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Home , Acetoxy group, Choline, Muscarine, Xanomeline

Topic 10 Drugs of the Nervous System

Ch 19,20 Patrick Part VI- Nervous system -Corey Contents

Part 1: & anticholinesterases

1. Transmission 2. 3. Transmission 4. receptors 4.1. Nicotinic 4.2. Muscarinic receptor - G Protein coupled receptor 5. Cholinergic 5.1. as an 5.2. and as cholinergic agonists 5.3. Requirements for cholinergic agonists 6. SAR for acetylcholine 7. Binding site (muscarinic) 8. Active conformation of acetylcholine 9. Instability of acetylcholine 10. Design of cholinergic agonists 11. Uses of cholinergic agonists CHOLINERGICCHOLINERGIC NERVOUSNERVOUS SYSTEMSYSTEM 1. Nerve Transmission

Peripheral nervous system

CNS

Brain

Peripheral

Muscle

Heart

Gastro- intestinal tract (GIT)

Spinal cord 1. Nerve Transmission

Peripheral nervous system

Skeletal muscle CNS (Somatic) Ach (N) CNS (Autonomic) Ach (N) NA Sympathetic

Adrenaline Ach Adrenal medulla Parasympathetic (N) Synapse AUTONOMIC

Ach Ach (M) (N) Smooth muscle Cardiac muscle 1. Nerve Transmission

Synapses

100-500A Receptors

Nerve impulse New signal

Nerve Nerve

Vesicles containing Release of Receptor binding neurotransmitters and new signal 2. Neurotransmitter

Acetylcholine (Ach)

O + C NMe 3 H3C O

Acetyl 3. Transmission process

Signal in nerve 1

Nerve 2 Nerve 1 .. Signal .. ..

Acetylcholinesterase . Acetylcholine Cholinergic receptor Vesicle 3. Transmission process

Vesicles fuse with membrane and release Ach

Nerve 2 Nerve 1

Signal 3. Transmission process

Nerve 2 3. Transmission process

• Receptor binds Ach 2o Message • Induced fit triggers 2o message • Triggers firing of nerve 2 • Ach undergoes no reaction

Nerve 2 3. Transmission process

• Ach departs receptor • Receptor reverts to resting state • Ach binds to

Nerve 2 3. Transmission process

Ach hydrolysed by acetylcholinesterase

O O

C C HO NMe3 Nerve 2 H3C O H3C OH + NMe3 Nerve 2 Acetylcholine Acetic acid Choline 3. Transmission process

Choline binds to carrier protein

Choline

Nerve 2 Nerve 1

Carrier protein for choline 3. Transmission process

Choline transported into nerve

Nerve 2 Nerve 1 3. Transmission process

Ach resynthesised

Nerve 2 Nerve 1

E 1 = Choline acetyltransferase

O O

C E 1 C NMe3 H C SCoA + HO CH CH NMe 3 2 2 3 H3C O Choline Acetylcholine 3. Transmission process

Ach repackaged in vesicles

Nerve 2 Nerve 1 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

HO

NMe

Me CH NMe O 2 3 N

Nicotine L-(+)-Muscarine Activates cholinergic Activates cholinergic receptors at nerve receptors on smooth and on skeletal muscle muscle and cardiac muscle Acetylcholine is natural messenger for both receptor types Peripheral nervous system

Skeletal Muscle CNS (Somatic) SOMATIC Ach (N) CNS (Autonomic) Synapse Ach (N) NA Sympathetic

Adrenaline Ach Adrenal medulla Parasympathetic (N) Synapse AUTONOMIC

Ach Ach (M) (N) Smooth Muscle Cardiac Muscle 4.1 Nicotinic receptor

Control of Cationic Channel:

Binding Receptor site Messenger

Induced Cell fit membrane ‘Gating’ (ion channel opens) Five glycoprotein subunits traversing cell membrane 4.1 Nicotinic receptor

The binding sites

Binding sites

Ion channel β α α γ

Cell γ membrane δ α β α δ

Two binding sites x subunits 2xα, β, γ, δ subunits mainly on α-subunits 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) messenger induced fit

closed open G-protein bound

G-protein split 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 • Intracellular reaction is catalysed

Enzyme Enzyme

active site active site (open) (closed) subunit Intracellular reaction 5. Cholinergic agonists

5.1 Acetylcholine as an agonist Advantages • Natural messenger • Easily synthesized

O HO NMe3 AcO NMe3 + NMe3 Ac2O

Disadvantages • Easily hydrolysed in stomach (acid catalysed ) • Easily hydrolysed in blood (esterases) • No selectivity between receptor types • No selectivity between different target organs 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 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 6. SAR for acetylcholine

Ethylene bridge

O

NMe3 Me O

Acetoxy 4 o Nitrogen

Quaternary nitrogen is essential

O O

CMe3 NMe2 H3C O H3C O

Bad for activity 6. SAR for acetylcholine bridge

O

NMe3 Me O

Acetoxy 4 o Nitrogen

• Distance from quaternary nitrogen to is important • Ethylene bridge must be retained

O O

H C H3C O 3 O NMe3 NMe3

Bad for activity 6. SAR for acetylcholine Ethylene bridge

O

NMe3 Me O

Acetoxy 4 o Nitrogen

Ester is important

NMe3 NMe3 H3C O H3C

Bad for activity 6. SAR for acetylcholine Ethylene bridge

O

NMe3 Me O

Acetoxy 4 o Nitrogen

Minimum of two methyl groups on quaternary nitrogen

O Et O Et N Me N Et H3C O H C 3 O Me Et

Bad for activity Active 6. SAR for acetylcholine Ethylene bridge

O

NMe3 Me O

Acetoxy 4 o Nitrogen

Methyl group of acetoxy group cannot be extended

O

H3C NMe3 O

Bad for activity 6. SAR for acetylcholine Ethylene bridge

O

NMe3 Me O

Acetoxy 4 o Nitrogen

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 7. Binding site (muscarinic)

hydrophobic pocket Trp-307 Asp311 CH 3 CH3 CO2

N CH3 hydrophobic O O pockets CH3 Trp-616 Trp-613

H H O N hydrophobic pocket

Asn-617 7. Binding site (muscarinic)

vdw Trp-307 Asp311 CH 3 CH3 Ionic bond CO2 N CH3 vdw O O vdw H-bonds CH3 Trp-616 Trp-613

H H O N

Asn-617 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

δ + δ + R NMe δ - 3 δ - 8. Active conformation of acetylcholine

O Me H H Me

C N Me O Me

H H

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

Rigid Analogues of acetylcholine

HO O O H NMe3

Me CH2NMe3 Me CH2NMe3 Me O O O MUSCARINE H

• Rotatable bonds ‘locked’ within ring • Restricts number of possible conformations • Defines separation of ester and N 4.4A 5.9A O O N N

Muscarinic Nicotinic receptor receptor 9. Instability of acetylcholine

Me3N

! " O

! + C

H3C O

• Neighbouring group participation • Increases electrophilicity of • Increases sensitivity to 10. Design of cholinergic agonists

Requirements

• Correct size

• Correct pharmacophore - ester and quaternary nitrogen

• Increased stability to acid and esterases

• Increased selectivity 10. Design of cholinergic agonists

Use of steric shields

Rationale

• Shields protect ester from nucleophiles and

• Shield size is important

• Must be large enough to hinder hydrolysis

• Must be small enough to fit binding site 10. Design of cholinergic agonists hinders binding to esterases and provides a shield to O Me nucleophilic attack NMe3 Me O * Properties asymmetric centre • 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

HO O Me O H

Me CH2NMe3 Me CH2NMe3 Me CH2NMe3 O O O H Me MUSCARINE (S) (R) 10. Design of cholinergic agonists

Use of electronic factors

• Replace ester with urethane • Stabilises the carbonyl group

! " O O O C C C = ! + H2N H2N H2N 10. Design of cholinergic agonists

O

C NMe3 H2N O

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 10. Design of cholinergic agonists

Steric + Electronic factors

O Me C NMe3 H2N O *

Properties • Very stable • Orally active • Selective for the muscarinic receptor • Used to stimulate GI tract and urinary bladder after surgery 10. Design of cholinergic agonists

Nicotinic selective agonist

O

C * NMe3 Me O * asymmetric centre

Me 11. 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 Peripheral nervous system

Skeletal Muscle CNS (Somatic) SOMATIC Ach (N) CNS (Autonomic) Synapse Ach (N) NA Sympathetic

Adrenaline Ach Adrenal medulla Parasympathetic (N) Synapse AUTONOMIC

Ach Ach (M) (N) Smooth Muscle Cardiac Muscle Contents

Part 1: Cholinergics & anticholinesterases

1. Nerve Transmission 2. Neurotransmitter 3. Transmission process 4. Cholinergic receptors 4.1. Nicotinic receptor 4.2. Muscarinic receptor - G Protein coupled receptor 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 7. Binding site (muscarinic) 8. Active conformation of acetylcholine 9. Instability of acetylcholine 10. Design of cholinergic agonists 11. Uses of cholinergic agonists CHOLINERGICCHOLINERGIC NERVOUSNERVOUS SYSTEMSYSTEM 1. Nerve Transmission

Peripheral nervous system

CNS

Brain

Peripheral nerves

Muscle

Heart

Gastro- intestinal tract (GIT)

Spinal cord 1. Nerve Transmission

Peripheral nervous system

Skeletal muscle CNS (Somatic) Ach (N) CNS (Autonomic) Synapse Ach (N) NA Sympathetic

Adrenaline Ach Adrenal medulla Parasympathetic (N) Synapse AUTONOMIC

Ach Ach (M) (N) Smooth muscle Cardiac muscle 1. Nerve Transmission

Synapses

100-500A Receptors

Nerve impulse New signal

Nerve Nerve

Vesicles containing Release of Receptor binding neurotransmitters neurotransmitters and new signal Contents

Part 2: Cholinergics & anticholinesterases

12. Cholinergic Antagonists (Muscarinic receptor) 12.1. 12.2. Hyoscine () 12.3. Comparison of atropine with acetylcholine 12.4. Analogues of atropine 12.5. Simplified Analogues 12.6. SAR for Antagonists 12.7. Binding Site for Antagonists 13. Cholinergic Antagonists (Nicotinic receptor) 13.1. 13.2. Binding 13.3. Analogues of tubocurarine 12. Cholinergic Antagonists (Muscarinic receptor)

• Drugs which bind to cholinergic receptor but do not activate it • Prevent acetylcholine from binding • Opposite clinical effect to agonists - lower activity of acetylcholine

Postsynaptic Postsynaptic nerve nerve

Ach Ach Ach

Antagonist 12. Cholinergic Antagonists (Muscarinic receptor)

Clinical Effects • Decrease of saliva and gastric secretions • Relaxation of smooth muscle • Decrease in motility of GIT and urinary tract • Dilation of pupils

Uses • Shutting down digestion for surgery • Ophthalmic examinations • Relief of peptic ulcers • Treatment of Parkinson’s Disease • Anticholinesterase poisoning • Motion sickness 12. Cholinergic Antagonists (Muscarinic receptor) Me 12.1 Atropine N easily racemised H CH2OH O CH C * O • Racemic form of • Source - roots of belladonna (1831) (deadly nightshade) • Used as a poison • Used as a medicine decreases GIT motility for anticholinesterase poisoning dilation of eye pupils • CNS side effects - hallucinations 12. Cholinergic Antagonists (Muscarinic receptor)

12.2 Hyoscine (scopolamine) Me N

H O CH2OH H O CH C * H O

• Source - thorn --jimsonweed • Medical use - treatment of motion sickness • CNS effects, hallucinations 12. Cholinergic Antagonists (Muscarinic receptor)

12.3 Comparison of atropine with acetylcholine Me N NMe3

CH 2 CHH2 CH2OH

O CH3 O C CH C O O • Relative positions of ester and nitrogen similar in both molecules • Nitrogen in atropine is ionised • and ester are important binding groups (ionic + H-bonds) • Aromatic ring of atropine is an extra binding group (vdW) • Atropine binds with a different induced fit - no activation • Atropine binds more strongly than acetylcholine 12. Cholinergic Antagonists (Muscarinic receptor)

12.4 Analogues of atropine

CH3 Br CH(CH3)2 NO3 H3C N H3C N

H H CH2OH CH2OH O CH O CH C C O O

Ipratropium Atropine methonitrate (bronchodilator & anti-asthmatic) (lowers GIT motility)

• Analogues are fully ionized • Analogues unable to cross the blood brain barrier • No CNS side effects 12. Cholinergic Antagonists (Muscarinic receptor)

12.5 Simplified Analogues Pharmacophore = ester + basic amine + aromatic ring

Et Me Me N CH Me Et CH2 CH2 O CH2CH2 N CH CH2 CH2OH HO C CH2CH2N(Et)3 Br O C Me O CH Me C O Cl O

Amprotropine bromide Propantheline 12. Cholinergic Antagonists (Muscarinic receptor)

12.5 Simplified Analogues Me N

N Me2N H O

CH CH CH 2 3 O CH N O OH CH O

CH2OH Benztropine (opthalmics) (opthalmics) (Parkinsons disease) O HN C

N N N C O

CH2 CH Benzhexol N (Parkinsons disease) (anti-ulcer) N

Me 12. Cholinergic Antagonists (Muscarinic receptor)

12.6 SAR for Antagonists R' R' = Aromatic or Heteroaromatic CH2 O CH R2N CH2 C R'

O Important features • Tertiary amine (ionised) or a quaternary nitrogen • Aromatic ring • Ester • N- groups (R) can be larger than methyl (unlike agonists) • Large branched • R’ = aromatic or heteroaromatic ring • Branching of aromatic/heteroaromatic rings is important 12. Cholinergic Antagonists (Muscarinic receptor)

12.6 SAR for Antagonists

Me Me CH Me O

O CH2CH2 N CH C O C Me Me CH2 O CH2CH2NR2 O Cl

Active Inactive 12. Cholinergic Antagonists (Muscarinic receptor)

12.6 SAR for Antagonists . Agonists

SAR for Antagonists SAR for Agonists

Tertiary amine (ionized) Quaternary nitrogen or quaternary nitrogen Aromatic ring Aromatic ring Ester Ester N-Alkyl groups (R) can be N-Alkyl groups = methyl larger than methyl R’ = aromatic or heteroaromatic R’ = H Branching of Ar rings important 12. Cholinergic Antagonists (Muscarinic receptor)

12.7 Binding Site for Antagonists

van der Waals binding regions for antagonists

Acetylcholine binding site

RECEPTOR SURFACE 12. Cholinergic Antagonists (Muscarinic receptor)

12.7 Binding Site for Antagonists

O

Me Me CH C O Me

O O CH2CH2 N CH C O Me H N Me 2 Asn O CH2 Cl CH2

NMeR2 CO2 13. Cholinergic Antagonists (Nicotinic receptor)

13.1 Curare • Extract from curare plant- Strychnos toxifera • Used for poison arrows • Causes paralysis (blocks acetylcholine signals to muscles) • Active principle = tubocurarine

MeO

Me N HO Me CH2 O H

CH H 2 Me O Tubocurarine H N OH

OMe 13. Cholinergic Antagonists (Nicotinic receptor)

Pharmacophore • Two quaternary centres at specific separation (1.15nm) • Different mechanism of action from atropine based antagonists • Different binding interactions

Clinical uses • Neuromuscular blocker for surgical operations • Permits lower and safer levels of general anaesthetic • Tubocurarine used (previously) as neuromuscular blocker but side effects 13. Cholinergic Antagonists (Nicotinic receptor)

13.2 Binding

protein complex (5 subunits) S diameter=8nm

8nm N N

9-10nm

a) Receptor dimer b) Interaction with tubocurarine

Probably not bridging Ach sites N N Tubocurarine Acetylcholine binding site 13. Cholinergic Antagonists (Nicotinic receptor)

13.3 Analogues of tubocurarine

O O

C C Me3N(CH2)10NMe3 Me3NCH2CH2 O CH2 CH2 O CH2CH2NMe3

Decamethonium Suxamethonium

• Long lasting • incorporated • Long recovery times • Shorter lifetime (5 min) • Side effects on heart • Fast onset and short duration • Side effects at autonomic ganglia 13. Cholinergic Antagonists (Nicotinic receptor)

13.3 Analogues of tubocurarine O

O Me Me Pancuronium (R=Me)

Vecuronium (R=H) Me H N N Me H H

O H

O Me

• Steroid acts as a spacer for the quaternary centres (1.09nm) • Acyl groups are added to introduce the Ach skeleton • Faster onset then tubocurarine but slower than suxamethonium • Longer duration of action than suxamethonium (45 min) • No effect on blood pressure and fewer side effects 13. Cholinergic Antagonists (Nicotinic receptor)

13.3 Analogues of tubocurarine

MeO OMe O O Me H N C C N MeO CH2 CH2 O (CH2)5 O CH2 CH2 OMe

Atracurium OMe MeO OMe OMe

• Design based on tubocurarine and suxamethonium • Lacks cardiac side effects • Rapidly broken down in blood both chemically and metabolically • Avoids patient variation in metabolic enzymes • Lifetime is 30 minutes • Administered as an i.v. drip • Self destruct system limits lifetime 13. Cholinergic Antagonists (Nicotinic receptor)

13.3 Analogues of tubocurarine

Me R -H Me R N N CH2 CH C H2C CH C H O O Ph Ph ACTIVE INACTIVE

Atracurium stable at acid pH Hofmann elimination at blood pH (7.4) 13. Cholinergic Antagonists (Nicotinic receptor)

13.3 Analogues of tubocurarine Mivacurium

MeO OMe Me O N O MeO O N O OMe H3C

OMe MeO OMe OMe

• Faster onset (2 min) • Shorter duration (15 min) 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 15. Anticholinesterases 15.1. 15.2. Mechanism of action 15.3. Physostigmine analogues 15.4. 15.5. Anticholinesterases as ‘Smart Drugs’ 14. Acetylcholinesterase 14.1 Role • Hydrolysis and deactivation of acetylcholine • Prevents acetylcholine reactivating receptor

Nerve 2 Nerve 1 .. Signal .. ..

Acetylcholinesterase enzyme 14. Acetylcholinesterase 14.2 Hydrolysis reaction catalysed

O O

C C HO NMe3 CH3 O CH3 OH + NMe3 Acetylcholine Acetic acid Choline

active inactive 14. Acetylcholinesterase 14.3 Effect of inhibition (Anticholinesterase)

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 Nerve 2 a cholinergic agonist 14. Acetylcholinesterase 14.4 Structure of enzyme complex

S S

Enzyme S S

S S S S S S S S S S Enzyme S S Enzyme S S

collagen 14. Acetylcholinesterase 14.5 Active site - binding interactions

Ester binding region Anionic binding region OH

Aspartate Histidine O N

C O N :O: : vdw Ionic O CH3 Me CH2 CH2 H-bond hydrophobic N H pockets O

vdwMe Me Tyrosine

• Anionic binding region similar to cholinergic receptor site • Binding and induced fit strains Ach and weakens bonds • Molecule positioned for reaction with His and Ser 14. Acetylcholinesterase 14.6 Active site - Mechanism of catalysis

O : CH3 C O CH2CH2NMe3 :O: R

CH3 C O

: NH NH : :N O H O : N H

Serine Histidine Histidine () (Base) (Base catalyst) : : O: : R :O: CH3 C O CH3 C NH OR NH :N O H N O H

Histidine Histidine Acid catalyst 14. Acetylcholinesterase 14.6 Active site - Mechanism of catalysis

H2O

: ROH :O: O CH3 C CH3 C

OR NH NH :N N O O H

Histidine Histidine : _ : O: O

CH3 C CH3 C OH H NH NH

O : :N O H : N : O H

Histidine Histidine Basic catalyst 14. Acetylcholinesterase 14.6 Active site - Mechanism of catalysis

_ : : :O: :O :

3 CH3 C OH CH C OH NH NH N :O : H N O H :

Histidine Histidine Basic catalyst (Acid catalyst) : _ :O: O

CH3 C OH CH C OH NH 3 NH O : N OH :N H

Histidine 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 µsecs of reaching active site

• An aspartate residue is also involved in the mechanism 14. Acetylcholinesterase The catalytic triad

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

:

H : N :O: : :N :O H O

Serine Histidine Aspartate (Nucleophile) (Base) 15. Anticholinesterases

• Inhibitors of acetylcholinesterase enzyme • Block hydrolysis of acetylcholine • Acetylcholine is able to reactivate cholinergic receptor • Same effect as a cholinergic agonist 15. Anticholinesterases 15.1 Physostigmine O Me C O Me N Pyrrolidine N H N N

Urethane or Me Me http://www.floradelaterre.com/index.php?id=19 • Natural product from the African calabar (ordeal) bean- Physostigma • Carbamate is essential (equivalent to ester of Ach) • Aromatic ring is important • Pyrrolidine N is important (ionized at blood pH) • Pyrrolidine N is equivalent to the quaternary nitrogen of Ach 15.2 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: : : 15.2 Mechanism of action

-ArOH O O :N :N NH MeNH C O Ar C NH H MeNH O :O: : Stable carbamoyl intermediate

O H :N Hydrolysis NH very slow

Rate of hydrolysis slower by 40 x 106 15.2 Mechanism of action

O O H C

H C : N O : N: O : Me Me Stable carbamoyl intermediate Carbonyl group 'deactivated' 15.3 Physostigmine analogues

O Me O

C O CH C O NMe3 Me Me N NMe2 N

H Me (ionised at blood pH)

Miotine

• Simplified analogue • Fully ionized • Susceptible to hydrolysis • Cannot cross BBB • Crosses BBB as free base • No CNS side effects • CNS side effects • More stable to hydrolysis • Extra N- increases stability 15.4 Organophosphates a) Nerve gases

iPrO O iPrO O P P iPrO F Me F

Dyflos (Diisopropyl fluorophosphonate)

• Agents developed in World War 2 • Agents irreversibly inhibit acetylcholinesterase • Permanent activation of cholinergic receptors by Ach • Results in death 15.4 Organophosphates b) Mechanism of action

-H Serine Serine

H O O

iPrO P O iPrO O iPrO - P F iPrO F STABLE

• Irreversible phosphorylation • P-O bond very stable 15.4 Organophosphates c) Medicinal

O

Me3N 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 15.4 Organophosphates d) Organophosphates as insecticides

EtO S MeO S P P CO2Et

EtO O NO2 MeO S CH

CH2CO2Et

• Relatively harmless to mammals • Agents act as in insects • Metabolised by insects to produce a toxic metabolite 15.4 Organophosphates d) Organophosphates as insecticides

MAMMALS INSECTS

EtO S EtO O P P Insect EtO O NO2 EtO O NO2 Oxidative PARATHION desulphurisation (Inactive ) Active drug Mammalian

EtO S Phosphorylates enzyme P EtO OH INACTIVE & excreted DEATH 15.4 Organophosphates e) Design of Organophosphate 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 + + ROH NH2OH RO P OR O P OR H N Hydroxylamine OR 2 OR

• Hydroxylamine is too toxic for clinical use • Increase selectivity by increasing binding interactions with active site 15.4 Organophosphates e) 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 • 1 million times more effective than hydroxylamine • Cannot act in CNS due to charge - cannot cross bbb 15.4 Organophosphates e) Design of Organophosphate Antidotes

O

N CH N H N CH N P OR O O Me Me OR O OR P OR O CO CO2 H 2 OH SER SER

Active Site (Blocked) Active Site (Free) 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 15.5 Anticholinesterases as ‘Smart Drugs’

• Act in CNS

• Must cross blood brain barrier

• Used to treat loss in Alzheimers disease

• Alzheimers causes deterioration of cholinergic receptors in brain

• Smart drugs inhibit Ach hydrolysis to increase activity at remaining receptors NMe2 Et 15.5 Anticholinesterases as ‘Smart Drugs’ CH O N 15.5 Anticholinesterases as ‘Smart Drugs’Me C Me

O (Exelon) Cl O (analogue of physostigmine) MeO

NH2 CH2 N

MeO H

Donepezil H N N N (Cognex) Toxic side effects O

P CCl3 Anabaseine MeO HO MeO (ants and marine worms) OH

O

MeO (organophosphate) S N N

N O Me Me

Galanthamine N Me (daffodil and snowdrop bulbs