This document outlines a lecture on medicinal chemistry related to the cholinergic system. It will review concepts of acetylcholine mimetics and acetylcholinesterase inhibitors. For muscarinic agonists, it will discuss biosynthesis and metabolism of acetylcholine, structure activity relationship studies of various muscarinic agonists. For acetylcholinesterase inhibitors, it will cover the mechanism of acetylcholine hydrolysis, reversible and irreversible inhibitors, and the antidote for irreversible inhibitors. Learning objectives and resources are also provided.

1. Medicinal Chemistry
Srikanth Kolluru, Ph.D.
Rm: 320; Ph. No:361‐593‐3034,
kolluru@pharmacy.tamhsc.edu

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. Learning objectives
After completing this topic students should be able to
1. Discuss biosynthesis and metabolism of acetylcholine (ACh)
2. Discuss muscarinic and nicotinic receptors sub types, tissue location and
their function
3. Identify properties of ACh which limits its use as therapeutic agent
4. Identify amino acid residues which interacts with ACh at muscarinic as
well as AChE catalytic site
5. 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 group
6. Identify therapeutic uses and metabolites of muscarinic agonists
7. Identify the hydrolysis products of acetylcholine and various
acetylcholine esterase inhibitors
8. Discuss the site/mechanism of action of irreversible AChEIs
9. Identify the important functional groups which serve as a basis in the
design of AChEIs
10. Discuss mechanism by which Pralidoxime chloride(2‐PAM)acts as
antidote for irreversible AChEIs.

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=000125901594
Required Reading:
 Foye’s Principles of Medicinal Chemistry, 6th Edition, Chapter
12
Additional reference:
 An Introduction to Medicinal Chemistry, Fourth Edition by
Graham L. Patrick; Oxford University Press: ISBN: 978‐0‐19‐
923447‐9

5. Review
 Nervous system
 Central
 Peripheral
 Somatic
 Autonomous
 Sympathetic
 Parasympathetic
 Enteric
 Neurotransmitters
 Acetylcholine
 Epinephrine
 Norepinephrine
 Cholinergic receptors
 Muscarinic receptors
 Nicotinic receptors

6. Nerve Transmission
Peripheral 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. Adrenergic and cholinergic
innervations in sympathetic and
parasympathetic nervous systems

8. Neurotransmission
Action potential
http://www.youtube.com/watch?v=U0NpTdge3aw&feature=related
Synaptic transmission of nerve impulse.
http://www.youtube.com/watch?v=HXx9qlJetSU

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

10. Neurotransmitter
Acetylcholine (Ach)
O
+
C NMe 3
H 3C O
Acetyl Choline

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

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

13. Transmission process
Nerve 2

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

16. Transmission process
Ach hydrolysed
by acetylcholinesterase
O O
C C HO
NMe3
H 3C O H 3C OH + NMe3
Acetylcholine Acetic acid Choline
Nerve 2

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

18. Transmission process
Choline transported into nerve
Nerve 2
Nerve 1

19. Transmission process
Ach resynthesized
Nerve 2
Nerve 1
E 1 = Choline acetyltransferase
O O
C C
E1 NMe3
H 3C SCoA + HO CH2 CH2 NMe3 H 3C O
Choline Acetylcholine

20. Transmission process
Ach repackaged in vesicles
Nerve 2
Nerve 1

21. Biosynthesis of acetylcholine
Serine
decarboxylase
Serine Ethanolamine
Choline‐N‐methyl S‐Adenosyl
transferase methionine
O N(CH3)3
Choline acetyl HO
N(CH3)3
H3C O transferase
Acetylcholine Choline

22. Metabolism of Acetylcholine
AChE‐Ser‐OH +
H2O
+ AChE‐Ser‐OH +
(inactive)

23. Cholinergic receptors
Acetylcholine
HO H
(S)
(R)
N
(S) (S) +
N (CH3)3 Cl
H3C O CH3
N
(‐)‐Muscarine S(‐)‐Nicotine
1. 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. Nerve Transmission
Peripheral 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. Muscarinic receptor subtypes and
functions
Receptor Tissue location Function
M1 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 on
M2 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 & analgesia
M3 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 oxide
M4 CNS Inhibition of autoreceptor‐ and heteroreceptor‐
mediated transmitter release in CNS, Analgesia,
Cataleptic activity;
Facilitates dopamine release
M5 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. Nicotinic receptor subtypes and
functions
Receptor Location Membrane Response
Skeletal 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 catecholamines
Central neuronal (CNS) CNS; pre‐ & postjunctional Pre‐ & postsynaptic
(α4)2(β4)3 (α‐bungarotoxin excitation; prejunctinal
insensitive) control of transmitter
release
(α7)5 (α‐bungarotoxin CNS; pre‐ and postsynaptic Same as central neuronal
sensitive)

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

28. Nicotinic receptor
The binding sites
Binding
sites
Ion channel
 
 
Cell  

 
membrane

Two ligand binding sites
x subunits mainly on -subunits

29. 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)
messenger
induced
fit
closed open
G-protein
bound
G-protein
split

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. 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. Acid catalyzed hydrolysis of ACh
O N+
H O
H
ACh
+H2O -H2O
H
O
O N+
O+
H H
Acetic acid Choline

33. Base catalyzed hydrolysis of ACh
H
O
O N+
O-
ACh
Acetic acid Choline

34. Cholinergic agonists
Nicotine 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. Cholinergic agonists
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

36. Structure Activity Relationship
(SAR) Studies
Modification 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. (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. Stereochemistry at ethylene bridge
Acetyl‐β‐methylcholine
(Methacholine) Acetyl‐α‐methylcholine
Methacholine:
 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. 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. 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. SAR summary
The 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. 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. 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. Specific muscarinic agonists
1. Methacholine chloride (Provocholine)
2. Carbachol chloride (Isopto carbachol)
3. Bethenechol chloride (Urecholine)
4. Pilocarpine hydrochloride (Isopto carpine)
5. Cevimeline hydrochloride (Evoxac)

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, Sjogren's syndrome, or
as a side effect of some psychotropic drugs.

46. Pilocarpine hydrochloride contd..
Pilocarpine stability CH3
N
(S) (R)
H3C
CH3 Hydrolysis
N
N
(S) (R) O OH OH
H3C 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. 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 Sjögren's syndrome.
 Before its approval, pilocarpine was the only drug for
this condition.

48. Uses of cholinergic agonists
Nicotinic selective agonists
Treatment of myasthenia gravis
‐ lack of acetylcholine at skeletal muscle causing weakness
Muscarinic 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

50. 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

51. The mechanism of action of
acetylcholinesterase

52. Mechanism of action of
acetylcholinesterase inhibitors
http://www.cnsforum.com/imagebank/item/Drug_neostig/default.aspx

53. Acetylcholinesterase
Active 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

54. 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

55. Acetylcholinesterase
Active 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

56. Acetylcholinesterase
Active 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

57. 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

58. 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

59. Acetylcholinesterase Inhibitors
1. 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

60. Reversible AChEIs
Ach metabolism by AChE
Fast
+ AChE-Ser-OH
~milliseconds +
HO-Ser-AChE
Carbamate 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

61. 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 :
:
:

62. 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 slow
Rate of hydrolysis slower by 40 x 106

63. Mechanism of action
O O
C H C
H
N O N O
Me
Me
Carbonyl group
'deactivated'

64. Reversible AChEIs contd..
Physostigmine
H3C
H Oxidation
N O H2O
H3C 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 Alzheimer's disease.

65. 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

66. Reversible AChEIs contd..
CH3 CH3
H3C
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.

67. Reversible AchEIs for treating
Alzheimer's disease (AD)
 Patients with AD are reported to have reduction in acetylcholine, serotonin,
norepinephrine, dopamine, and glutamate levels
Tacrine Donepezil Rivastigmine
Tacrine (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

68. Reversible AchEIs for treating Alzheimer's 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

69. 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.

70. 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

71. 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

72. Organophosphates
Design 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

73. Organophosphates
Design 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

74. Organophosphates
Design 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)

75. Organophosphates
Design of Organophosphate Antidotes
H H
ProPAM
NOH
N
CH3
• Prodrug for pralidoxime
• Passes through BBB as free base
• Oxidised in CNS to pralidoxime

76. Questions ??