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

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Patrick Patrick An Introduction to Medicinal An Introduction to Medicinal

ChemistryChemistry 3/e 3/e

Chapter 19Chapter 19

CHOLINERGICS, CHOLINERGICS, ANTICHOLINERGICSANTICHOLINERGICS

& ANTICHOLINESTERASES& ANTICHOLINESTERASES

Part 3: Cholinergics & anticholinesterasesPart 3: Cholinergics & anticholinesterases

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ContentsContents

Part 3: Cholinergics & anticholinesterases

14. Acetylcholinesterase14.1. Role 14.2. Hydrolysis reaction catalysed14.3. Effect of inhibition14.4. Structure of enzyme complex14.5. Active site - binding interactions14.6. Active site - Mechanism of catalysis (3 slides)

15. Anticholinesterases15.1. Physostigmine15.2. Mechanism of action (3 slides)15.3. Physostigmine analogues15.4. Organophosphates (9 slides)15.5. Anticholinesterases as ‘Smart Drugs’ (2 slides)

[30 slides]

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14. Acetylcholinesterase14. Acetylcholinesterase

Acetylcholinesterase enzymeAcetylcholinesterase enzyme

......

...

Nerve 1Nerve 1Nerve 2Nerve 2

SignalSignal

14.1 Role14.1 Role • Hydrolysis and deactivation of acetylcholineHydrolysis and deactivation of acetylcholine• Prevents acetylcholine reactivating receptorPrevents acetylcholine reactivating receptor

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activeactive inactiveinactive

14.2 Hydrolysis reaction catalysed14.2 Hydrolysis reaction catalysed

Acetylcholine

CH3

C

O

O

Choline

+ NMe3

C

O

CH3 OH

Acetic acid

NMe3HO

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• Inhibitor blocks acetylcholinesteraseInhibitor blocks acetylcholinesterase• Ach is unable to bindAch is unable to bind• Ach returns to receptor and reactivates Ach returns to receptor and reactivates

itit• Enzyme inhibitor has the same effect as Enzyme inhibitor has the same effect as

a cholinergic agonista cholinergic agonist

22oo Message Message

Nerve 2Nerve 2

Enzyme inhibitorEnzyme inhibitor(Anticholinesterase)(Anticholinesterase)

14.3 Effect of inhibition14.3 Effect of inhibition

AchAch

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14.4 Structure of enzyme complex14.4 Structure of enzyme complex

S

S

SS

S

SS

S

S S

S

S

S

S

S S

S

S

Enzyme

EnzymeEnzyme

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: ::OO

Serine

Histidine

Tyrosine

OH

N

N

HO

Aspartate


14.5 Active site - binding interactions14.5 Active site - binding interactions

• Anionic binding region similar to cholinergic receptor siteAnionic binding region similar to cholinergic receptor site• Binding and induced fit strains Ach and weakens bondsBinding and induced fit strains Ach and weakens bonds• Molecule positioned for reaction with His and SerMolecule positioned for reaction with His and Ser

vdwvdw

vdwvdw

hydrophobic pockets

Anionic binding regionEster binding region

Me

N

CH2 CH2O

C

CH3

O

Me

Me

H-bondIonic

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14.6 Active site - Mechanism of catalysis14.6 Active site - Mechanism of catalysis

O H NNH

:

Serine Histidine(Nucleophile) (Base)

:

:

CH3 C

O

O CH2CH2NMe3

NNH

O

O

R

C

O

CH3

H

:

:Histidine(Base catalyst)

::

NNH

O

OR

C

O

CH3

H

:

Histidine

:: :

:

COR

O

CH3

O NNH

H

:

:

Histidine

Acid catalyst

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:

:

COR

O

CH3

O NNH

H

:

:

Histidine

H2O

NNH

OO

H

CCH3

O

H::

:

Histidine

ROH

CO

CH3

ON

NH

Histidine

C

O

OHCH3

O NNH

: _

H :

::

HistidineBasic catalyst

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C

O

OHCH3

O NNH

::

:

: H:

Histidine(Acid catalyst)

C

O

OHCH3

O NNH

: _

H

:

Histidine

::

C

O

OHCH3

O NNH

: _

H :

::

HistidineBasic catalyst

OHC

O

CH3

OH NNH

:

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• Serine and water are poor nucleophilesSerine and water are poor nucleophiles

• Mechanism is aided by histidine acting as a basic catalystMechanism is aided by histidine acting as a basic catalyst

• Choline and serine are poor leaving groupsCholine and serine are poor leaving groups

• Leaving groups are aided by histidine acting as an acid catalystLeaving groups are aided by histidine acting as an acid catalyst

• Very efficient - 100 x 10Very efficient - 100 x 1066 faster than uncatalysed hydrolysis faster than uncatalysed hydrolysis

• Acetylcholine hydrolysed within 100 Acetylcholine hydrolysed within 100 secs of reaching active secs of reaching active sitesite

• An aspartate residue is also involved in the mechanismAn aspartate residue is also involved in the mechanism

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The catalytic triadThe catalytic triad

• An aspartate residue interacts with the imidazole ring of An aspartate residue interacts with the imidazole ring of histidine to orientate and activate ithistidine to orientate and activate it

Serine Histidine(Nucleophile) (Base)

O H

NN

H

O

O

Aspartate

:

:::

::

:

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15. Anticholinesterases15. Anticholinesterases

• Inhibitors of acetylcholinesterase enzymeInhibitors of acetylcholinesterase enzyme

• Block hydrolysis of acetylcholineBlock hydrolysis of acetylcholine

• Acetylcholine is able to reactivate cholinergic receptorAcetylcholine is able to reactivate cholinergic receptor

• Same effect as a cholinergic agonistSame effect as a cholinergic agonist

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15. Anticholinesterases15. Anticholinesterases

15.1 Physostigmine15.1 Physostigmine

• Natural product from the African calabar beanNatural product from the African calabar bean• Carbamate is essential (equivalent to ester of Ach)Carbamate is essential (equivalent to ester of Ach)• Aromatic ring is important Aromatic ring is important • Pyrrolidine N is important (ionised at blood pH)Pyrrolidine N is important (ionised at blood pH)• Pyrrolidine N is equivalent to the quaternary nitrogen of AchPyrrolidine N is equivalent to the quaternary nitrogen of Ach

N N

MeOC

O

NMe

H

Me MeUrethane orCarbamate

Pyrrolidine N

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Physostigmine

O:N

NH

H

MeNHC

O

O Ar

::

15.2 Mechanism of action15.2 Mechanism of action

O ArC

O

MeNH

O

NHN:

:

H

: :

O ArC

O

MeNH

O

NHNH

: : :

::

:

: :

O ArC

O

MeNH

O

NHN

:H

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:

: :

O ArC

O

MeNH

O

NHN

:H


Rate of hydrolysis slower by 40 x 10Rate of hydrolysis slower by 40 x 1066

Stable carbamoylintermediate

O H

NH:N

Hydrolysisvery slow

OC

O:N

NH

MeNH

-ArOH

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Carbonyl group'deactivated'

:

O

CON

Me

H: ::N

H

Me

CO

O

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15.3 Physostigmine analogues15.3 Physostigmine analogues

• Simplified analogueSimplified analogue• Susceptible to hydrolysisSusceptible to hydrolysis• Crosses BBB as free baseCrosses BBB as free base• CNS side effectsCNS side effects

• Fully ionisedFully ionised• Cannot cross BBBCannot cross BBB• No CNS side effectsNo CNS side effects• More stable to More stable to

hydrolysishydrolysis• Extra Extra NN-methyl group -methyl group

increases stabilityincreases stability

MiotineMiotine

C

N

O

O

H

Me

Me

CHNMe2

(ionised at blood pH)

NeostigmineNeostigmine

NMe3OMe

N

Me

C

O

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CN

O

OAr

H

Me

-

+:

Me

N

H

C OAr

O

OH

::

MeN

H

C

OH

OMeNH2

+CO2

Water

Hydrolysis mechanismsHydrolysis mechanisms

Possible mechanism 1Possible mechanism 1

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Me

N

H

COAr

O-H

CN O

Me MeNH2

+CO2

Compare:

CN

O

OAr

Me

Me-Me

No hydrolysis

Too reactive

Hydrolysis mechanismsHydrolysis mechanisms

Possible mechanism 2Possible mechanism 2

CN

O

OH

H

MeHH22OO

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15.4 Organophosphates15.4 Organophosphates

a) Nerve gasesa) Nerve gases

• Agents developed in World War 2Agents developed in World War 2• Agents irreversibly inhibit acetylcholinesteraseAgents irreversibly inhibit acetylcholinesterase• Permanent activation of cholinergic receptors by AchPermanent activation of cholinergic receptors by Ach• Results in deathResults in death

DyflosDyflos(Diisopropyl fluorophosphonate)(Diisopropyl fluorophosphonate)

iPrO

P

O

FiPrO

SarinSarin

P

Me F

OiPrO

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b) Mechanism of actionb) Mechanism of action

• Irreversible phosphorylationIrreversible phosphorylation• P-O bond very stableP-O bond very stable

PiPrO F

OiPrO

STABLE

-H

O

P O

iPrO

iPrO

Serine

OH

Serine

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c) Medicinal organophosphatec) Medicinal organophosphate

• Used to treat glaucomaUsed to treat glaucoma• Topical applicationTopical application• Quaternary N is added to improve binding interactionsQuaternary N is added to improve binding interactions• Results in better selectivity and lower, safer dosesResults in better selectivity and lower, safer doses

EcothiopateEcothiopateMe3N CH2 CH2 S P

O

OEt

OEt


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d) Organophosphates as insecticidesd) Organophosphates as insecticides

• Relatively harmless to mammalsRelatively harmless to mammals• Agents act as prodrugs in insectsAgents act as prodrugs in insects• Metabolised by insects to produce a toxic metaboliteMetabolised by insects to produce a toxic metabolite

ParathionParathion

EtO

P

S

EtO O NO2

MalathionMalathion

CH

CH2CO2Et

CO2Et

S

P

MeO

SMeO


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d) Organophosphates as insecticidesd) Organophosphates as insecticides

MAMMALS INSECTS

NO2O

P

EtO

SEtO

PARATHION(Inactive Prodrug)

Active drug

Phosphorylates enzyme

DEATHDEATH

Mammalian Metabolism

EtO

P

S

EtO OH

INACTIVE& excreted

EtO

P

O

EtO O NO2InsectOxidative

desulphurisation


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e) Design of Organophosphate Antidotese) Design of Organophosphate Antidotes

StrategyStrategy• Strong nucleophile required to cleave strong P-O bondStrong nucleophile required to cleave strong P-O bond• Find suitable nucleophile capable of cleaving phosphate estersFind suitable nucleophile capable of cleaving phosphate esters• Water is too weak as a nucleophileWater is too weak as a nucleophile• Hydoxylamine is a stronger nucleophileHydoxylamine is a stronger nucleophile

• Hydroxylamine is too toxic for clinical useHydroxylamine is too toxic for clinical use• Increase selectivity by increasing binding interactions with active Increase selectivity by increasing binding interactions with active

sitesite

Hydroxylamine

+ +RO P

O

OR

ORH2N

O P

O

OR

OR

NH2OH ROH


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• Quaternary N is added to bind to the anionic regionQuaternary N is added to bind to the anionic region

• Side chain is designed to place the hydroxylamine moiety in Side chain is designed to place the hydroxylamine moiety in the correct position relative to phosphorylated serinethe correct position relative to phosphorylated serine

• Pralidoxime 1 million times more effective than Pralidoxime 1 million times more effective than hydroxylaminehydroxylamine

• Cannot act in CNS due to charge - cannot cross bbbCannot act in CNS due to charge - cannot cross bbb

PralidoximePralidoximeN

CH3

CH N

OH


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H

N CH NO

H

Me Me

NO

PNCH

O

OR

OR

CO2 OHSER

Active Site (Free)

CO2 O

P

OOR

OR

SER

Active Site (Blocked)


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• Prodrug for pralidoximeProdrug for pralidoxime• Passes through BBB as free basePasses through BBB as free base• Oxidised in CNS to pralidoximeOxidised in CNS to pralidoxime

ProPAMProPAM

N

HH

CH3

NOH


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15.5 Anticholinesterases as ‘Smart Drugs’15.5 Anticholinesterases as ‘Smart Drugs’

• Act in CNS Act in CNS

• Must cross blood brain barrierMust cross blood brain barrier

• Used to treat memory loss in Alzheimers diseaseUsed to treat memory loss in Alzheimers disease

• Alzheimers causes deterioration of cholinergic receptors in Alzheimers causes deterioration of cholinergic receptors in brainbrain

• Smart drugs inhibit Ach hydrolysis to increase activity at Smart drugs inhibit Ach hydrolysis to increase activity at remaining receptorsremaining receptors

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15.5 Anticholinesterases as ‘Smart Drugs’15.5 Anticholinesterases as ‘Smart Drugs’

Donepezil

Tacrine (Cognex)Toxic side effects

Rivastigmine (Exelon)(analogue of physostigmine)

Galanthamine(daffodil and snowdrop bulbs

Metrifonate(organophosphate)

Xanomeline

Anabaseine(ants and marine worms)

N

NH2

MeO

MeO

O

CH2 N

H

Cl

CH OC

N

O

Me

Et

Me

NMe2

MeOP CCl3

O

MeOOH

HN N

NMe

N

S

N

O MeN

O

MeO

Me

HO

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