Structure-activity relationships of novel anti-diabetic ruthenium compounds : synthesis, characterization, mechanistic and in vitro studies.

Abstract

Type 2 Diabetes Mellitus (T2DM) is a chronic metabolic disorder which is globally responsible for millions of fatalities per year. Management of T2DM typically involves orally administered anti-hyperglycaemic drugs in conjunction with dietary interventions. However, the current conventional therapy seems to be largely ineffective as patients continue to develop complications such as cardiovascular diseases, blindness and kidney failure. Existing alternative treatment entails the administration of organic therapeutic pharmaceuticals, but these drugs have various side effects such as nausea, headaches, weight gain, and respiratory and liver damage. Transition metal complexes have shown promise as anti-diabetic agents owing to their diverse mechanisms of activity. In particular, selected ruthenium compounds have exhibited intriguing biological behaviours as Protein Tyrosine Phosphatase (PTP) 1B and Glycogen Synthase Kinase 3 (GSK-3) inhibitors, as well as aggregation suppressants for the human islet amyloid polypeptide (hIAPP). The introduction chapter served as a survey on studies pertaining to ruthenium compounds as metallo-drugs for T2DM. Herein, we also provide perspectives on directions to fully elucidate in vivo functions of this class of potential metallopharmaceuticals. More specifically, there is still a need to investigate the pharmacokinetics of ruthenium drugs in order to establish their biodistribution patterns which will affirm whether these metal complexes are substitutionally inert or serve as pro-drugs. In addition, embedding oral-administered ruthenium complexes into bio-compatible polymers can be a prospective means of enhancing stability during drug delivery. This chapter was concluded with a descriptive rationale of the research study as well as specifying the specific research aims and objectives. Our prior studies have illustrated that the uracil ruthenium(II) diimino complex, [Ru(H3ucp)Cl(PPh3)] (1) (H4ucp = 2,6-bis-((6-amino-1,3-dimethyluracilimino)methylene)pyridine) displayed high hypoglycaemic effects in diet-induced diabetic rats. To rationalize the anti-diabetic effects of 1 in the first experimental chapter, three new derivatives have been prepared, cis-[Ru(bpy)2(urdp)]Cl2 (2) (urdp = 2,6-bis-((uracilimino)methylene)pyridine), trans-[RuCl2(PPh3)(urdp)] (3), and cis-[Ru(bpy)2(H4ucp)](PF6)2 (4). Various physicochemical techniques were utilized to characterize the structures of the novel ruthenium compounds. Prior to biomolecular interactions or in vitro studies, the stabilities of 1 – 4 were monitored in anhydrous DMSO, aqueous phosphate buffer containing 2% DMSO, and dichloromethane (DCM) via UV-Vis spectrophotometry. Time-dependent stability studies showed ligand exchange between DMSO nucleophiles and chloride co-ligands of 1 and 3, which was suppressed in the presence of an excess amount of chloride ions. In addition, the metal complexes 1 and 3 are stable in both DCM and an aqueous phosphate buffer containing 2% DMSO. In the case of compounds 2 and 4 with no chloride co-ligands within their coordination spheres, high stability in aqueous phosphate buffer containing 2% DMSO was observed. Fluorescence emission titrations of the individual ruthenium compounds with bovine serum albumin (BSA) showed that the metal compounds interact non-discriminately within the protein's hydrophobic cavities as moderate to strong binders. The metal complexes were capable of disintegrating mature amylin amyloid fibrils. In vivo, glucose metabolism studies in the liver (Chang) cell lines confirmed enhanced glucose metabolism as evidenced by the increased glucose utilization and glycogen synthesis in liver cell lines in the presence of complexes 2 – 4. The second experimental chapter, reports on the formation and characterization of new diamagnetic ruthenium uracil mono-imine compounds: [(η6-p-cymene)RuII(L)Cl] (L = urpda = 5-((pyridin-2-yl)methyleneamino)-6-aminouracil) for 1, urdpy = 6-amino-1,3-dimethyl-5-((pyridin-2-ylmethylene)amino)uracil) for 2 or urqd = 5-((quinolin-2-yl)methyleneamino)-6-aminouracil) for 3); cis-[RuII(L)(bipy)2] (L = urpy = 5-((pyridin-2-yl)methyleneamino)uracil) for 4 and H2dadp = 5,6-diaminouracil for 5); trans-[RuII(L)(PPh3)Cl2] (L = urpda for 6) are described. Various physicochemical techniques were utilized to characterize the structures of the novel ruthenium compounds. Furthermore, the DPPH and NO radical scavenging capabilities of metal complexes (2 – 10) were investigated. UV-Vis spectrophotometry data of the time-dependent (for 24 h) studies show that 4 and 5 are stable in aqueous phosphate buffer containing 2% DMSO. Similarly, the stabilities of 1 - 3 and 6 monitored in chloro-containing and non-coordinating solvent dichloromethane show that they are kinetically inert, whereas, in a high nucleophilic environment, the chloride co-ligands of 1 - 3 and 6 were rapidly substituted by DMSO. In contrast, the substitution of the labile ligand of the complexes by DMSO molecules from its solution with a high chloride content was suppressed. Solution chemical reactivities of the different metal complexes were rationalized by density functional theory computations. Furthermore, the binding affinities and strengths between BSA and the respective metal complexes were monitored using fluorescence spectroscopy. Mutually, these metal complexes showed comparable capabilities of denaturing mature BSA aggregates which was established by fluorescence spectroscopy and Transmission Electron Microscopy (TEM). The final experimental chapter entails the encapsulation of the ruthenium complexes 1 - 10 into separate organic chitosan (CS)-polyvinyl alcohol (PVA) blends and the subsequent nanofabrication of their electrospun nanofiber (ENF) conjugates, Ru-CS-PVA ENFs. Intravenous injections of insulin can be regarded as a primitive method for Diabetes Mellitus management which characteristically leads to patients developing insulin resistance while oral-administered anti-diabetic organodrugs such as Metformin have exhibited low bio-availability and typically induce gastrointestinal (GI) side-effects. Although the intravenous injections of selected metal compounds in Streptozocin (STZ)-diabetic results have delivered promising results, limited work has been done to evaluate their efficiencies during oral administration. Herein, the fabricated chitosan (CS)-polyvinyl alcohol (PVA) electrospun nanofibers (CS-PVA ENFs) of the leading insulin-enhancing ruthenium complex 1, cis-[Ru(bipy)2(H4ucp)](PF6)2 and its analogs: 2 – 10. The Ru-CS-PVA ENFs nanocomposites were characterized by using (SEM-EDX), powder X-ray diffraction, and FTIR spectroscopy. The Ru-CS-PVA ENF nanohybrids exhibited randomly oriented fiber mat morphology with mean diameters in the range of 118 - 280 nm. Metal-based drug release kinetics of 1 - 10 from the ENF polymer matrix were measured spectrophotometrically at pH 1.5 and 7.4, respectively. Electronic spectral trends and data analysis over a 24-hour data collection period reveals variable dissolution rates with first-order rate (kobs) constants ranging from 0.0146 to 2.74 μM h-1 with accompanying hyperchromism effects between 5.69 to 37.6% at a pH of 1.5 while at a pH of 7.4, kobs value limits were 0.0104 and 3.89 μM h-1 rendering corresponding 19.14 and 87.32% hypochromic shifts. The release kinetics data of 1 - 10 were spontaneously released into the aqueous media from the Ru-CS-PVA ENFs, with the highest and releasing rates recorded for complexes 8 and 4, respectively.