Cholinergic Drug Lecture 4-7 By Nohad A AlOmari Atrushi 28/10/2014 2/11/2014 4/10/2014

Contents: 1. Review to lecture 1 & 2 2. SAR of ACH 3. Binding affinity of ACH to Cholinergic receptor : 4. COLINERGIC DRUGS Classifications

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

Cholinergic agonists Acetylcholine as an agonist Advantages Natural messenger Easily synthesised Disadvantages Easily hydrolysed in stomach (acid catalysed hydrolysis) Easily hydrolysed in blood (esterases) No selectivity between receptor types No selectivity between different target organs

.SAR for acetylcholine Quaternary nitrogen is essential Acetoxy Ethylene bridge Nitrogen Acetoxy 4 o Nitrogen Quaternary nitrogen is essential Bad for activity

SAR for acetylcholine Acetoxy Ethylene bridge Nitrogen Acetoxy Ethylene bridge 4 o Nitrogen Distance from quaternary nitrogen to ester is important Ethylene bridge must be retained Bad for activity

SAR for acetylcholine Ester is important Bad for activity Acetoxy Ethylene bridge Nitrogen Acetoxy Ethylene bridge 4 o Nitrogen Ester is important Bad for activity

SAR for acetylcholine Acetoxy Ethylene bridge 4 o Nitrogen Acetoxy Ethylene bridge 4 o Nitrogen Minimum of two methyl groups on quaternary nitrogen Bad for activity Active

SAR for acetylcholine Methyl group of acetoxy group cannot be extended Ethylene bridge Nitrogen Acetoxy Ethylene bridge 4 o Nitrogen Methyl group of acetoxy group cannot be extended Bad for activity

SAR for acetylcholine Conclusions: Acetoxy Ethylene bridge 4 o Nitrogen Conclusions: Tight fit between Ach and binding site Methyl groups fit into small hydrophobic pockets Ester interacting by H-bonding Quaternary nitrogen interacting by ionic bonding

Binding site (muscarinic) Trp-307 Asp311 Trp-613 Trp-616 Asn-617 hydrophobic pocket pockets

Binding site (muscarinic) Trp-307 Asp311 Trp-613 Trp-616 Asn-617 vdw Ionic bond H-bonds

Binding site (muscarinic) Possible induced dipole dipole interaction between quaternary nitrogen and hydrophobic aromatic rings in binding site N+ induces dipole in aromatic rings d + d -

Active conformation of acetylcholine Several freely rotatable single bonds Large number of possible conformations Active conformation does not necessarily equal the most stable conformation

Active conformation of acetylcholine Rigid Analogues of acetylcholine Rotatable bonds ‘locked’ within ring Restricts number of possible conformations Defines separation of ester and N Muscarinic receptor Nicotinic receptor

Instability of acetylcholine Neighbouring group participation Increases electrophilicity of carbonyl group Increases sensitivity to nucleophiles

Requirements Design of cholinergic agonists Correct size Correct pharmacophore - ester and quaternary nitrogen Increased stability to acid and esterases Increased selectivity

Design of cholinergic agonists Use of steric shields Rationale Shields protect ester from nucleophiles and enzymes Shield size is important Must be large enough to hinder hydrolysis Must be small enough to fit binding site

Design of cholinergic agonists hinders binding to esterases and provides a shield to nucleophilic attack Methacholine asymmetric centre Properties Three times more stable than acetylcholine Increasing the shield size increases stability but decreases activity Selective for muscarinic receptors over nicotinic receptors S-enantiomer is more active than the R-enantiomer Stereochemistry matches muscarine Not used clinically

Pharmacologic manipulation of the cholinergic system Ca2+ Na+ Muscarinic Receptor ACH Choline Acetyltransferase Acetylcholinesterase Acetyl CoA + Acetylcholine Action Potential Choline a b Na+ H+ Nicotinic Receptor ACH Choline Acetate Choline Presynaptic neuron Postsynaptic target

Receptor agonists activate signal transduction pathways 3 NH 3 COOH G q Phospho - lipase C (+) PIP 2 IP Diacylglycerol Increase Ca 2+ Activate Protein Kinase C Response C C H 2 C H 2 N C H C H O 3 3 Acetylcholine C H 3 M3 muscarinic receptor

Molecular actions of acetylcholine at muscarinic receptors VI Tyr381 I VII Thr189 V II IV III Asp105

ANS ANTICHOLINESTERASES AGENTS Reversible : Short : Edrophonium Medium : Neostigmine, Physostigmine, Pyridostigmine, Tacrine

ANS ANTICHOLINESTERASES

ANS ANTI-CHOLINESTERASES AGENTS Mechanism of action : Acetyl cholinesterase (AchE) is an enzyme with anionic and esteratic site. Acetylcholine (Ach ) involves attraction of the positive charge N+ of Ach and anionic site; acetylation of serine leading to the acetylated enzyme. The acetylated enzyme reacts with the water to produce acetic acid and free enzyme within milliseconds.

ANS CHOLINESTERASES

Hydrolysis of acetylcholine by AChE Glu 327 Phe 338 His 440 Anionic site Trp 86 Ser 203 Esteratic site

Hydrolysis of acetylcholine by AChE Glu 327 His 440 Phe 338 choline Anionic site Trp 86 Ser 203 Esteratic site

Hydrolysis of acetylcholine by AChE Glu 327 His 440 Phe 338 Anionic site Trp 86 Ser 203 Esteratic site

Hydrolysis of acetylcholine by AChE Glu 327 His 440 Phe 338 acetate Anionic site Trp 86 Ser 203 Esteratic site

Pharmacologic manipulation of AChE: No inhibition Ca2+ Na+ Muscarinic Receptor ACH ACH Acetylcholinesterase ACH ACH Action Potential ACH ACH ACH ACH ACH ACH ACH Choline Acetate Presynaptic neuron Postsynaptic target

Pharmacologic manipulation of AChE: Inhibition by drugs Ca2+ ACH ACH Na+ ACH Muscarinic Receptor ACH ACH Acetylcholinesterase ACH ACH ACH Action Potential ACH ACH ACH ACH ACH ACH ACH ACH ACH Presynaptic neuron Postsynaptic target

Acetylcholinesterase inhibitors Tetraalkylammonium ions Simplest structures Bind to anionic site and block ACh binding Reversible Non-covalent R CH3 C2H5 C3H7 C4H9 Relative Potency 1.0 5.0 100 50

Acetylcholinesterase inhibitors Quaternary ammonium alcohol Simplest structures Bind to anionic site and block ACh binding Reversible Non-covalent

Acetylcholinesterase inhibitors Carbamates Quaternary or tertiary ammonium groups Reversible Covalent modification to AChE Most basic Nitrogen; protonated at physiological pH.

ANS ANTICHOLINESTERASES

ANS Reversible anticholinesterases : Edrophonium, Neostigmine and Physostigmine They combine with the ChE and carbamylated enzyme is slow to hydrolyze and free the enzyme (~30 mins).

Inhibition of AChE by Neostigmine Glu 327 Phe 338 His 440 Anionic site Trp 86 Ser 203 Esteratic site

Inhibition of AChE by Neostigmine Glu 327 Phe 338 His 440 Anionic site Trp 86 Ser 203 Esteratic site

Inhibition of AChE by Neostigmine Glu 327 Phe 338 His 440 Anionic site Trp 86 Ser 203 Esteratic site

Inhibition of AChE by Neostigmine Glu 327 Phe 338 His 440 Anionic site Trp 86 Ser 203 Esteratic site

Inhibition of AChE by Neostigmine Glu 327 Phe 338 His 440 Anionic site Trp 86 Ser 203 Esteratic site

Acetylcholinesterase inhibitors Organophosphates Irreversible Covalent modification to AChE Longer acting Used in the treatment of glaucoma

Acetylcholinesterase inhibitors Organophosphates Nerve gases Irreversible Covalent modification to AChE

Acetylcholinesterase inhibitors Organophosphates Insecticides Irreversible Covalent modification to AChE Rapidly inactivated in mammals

Biotransformation of insecticides Cyt P450 Insects Carboxyesterase Mammals, Birds

Inhibition of AChE by Organophosphates Glu 327 Phe 338 His 440 Anionic site Trp 86 Ser 203 Esteratic site

Inhibition of AChE by Organophosphates Glu 327 Phe 338 His 440 Anionic site Trp 86 Ser 203 Esteratic site

Inhibition of AChE by Organophosphates Aging Glu 327 Phe 338 His 440 Anionic site Trp 86 Ser 203 Esteratic site

Antidote for AChE “poisoning” Pralidoxime Chloride (Protopam; 2-pyridine aldoxime methyl chloride; 2-PAM) Antidote for pesticide or nerve gas poisoning Most effective if given within a few hours of exposure

Regeneration of AChE by Pralidoxime Glu 327 Phe 338 His 440 Anionic site Trp 86 Ser 203 Esteratic site

Regeneration of AChE by Pralidoxime Glu 327 Phe 338 His 440 Anionic site Trp 86 Ser 203 Esteratic site

Regeneration of AChE by Pralidoxime Glu 327 His 440 Phe 338 Anionic site Trp 86 Ser 203 Esteratic site

Clinical pharmacology of acetylcholinesterase inhibitors Type of Route of Drug inhibition administration Clinical Use Edrophonium Rev IM or IV Diagnostic for Myasthenia Gravis Neostigmine Rev IM, IV, or oral Myasthenia Gravis, post-operative ileus and bladder distention, surgical adjunct Physostigmine Rev IM, IV, or local Glaucoma, Alzheimer’s disease, antidote to anticholinergic overdose Tacrine Rev Oral Alzheimer’s disease Donepezil Rev Oral Alzheimer’s disease Isofluorophate Irrev Local Glaucoma Echothiophate Irrev Local Glaucoma

ANS Irreversible Anti-cholinesterases: Organophosphorus compounds - Parathion, Malathion The organophosphorus compounds reacts with esteratic site which is hydrolyzed extremely slowly with water or not at all . The phosphorylated enzyme undergo aging by the loss of one of the alkyl groups and become totally resistant to hydrolysis.

Synthesis of Ecothiophosphate

ANS CHOLINESTERASE REACTIVATORS : The phosphorylated ChE reacts very slowly or not at all with the water. If more OH groups in the form of Oximes are provided, reactivation occurs faster. Pralidoxime (PAM) attaches to the anionic site in presence of Organophosphorus compounds and set the enzyme free. PAM is ineffective in case of physostigmine poisoning as anionic site is not free.

Treatment of Alzheimer’s Disease Bind to anionic site and block ACh binding Reversible Non-covalent Enhances cognitive ability Does not slow progression of disease Newer agent: Donepezil (Aricept)

Treatment of Alzheimer’s Disease Reversible carbamate AChE inhibitor Enhances cognitive ability by increasing cholinergic function Loses effectiveness as disease progresses Side Effects: Nausea, vomiting, anorexia, and weight loss Newer long-acting carbamate: Eptastigmine

Treatment of Alzheimer’s Disease Reversible competitive AChE inhibitor Extract from daffodil (Narcissus pseudonarcissus) bulbs Loses effectiveness as disease progresses May be a nicotinic receptor agonist Inhibitors of P450 enzymes (3A4, 2D6) will increase galantamine bioavailability

Treatment of Alzheimer’s Disease N-methyl-D-aspartate (NMDA) receptor antagonist NMDA receptors are activated by glutamate in the CNS in areas associated with cognition and memory Neuronal loss in Alzheimer’s may be related to increased activity of glutamate May slow progression of the disease Favorable adverse effect profile

Cholinergic Antagonists By Nohad A AlOmari

Synthesis of Atropine

Cholinergic Antagonists By Nohad A AlOmari