Patrick An Introduction to Medicinal Chemistry 3/e Chapter 19 CHOLINERGICS, ANTICHOLINERGICS

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

2. Contents Part 1: Cholinergics & anticholinesterases 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]

3. CHOLINERGIC NERVOUS SYSTEM

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

5. Sympathetic nervous system = ‘Fight or Flight Response’ Parasympathetic nervous system = ‘Rest and Digest Response’ Link Link

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

8. Dual Innervation

9. http://entochem.tamu.edu/neurobiology/index.html

10. 100-500A Receptors New signal Nerve impulse Nerve Nerve Vesicles containing neurotransmitters 1. Nerve Transmission Synapses Release of neurotransmitters Receptor binding and new signal

11. O + C N M e 3 H C O 3 Acetyl Choline 2. Neurotransmitter Acetylcholine (Ach)

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

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

14. Nerve 2 3. Transmission process

15. 2o Message Nerve 2 3. Transmission process • Receptor binds Ach • Induced fit triggers 2o message • Triggers firing of nerve 2 • Ach undergoes no reaction

16. Nerve 2 3. Transmission process • Ach departs receptor • Receptor reverts to resting state • Ach binds to acetylcholinesterase

17. Nerve 2 3. Transmission process Ach hydrolysed by acetylcholinesterase

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

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

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

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

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

23. Nicotine comprises up the 3% the dry weight of the tobacco leaf • When inhaled, it rapidly crosses the blood-brain barrier where it activates (acts as an agonist at) the acetylcholine receptors • Addiction to nicotine is reported to be one of the hardest addictions to break.

24. Muscarine is the active poisonous ingredient in several species of mushrooms • Ingestion causes severe nausea and diarrhea as the muscarine acts as an acetylcholine agonist. Also causes perspiration and lacrimation (tearing). • The antidote is atropine, an acetycholine antagonist at the muscarinic receptor.

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

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

27. a g Binding sites a b d Ion channel a b g d a Cell membrane Two ligand binding sites mainly on a-subunits 4.1 Nicotinic receptor The binding sites 2xa, b, g, d subunits

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

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

30. 5. Cholinergic agonists 5.1 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

31. 5. Cholinergic agonists 5.2 Nicotine and muscarine as cholinergic agonists • Advantages • More stable than Ach • Selective for main cholinergic receptor types • Selective for different organs • Disadvantages • Activate receptors for other chemical messengers • Side effects

32. 5. Cholinergic agonists 5.3 Requirements for cholinergic agonists • Stability to stomach acids and esterases • Selectivity for cholinergic receptors • Selectivity between muscarinic and nicotinic receptors • Knowledge of binding site • SAR for acetylcholine

33. Ethylene bridge Ethylene bridge o Acetoxy 4 Nitrogen Acetoxy Nitrogen Bad for activity 6. SAR for acetlcholine Quaternary nitrogen is essential

34. Ethylene bridge Ethylene bridge o Acetoxy 4 Nitrogen Acetoxy Nitrogen Bad for activity 6. SAR for acetylcholine • Distance from quaternary nitrogen to ester is important • Ethylene bridge must be retained

35. Ethylene bridge Ethylene bridge o Acetoxy 4 Nitrogen Acetoxy Nitrogen Bad for activity 6. SAR for acetylcholine Ester is important

36. Ethylene bridge Ethylene bridge o Acetoxy 4 Nitrogen o Acetoxy 4 Nitrogen Bad for activity Active 6. SAR for acetylcholine Minimum of two methyl groups on quaternary nitrogen

37. Ethylene bridge Ethylene bridge o Acetoxy 4 Nitrogen Acetoxy Nitrogen Bad for activity 6. SAR for acetylcholine Methyl group of acetoxy group cannot be extended

38. Ethylene bridge o Acetoxy 4 Nitrogen 6. SAR for acetylcholine • 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

39. hydrophobic pocket Trp-307 Asp311 hydrophobic pockets Trp-616 Trp-613 hydrophobic pocket Asn-617 7. Binding site (muscarinic)

40. vdw Trp-307 Asp311 vdw vdw Trp-616 Trp-613 Asn-617 7. Binding site (muscarinic) Ionic bond H-bonds

41. d + d + d - d - 7. Binding site (muscarinic) • Possible induced dipole dipole interaction between quaternary nitrogen and hydrophobic aromatic rings in binding site • N+ induces dipole in aromatic rings

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

43. Muscarinic receptor Nicotinic receptor 8. Active conformation of acetylcholine Rigid Analogues of acetylcholine • Rotatable bonds ‘locked’ within ring • Restricts number of possible conformations • Defines separation of ester and N

44. 9. Instability of acetylcholine • Neighbouring group participation • Increases electrophilicity of carbonyl group • Increases sensitivity to nucleophiles

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

46. 10. 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

47. hinders binding to esterases and provides a shield to nucleophilic attack asymmetric centre 10. Design of cholinergic agonists Methacholine • 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

48. The primary clinical use of methacholine is as an acetylcholine agonist at the muscarinic receptor • As such, it is utilized in a test for asthma, called the ‘bronchial challenge test’ • The methacholine provokes bronchoconstriction • Asthmatic patients, which already have airway hyperactivity, are more sensitive to the effect of methacholine, and this reaction can be quantified using a breathing test called spirometry..

49. = 10. Design of cholinergic agonists • Use of electronic factors • Replace ester with urethane • Stabilises the carbonyl group

50. Carbachol 10. Design of cholinergic agonists • Properties • Resistant to hydrolysis • Long lasting • NH2 and CH3 are equal sizes. Both fit the hydrophobic pocket • NH2 = bio-isostere • Muscarinic activity = nicotinic activity • Used topically for glaucoma