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Patrick
An Introduction to Medicinal Chemistry 3/e
Chapter 19
CHOLINERGICS, ANTICHOLINERGICS
& ANTICHOLINESTERASES
Part 3: Cholinergics & anticholinesterases
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Contents
Part 3: Cholinergics & anticholinesterases
14. Acetylcholinesterase
14.1.
Role
14.2.
Hydrolysis reaction catalysed
14.3.
Effect of inhibition
14.4.
Structure of enzyme complex
14.5.
Active site - binding interactions
14.6.
Active site - Mechanism of catalysis (3 slides)
15. Anticholinesterases
15.1.
Physostigmine
15.2.
Mechanism of action (3 slides)
15.3.
Physostigmine analogues
15.4.
Organophosphates (9 slides)
15.5.
Anticholinesterases as ‘Smart Drugs’ (2 slides)
[30 slides]
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14. Acetylcholinesterase
14.1 Role
• Hydrolysis and deactivation of acetylcholine
• Prevents acetylcholine reactivating receptor
...
Nerve 1
Signal
...
Nerve 2
...
Acetylcholinesterase enzyme
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14. Acetylcholinesterase
14.2 Hydrolysis reaction catalysed
O
O
C
CH3
O
CH3
Ac e tylcholine
active
HO
C
NMe3
OH
+
NMe3
Choline
Ac e tic acid
inactive
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14. Acetylcholinesterase
Enzyme inhibitor
(Anticholinesterase)
14.3 Effect of inhibition
2o Message
Ach
•
•
•
•
Inhibitor blocks acetylcholinesterase
Ach is unable to bind
Ach returns to receptor and reactivates
it
Enzyme inhibitor has the same effect as
a cholinergic agonist
Nerve 2
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14. Acetylcholinesterase
14.4 Structure of enzyme complex
S
S
Enzyme
S
S
S
Enzyme
S
S
S
S
S
S
S
S
S
S
S
S
Enzyme
S
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14. Acetylcholinesterase
14.5 Active site - binding interactions
Ester binding region
Anionic binding region
Serine
OH
Aspartate
Histidine
N
hydrophobic
pockets
:
vdw
:O:
Me
N
Ionic
C
N
O
CH2
CH2
O
O
H-bond
H
CH3
O
vdwMe
Me
Tyrosine
•
•
•
Anionic binding region similar to cholinergic receptor site
Binding and induced fit strains Ach and weakens bonds
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Molecule positioned for reaction with His and Ser
14. Acetylcholinesterase
14.6 Active site - Mechanism of catalysis
O
O
:O :
CH2CH2NMe3
:N
:
:O
C
:
CH3
H
CH3
NH
C
R
O
:N
O
NH
H
Se rine
(Nucle ophile)
His tidine
(Ba se cata lys t)
:
: O:
His tidin e
(Ba s e)
C
O
:
CH3
:O :
R
CH3
O
C
OR
NH
H N
O
:N
NH
H
H istidine
Acid catalyst
Histidine
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14. Acetylcholinesterase
14.6 Active site - Mechanism of catalysis
H2O
ROH
:
:O :
CH3
O
C
OR
:N
O
NH
CH3
C
N
O
H
NH
Histidine
Histidine
_
:
: O:
O
CH3
C
H
O
::
O
NH
:N
CH3
C
O
OH
H
NH
:N
H
H is tidine
Histidine
Basic catalyst
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14. Acetylcholinesterase
14.6 Active site - Mechanism of catalysis
_
:
:O :
:O :
CH3
C
CH3
OH
O
H
NH
C
OH
NH
:O :
:N
H
N
Histidine
Basic catalyst
Histidine
(Acid catalyst)
_
:
:O :
CH3
C
O
OH
NH
:N
O
CH3
C
OH
OH
:N
NH
H
His tidine
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14. 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 uncatalysed hydrolysis
•
Acetylcholine hydrolysed within 100 msecs of reaching active
site
•
An aspartate residue is also involved in the mechanism
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14. Acetylcholinesterase
The catalytic triad
•
An aspartate residue interacts with the imidazole ring of
histidine to orientate and activate it
:
:
:O
H
:N
:
H
:O:
N
O
Serine
(Nucleophile)
Histidine
(Base)
Aspartate
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15. Anticholinesterases
•
Inhibitors of acetylcholinesterase enzyme
•
Block hydrolysis of acetylcholine
•
Acetylcholine is able to reactivate cholinergic receptor
•
Same effect as a cholinergic agonist
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15. Anticholinesterases
15.1 Physostigmine
O
C O
Me
N
H
Urethane
or
Carbamate
•
•
•
•
•
Me
N
N
Me
Me
Pyrrolidine N
Natural product from the African calabar bean
Carbamate is essential (equivalent to ester of Ach)
Aromatic ring is important
Pyrrolidine N is important (ionised at blood pH)
Pyrrolidine N is equivalent to the quaternary nitrogen of Ach
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: :
15.2 Mechanism of action
O
MeNH
H
H
O
:N
NH
MeNH
O Ar
C O
:N
Ar
NH
:O :
:
C
O
Physostigmine
O
:O :
Ar
N
NH
MeNH
C
:O :
:N
O Ar
NH
H
:
: :
C O
:
MeNH
O
H
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15.2 Mechanism of action
-ArOH
O
MeNH
C
O Ar
O
:N
NH
C
MeNH
H
O
:
:O :
:N
Stable carbamoyl
intermediate
O
Hydrolysis
very slow
H
:N
NH
Rate of hydrolysis slower by 40 x 106
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NH
15.2 Mechanism of action
O
O
H
H
C
N
Me
O
C
N
O
Me
Carbonyl group
'deactivated'
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15.3 Physostigmine analogues
Me
O
C
Me
O
CH
NMe2
N
H
•
•
•
•
O
Me
C
O
NMe3
N
Me
(ionised at blood pH)
Miotine
Neostigmine
Simplified analogue
Susceptible to hydrolysis
Crosses BBB as free base
CNS side effects
•
•
•
•
•
Fully ionised
Cannot cross BBB
No CNS side effects
More stable to
hydrolysis
Extra N-methyl group
increases stability
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Hydrolysis mechanisms
Possible mechanism 1
N
:
Me
 -O
+C
: O:
Me
N
OAr
H
Water
H
O
C
Me
N
OH
C
OAr
OH
MeNH2
+
CO 2
H
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Hydrolysis mechanisms
Possible mechanism 2
O
Me
-H
C
N
H2O
Me
N
OAr
C
O
MeNH2
+
CO 2
Me
C
O
N
OH
H
H
Too reactive
Compare:
O
Me
-Me
No hydrolysis
C
N
OAr
Me
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15.4 Organophosphates
a) Nerve gases
i
PrO
O
i
PrO
P
i
PrO
P
F
Dyflos
(Diisopropyl fluorophosphonate)
•
•
•
•
O
Me
F
Sarin
Agents developed in World War 2
Agents irreversibly inhibit acetylcholinesterase
Permanent activation of cholinergic receptors by Ach
Results in death
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15.4 Organophosphates
b) Mechanism of action
-H
Serine
Serine
H
O
O
i
PrO
i
PrO
O
P
i
PrO
•
•
F
P
O
i
PrO
STABLE
Irreversible phosphorylation
P-O bond very stable
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15.4 Organophosphates
c) Medicinal organophosphate
O
Me 3N
CH2
CH2
S
P
OEt
Ecothiopate
OEt
•
•
•
•
Used to treat glaucoma
Topical application
Quaternary N is added to improve binding interactions
Results in better selectivity and lower, safer doses
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15.4 Organophosphates
d) Organophosphates as insecticides
EtO
MeO
S
CO2Et
P
P
EtO
S
O
NO2
MeO
S
CH
CH2 CO2Et
Parathion
•
•
•
Malathion
Relatively harmless to mammals
Agents act as prodrugs in insects
Metabolised by insects to produce a toxic metabolite
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15.4 Organophosphates
d) Organophosphates as insecticides
MAMMALS
EtO
INSECTS
S
EtO
P
EtO
O
P
O
NO2
PARATHION
(Inactive Prodrug)
Insect
Oxidative
desulphurisation
EtO
O
NO2
Active drug
Mammalian
Metabolism
EtO
S
Phosphorylates enzyme
P
EtO
OH
INACTIVE
& excreted
DEATH
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15.4 Organophosphates
e) 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
• Hydoxylamine is a stronger nucleophile
O
O
NH 2OH + RO P OR
Hydroxylamine
•
•
OR
O P OR
H2 N
+
ROH
OR
Hydroxylamine is too toxic for clinical use
Increase selectivity by increasing binding interactions with active
site
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15.4 Organophosphates
e) Design of Organophosphate Antidotes
Pralidoxime
N
CH3
•
•
•
•
CH N
OH
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
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15.4 Organophosphates
e) Design of Organophosphate Antidotes
O
N
CH N
N
H
O
O
Me
CH N
Me
O
P
OR
OR
OR
P
OR
CO 2
H
Active Site (Blocked)
CO 2
O
OH
SER
SER
Active Site (Free)
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15.4 Organophosphates
e) Design of Organophosphate Antidotes
H
H
ProPAM
NOH
N
CH3
•
•
•
Prodrug for pralidoxime
Passes through BBB as free base
Oxidised in CNS to pralidoxime
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15.5 Anticholinesterases as ‘Smart Drugs’
•
Act in CNS
•
Must cross blood brain barrier
•
Used to treat memory loss in Alzheimers disease
•
Alzheimers causes deterioration of cholinergic receptors in
brain
•
Smart drugs inhibit Ach hydrolysis to increase activity at
remaining receptors
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NMe2
15.5 Anticholinesterases as ‘Smart Drugs’ Me
Et
CH
O
N
C
Me
O
Cl
O
MeO
NH2
CH2
Rivastigmine (Exelon)
(analogue of physostigmine)
N
H
MeO
Donepezil
H
N
N
Tacrine (Cognex)
Toxic side effects
N
O
P
MeO
MeO
HO
Anabaseine
(ants and marine worms)
CCl3
OH
Metrifonate
(organophosphate)
O
MeO
S
N
N
N
Galanthamine
(daffodil and snowdrop bulbs
Me
O
Me
N
Me
Xanomeline
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