284 292 302 284 288 294 A3 298 316 318 316 316 308 A4 322 348 348 338 340 338 A5 286 302 310 302 304 300 AVG 296 311 316 304 306 305 SEM 7 10 9 11 10 9 BEOV STZ t = 0 t= 12h t = 24h t = 48h t = 72h B6 308 360 360 360 362 346 B7 268 296 272 274 B8 316 322 312 310 324 310 B9 306 336 316 314 BIO 276 308 308 304 300 300 AVG 295 324 314 312 329 319 SEM 10 11 14 14 18 14 metformin STZ t = 0 t = 12 h t = 24h t = 48h t = 72h C l l 298 316 338 322 326 318 C12 284 306 318 306 304 302 C13 280 316 320 308 312 304 C14 274 298 310 298 292 284 C15 304 324 336 306 314 308 C16 330 356 364 354 350 346 AVG 295 319 331 316 316 310 SEM 8 8 8 8 8 8 VO(met)2 STZ t = 0 t= 12 h t = 24h t = 48h t = 72h D17 320 322 308 306 300 306 D18 318 346 330 326 328 332 D19 304 316 300 306 296 286 D20 322 352 326 334 338 330 D21 302 300 302 278 290 292 D22 274 294 292 282 280 278 AVG 307 322 310 305 305 304 SEM 7 10 6 9 9 9 62 


Citation Scheme:


Citations by CSL (citeproc-js)

Usage Statistics



Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            async >
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:


Related Items