1 © Patrick An Introduction to Medicinal Chemistry 3/e Chapter 19 CHOLINERGICS, ANTICHOLINERGICS & ANTICHOLINESTERASES Part 3: Cholinergics & anticholinesterases

1 © Contents 14.Acetylcholinesterase 14.1.Role 14.2.Hydrolysis reaction catalysed 14.3.Effect of inhibition 14.4.Structure of enzyme complex 14.5.Active site - binding interactions 14.6.Active site - Mechanism of catalysis (3 slides) 15.Anticholinesterases 15.1.Physostigmine 15.2.Mechanism of action (3 slides) 15.3.Physostigmine analogues 15.4.Organophosphates (9 slides) 15.5.Anticholinesterases as ‘Smart Drugs’ (2 slides) [30 slides]

1 © 14. Acetylcholinesterase Acetylcholinesterase enzyme Nerve 1 Nerve 2 Signal 14.1 Role Hydrolysis and deactivation of acetylcholineHydrolysis and deactivation of acetylcholine Prevents acetylcholine reactivating receptorPrevents acetylcholine reactivating receptor

1 © 14. Acetylcholinesterase activeinactive 14.2 Hydrolysis reaction catalysed

1 © 14. Acetylcholinesterase Inhibitor blocks acetylcholinesteraseInhibitor blocks acetylcholinesterase Ach is unable to bindAch is unable to bind Ach returns to receptor and reactivates itAch returns to receptor and reactivates it Enzyme inhibitor has the same effect as a cholinergic agonistEnzyme inhibitor has the same effect as a cholinergic agonist 2 o Message Nerve 2 Enzyme inhibitor (Anticholinesterase) 14.3 Effect of inhibition Ach

1 © 14. Acetylcholinesterase 14.4 Structure of enzyme complex

1 © Serine Histidine Tyrosine Aspartate 14. Acetylcholinesterase 14.5 Active site - binding interactions Anionic binding region similar to cholinergic receptor siteAnionic binding region similar to cholinergic receptor site Binding and induced fit strains Ach and weakens bondsBinding and induced fit strains Ach and weakens bonds Molecule positioned for reaction with His and SerMolecule positioned for reaction with His and Ser vdwvdw hydrophobic pockets Anionic binding region Ester binding region H-bond Ionic

1 © 14. Acetylcholinesterase 14.6 Active site - Mechanism of catalysis Acid catalyst

1 © 14. Acetylcholinesterase 14.6 Active site - Mechanism of catalysis ROH C O CH 3 O N NH Histidine Basic catalyst

1 © 14. Acetylcholinesterase 14.6 Active site - Mechanism of catalysis C O OHCH 3 O N NH : : : : H : Histidine (Acid catalyst) C O OHCH 3 O N NH : _ H : :: Histidine Basic catalyst

1 © 14. Acetylcholinesterase Serine and water are poor nucleophilesSerine and water are poor nucleophiles Mechanism is aided by histidine acting as a basic catalystMechanism is aided by histidine acting as a basic catalyst Choline and serine are poor leaving groupsCholine and serine are poor leaving groups Leaving groups are aided by histidine acting as an acid catalystLeaving groups are aided by histidine acting as an acid catalyst Very efficient x 10 6 faster than uncatalysed hydrolysisVery efficient x 10 6 faster than uncatalysed hydrolysis Acetylcholine hydrolysed within 100  secs of reaching active siteAcetylcholine hydrolysed within 100  secs of reaching active site An aspartate residue is also involved in the mechanismAn aspartate residue is also involved in the mechanism

1 © 14. Acetylcholinesterase The catalytic triad An aspartate residue interacts with the imidazole ring of histidine to orientate and activate itAn aspartate residue interacts with the imidazole ring of histidine to orientate and activate it

1 © 15. Anticholinesterases Inhibitors of acetylcholinesterase enzymeInhibitors of acetylcholinesterase enzyme Block hydrolysis of acetylcholineBlock hydrolysis of acetylcholine Acetylcholine is able to reactivate cholinergic receptorAcetylcholine is able to reactivate cholinergic receptor Same effect as a cholinergic agonistSame effect as a cholinergic agonist

1 © 15. Anticholinesterases 15.1 Physostigmine Natural product from the African calabar beanNatural product from the African calabar bean Carbamate is essential (equivalent to ester of Ach)Carbamate is essential (equivalent to ester of Ach) Aromatic ring is importantAromatic ring is important Pyrrolidine N is important (ionised at blood pH)Pyrrolidine N is important (ionised at blood pH) Pyrrolidine N is equivalent to the quaternary nitrogen of AchPyrrolidine N is equivalent to the quaternary nitrogen of Ach Pyrrolidine N

1 © Physostigmine 15.2 Mechanism of action

1 © Rate of hydrolysis slower by 40 x 10 6 Stable carbamoyl intermediate Hydrolysis very slow O C O :N NH MeNH -ArOH

1 © 15.2 Mechanism of action Carbonyl group 'deactivated' : O C ON Me H : : : N H Me C O O

1 © 15.3 Physostigmine analogues Simplified analogueSimplified analogue Susceptible to hydrolysisSusceptible to hydrolysis Crosses BBB as free baseCrosses BBB as free base CNS side effectsCNS side effects Fully ionisedFully ionised Cannot cross BBBCannot cross BBB No CNS side effectsNo CNS side effects More stable to hydrolysisMore stable to hydrolysis Extra N-methyl group increases stabilityExtra N-methyl group increases stability Miotine (ionised at blood pH) Neostigmine

1 © Water Hydrolysis mechanisms Possible mechanism 1

1 © -H Compare: -Me No hydrolysis Too reactive Hydrolysis mechanisms Possible mechanism 2 H2OH2OH2OH2O

1 © 15.4 Organophosphates a) Nerve gases Agents developed in World War 2Agents developed in World War 2 Agents irreversibly inhibit acetylcholinesteraseAgents irreversibly inhibit acetylcholinesterase Permanent activation of cholinergic receptors by AchPermanent activation of cholinergic receptors by Ach Results in deathResults in death Dyflos (Diisopropyl fluorophosphonate) Sarin

1 © 15.4 Organophosphates b) Mechanism of action Irreversible phosphorylationIrreversible phosphorylation P-O bond very stableP-O bond very stable STABLE -H Serine

1 © c) Medicinal organophosphate Used to treat glaucomaUsed to treat glaucoma Topical applicationTopical application Quaternary N is added to improve binding interactionsQuaternary N is added to improve binding interactions Results in better selectivity and lower, safer dosesResults in better selectivity and lower, safer doses Ecothiopate 15.4 Organophosphates

1 © d) Organophosphates as insecticides Relatively harmless to mammalsRelatively harmless to mammals Agents act as prodrugs in insectsAgents act as prodrugs in insects Metabolised by insects to produce a toxic metaboliteMetabolised by insects to produce a toxic metabolite Parathion Malathion 15.4 Organophosphates

1 © d) Organophosphates as insecticides MAMMALSINSECTS PARATHION (Inactive Prodrug) Active drug Phosphorylates enzyme DEATH Mammalian Metabolism INACTIVE & excreted Insect Oxidative desulphurisation 15.4 Organophosphates

1 © e) Design of Organophosphate Antidotes Strategy Strong nucleophile required to cleave strong P-O bondStrong nucleophile required to cleave strong P-O bond Find suitable nucleophile capable of cleaving phosphate estersFind suitable nucleophile capable of cleaving phosphate esters Water is too weak as a nucleophileWater is too weak as a nucleophile Hydoxylamine is a stronger nucleophileHydoxylamine is a stronger nucleophile Hydroxylamine is too toxic for clinical useHydroxylamine is too toxic for clinical use Increase selectivity by increasing binding interactions with active siteIncrease selectivity by increasing binding interactions with active site Hydroxylamine 15.4 Organophosphates

1 © e) Design of Organophosphate Antidotes Quaternary N is added to bind to the anionic regionQuaternary N is added to bind to the anionic region Side chain is designed to place the hydroxylamine moiety in the correct position relative to phosphorylated serineSide chain is designed to place the hydroxylamine moiety in the correct position relative to phosphorylated serine Pralidoxime 1 million times more effective than hydroxylaminePralidoxime 1 million times more effective than hydroxylamine Cannot act in CNS due to charge - cannot cross bbbCannot act in CNS due to charge - cannot cross bbb Pralidoxime 15.4 Organophosphates

1 © e) Design of Organophosphate Antidotes SER Active Site (Free) SER Active Site (Blocked) 15.4 Organophosphates

1 © e) Design of Organophosphate Antidotes Prodrug for pralidoximeProdrug for pralidoxime Passes through BBB as free basePasses through BBB as free base Oxidised in CNS to pralidoximeOxidised in CNS to pralidoxime ProPAM 15.4 Organophosphates

1 © 15.5 Anticholinesterases as ‘Smart Drugs’ Act in CNSAct in CNS Must cross blood brain barrierMust cross blood brain barrier Used to treat memory loss in Alzheimers diseaseUsed to treat memory loss in Alzheimers disease Alzheimers causes deterioration of cholinergic receptors in brainAlzheimers causes deterioration of cholinergic receptors in brain Smart drugs inhibit Ach hydrolysis to increase activity at remaining receptorsSmart drugs inhibit Ach hydrolysis to increase activity at remaining receptors

1 © 15.5 Anticholinesterases as ‘Smart Drugs’ Donepezil Tacrine (Cognex) Toxic side effects Rivastigmine (Exelon) (analogue of physostigmine) Galanthamine (daffodil and snowdrop bulbs Metrifonate (organophosphate) Xanomeline Anabaseine (ants and marine worms)