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

1 © Contents 1.Nerve Transmission (3 slides) 2.Neurotransmitter 3.Transmission process (10 slides) 4.Cholinergic receptors (2 slides) 4.1.Nicotinic receptor (2 slides) 4.2.Muscarinic receptor - G Protein coupled receptor (2 slides) 5.Cholinergic agonists 5.1.Acetylcholine as an agonist 5.2.Nicotine and muscarine as cholinergic agonists 5.3.Requirements for cholinergic agonists 6.SAR for acetlcholine (6 slides) 7.Binding site (muscarinic) (3 slides) 8.Active conformation of acetylcholine (2 slides) 9.Instability of acetylcholine 10.Design of cholinergic agonists (7 slides) 11.Uses of cholinergic agonists (2 slides) [46 slides]

1 © CHOLINERGICNERVOUSSYSTEM

1 © 1. Nerve Transmission Peripheral nervous system Peripheral nerves Muscle Gastro- intestinal tract (GIT) Brain Spinal cord CNS Heart

1 © 1. Nerve Transmission Peripheral nervous system CNS (Somatic) CNS (Autonomic) Sympathetic Parasympathetic NA Ach (N) Synapse Ach (N) Ach (N) Ach (N) Ach (M) Adrenal medulla Adrenaline Skeletal muscle Synapse AUTONOMIC Smooth muscle Cardiac muscle

1 © Nerve 1. Nerve Transmission Synapses A Receptors Release of neurotransmitters Receptor binding and new signal Nerve impulse New signal Vesicles containing neurotransmitters

1 © 2. Neurotransmitter Acetylcholine (Ach) CholineAcetyl H 3 CO CNMe 3 O +

1 © 3. Transmission process Signal in nerve 1 Acetylcholinesterase enzyme Cholinergic receptor.Acetylcholine Vesicle Nerve 1 Nerve 2 Signal

1 © 3. Transmission process Vesicles fuse with membrane and release Ach Nerve 1 Nerve 2 Signal

1 © 3. Transmission process Nerve 2

1 © 3. Transmission process Receptor binds AchReceptor binds Ach Induced fit triggers 2 o messageInduced fit triggers 2 o message Triggers firing of nerve 2Triggers firing of nerve 2 Ach undergoes no reactionAch undergoes no reaction 2 o Message

1 © 3. Transmission process Ach departs receptorAch departs receptor Receptor reverts to resting stateReceptor reverts to resting state Ach binds to acetylcholinesteraseAch binds to acetylcholinesterase Nerve 2

1 © 3. Transmission process Ach hydrolysed by acetylcholinesterase Nerve 2

1 © 3. Transmission process Choline binds to carrier protein Carrier protein for choline Nerve 1 Nerve 2 Choline

1 © 3. Transmission process Choline transported into nerve Nerve 1 Nerve 2

1 © 3. Transmission process Ach resynthesised Nerve 1 Nerve 2 E 1 = Choline acetyltransferase

1 © 3. Transmission process Ach repackaged in vesicles Nerve 1 Nerve 2

1 © 4. Cholinergic receptors Receptor types Not all cholinergic receptors are identicalNot all cholinergic receptors are identical Two types of cholinergic receptor - nicotinic and muscarinicTwo types of cholinergic receptor - nicotinic and muscarinic Named after natural products showing receptor selectivityNamed after natural products showing receptor selectivity Acetylcholine is natural messenger for both receptor types Activates cholinergic receptors at nerve synapses and on skeletal muscle Activates cholinergic receptors on smooth muscle and cardiac muscle Nicotine L-(+)-Muscarine

1 © Peripheral nervous system CNS (Somatic) CNS (Autonomic) Sympathetic Parasympathetic NA Ach (N) Synapse Ach (N) Ach (N) Ach (N) Ach (M) Adrenal medulla Adrenaline Skeletal Muscle SOMATIC Synapse AUTONOMIC Smooth Muscle Cardiac Muscle

1 © Cell membrane Five glycoprotein subunits traversing cell membrane Receptor Binding site Messenger Control of Cationic Ion Channel: 4.1 Nicotinic receptor Cell membrane Induced fit ‘Gating’ (ion channel opens)

1 © The binding sites      Two ligand binding sites mainly on  -subunits Ion channel  x  subunits Bindingsites 4.1 Nicotinic receptor Cell membrane    

1 © Activation of a signal protein Receptor binds messenger leading to an induced fitReceptor binds messenger leading to an induced fit Opens a binding site for a signal protein (G-protein)Opens a binding site for a signal protein (G-protein) closed messenger induced fit open 4.2 Muscarinic receptor - G Protein coupled receptor G-protein bound G-protein split

1 © Activation of membrane bound enzyme G-Protein is split and subunit activates a membrane bound enzymeG-Protein is split and subunit activates a membrane bound enzyme Subunit binds to an allosteric binding site on enzymeSubunit binds to an allosteric binding site on enzyme Induced fit results in opening of an active siteInduced fit results in opening of an active site Intracellular reaction is catalysedIntracellular reaction is catalysed active site (closed) active site (open) Enzyme Intracellular reaction 4.2 Muscarinic receptor - G Protein coupled receptor subunit

1 © 5. Cholinergic agonists 5.1 Acetylcholine as an agonist Advantages Natural messengerNatural messenger Easily synthesisedEasily synthesised Disadvantages Easily hydrolysed in stomach (acid catalysed hydrolysis)Easily hydrolysed in stomach (acid catalysed hydrolysis) Easily hydrolysed in blood (esterases)Easily hydrolysed in blood (esterases) No selectivity between receptor typesNo selectivity between receptor types No selectivity between different target organsNo selectivity between different target organs

1 © 5. Cholinergic agonists 5.2 Nicotine and muscarine as cholinergic agonists Advantages More stable than AchMore stable than Ach Selective for main cholinergic receptor typesSelective for main cholinergic receptor types Selective for different organsSelective for different organs Disadvantages Activate receptors for other chemical messengersActivate receptors for other chemical messengers Side effectsSide effects

1 © 5. Cholinergic agonists 5.3 Requirements for cholinergic agonists Stability to stomach acids and esterasesStability to stomach acids and esterases Selectivity for cholinergic receptorsSelectivity for cholinergic receptors Selectivity between muscarinic and nicotinic receptorsSelectivity between muscarinic and nicotinic receptors Knowledge of binding siteKnowledge of binding site SAR for acetylcholineSAR for acetylcholine

1 © AcetoxyEthylenebridge4 o Nitrogen Nitrogen AcetoxyEthylenebridge 6. SAR for acetlcholine Quaternary nitrogen is essential Bad for activity

1 © Distance from quaternary nitrogen to ester is importantDistance from quaternary nitrogen to ester is important Ethylene bridge must be retainedEthylene bridge must be retained 6. SAR for acetylcholine Bad for activity AcetoxyEthylenebridge4 o Nitrogen Nitrogen AcetoxyEthylenebridge

1 © Ester is important 6. SAR for acetylcholine Bad for activity AcetoxyEthylenebridge4 o Nitrogen Nitrogen AcetoxyEthylenebridge

1 © Minimum of two methyl groups on quaternary nitrogen 6. SAR for acetylcholine Bad for activity Active AcetoxyEthylenebridge4 o Nitrogen Nitrogen AcetoxyEthylenebridge4 o

1 © Methyl group of acetoxy group cannot be extended 6. SAR for acetylcholine Bad for activity AcetoxyEthylenebridge4 o Nitrogen Nitrogen AcetoxyEthylenebridge

1 © Conclusions: Tight fit between Ach and binding siteTight fit between Ach and binding site Methyl groups fit into small hydrophobic pocketsMethyl groups fit into small hydrophobic pockets Ester interacting by H-bondingEster interacting by H-bonding Quaternary nitrogen interacting by ionic bondingQuaternary nitrogen interacting by ionic bonding 6. SAR for acetylcholine AcetoxyEthylenebridge4 o Nitrogen Nitrogen

1 © Trp-307 Asp311 Trp-613 Trp-616 Asn Binding site (muscarinic) hydrophobic pocket hydrophobic pocket hydrophobic pockets

1 © Trp-307 Asp311 Trp-613 Trp-616 Asn Binding site (muscarinic) H-bonds Ionic bond vdw

1 © Possible induced dipole dipole interaction between quaternary nitrogen and hydrophobic aromatic rings in binding sitePossible induced dipole dipole interaction between quaternary nitrogen and hydrophobic aromatic rings in binding site N + induces dipole in aromatic ringsN + induces dipole in aromatic rings 7. Binding site (muscarinic)  -  +

1 © Several freely rotatable single bondsSeveral freely rotatable single bonds Large number of possible conformationsLarge number of possible conformations Active conformation does not necessarily equal the most stable conformationActive conformation does not necessarily equal the most stable conformation 8. Active conformation of acetylcholine

1 © Rigid Analogues of acetylcholine Rotatable bonds ‘locked’ within ringRotatable bonds ‘locked’ within ring Restricts number of possible conformationsRestricts number of possible conformations Defines separation of ester and NDefines separation of ester and N 8. Active conformation of acetylcholine Muscarinic receptor Nicotinic receptor

1 © Neighbouring group participationNeighbouring group participation Increases electrophilicity of carbonyl groupIncreases electrophilicity of carbonyl group Increases sensitivity to nucleophilesIncreases sensitivity to nucleophiles 9. Instability of acetylcholine

1 © 10. Design of cholinergic agonists Requirements Correct sizeCorrect size Correct pharmacophore - ester and quaternary nitrogenCorrect pharmacophore - ester and quaternary nitrogen Increased stability to acid and esterasesIncreased stability to acid and esterases Increased selectivityIncreased selectivity

1 © Use of steric shields Rationale Shields protect ester from nucleophiles and enzymesShields protect ester from nucleophiles and enzymes Shield size is importantShield size is important Must be large enough to hinder hydrolysisMust be large enough to hinder hydrolysis Must be small enough to fit binding siteMust be small enough to fit binding site 10. Design of cholinergic agonists

1 © Methacholine Properties Three times more stable than acetylcholineThree times more stable than acetylcholine Increasing the shield size increases stability but decreasesIncreasing the shield size increases stability but decreasesactivity Selective for muscarinic receptors over nicotinic receptorsSelective for muscarinic receptors over nicotinic receptors S-enantiomer is more active than the R-enantiomerS-enantiomer is more active than the R-enantiomer Stereochemistry matches muscarineStereochemistry matches muscarine Not used clinicallyNot used clinically asymmetric centre hinders binding to esterases and provides a shield to nucleophilic attack

1 © 10. Design of cholinergic agonists Use of electronic factors Replace ester with urethaneReplace ester with urethane Stabilises the carbonyl groupStabilises the carbonyl group =

1 © 10. Design of cholinergic agonists Properties Resistant to hydrolysisResistant to hydrolysis Long lastingLong lasting NH 2 and CH 3 are equal sizes. Both fit the hydrophobic pocketNH 2 and CH 3 are equal sizes. Both fit the hydrophobic pocket NH 2 = bio-isostereNH 2 = bio-isostere Muscarinic activity = nicotinic activityMuscarinic activity = nicotinic activity Used topically for glaucomaUsed topically for glaucoma Carbachol

1 © 10. Design of cholinergic agonists Steric + Electronic factors Properties Very stableVery stable Orally activeOrally active Selective for the muscarinic receptorSelective for the muscarinic receptor Used to stimulate GI tract and urinary bladder after surgeryUsed to stimulate GI tract and urinary bladder after surgery Bethanechol

1 © 10. Design of cholinergic agonists Nicotinic selective agonist

1 © 11. Uses of cholinergic agonists Nicotinic selective agonists Treatment of myasthenia gravis - lack of acetylcholine at skeletal muscle causing weakness - lack of acetylcholine at skeletal muscle causing weakness Muscarinic selective agonists Treatment of glaucomaTreatment of glaucoma Switching on GIT and urinary tract after surgerySwitching on GIT and urinary tract after surgery Treatment of certain heart defects. Decreases heart muscle activity and decreases heart rateTreatment of certain heart defects. Decreases heart muscle activity and decreases heart rate

1 © Peripheral nervous system CNS (Somatic) CNS (Autonomic) Sympathetic Parasympathetic NA Ach (N) Synapse Ach (N) Ach (N) Ach (N) Ach (M) Adrenal medulla Adrenaline Skeletal Muscle SOMATIC Synapse AUTONOMIC Smooth Muscle Cardiac Muscle