Potential therapeutic effects of oximes 1

Chapter Introduction 1.1. Design of Oxime Compounds With Non-Reactivation Therapeutic Effects Against Nerve Agent Soman 1.1.1. Toxicology of Nerve Agent Poisoning Nerve agents belong to a group of organophosphorus compounds with high acute mammalian toxicity. It can be broadly divided into two subclasses, the G- and V- agents (Somani et al. 1992, Marrs et al. 1996). The G-agents are derivatives of phosphoric acids, with the leaving group linked directly to the phosphorus atom (Fest et al. 1982). The leaving group is usually a fluorine atom except in the case of tabun, which has a nitrile functional group as the leaving group. V-agents are phosphonothioates of the P=O type, with the leaving group linked to the phosphorus atom through a sulphur atom (Table 1a & b). Nerve agents are colourless liquids when pure, but take on a yellowbrown colour when present in an impure form. Detailed physicochemical properties of nerve agents are tabulated in Table 2. Important physical characteristics contributing to their application as chemical weapons are their high volatility and lipophilicity. Having a high volatility facilitates their dispersion in the battlefield while their lipophilicity enhances penetration through skin. G-agents differ from V-agents in having higher volatility. Nerve agents are degraded in water with the rate of degradation correlated to their water solubility. Sarin, the most soluble nerve agent, has a half-life of only 15 minutes in water as compared to the half-life of 577 for the more lipophilic soman. For the same reason, sarin has a faster rate of detoxification in the body compared to soman, which is comparatively more persistent in vivo. The persistence of soman in the body is unique among nerve agents and has been postulated to be due to existence of storage depots for soman in the body. Stored soman is protected from degradation in tissues and is released slowly into the circulatory system, resulting in the reappearance of signs of intoxication and death (Wolthuis 1981, Van Helden et al. 1983, Somani et al. 1992). Nerve agents exert their effect through irreversible inhibition of the synaptic nerve enzyme, acetylcholinesterase (AChE) (Somani et al. 1992, Marrs et al. 1996). Acetylcholinesterase neurotransmitter (AChE) acetylcholine (ACh) catalyzes and the hydrolysis terminates neural of the impulse transmission at the cholinergic synapses (Figure 1). When a critical proportion of the synaptic AChEs are inactivated by phosphorylation, signs of OP poisoning would be manifested. Inhibition of AChE will result in the accumulation of acetylcholine at neural synapses to produce depolarisation block (Karalliedde et al. 1993). Intoxication will result in depression of the brainstem respiratory center leading to loss of central respiratory drive (Rickett et al. 1986). Peripheral neuromuscular blockade from prolonged ACh-receptor interactions leading to receptor desensitization (i.e., desensitization block), will exacerbate respiratory paralysis leading to respiratory insufficiency and Table 1a Structures of Nerve Agents (G-Agents) Abbreviation Common IUPAC Name Molecular Formulae Name GA Tabun Ethyl N-dimethylphosphoramido – H3C CH2 cyanidate O P O C N N H3C GB Sarin CH3 Isopropyl methylphosphono - O H3C P fluoridate F O H3C CH3 H GD Soman Pinacolyl methylphosphono - O fluoridate H3C P F O H3C C CH3 CH CH3 H GE - Isopropyl H3C ethylphosphonofluoridate O H2C P F O H3C CH3 H GF - Cyclohexyl methylphosphono fluoridate H3C P O F O H GV - 2-Dimethylaminoethyldimethyl CH3 -amido phosphonofluoridate CH3 N H2C CH2 O P N H3C O F CH3 Table 1b Abbreviation Formulae of Nerve Agents (V-Agents) Common IUPAC Name Molecular Formulae Name VX O-Ethyl-S-[2- H3C (diisopropylamino)ethyl] CH2 O P methylphosphonothioate CH3 O CH3 S H3C N CH3 VE - CH3 O-Ethyl-S-[2-(diethylamino)ethyl] ethylphosphonothioate H3C CH2 O P CH3 O H2C S N CH3 H3C VG - O,O-Diethyl-S-[2(diethylamino)ethyl] H3C CH2 O P phosphorothioate H2C CH3 O S O N CH3 H3C VM - O-Ethyl-S-[2-(diethylamino)ethyl] methylphosphonothioate H3C CH2 O P H3C CH3 O S N CH3 Figure Photomicrograph (Upper) Of Neuromuscular Junction And Schematic Diagram (Lower; Adapted From FOI Chemical Defence Handbook 1996) Illustrating Effects Of Nerve Agent Inhibition Of Acetylcholinesterase Leading To An Excess Of Acetylcholine At Nerve Synapse. If Left Untreated, Manifestations Of Cholinergic Toxicity As Indicated In Table Would Ensue. Table Physicochemical Properties of Classical Nerve Agents Properties Appearance Tabun Sarin Soman GF Colorless liquid (pure); Yellow-brown liquid (impure) Odour Fruity Odourless Odourless Odourless Odourless Molecular Wt. 162.1 140.1 182.2 180.2 267.4 Density (g/cm3) at 25OC 1.073 1.089 1.022 1.12 1.008 Boiling Point (OC) (760 mm Hg) Melting Point (OC) 247 158 167 92 (10 mm Hg) 300 -50 -56 -42 40 following seizure onset) increases in extracellular dopamine levels has also been reported by other research groups (Fosbraey et al. 1990, Jacobsson et al. 1997, 1999). As dopamine serves as a major source of free radicals in the brain, massive release of dopamine has also been suggested to generate oxidative stress on striatal neurons leading to increased neuronal loss in the striatum region (Pazdernik et al. 2001). In addition, besides the initial cholinergic hyperexcitation during nerve agent poisoning, sustained cholinergic excitation of locus coeruleus (LC) norepinephrine-containing neurons has also been reported by other research 10 HI-6 in aqueous solution has necessitated the creation of new binary autoinjector for their application in the field (Brodin et al. 1992). Such autoinjectors maintain HI-6 in its dry state in a separate compartment from the atropine aqueous solution. The two components are only mixed at the point of injection. Unlike atropine, which has been universally adopted as the antagonist of choice in nerve agent therapy, countries differ in their selection of an oxime reactivator. Countries like the UK and USA prefer PAM while European countries like Germany and Netherlands adopted obidoxime as the drug of choice. Recently, HI-6 has become the drug of choice in both Canada and Sweden. Thus, despite being one of the most intensely investigated areas in nerve agent-related pharmacology, the question of which oxime to use remains controversial in both the military and scientific arena. Current opinion holds that the efficacy of oxime antidotes varies with the nature of the nerve agent involved in the intoxication and none of these oximes are regarded as broad-spectrum reactivators against all classical nerve agents – sarin (GB), soman (GD), tabun (GA) and VX (Worek et al. 1998). Thus the choice and dose of oximes remains an important area being investigated for enhancing the survival of nerve agent casualties. For ethical reasons, the antidote efficacy of oximes against high lethal chemicals such as nerve agents cannot be investigated in humans. Hence, data from animal experiments have been the main basis for the licensing of oximes by regulatory authorities. On the basis of data obtained from animal experimentation (Clement et al. 1981), all three oximes are able to reactivate sarin and VX-inhibited 25 acetylcholinesterase, while only obidoxime would reactivate tabun-inhibited acetylcholinesterase (Inns et al. 1983, Bismuth et al. 1992, Dawson 1994) and HI-6 as the only oxime effective against soman. It is also reported from some animal experiments that HI-6 is superior in antidotal effects, compared to obidoxime and PAM, against soman, sarin and VX (Clement et al. 1981, Shiloff et al. 1987, Dawson 1994). Pralidoxime, on the other hand, was generally found to be less potent compared to both obidoxime and HI-6 (Bismuth et al. 1992, Dawson 1994) Before these results may be extrapolated to support the superiority of HI-6 in soman poisoning, it should be worthwhile to note that while HI-6 is equally effective in both sarin-treated intercostal muscles from both animals and humans, it restored neuromuscular transmission in soman-poisoned intercostal muscle preparations from rodents and dogs but not those human intercostal muscle preparations (Inns et al. 1983, Dawson 1994). However, it was also noted that HI-6 oxime-induced recovery of neuromuscular transmission was consistently greater in the diaphragm muscles of rodents as compared to their intercostal muscles (Wolthuis et al. 1981a). This would suggest that inhibited cholinesterase in human diaphragm may be more susceptible to reactivation by HI-6 than those found in intercostal muscles. However, as human diaphragm muscle preparations could not be obtained for in vitro studies, such postulations remained unproven. Recent in vitro studies to determine the reactivation rate constants of oximes on human and animal acetylcholinesterases inhibited by nerve agents, 26 conducted by Worek’s group, confirmed that there exists substantial species differences in toxicokinetics and kinetic properties of AChE that would hinder the extrapolation of in vivo data from animal to human (Worek et al. 2002). In particular, there was substantial resistant to HI-6 reactivation with guinea pig acetycholinesterase inhibited by nerve agents as compared to human acetylcholinesterase. This finding was critical as the guinea-pig model, with its similar low blood carboxylesterase level as observed in human blood (Maxwell 1987), has often been used and cited in literature (Dawson 1994) as the most appropriate model for extrapolation of oxime antidotal effectiveness to human situation. While the use of kinetic parameters helped to ascertain efficacy of oximes on human acetylcholinesterase, it should also be noted the reactivation potency of certain oximes is markedly reduced by the formation of stable phosphorylated oximes (POXs) at high OP-AChE conjugate concentration, especially those with an oxime functional group located on position of the pyridinium ring (e.g. obidoxime, PAM) (Luo et al. 1999). The interference of POXs was intentionally minimized with current in vitro assays where the concentration of OP-AChE conjugate were kept low (0.03 nM) when measuring the reactivation kinetics of oximes that in such in vitro experiments to assume pseudo-first order kinetics. These kinetic results, therefore, may not reflect the true reactivation potency of oximes under in vivo conditions where concentration of AChE is much higher at the synapses and in blood (~ - 10 nM). 27 To complicate matters, during the course of nerve agent intoxication, the inhibited acetylcholinesterase is changed by a process known as “ageing”, into a complex in which the inhibition can no longer be reversed by antidotes (Wolthuis et al. 1981b, Somani et al. 1992, Marrs et al. 1996). The rate of ageing depends on the nature of the organophosphate agent inhibiting the enzyme; VX, sarin and tabun are generally placed in one category while soman is placed in another (Table 4). In addition, soman intoxication is a difficult nerve agent poisoning to treat as soman-inhibited human acetylcholinesterase rapidly (half-life of min) becomes non-reactivatable in a phenomenon known as “aging” (Clement et al. 1981). Post-intoxication administration of HI-6 after a four half-life (i.e., 16 min) delay is expected to regenerate a maximum of 48 hr Tabun 14 hr Sarin hr Soman 2-6 28 1.1.2.5 Non-Reactivation Pharmacological Effects Of Oximes However, in the last decade, there have been interesting results coming out from the laboratories in Netherlands (i.e., TNO) and Canada (DRES) indicating that oximes like HI-6 owed their therapeutic effects to other direct therapeutic mechanism not related to acetylcholinesterase reactivation (van Helden et al. 1991, 1994, 1996). This conclusion arises from observations that non-human primates survived soman and tabun intoxication following administration of HI-6 although reactivation of inhibited AChE was not observed (Hamilton et al. 1989). These direct effects of bispyridinium oximes as exemplified by HI-6 could lead to the recovery of neuronal transmission in the respiratory centres of the brain as well as recovery of neuromuscular transmission in the diaphragm. These results suggested that oxime therapeutic effects may have contributions from other direct therapeutic mechanism(s) not related to acetylcholinesterase reactivation. There has since been a multitude of findings associating various pharmacological effects with oxime reactivators (French et al. 1983, Kloog et al. 1985a, Su et al. 1986, Alkondon 1988, 1989, Tattersall 1993). Most of the reported research work on “direct effects” of oximes have largely centred on modifications of the bispyridinium oxime structures, where substation of various functional groups on either of the pyridinium rings were created. Alternatively, changes have also been made to the short ether linkage between the bispyridinium rings to alter their reactivation and non-reactivation pharmacological properties. While such structural changes have considerable impact on the resultant pharmacological and toxicological properties of the 29 resultant oxime compounds, resulting in a myriad of reported pharmacological effects, which include presynaptic inhibition of acetylcholine release and nicotinic channel blocking activities, none has correlated well with the observed protection effects of these oximes against soman poisoning. Subsequent studies have since shifted to improving the reactivation efficacy of oximes against all classical nerve agents using various modifications of mono- and bis-disubstituted 2-[(hydroxyimino)methyl]imidazolium halides (Bedford et al. 1989). However, such correlation studies of oxime "direct effects" with observed protective capabilities remain important as primate studies with high doses of the oxime HI-6 have verified the survival from supra-lethal nerve agent poisoning without the need for acetylcholinesterase reactivation (Hamilton et al. 1989). Further research into such "non-reactivation therapeutic effects" may lead to discovery and/or optimisation of hitherto unexploited therapeutic mechanisms that could be at least as important as cholinesterase reactivation effects (Ellin 1982, Jepsen et al. 1988, Stalc et al. 1990). A different understanding of how oximes counter the effects of nerve agent intoxication could enhance the current repertoire of medical countermeasures against nerve agent poisoning by introducing new treatment approaches and/or assist in the regulatory approval of the Hagedorn class of bispyridinium oximes. While much efforts have been expended on modifying the bispyridinium ring structure to enhance its “direct therapeutic effects’ against nerve agents like soman, effects from modifications to the monopyridinium pralidoxime ring, 30 located within the HI-6 structure, on possible “non-reactivation therapeutic” effects is relatively less well studied. Pralidoxime itself is known to possess similar weak anti-muscarinic and AChE inhibition effects as HI-6 (Shih et al. 1991b and Loke et al. 2002), yet it possesses little of HI-6 protective effects against soman poisoning. Since the oxime ether (i.e., O-alkyl substituted oxime group) functionality shares common electrostatic properties and hence been successfully deployed as a bioisostere for the ester group in muscarinic ligands, it is possible to enhance the anti-muscarinic characteristics of the pralidoxime structure by adding alkyl groups of increasing size to the oxime functional group. While the resultant compounds would have lost their reactivation capability, they would also have gained additional anti-muscarinic properties that could directly antagonise against the cholinergic toxicity induced by nerve agent poisoning. Since central muscarinic pathways are intimately involved in the ensuing neurotoxicity and hence lethality of nerve agents, I decided to study whether modifications to the oxime functional group in a mono-pyridinium ring structure to enhance its muscarinic properties could result in novel “direct” therapeutic effects against nerve agent poisoning. If therapeutic effects were indeed observed, they would likely to be nonreactivation in nature since the O-alkyl substitution would deprive the oxime functional group of its enzyme reactivation capability. The objective for the main section of this thesis (Chapters 2-4) is hence to synthesize a series of structurally-related O-alkyl substituted 2-PAM derivatives and evaluate the possibility of increasing their anti-cholinergic and hence “direct” effects, of pralidoxime through attachment of alkyl groups of 31 increasing chain length to the oxime functional group. Addition of such alkyl groups were also intended to enhance the lipophilicity of the resultant oxime compounds and hopefully aid in their penetration through the blood-brainbarrier where they could exert their therapeutic effects against the central actions of nerve agents. From these studies, new understanding on novel therapeutic mechanisms by oxime compounds against the toxic effects induced rapidly “aging” nerve agent compounds like soman may be realised. 1.2. Muscarinic Receptor Agonist-Based Treatment of Alzheimer’s Disease 1.2.1. Biology of Alzheimer’s Disease Dementia Alzheimer’s disease (AD) is the most common form of degenerative dementia of the human central nervous system and is clinically characterized by a progressive impairment in intellectual abilities concomitant with drastic alternations in personality from mid- to late-adult life (Auld et al. 2002) . In its more advanced form, AD is typified by severe and wide-ranging cognitive deficits. AD is characterised by several distinct neuropathological features, which includes extracellular deposition of plaques, intracellular phosphorylated neurofibrillary -amyloid (A ) peptide-containing tangles composed of abnormally and degeneration of cholinergic neurons of the basal forebrain. The basal forebrain cholinergic system, which comprised of the nucleus basalis of Meynert (NBM), the horizontal and vertical diagonal bands of Broca (HDBB and VDBB) and the medial septal nucleus (MS), provides the primary cholinergic innervations to both limbic and cortical brain structures such 32 as the hippocampus, anterior cingulate cortex, olfactory bulb, amygdala, frontal, parietal and temporal cortex. Post-mortem studies on the AD brain confirmed the loss of ChAT activity, which is reflective of the observed loss of cholinergic neurons in the basal forebrain region. While cholinergic denervation is accepted as a major component of neuropathology of AD, it remains to be fully established whether the observed cholinergic neurodegeneration plays a key role in the observed decrements in cognitive symptoms of AD or in the pathogenesis of AD. 1.2.2. Cholinergic Hypothesis of Memory Dysfunction in Alzheimer’s Disease AD is characterized by A deposits in the brain, cholinergic deficits and cognitive impairment, hence the postulation arose that A -induced cholinergic dysfunction have play a role in the appearance of cognitive impairment in AD. Amyloid deposits in the AD brain, in the from of 40-43 peptide residues, is derived from proteolytic cleavage of a transmembrane glycoprotein family known as amyloid precursor protein (APP). A is reported to be a potent negative modulator of acetylcholine synthesis and release, interferes with normal signal transduction mechanisms mediated by muscarinic and reduces nicotinic currents. Reduction of acetylcholine release and cholinergic signalling reportedly results in feedback dysregulation of Nerve Growth Factor (NGF) and disrupt trophic factor homeostasis that is extremely important for the maintenance of a functional basal forebrain cholinergic system (Hellweg et al. 1997). Interestingly, acetylcholine is also known to promote the non- amyloidogenic processing of APP (Hellstrom-Lindahl et al. 2000). Hence, 33 reduction of acetylcholine release by A increases A may initiate a feedback loop that production through altered APP processing, thus starting a vicious destructive cycle resulting in progressively declining cholinergic activity in the basal forebrain. As there exists considerable psychopharmacological evidence indicating that systemic administration of cholinergic antagonists (e.g. scopolamine) to effect cholinergic blockade interferes with the memory function in rodents, non-human primates and humans, this led to the formation of the “cholinergic hypothesis of geriatric memory dysfunction” in AD (Bartus 2000). Support for this “cholingeric hypothesis came from studies that indicated that drugs that potentiate central cholinergic functions have thus far proven to be the most effective therapy against AD. 1.2.3. Cholinergic Strategies For Treatment of Alzheimer’s Disease Induced Cognitive Impairments From observations that correlated severity of basal forebrain cholinergic deficits in AD with extend of cognitive impairments observed in this disease, the natural progression of such observations led to the development of cholinergic strategies for the treatment of Alzheimer’s disease. While the ultimate intention is to boost cholinergic function in the AD brain, the strategies attempted are different and could be classified into the categories described briefly below: 1.2.3.1. Cholinergic Precursors Initial attempts at overcoming cholinergic deficit in AD patients looked into replacement therapy with acetylcholine precursors such as choline, lecithin and acetyl-L-carnitine. However, majority of studies that took this approach failed to 34 provide clear beneficial effects on ameliorating AD symptoms. The failure of this approach was not surprising as acetylcholine synthesis precursors are usually not the rate-limiting factor in acetylcholine synthesis. The limited penetration of such cholinergic precursors through the blood-brain barrier and their brevity in systemic circulation are further factors that limit the utility of this approach. 1.2.3.2. Acetylcholinesterase Inhibitors A different and more successful approach in AD treatment was the application of acetylcholinesterase inhibitors (AChEI), which prevent acetylcholine degradation in the synaptic cleft and hence extend the half-life of acetylcholine at the synapses. The extended duration of action of acetylcholine at postsynaptic muscarinic receptors would help ameliorate A -induced reduction in muscarinic M1 signalling, especially since A achieved this effect without modifying receptor binding kinetics of acetylcholine (Kelly et al. 1996). Three generations of AChEI have been developed and marketed for successful clinical treatment of AD cognitive dysfunctions. The first generation of AChEI, Tacrine and physostigmine, have major clinical limitations due to low bioavailability and undesirable side-effects that accompanied only moderate restorations of cognitive status. Second generation AChEIs such as Donepezil (Aricept, Pfizer Inc), Rivastigmine (Exelon, Novartis Pharmaceuticals) and Galantamine (Reminyl Janssen-Cigal) obtained FDA approval over the period of 1996-2001. These new AChEI are all reversible and well-tolerated inhibitors of acetylcholinesterase with improved potency, favourable pharmacokinetics and safety profiles compared to the first generation AChEIs. Third generation 35 AChEIs, which includes Huperzine A and Metrifonate, intends to improve on the modest therapeutic improvements in cognition achieved by the second generation drugs. However, there appears to be a limitation in this approach of increasing local acetylcholine levels through inhibition of synaptic acetylcholinesterase as repeated doses of AChEI would lead to the development of tolerance through either up-regulation in synthesis of acetylcholinesterase or further down-regulation of post-synaptic muscarinic receptors. Nonetheless, AChEI is presently the only approved FDA therapeutic option for AD. 1.2.3.3. Modulation of Nicotinic Receptors While there is at present no firm evidence that nicotinic agonists are useful in treatment of AD cognitive impairment, there has been substantial research ongoing to develop allosterically potentiating ligands that bind to a site on nicotinic receptors distinct from acetylcholine. The intention is to enhance the excitability of the nicotinic receptor to acetylcholine. Galathamine has a dual action in that besides acting as a AChEI, it also acts as an allosteric modulator on nicotinic receptors to enhance neurotransmission. Besides Galathamine, the other potential drug candidates are currently only at Phase I studies and it remained to be seen what is the true potential of this approach. 1.2.3.4. Modulation of Muscarinic Receptors Last but not least, as postsynaptic M1 muscarinic receptor subtype is relatively spared in the AD brain (Araujo et al. 1988), development of M1 specific muscarinic agonists to target these sites have become an important research 36 area. While current M1 agonist drug candidates such as xanomeline, sabcomeline and alavameline possess low agonist activity, low oral bioavailability, rapid metabolism and hence clearance in the body, lack of selectivity for M1 receptor resulting in serious side effects at clinically effective dose to treat AD cognitive dysfunctions and hence disappointing clinical outcomes, there remained research interest in this field for two reasons. The first and most obvious reason is that muscarinic M1-selective agonist may be effective in symptomatic treatment of AD regardless of extent of degeneration of presynaptic cholinergic projects in the brain and hence may present a viable option for AD management when acetylcholinesterase inhibitors no longer work due to development of innate tolerance to the actions of these inhibitors. The second reason is that cholinergic stimulation of M1 receptor has been reported to increase the cleavage of amyloid precursor proteins (APP), by a yet identified protease designated as -secretase, to produce the secreted non- amyloidogenic APP, this preventing further formation of A (Fisher 1999). Hence, it is postulated that maintaining cholinergic stimulation at M1 receptor would provide protective actions against the initiation of the vicious feedback loop that increases A production in the absence of cholinergic stimulation leading to progressively declining cholinergic activity in the basal forebrain. Because of such new findings, there is still continued hope that improved versions of M1 agonist drug candidates such as Cevimeline (Phase III Trials), may represent the next generation of therapies in Alzheimer’s disease. 37 1.2.4. Design of Full Muscarinic Agonists Most of the muscarinic agonist identified to date possess features found in acetylcholine. Consequently, structural modifications of existing muscarinic agonists such as arecoline, pilocarpine, acelidine, muscarine and even acetylcholine (Table 5) have been used as a starting point for synthesis. From these studies, the pharmacophore for muscarinic agonist properties have been identified to include the following features : a. A basic N-atom are or quaternary derivative Structures with the basic N-atom or non-quaternary amines have better penetration through the blood-brain barrier but relatively lower agonist properties whereas structures with the positive charged quaternary amine structure have better agonist properties but are unable to reach the brain; b. A Small Lipophilic Region Muscarinic agonists not tolerate steric bulk (Tecle et al. 1993), hence addition of bulky substituents often convert potential agonist structures into muscarinic antagonist; c. Heteroatom Sites As Hydrogen Bond Acceptor Regions In acetylcholine, the ether and carbonyl oxygen atoms provide electronegative potential zones that could function as electron donors to facilitate hydrogen bonding with the muscarinic active sites. Several bioisosteres of the ester function has been identified as potential replacement for the labile ester functional group on the basis of similar distribution in electronegative potentials as the ester functional group 38 Table History of Synthetic Approaches Used in Creation of M1 Selective Agonists By Modifications of Known Muscarinic Agonists or Heterocyclic Structures (adapted from ref: Fisher 1999) Prototype Agonist Arecoline Analogues of 1,2,5-Thiadiazole Lead Compounds Xanomelinea Conformational Rigidity Flexible Oxadiazole L-687, 306 Flexible Tetrazole Lu 25109 Flexible Oxime Milameline (CI 979) PD14250b Flexible PD 151832b Semi-Rigid CDD 0078 Flexible CDD0097 Flexible CDD 0102 Flexible Thiopilocarpine (SDZ ENS 163) YM 796 Flexible Oxime with Extended Alkyl or Alkynyl Chain Amidine Pilocarpine Thuipilocarpine Muscarone Isoxazolidine Carbachol Semi-Rigid Semi-Rigid Carbamate WAY 131256 Flexible Quinuclidine Sabcomeline (SB 202023) Tasaclidine (WAL 2014) AF102B Semi-rigid Acetylcholine/Aceclidine Oxathiolane Quinuclidine Acetylcholine Semi-Rigid Semi-Rigid Rigid Rigid Oxazoline AF105b Semi-Rigid Isooxazoline AF151 Semi-Rigid Thiazoline AF150 (S) Semi-Rigid Isothiazoline AF151 (S) Semi-Rigid a : More Potent M4 Than M1 Receptor Agonist b : Shows a predominant Muscarinic M1 Selectivity (as an agonist; Tecle et al. 1993), 39 Previous investigations have proposed that the oxime ether is a viable alternative bioisostere to the chemically labile ester functional group, present in naturally occurring muscarinic agonists, on the basis of similar electrostatic potential maps of the methyl carboxylate ester and the aldoxime/ketoxime methyl ether groups (Bromidge et al. 1995) . In comparison, the ether functional group is stable and readily introduced into synthetic pathways and hence offers a convenient handle for structural and physicochemical modifications. Moreover, oxime derivatives have been suggested as muscarinic ligands with a potential M1 profile (Plate et al. 1996). Recent studies that have utilised the oxime functional group as the basis for constructing structural analogues of muscarinic agonists have reported successful attainment of muscarinic agonist properties (Xu et al. 1998, Angeli et al. 2002, Somanadhan et al. 2002). Such agonist properties were obtained when the oxime functional group attached to a small molecule such as deoxamuscarine, or to a rigid parent structure such as quinuclidinone, or even the more flexible tropinone structure. The presence of agonist behaviour with the tropinone oxime (Xu 1998) was intriguing as rigid ring structures have consistently been associated with good muscarinic agonist activity. In the last section of this thesis, we re-investigated the requirement for rigidity in the basic nitrogen ring system as an absolute requirement for muscarinic agonist activity by evaluating the muscarinic properties of two series of oxime derivatives attached respectively to a rigid quinuclidinone and flexible tropinone structure. 40 [...]... al 19 86, 19 87, 19 88a, 19 88b, 19 96, Loke et al 20 01, 2004) There could hence be more than one neurochemical mechanisms responsible for induction and propagation of neuropathology following nerve agent mediated seizures 1. 1.2 Medical Countermeasures For Nerve Agent Poisoning Medical countermeasure for nerve agent poisoning can be divided into four stages: 1. 1.2 .1 Pre-treatment, 1. 1.2.2 Administration of. .. been a multitude of findings associating various pharmacological effects with oxime reactivators (French et al 19 83, Kloog et al 19 85a, Su et al 19 86, Alkondon 19 88, 19 89, Tattersall 19 93) Most of the reported research work on “direct effects of oximes have largely centred on modifications of the bispyridinium oxime structures, where substation of various functional groups on either of the pyridinium... reactivation (Hamilton et al 19 89) Further research into such "non-reactivation therapeutic effects" may lead to discovery and/or optimisation of hitherto unexploited therapeutic mechanisms that could be at least as important as cholinesterase reactivation effects (Ellin 19 82, Jepsen et al 19 88, Stalc et al 19 90) A different understanding of how oximes counter the effects of nerve agent intoxication... that oximes like HI-6 owed their therapeutic effects to other direct therapeutic mechanism not related to acetylcholinesterase reactivation (van Helden et al 19 91, 19 94, 19 96) This conclusion arises from observations that non-human primates survived soman and tabun intoxication following administration of HI-6 although reactivation of inhibited AChE was not observed (Hamilton et al 19 89) These direct effects. .. clinicians on the usefulness on the new Hagedorn class of oximes (obidoxime/HI-6) for management of nerve agent-poisoned patients Table 4 Rate of Aging of Nerve Agent-RBC Acetylcholinesterase Complex Nerve Agent Biological half-life of Agent-AChE Complex VX > 48 hr Tabun 14 hr Sarin 5 hr Soman 2-6 min 28 1. 1.2.5 Non-Reactivation Pharmacological Effects Of Oximes However, in the last decade, there have been... 19 87), has often been used and cited in literature (Dawson 19 94) as the most appropriate model for extrapolation of oxime antidotal effectiveness to human situation While the use of kinetic parameters helped to ascertain efficacy of oximes on human acetylcholinesterase, it should also be noted the reactivation potency of certain oximes is markedly reduced by the formation of stable phosphorylated oximes. .. G.P H et al 19 95) 11 A Piriform Cortex - Control B Hippocampus - Control III II I hilus C Piriform Cortex – 1 day post soman treatment D Hippocampus – 1 day post soman treatment E Piriform Cortex – 1 week post soman treatment F Hippocampus – 1 week post soman treatment Figure 3 Cellular Pathology in Soman (1. 6 x LD50) Treatment in Piriform Cortex (PC) - Control (A), 1 day Post-Soman (C), 1 week Post-Soman... divided into four stages: 1. 1.2 .1 Pre-treatment, 1. 1.2.2 Administration of Anticholinergic Drugs, 1. 1.2.3 Anticonvulsant and Neuroprotection Therapy, 1. 1.2.4 Administration of Oxime Reactivators 13 The nature of medical therapy in each stage will be further elaborated in the following sections: 1. 1.2 .1 Pyridostigmine Pretreatment in Nerve Agent Poisoning Pre-treatment for nerve agent intoxication is... gastrointestinal tract with a bioavailability of the administered dose at 5 -10 % Despite its poor bioavailability, enough of the drug is absorbed to cause an inhibition of 2579% of serum butyrylcholinesterase activity A single dose of 30 mg pyridostigmine is able to achieve 25% reversible inhibition of acetylcholinesterase (AChE) within 5h of administration (Sharab et al 19 91) The time taken by the drug to reach... elimination half-life of 2.6-4.3 h and an apparent volume of distribution (VD) of 1- 1.7 l/kg while its clearance rate is 5.9-6.8 ml/kg per min The elimination half-life is longer in children under 2 years of age and in the elderly where half-life may be prolonged to 10 -30 h due to reduced clearance (Heath et al 19 92) Although the adequacy of atropinization is usually determined by drying of secretions, there . Molecular Wt. 16 2 .1 14 0 .1 18 2.2 18 0.2 267.4 Density (g/cm 3 ) at 25 O C 1. 073 1. 089 1. 022 1. 12 1. 008 Boiling Point ( O C) (760 mm Hg) 247 15 8 16 7 92 (10 mm Hg) . 1 Chapter 1 Introduction 1. 1. Design of Oxime Compounds With Non- Reactivation Therapeutic Effects Against Nerve Agent Soman 1. 1 .1. Toxicology of Nerve Agent Poisoning . et al. 19 94, J.H. McDonough et al. 19 86, 19 95, 19 96) with the pattern of injury resembling those described for status epilepticus (Oxbury et al. 19 71, Meldrum 19 73, Corsellis et al. 19 83, Shorvon