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Medicinal Chemistry     Srikanth Kolluru, Ph.D. Rm: 320; Ph. No:361‐593‐3034, kolluru@pharmacy.tamhsc.edu
Lecture overview•   Review of concepts•   Acetylcholine mimetics (muscarinic agonists)    – Biosynthesis and metabolism of...
Learning objectives     After completing this topic students should be able to1.         Discuss biosynthesis  and metabol...
Useful Resources   Action potential http://www.youtube.com/watch?v=SCasruJT‐DU&NR=1   Synaptic transmission of nerve imp...
Review   Nervous system     Central     Peripheral       Somatic       Autonomous          Sympathetic         Para...
Nerve TransmissionPeripheral nervous system                                                                   Skeletal    ...
Adrenergic and cholinergicinnervations in sympathetic andparasympathetic nervous systems
NeurotransmissionAction potentialhttp://www.youtube.com/watch?v=U0NpTdge3aw&feature=relatedSynaptic transmission of nerve ...
Nerve Transmission    Synapses          100-500A                     ReceptorsNerve impulse                               ...
NeurotransmitterAcetylcholine (Ach)                   O                                     +                   C         ...
Transmission processSignal in nerve 1                                                           Nerve 2    Nerve 1        ...
Transmission processVesicles fuse with membrane and release Ach                                              Nerve 2    Ne...
Transmission process                       Nerve 2
Transmission process•   Ach binds to the receptor           2o Message•   Induced fit triggers 2o message•   Triggers firi...
Transmission process•    Ach departs receptor•    Receptor reverts to resting state•    Ach binds to acetylcholinesterase ...
Transmission process  Ach hydrolysed  by acetylcholinesterase       O                                 O       C           ...
Transmission processCholine binds to carrier protein                                  Choline                             ...
Transmission processCholine transported into nerve                                 Nerve 2   Nerve 1
Transmission process Ach resynthesized                                                                                    ...
Transmission processAch repackaged in vesicles                             Nerve 2   Nerve 1
Biosynthesis of acetylcholine                            Serine                         decarboxylase          Serine     ...
Metabolism of AcetylcholineAChE‐Ser‐OH +                           H2O           + AChE‐Ser‐OH                      +     ...
Cholinergic receptors                                       Acetylcholine       HO         H                              ...
Nerve TransmissionPeripheral nervous system                                                                   Skeletal    ...
Muscarinic receptor subtypes and                      functionsReceptor     Tissue location                               ...
Nicotinic receptor subtypes and                functions          Receptor                     Location              Membr...
Nicotinic receptor Control of Cationic Ion Channel: Ionotropic receptor              Binding   Receptor   site            ...
Nicotinic receptorThe binding sites                Binding                sites                                           ...
Muscarinic receptor - G Protein coupled receptorActivation of a signal protein• Receptor binds messenger leading to an ind...
Muscarinic receptor - G Protein coupled receptor  Activation of membrane bound enzyme  • G-Protein is split and subunit ac...
Muscarinic agonists  Acetylcholine                                                                    Imparts excellent wa...
Acid catalyzed hydrolysis of ACh                                       O        N+                          H   O         ...
Base catalyzed hydrolysis of ACh                               H                          O                               ...
Cholinergic agonistsNicotine and muscarine as cholinergic agonists     HO         H                                       ...
Cholinergic agonistsRequirements for cholinergic agonists •   Stability to stomach acids and esterases •   Selectivity for...
Structure Activity Relationship            (SAR) StudiesModification of the quaternary ammonium group                     ...
(SAR) Studies contd..      Modification of the ethylene bridge    Methyl substitution affords acetyl‐β‐methylcholine      ...
Stereochemistry at ethylene bridge       Acetyl‐β‐methylcholine            (Methacholine)                           Acetyl...
Modification of the acyloxy group    Choline esters of aromatic or higher‐      molecular‐weight acids possess       choli...
Modification of the acyloxy group contd..           Carbachol                             Bethanechol Bethanechol:  Orall...
SAR summaryThe molecule should have an oxygen atom,                                                The molecule must posse...
Binding site (muscarinic)              hydrophobic              pocket                      Trp-307                       ...
Binding site (muscarinic)                   vdw                       Trp-307                                             ...
Specific muscarinic agonists1.   Methacholine chloride (Provocholine)2.   Carbachol chloride (Isopto carbachol)3.   Bethen...
Pilocarpine hydrochloride                  (Isopto carpine)                                                 CH3           ...
Pilocarpine hydrochloride contd..                 Pilocarpine stability                                           CH3     ...
Cevimeline hydrochloride (Evoxac)                                                         O                               ...
Uses of cholinergic agonistsNicotinic selective agonistsTreatment of myasthenia gravis     ‐ lack of acetylcholine at skel...
Cholinesterases   Two types in humans:     Differs in their location in the body and substrate specificity.   Acetylcho...
The mechanism of action of    acetylcholinesterase
Mechanism of action ofacetylcholinesterase inhibitors     http://www.cnsforum.com/imagebank/item/Drug_neostig/default.aspx
AcetylcholinesteraseActive site - binding interactions                                                   Ester binding reg...
Acetylcholinesterase Active site - mechanism of catalysis                 O                                               ...
AcetylcholinesteraseActive site - Mechanism of catalysis                                                                  ...
AcetylcholinesteraseActive site - Mechanism of catalysis                _            :            :O :                    ...
Acetylcholinesterase  Serine and water are poor nucleophiles  Mechanism is aided by histidine acting as a basic catalyst...
AChEIs   Commonly referred to as anticholinesterases   Classified as indirect cholinomimetics     Principle mechanism o...
Acetylcholinesterase Inhibitors1.        Reversible AChEIs                2. Irreversible AChEIs     1.     Physostigmine ...
Reversible AChEIsAch metabolism by AChE                                                             Fast                  ...
Mechanism of action                                                           H               : :               O         ...
Mechanism of action                                          -ArOH             O                                          ...
Mechanism of action          O                    O          C           H        C  H      N       O            N       O...
Reversible AChEIs contd..                                   Physostigmine                       H3C      H                ...
Reversible AChEIs contd..    Neostigmine (Prostigmin)                             CH3                           CH3       ...
Reversible AChEIs contd..      CH3                                                CH3H3C      N+            CH3           ...
Reversible AchEIs for treating           Alzheimers disease (AD)  Patients with AD are reported to have reduction in acet...
Reversible AchEIs for treating Alzheimers disease (AD) contd.. Galantamine                                           OH   ...
Irreversible inhibitors of AChE   Designed based on chemical logic that “phosphate esters are     more stable to hydrolys...
Irreversible inhibitors of AChE as insecticides Irreversible AChEI insecticides is beneficial to agricultural production ...
Organophosphates  Organophosphates as insecticides                       MAMMALS                                          ...
OrganophosphatesDesign of Organophosphate Antidotes Strategy •    Strong nucleophile required to cleave strong P-O bond • ...
OrganophosphatesDesign of Organophosphate Antidotes                                         Pralidoxime            N      ...
OrganophosphatesDesign of Organophosphate Antidotes                                                                    O  ...
OrganophosphatesDesign of Organophosphate Antidotes                 H       H                                        ProPA...
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Cholinergic agonists  -  medicinal chemistry updated
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Cholinergic agonists - medicinal chemistry updated

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Cholinergic agonists - medicinal chemistry updated

  1. 1. Medicinal Chemistry Srikanth Kolluru, Ph.D. Rm: 320; Ph. No:361‐593‐3034, kolluru@pharmacy.tamhsc.edu
  2. 2. Lecture overview• Review of concepts• Acetylcholine mimetics (muscarinic agonists) – Biosynthesis and metabolism of acetylcholine – SAR (Structure Activity Relationship) studies – Various muscarinic agonists• Acetylcholinesterase inhibitors (Anticholinesterases) – Mechanism of acetylcholine esterase hydrolysis – Reversible and irreversible inhibitors of acetylocholineesterase – Antidotes for irreversible inhibitors of acetylcholine
  3. 3. Learning objectives After completing this topic students should be able to1. Discuss biosynthesis  and metabolism of acetylcholine (ACh)2. Discuss muscarinic and nicotinic receptors sub types, tissue location and  their function3. Identify properties of ACh which limits its use as therapeutic agent4. Identify amino acid residues which interacts with ACh at muscarinic as  well as AChE catalytic site5. Discuss structural features that affects activity of muscarinic agonists  1. Substitution on nitrogen 2. Substitution on ethylene bridge and stereochemistry associated with it 3. Changes on the acyloxy group6. Identify therapeutic uses and metabolites of muscarinic agonists7. Identify the hydrolysis products of acetylcholine and various  acetylcholine esterase inhibitors8. Discuss the site/mechanism of action of irreversible AChEIs9. Identify the important functional groups which serve as a basis in the  design of AChEIs10. Discuss mechanism by which Pralidoxime chloride(2‐PAM)acts as  antidote for irreversible AChEIs.
  4. 4. Useful Resources Action potential http://www.youtube.com/watch?v=SCasruJT‐DU&NR=1 Synaptic transmission of nerve impulse.  http://www.youtube.com/watch?v=HXx9qlJetSU http://video.search.yahoo.com/video/play?p=agonists+and+antagonists&ei=UTF‐ 8&fr=slv8‐msgr&fr2=tab‐web&tnr=21&vid=000125901594Required Reading: Foye’s Principles of Medicinal Chemistry, 6th Edition, Chapter  12Additional reference: An Introduction to Medicinal Chemistry, Fourth Edition by  Graham L. Patrick; Oxford University Press: ISBN: 978‐0‐19‐ 923447‐9
  5. 5. Review Nervous system  Central  Peripheral  Somatic  Autonomous   Sympathetic  Parasympathetic  Enteric Neurotransmitters  Acetylcholine  Epinephrine  Norepinephrine Cholinergic receptors  Muscarinic receptors  Nicotinic receptors
  6. 6. Nerve TransmissionPeripheral nervous system Skeletal muscle CNS (Somatic) Ach (N) CNS (Autonomic) Synapse Ach (N) NA Sympathetic Adrenaline Ach Adrenal (N) medulla AUTONOMIC Parasympathetic Synapse Ach Ach (M) (N) Smooth muscle Cardiac muscle
  7. 7. Adrenergic and cholinergicinnervations in sympathetic andparasympathetic nervous systems
  8. 8. NeurotransmissionAction potentialhttp://www.youtube.com/watch?v=U0NpTdge3aw&feature=relatedSynaptic transmission of nerve impulse. http://www.youtube.com/watch?v=HXx9qlJetSU
  9. 9. Nerve Transmission Synapses 100-500A ReceptorsNerve impulse New signalNerve Nerve Vesicles containing Release of Receptor binding neurotransmitters neurotransmitters and new signal
  10. 10. NeurotransmitterAcetylcholine (Ach) O + C NMe 3 H 3C O Acetyl Choline
  11. 11. Transmission processSignal in nerve 1 Nerve 2 Nerve 1 .. . Signal .. . .. . . Acetylcholine Acetylcholinesterase enzyme Vesicle Cholinergic receptor
  12. 12. Transmission processVesicles fuse with membrane and release Ach Nerve 2 Nerve 1 Signal
  13. 13. Transmission process Nerve 2
  14. 14. Transmission process• Ach binds to the receptor 2o Message• Induced fit triggers 2o message• Triggers firing of nerve 2• Ach undergoes no reaction Nerve 2
  15. 15. Transmission process• Ach departs receptor• Receptor reverts to resting state• Ach binds to acetylcholinesterase Nerve 2
  16. 16. Transmission process Ach hydrolysed by acetylcholinesterase O O C C HO NMe3H 3C O H 3C OH + NMe3 Acetylcholine Acetic acid Choline Nerve 2
  17. 17. Transmission processCholine binds to carrier protein Choline Nerve 2 Nerve 1 Carrier protein for choline
  18. 18. Transmission processCholine transported into nerve Nerve 2 Nerve 1
  19. 19. Transmission process Ach resynthesized Nerve 2 Nerve 1E 1 = Choline acetyltransferase O O C C E1 NMe3 H 3C SCoA + HO CH2 CH2 NMe3 H 3C O Choline Acetylcholine
  20. 20. Transmission processAch repackaged in vesicles Nerve 2 Nerve 1
  21. 21. Biosynthesis of acetylcholine Serine decarboxylase Serine Ethanolamine Choline‐N‐methyl  S‐Adenosyl transferase methionine O N(CH3)3 Choline acetyl  HO N(CH3)3H3C O transferase Acetylcholine Choline
  22. 22. Metabolism of AcetylcholineAChE‐Ser‐OH + H2O + AChE‐Ser‐OH + (inactive)
  23. 23. Cholinergic receptors Acetylcholine HO H (S) (R) N (S) (S) + N (CH3)3 Cl H3C O CH3 N (‐)‐Muscarine S(‐)‐Nicotine1. Muscarinic receptors 2. Nicotinic receptors Activates cholinergic Activates cholinergic receptors on smooth receptors at nerve synapses muscle and cardiac muscle and on skeletal muscle
  24. 24. Nerve TransmissionPeripheral nervous system Skeletal muscle CNS (Somatic) Ach (N) CNS (Autonomic) Synapse Ach (N) NA Sympathetic Adrenaline Ach Adrenal (N) medulla AUTONOMIC Parasympathetic Synapse Ach Ach (M) (N) Smooth muscle Cardiac muscle
  25. 25. Muscarinic receptor subtypes and functionsReceptor Tissue location FunctionM1 CNS, gastric and salivary glands,  ↑ Cogni ve func on autonomic ganglia, enteric nerves ↑Seizure ac vity, ↑Secre ons ↑ Autonomic ganglia depolariza on ↓ DA release and locomo onM2 Autonomic nerve terminals; CNS; heart;  ↑ Smooth muscle contrac on smooth muscle Neural inhibition in periphery via autoreceptors and heteroreceptor ↓ Ganglionic transmission Neural inhibi on in CNS, ↓ Heart rate ↑ Tremors hypothermia & analgesiaM3 CNS (< other mAChRs), smooth muscle,  ↑ Smooth muscle contrac on (e.g., bladder) glands, heart ↑ Salivary gland secre on ↑ Food intake, body fat deposits Inhibits dopamine release Synthesis of nitric oxideM4 CNS Inhibition of autoreceptor‐ and heteroreceptor‐ mediated transmitter release in CNS, Analgesia,  Cataleptic activity; Facilitates dopamine releaseM5 Low levels in CNS & periphery; predominate  Mediates dilation of cerebral arteries mAChRs in dopaminergic neurons of substantia Facilitates dopamine release nigra & ventral tegmentum area Augments drug seeking behavior and reward
  26. 26. Nicotinic receptor subtypes and functions Receptor Location Membrane ResponseSkeletal muscle (NM)  Skeletal neuromuscular  Excitatory; end plate (α1)2β1 εδ  junction (post‐junctional) depolarization; contraction (α1)2β1 γδ (skeletal muscle)Peripheral neuronal (NN)  Autonomic ganglia; adrenal  Excitatory; depolarization (α3)2(β4)3 medulla firing of postganglionic  neuron; depolarization &  secretion of catecholaminesCentral neuronal (CNS)  CNS; pre‐ & postjunctional Pre‐ & postsynaptic (α4)2(β4)3 (α‐bungarotoxin excitation; prejunctinalinsensitive) control of transmitter  release(α7)5 (α‐bungarotoxin CNS; pre‐ and postsynaptic Same as central neuronalsensitive)
  27. 27. Nicotinic receptor Control of Cationic Ion Channel: Ionotropic receptor Binding Receptor site Messenger InducedCell fit Cellmembrane membrane ‘Gating’ (ion channel opens) Five glycoprotein subunits traversing cell membrane
  28. 28. Nicotinic receptorThe binding sites Binding sites Ion channel     Cell      membrane  Two ligand binding sites x subunits mainly on -subunits
  29. 29. Muscarinic receptor - G Protein coupled receptorActivation of a signal protein• Receptor binds messenger leading to an induced fit• Opens a binding site for a signal protein (G-protein) messenger induced fit closed open G-protein bound G-protein split
  30. 30. 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 Enzyme Enzyme active site active site (open) (closed) subunit Intracellular reaction
  31. 31. Muscarinic agonists Acetylcholine Imparts excellent water Undergoes rapid hydrolysis by  solubility, but poorly  acid in GI track (oral  absorbed through lipid  administration) and  membranes because of  pseudocholinesterase in  high hydrophilic and ionic  serum  character Quarternary  Acyloxy  Ethylene  Ammonium  group group group 1. Prototype muscarinic (and nicotinic) agonist 2. Used in ocular surgery to produce miosis, but needs to be reconstituted  immediately before injection to anterior chamber due to aqueous instability. 3. It cannot be administered topically, because it is not lipophilic enough to  penetrate the cornea.
  32. 32. Acid catalyzed hydrolysis of ACh O N+ H O H ACh +H2O -H2O H O O N+ O+ H H Acetic acid Choline
  33. 33. Base catalyzed hydrolysis of ACh H O O N+ O- ACh Acetic acid Choline
  34. 34. Cholinergic agonistsNicotine and muscarine as cholinergic agonists HO H (S) (R) N (S) (S) + N (CH3)3 Cl H3C O CH3 N (‐)‐Muscarine S(‐)‐Nicotine Advantages • More stable than Ach • Selective for main cholinergic receptor types Disadvantages • Activate receptors for other chemical messengers • Side effects
  35. 35. Cholinergic agonistsRequirements 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
  36. 36. Structure Activity Relationship (SAR) StudiesModification of the quaternary ammonium group Replacing nitrogen atom by arsenic,  phosphorus, or sulfur resulted in less active  compounds and are not used clinically Replacing all three methyl groups on the nitrogen  by larger alkyl groups resulted in inactive agonists Replacing all three methyl groups with ethyl groups  resulted in antagonist Replacement of only one methyl group by an ethyl or propyl group affords a  compound that is active, but much less so than acetylcholine  Successive replacement of one, two, or three of the methyl groups with hydrogen  atoms to afford a tertiary, secondary, or primary amine, respectively, leads to  successively diminishing muscarinic activity
  37. 37. (SAR) Studies contd.. Modification of the ethylene bridge Methyl substitution affords acetyl‐β‐methylcholine HO H (methacholine), which has muscarinic potency almost  (R) equivalent to that of acetylcholine and much greater  (S) (S) N+(CH3)3 Cl muscarinic than nicotinic selectivity. H3C O (‐)‐Muscarine Methyl substitution affords acetyl‐α‐methylcholine,  which has reduced muscarinic and nicotinic potency  to that of acetylcholine  But has greater nicotinic than muscarinic selectivity.  Addition of methyl groups to either one or both of the ethylene  carbons results in chiral molecules. Muscarinic receptors display  stereoselectivity for the enantiomers of methacholine. 
  38. 38. Stereochemistry at ethylene bridge Acetyl‐β‐methylcholine  (Methacholine) Acetyl‐α‐methylcholineMethacholine: The S‐(+)‐enantiomer is equipotent with acetylcholine, and the R‐(–)‐enantiomer  is approximately 20‐fold less potent.  Acetylcholinesterase hydrolyzes the S‐(+)‐isomer much slower (approximately half  the rate) than acetylcholine.  The R‐(–)‐isomer is not hydrolyzed by AChE and even acts as a weak competitive  inhibitor of the enzyme. This stability toward AChE hydrolysis as well as the AChE  inhibitory effect of the R‐(–)‐enantiomer may explain why racemic methacholine  produces a longer duration of action than acetylcholine.  Acetyl‐α‐methylcholine: The nicotinic receptor and AChE exhibit little  stereoselectivity for the optical isomers of acetyl‐α‐methylcholine.
  39. 39. Modification of the acyloxy group Choline esters of aromatic or higher‐ molecular‐weight acids possess  cholinergic antagonist activity. Replacing methyl with amine group  results in carbamate (carbachol) which is more resistant to hydrolysis than ester  group  Carbachol is a potent cholinergic agonist possessing both muscarinic and  nicotinic activity Carbachol is used topically for glaucoma Carbachol is less readily hydrolyzed by gastric acid, AChE, or  butyrylcholinesterase than acetylcholine is, and it can be administered orally.
  40. 40. Modification of the acyloxy group contd.. Carbachol Bethanechol Bethanechol:  Orally active   Selective for muscarinic receptor  Used to stimulate GI tract and urinary bladder after surgery  Similar to methacholine, the S‐(+)‐enantiomer exhibits greater  binding affinity at muscarinic receptors than the R‐(–)‐enantiomer   Modification of ester group in Ach with ether or ketone resulted in  potent muscarinic agonists, but these are not clinically used.
  41. 41. SAR summaryThe molecule should have an oxygen atom,  The molecule must possess a nitrogen preferably an ester‐like oxygen, capable of  atom capable of bearing a positive charge, participating in a hydrogen bond preferably a quaternary ammonium salt. For maximum potency, the size of the alkyl  groups substituted on the nitrogen should  not exceed the size of a methyl group There should be a two‐carbon unit  between the oxygen atom and the  nitrogen atom
  42. 42. Binding site (muscarinic) hydrophobic pocket Trp-307 Asp311 CH3 CH3 CO 2 N CH3 hydrophobic O O pockets CH3 Trp-616 Trp-613 H H O N hydrophobic pocket Asn-617
  43. 43. Binding site (muscarinic) vdw Trp-307 Asp311 CH3 CH3 CO 2 Ionic bond N CH3 vdw O O H-bonds CH3 vdw Trp-616 Trp-613 H H O N Asn-617
  44. 44. Specific muscarinic agonists1. Methacholine chloride (Provocholine)2. Carbachol chloride (Isopto carbachol)3. Bethenechol chloride (Urecholine)4. Pilocarpine hydrochloride (Isopto carpine)5. Cevimeline hydrochloride (Evoxac)
  45. 45. Pilocarpine hydrochloride (Isopto carpine) CH3 N (S) (R) H3C N O O Pilocarpine Pilocarpine is marketed as tablets (Salogen), an ophthalmic solution, and  gel. It penetrates the eye wall and is the miotic of choice for open‐angle  glaucoma and to terminate acute angle closure attacks.  It also is used for the treatment of xerostomia (dryness of the mouth)  caused by radiation therapy of the head and neck, Sjogrens syndrome, or  as a side effect of some psychotropic drugs.
  46. 46. Pilocarpine hydrochloride contd.. Pilocarpine stability CH3 N (S) (R) H3C CH3 Hydrolysis N N (S) (R) O OH OHH3C Inactive Pilocarpicacid N O O Pilocarpine Epimerization (base catalyzed) Isopilocarpine Inactive  Epimerization is not believed to be a serious problem if the drug is  properly stored.   Its solutions can be stored at room temperature, but the gel should be  refrigerated and labeled with a 2‐week expiration date when dispensed.
  47. 47. Cevimeline hydrochloride (Evoxac) O S H S S CYP2D6 H H CYP3A4 O CH3 + O CH3 O CH3 N N N Inactive O Cevimeline hydrochloride is available as an oral capsule  for the treatment of xerostomia (dry mouth) associated  with Sjögrens syndrome.  Before its approval, pilocarpine was the only drug for  this condition.
  48. 48. Uses of cholinergic agonistsNicotinic selective agonistsTreatment of myasthenia gravis  ‐ lack of acetylcholine at skeletal muscle causing weaknessMuscarinic selective agonists• Treatment of glaucoma• Switching on GIT and urinary tract after surgery• Treatment of certain heart defects.  Decreases heart muscle  activity and decreases heart rate
  49. 49. Cholinesterases Two types in humans:  Differs in their location in the body and substrate specificity. Acetylcholinesterase (AChE):  Associated with glial cells in the synapse  Catalyzes the hydrolysis of Acetylcholine (serine hydrolase) Butyrylcholinesterase (BuChE):  Located in human plasma (also called pseudocholinesterase)  Broader substrate specificity for esters  May hydrolyze dietary ester and drug molecules in the blood
  50. 50. The mechanism of action of acetylcholinesterase
  51. 51. Mechanism of action ofacetylcholinesterase inhibitors http://www.cnsforum.com/imagebank/item/Drug_neostig/default.aspx
  52. 52. AcetylcholinesteraseActive site - binding interactions Ester binding region Anionic binding region Serine OH Aspartate Histidine O N N C O :O: : vdw O CH3 Me Ionic CH2 CH2 H-bond hydrophobic N H pockets O vdwMe Me Tyrosine  Anionic binding region is similar to the cholinergic receptor site  Binding and induced fit strains Ach and weakens bonds  Molecule is positioned for reaction with His and Ser
  53. 53. Acetylcholinesterase Active site - mechanism of catalysis O : CH3 C O CH2 CH2 NMe3 :O : R NH CH3 C O NH : :O H :N :N O H Serine Histidine Histidine (Nucleophile) (Base) (Base catalyst) : : O: : R :O : CH3 C O CH3 C NH OR NH H N O :N O H Histidine Histidine Acid catalyst
  54. 54. AcetylcholinesteraseActive site - Mechanism of catalysis H2O ROH : :O : O CH3 C CH3 C OR NH NH O :N N O H Histidine Histidine _ : : O: O CH3 C CH3 C OH H NH NH O :N H :N O O :: H Histidine Histidine Basic catalyst
  55. 55. AcetylcholinesteraseActive site - Mechanism of catalysis _ : :O : :O : CH3 C OH CH3 C OH NH NH O :N :O : H N H Histidine Histidine Basic catalyst (Acid catalyst) _ : :O : O CH3 C OH CH3 C OH NH NH O :N OH :N H Histidine
  56. 56. Acetylcholinesterase  Serine and water are poor nucleophiles  Mechanism is aided by histidine acting as a basic catalyst  Choline and serine are poor leaving groups  Leaving groups are aided by histidine acting as an acid catalyst  Very efficient - 100 x 106 faster than the uncatalysed hydrolysis  Acetylcholine hydrolysed within 100 secs of reaching active site
  57. 57. AChEIs Commonly referred to as anticholinesterases Classified as indirect cholinomimetics  Principle mechanism of action does not involve binding to  cholinergic receptors Act by interfering with the metabolism of ACh  Response is non‐selective resulting in activity at both muscarinic and  nicotinic receptors  AChE inhibitors are useful in the treatment of myasthenia gravis  (muscular fatigue / weakness), atony in the gastrointestinal tract  and glaucoma.  Also useful as agricultural insecticides and nerve gas warfare agents.  Investigational therapy for Alzheimer’s disease and other cognitive  disorders
  58. 58. Acetylcholinesterase Inhibitors1. Reversible AChEIs 2. Irreversible AChEIs 1. Physostigmine 1. Echothiophate 2. Neostigmine 2. Malathion  3. Pyridostigmine 4. Carboaryl 5. Edrophonium chloride 3. Antidotes for  6. Tacrine HCl 7. Donepezil Approved by  irreversible AChEIs 8. Rivastigmine FDA to treat AD 1. Pralidoxime 9. Galantamine
  59. 59. Reversible AChEIsAch metabolism by AChE Fast + AChE-Ser-OH ~milliseconds + HO-Ser-AChECarbamate metabolism by AChE Very slow + AChE-Ser-OH ~minutes + HO-Ser-AChE Half life for the methylcarbamated enzyme = ~ 15 minutes Carbamates are reversible AChEIs Aryl carbamates are more potent than alkyl carbamates because  phenoxide anions are more stable and better leaving groups than  alkoxide anions Phenoxide anions are stabilized by resonance 
  60. 60. Mechanism of action H : : O H O :N :N NH MeNH C O Ar NH MeNH O Ar :O : C : O Physostigmine O O H N :N : : MeNH C O Ar NH MeNH C O Ar NH H :O : :O : : :
  61. 61. Mechanism of action -ArOH O O :N :N MeNH C O Ar NH NH C H MeNH O :O : : Stable carbamoyl intermediate O H :N Hydrolysis NH very slowRate of hydrolysis slower by 40 x 106
  62. 62. Mechanism of action O O C H C H N O N O Me Me Carbonyl group deactivated
  63. 63. Reversible AChEIs contd.. Physostigmine H3C H Oxidation N O H2OH3C N CH3 O N light CH3 Physostigmine Eserine Rubreserine inactive as AChEIs  Acetylcholinesterase is carbamylated at a slow rate and the carbamylated  AChE also is regenerated quite slowly  Because physostigmine is a tertiary amine rather than a quaternary  ammonium salt, it is more lipophilic than other AChEIs and can diffuse across  the blood‐brain barrier.  It is investigated for use in the treatment of Alzheimers disease.
  64. 64. Reversible AChEIs contd.. Neostigmine (Prostigmin) CH3 CH3 O N O N CH3 CH3 O O N Br Br N+ CH3 H3C CH3 CH3 Pyridostigmine Neostigmine Fully ionised Cannot cross BBB No CNS side effects More stable to hydrolysis Extra N-methyl group increases stability
  65. 65. Reversible AChEIs contd.. CH3 CH3H3C N+ CH3 H3C N+ CH3 Demecarium (Humorsol, Tosmilen)- bridged diester with a decamethylene O O bridge. (CH2)10 O N N O CH3 CH3 Ambenonium (Mytelase)- has a long DOA and is used when patients don’t respond to Neostigmine or Pyridostigmine. CH3 Edrophonium (Antirex, Reversol)- very short DOA HO N CH3 (minutes) also very fast onset. Used to diagnose CH3 myasthenia gravis. Also used as an antidote to Curare.
  66. 66. Reversible AchEIs for treating Alzheimers disease (AD)  Patients with AD are reported to have reduction in acetylcholine, serotonin,  norepinephrine, dopamine, and glutamate levels Tacrine Donepezil RivastigmineTacrine (Cognex) Donepezil (Aricept) Rivastigmine (Exelon) Nonclassical AChEI.  Selective non‐competitive   Pseudo‐irreversible AChEI Effective in only about  AChEI  Duration of action of 10  20% of treated patients  1000x more selective for  hours Blocks both AChE and  AChE than BuChE  Low hepatotoxicity BuChE  Has greater affinity for  Usage is limited due to  AChE in the brain than the  hepatotoxicity periphery  Low hepatotoxicity
  67. 67. Reversible AchEIs for treating Alzheimers disease (AD) contd.. Galantamine OH O‐desmethyl compound is  Because it is a tertiary amine and can  (R) major metabolite cross the blood‐brain barrier (Z) O (S) (S) H3CO N Des‐methyl compound is  CH3 another major metabolite  Dual cholinergic action  By allosterically binding to nicotinic receptors  By inhibiting AChE (Selective)  No hepatotoxicity
  68. 68. Irreversible inhibitors of AChE Designed based on chemical logic that “phosphate esters are  more stable to hydrolysis than carboxylate ester or an amide Sarin (Chemical warfare agent) Ecothiophate iodide  Rate of hydrolysis of phosphorylated enzyme is much slower due to  aging (t1/2 for diethyl phosphate is about ~8h)  These agents are used as insecticides  Echothiophate is used by topical application to treat glaucoma.
  69. 69. Irreversible inhibitors of AChE as insecticides Irreversible AChEI insecticides is beneficial to agricultural production throughout  the world To be used with extreme caution in the presence of humans and other mammals  to prevent inhalation of the vapors and their absorption through the skin.  Both routes of exposure cause a number of poisoning accidents every year,  some of which are fatal
  70. 70. Organophosphates Organophosphates as insecticides MAMMALS INSECTS EtO S EtO O P P EtO O NO2 Insect EtO O NO2 Oxidative PARATHION desulphurisation(Inactive Prodrug) Active drug Mammalian Metabolism EtO S Phosphorylates enzyme P EtO OH INACTIVE & excreted DEATH
  71. 71. OrganophosphatesDesign of Organophosphate Antidotes Strategy • Strong nucleophile required to cleave strong P-O bond • Find suitable nucleophile capable of cleaving phosphate esters • Water is too weak as a nucleophile • Hydroxylamine is a stronger nucleophile O O NH 2 OH + RO P OR O P OR + ROH H2 N Hydroxylamine OR OR • Hydroxylamine is too toxic for clinical use • Increase selectivity by increasing binding interactions with active site
  72. 72. OrganophosphatesDesign of Organophosphate Antidotes Pralidoxime N CH N OH CH3 Quaternary 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 serine Pralidoxime 1 million times more effective than hydroxylamine Cannot act in CNS due to charge - cannot cross bbb
  73. 73. OrganophosphatesDesign of Organophosphate Antidotes O N CH N H N CH N P OR O O Me Me OR O OR P OR CO2 O CO2 OH H SER SER Active Site (Blocked) Active Site (Free)
  74. 74. OrganophosphatesDesign of Organophosphate Antidotes H H ProPAM NOH N CH3• Prodrug for pralidoxime• Passes through BBB as free base• Oxidised in CNS to pralidoxime
  75. 75. Questions ??

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