Drugs acting on the NS

Medicinal Chemistry I
By: Abiyot M.
 Introduction to the Autonomic Nervous system

• The peripheral nervous system

• The peripheral nervous system (PNS) is so called because it
is peripheral to the central nervous system (CNS; the brain
and spinal column).
• There are two subdivisions of the peripheral system:
– sensory nerves take messages from the body to the CNS;
– motor nerves carry messages from the CNS to the rest of
the body
• Motor nerves of the peripheral nervous system
– These nerves take messages from the CNS to various
parts of the body such as skeletal muscle, smooth muscle,
cardiac muscle, and glands.
– Motor nerves of the peripheral nervous system have been
divided into two sub-systems
• The somatic motor nervous system
• The autonomic motor nervous system
• Somatic motor nervous system
– These are nerves which carry messages from the CNS to
skeletal muscles
– There are no synapses (junctions) and the neurotransmitter at
the neuromuscular junction is acetylcholine
– Its binding to cholinergic receptors within the cell membranes
of muscle cells and the final result is contraction of skeletal
muscle.
• The autonomic motor nervous system
– The autonomic motor nerves carry messages from the CNS to
smooth muscle, cardiac muscle, and the adrenal medulla.
– ACh serves as a neurotransmitter at both
• sympathetic and parasympathetic preganglionic nerve
endings, postganglionic nerve fibers in the parasympathetic
division, and some postganglionic fibers (e.g., salivary and
sweat glands) in the sympathetic division of the autonomic
nervous system.
– The autonomic nervous system regulates the activities of
smooth muscle and glandular secretions.
– These, as a rule, function below the level of consciousness
• The two divisions have contrasting effects on the
internal environment of the body.
– The sympathetic division frequently discharges as a unit,
especially during conditions of rage or fright, and expends
energy.
– The parasympathetic division is organized for discrete and
localized discharge and stores and conserves energy.
• Sympathetic nerves
– These leave the CNS, but almost immediately synapse
with a second nerve (neurotransmitter acetylcholine)
which then proceeds to the same target organs as the
parasympathetic nerves.
– However, they synapse with different receptors on the
target organs and use a different neurotransmitter—
noradrenaline
• The neurotransmitters
– There are a large variety of neurotransmitters in the CNS,
but as far as the peripheral nervous system is concerned
we need only consider two—acetylcholine and
noradrenaline
• Actions of the peripheral nervous system
– Somatic
• Stimulation leads to the contraction of skeletal
muscle.
• B. Autonomic
– Sympathetic.
• Contraction of cardiac muscle and an increase in
heart rate.
• Relaxes smooth muscle and reduces the
contractions of the GIT and urinary tracts
• Reduces salivation and reduces dilation of the
peripheral blood vessels.
• Parasympathetic.
– The stimulation of the parasympathetic system leads to
the opposite effects from those of the sympathetic system.
– Rest and digest role of the body (house keeping role)
– Acetylcholine is released at the target organs and reacts
with receptors specific to it and not to noradrenaline
• The first stage involves the biosynthesis of acetylcholine from
– choline and acetyl coenzyme A at the end of the presynaptic neuron.
• The reaction is catalysed by the enzyme choline
acetyltransferase .

• It is incorporated into membrane-bound vesicles by means of a specific

transport protein.
• The arrival of a nerve signal leads to an opening of calcium ion channels
and an increase in intracellular calcium concentration.
– This induces the vesicles to fuse with the cell membrane and release
the transmitter into the synaptic gap.
• it crosses the synaptic gap and binds to the cholinergic receptor,
resulting in stimulation of the second neuron
• It moves to an enzyme called acetylcholinesterase , which is situated
on the postsynaptic neuron, and which catalyses the hydrolysis of
acetylcholine to produce choline and acetic acid (ethanoic acid)
• Choline is taken up into the presynaptic neuron by a transport protein to
continue the cycle.
• Agonists at the cholinergic receptor
– If there is a lack of acetylcholine acting at a certain part of
the body, why do not we just give the patient more
acetylcholine?
– After all, it is easy enough to make in the laboratory.

– There are three reasons why this is not feasible.

• Acetylcholine is easily hydrolysed in the stomach by
acid catalysis and cannot be given orally.
• Acetylcholine is easily hydrolysed in the blood, both
chemically and by enzymes (esterases and
acetylcholinesterase).
• There is no selectivity of action. Acetylcholine will
• Therefore, we need analogues of acetylcholine which are
– More stable to hydrolysis
– More selective with respect to where they act in the body.
• There are two ways in which selectivity can be
achieved.
– Firstly, some drugs might be distributed more efficiently
to one part of the body than another.
– Secondly, cholinergic receptors in various parts of the
body might be slightly different.
• How do we know if there are different subtypes of
acetylcholine receptor?
– The first clue came from the action of natural compounds.
– Compounds nicotine and muscarine were both
acetylcholine agonists
– Nicotine was found to be active at the synapses between
• two different nerves and
• at the synapses between nerves and skeletal muscle, but
– had poor activity elsewhere.
– Muscarine was active at the synapses of nerves with
smooth muscle and cardiac muscle, but showed poor
activity at the sites where nicotine was active.
• From these results:
– There is one type of acetylcholine receptor on skeletal
muscles and at nerve synapses
• Known as nicotinic receptor, N
– There is a different sort of acetylcholine receptor on
smooth and cardiac muscles
• Known as muscarinic receptor, M
• Unfortunately, these two compounds are not suitable as
medicines since they have undesirable side-effects.
• But they have served as a lead compound for further
development
• Acetylcholine: Structure, SAR, and receptor
binding
 Modification of the quaternary ammonium group
• Analogues of acetylcholine in which the nitrogen atom was
replaced by arsenic, phosphorus, or sulfur have been
synthesized
• Although they exhibited some of the activity of acetylcholine,
these compounds are less active and are not used clinically.
– It was concluded that only compounds possessing a positive charge on the
atom in the position of the nitrogen had appreciable muscarinic activity.
• Compounds in which all three methyl groups on the nitrogen are
replaced by larger alkyl groups are inactive as agonists.
• When the methyl groups are replaced by three ethyl groups, the
resulting compound is a cholinergic 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
• Furthermore, successive replacement of one, two, or three of
the methyl groups with hydrogen atoms to af ford a tertiary,
secondary, or primary amine, respect ively, leads to
successively diminishing muscarinic activity
• Modification of the ethylene bridge
• Synthesis of acetic acid esters of quaternary ammonium
alcohols of greater length than choline led to a series of
compounds with activity that was rapidly reduced as the
chain length increased.
– This observation led Ing to postulate his Rule of Five.
– This rule suggests that there should be no more than five
atoms between the nitrogen and the terminal hydrogen
atom for maximal muscarinic potency.
– Present concepts suggest that the muscarinic receptor
cannot successfully accommodate molecules larger than
acetylcholine and still produce its physiologic effect .
– Although larger molecules may bind to the receptor, they
lack efficacy and demonstrate antagonist properties.
• Replacement of the hydrogen atoms of the ethylene bridge
by alkyl groups larger than methyl affords compounds that
are much less active than acetylcholine
• Introduction of a methyl group on the carbon β to the
quaternary nitrogen affords
• A methyl group on the carbon α to the quaternary nitrogen affords
acetyl-α-methylcholine.
– Al though activity relative to acetylcholine is reduced at both
muscarinic and nicotinic receptors, it exhibits greater nicotinic
than muscarinic potency.
• This compound is not currently used as a therapeutic agent .
• 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.
• 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.
• The nicotinic receptor and AChE exhibit little stereoselectivity
for the optical isomers of acetyl-α-methylcholine
• Conclusions:
– Clearly, there is a tight fit between acetylcholine and its
binding site which leaves little scope for variation.

• Shortening or lengthening the chain of atoms that separates

the ester group from the onium moiety reduces muscarinic
activity.
• Hydrolysis by AChE is more affected by substitutions on the
than the carbon.
• The hydrolysis rate of racemic acetyl –β-methylacetylcholine
is about 50% of that of ACh; racemic acetyl-α-ACh is
hydrolyzed about 90% as fast.
• Modification of the acyloxy group
• As would be predicted by the Rule of Five , when the acetyl
group is replaced by higher homologues (i .e. , the propionyl
or butyryl groups), the resulting esters are less potent than
acetylcholine.
• Choline esters of aromatic or higher-molecular-weight acids
possess cholinergic antagonist act ivity.
• Replacement of the acetyloxy functional group of ACH with a
functional group more resistant to hydrolysis.
• This led to synthesis of the carbamic acid ester of choline
(carbachol),
– a potent cholinergic agonist possessing both muscarinic
and nicotinic activity.
– they are more stable than carboxylate esters to
hydrolysis.
– Carbachol is less readily hydrolyzed by gastric acid, AChE,
or butyrylcholinesterase than acetylcholine is, and i t can
be administered orally.
• The classic SAR for muscarinic agonist activity can be
summarized as follows:
– The molecule must possess a nitrogen atom capable of bearing a
positive charge, 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.
– The molecule should have an oxygen atom, preferably
an ester-like oxygen, capable of part- icipating in a
hydrogen bond.
– There should be a two-carbon unit between the oxygen
atom and the nitrogen atom.
 Carbachol chloride (Isopto Carbachol)
Methacholine chloride (Provocholine) • It is the carbamate analogue of
• Methacholine, acetyl-β- acetylcholine,
methylcholine, is marketed as • shows no selectivity for
the racemic mixture. muscarinic or nicotinic receptors.
• It is a selective muscarinic • It is more resistant toward acid-,
agonist with very little activity base-, or enzyme (AChE)-
at nicotinic receptors. catalyzed hydrolysis than
• Al though it is marketed as the acetylcholine.
racemic mixture, the S-(+)- • It also is reported to exhibit weak
enantiomer is anticholinesterase activity.
– 240-fold more potent than – Both of these actions work to
prolong the duration of act ion
the R-(–)-isomer at
of carbachol .
muscarinic receptors.
• Because of erratic absorption and
– In addition, AChE hydrolyzes its actions at nicotinic receptors,
S-(+)–methacholine at use of carbachol has been limited
approximately 54% the rate to the treatment of
of acetylcholine, whereas – glaucoma and for the induction
the R-(–)–enantiomer is a of miosis in ocular surgery.
weak inhibitor. • It is available as an intraocular
• It is used via inhalation for solution and an ophthalmic
solution.
• The instability of acetylcholine
• Why is this and how can the stability being improved?

• The positively charged nitrogen interacts with the

carbonyl oxygen and has an electron withdrawing
effect.
• This influence of the nitrogen ion is known as
neighboring group participation or anchimeric
assistance.
• Design of acetylcholine analogues
– In order to tackle the inherent instability of acetylcholine,
two approaches are possible:
• Steric hindrance
• Electronic stabilization
• I. Steric hindrance
– The principle involved here can be demonstrated with
methacholine which is three times more stable to
hydrolysis than acetylcholine.
– Why don’t we put on a bigger alkyl group? Alternatively,
why not put a bulky group on the acyl half of the
molecule?
• In conclusion, attempts to increase the steric shield beyond
the methyl group certainly increase both chemical and
enzymatic stability of the molecule, but decrease its activity
since it cannot fit the cholinergic receptor.
• One other very useful result was obtained from
methacholine.
• It was discovered that the introduction of the methyl group
led to significant muscarinic activity and very little nicotinic
activity.
• This result is perhaps more important than the gain in
stability.
• The good binding to the muscarinic receptor can be
explained if we compare the active conformation of
methacholine with muscarine. Which isomer is more
active???
• This is only possible for the S -enantiomer of methacholine
and when the two enantiomers of methacholine were
separated, it was found that the S- enantiomer was, indeed,
the more active enantiomer. It is not used therapeutically,
however.
• Electronic effects
– The best example of this approach is provided by
carbachol, a long acting cholinergic agent which is
resistant to hydrolysis.
– In carbachol, the acyl methyl group has been replaced by
an NH2 group which is of comparable size and can
therefore fit the receptor
– The resistance to hydrolysis is due to the electronic effect
of the carbamate group.
• Since the methyl group of acetylcholine has been replaced
with an amino group without affecting the biological activity,
we can call the amino group a 'bioisostere' of the methyl
group.
– Therefore, the inclusion of an electron donating group
such as the amino group has greatly increased the
chemical and enzymatic stability of our cholinergic
agonist.
– It is found that carbachol shows very little selectivity
between the muscarinic and nicotinic sites.
– Carbachol is used clinically for the treatment of glaucoma.
– The drug is applied locally and so selectivity is not a great
problem.
• Combining steric and electronic effects
– Only selective for muscarinic receptor
– Bethanechol is used therapeutically in stimulating the
gastrointestinal tract and urinary bladder after surgery.
• Both these organs are 'shut down' with drugs during surgery
• The drug is used orally and by subcutaneous injection.
• It must never be administered by intramuscular or
intravenous injection
• Clinical uses of cholinergic agonists
• Muscarinic agonists:
– Treatment of glaucoma.
– 'Switching on' the GIT and urinary tract after surgery.
– Treatment of certain heart defects by decreasing heart muscle activity
and heart rate.
• Nicotinic agonists:
– Treatment of myasthenia gravis
• Pilocarpine
– is an example of a muscarinic agonist which is used in
the treatment of glaucoma.
– It is an alkaloid obtained from the leaves of shrubs
belonging to the genus Pilocarpus .
– Although there is no quaternary ammonium group
present in pilocarpine, it is assumed that the drug is
protonated before it interacts with the muscarinic
receptor.
– Molecular modelling shows that pilocarpine can adopt a
conformation having the correct pharmacophore for the
muscarine receptor; i.e. a separation between nitrogen
and oxygen of 4.4 A.
– It is also being considered for the treatment of
Alzheimer's disease, as are other muscarinic agonists
such as oxotremorine and various arecoline analogues
• Because pilocarpine is a lactone, its solutions are
subject to hydrolysis to afford the pharmacologically
inactive pilocarpic acid and to base-catalyzed
epimerization at C3 in the lactone to give
isopilocarpine, an inactive stereoisomer of pilocarpine.
• NB: 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.
 Acetylcholinestrase inhibitors (indirectly
acting cholinergic agonists)
• Acetylcholinesterase is the enzyme which hydrolyses
acetylcholine.
• Anticholinesterases are antagonists of this enzyme
• Antagonist at the acetylcholinesterase enzyme will have the
same biological effect as an agonist at the cholinergic
receptor.
• Action of anticholiesterase is shown below
• Therapeutic Application
– These agents are used therapeutically to improve muscle
strength in myasthenia gravis.
– They also are used in open-angle glaucoma to decrease
intraocular pressure by stimulating contract ion of the
ciliary muscle and sphincter of the iris.
• This facilitates out flow of aqueous humor via the canal
of Schlemm.
– Recently, AChEIs have found use in the treatment of
symptoms of Alzheimer's disease and similar cognitive
disorders
• which are conditions characterized by a cholinergic
deficiency in the cortex and basal forebrain.
– They are used extensively as insecticides and are in
military arsenals for use as chemical warfare agents.
• Structure of the acetylcholinesterase enzyme
– Has a fascinating tree-like structure
– The trunk of the tree is a collagen molecule which is
anchored to the cell membrane
– There are three branches (disulfide bridges) leading off
from the trunk
– The enzyme itself is made up of four protein subunits,
each of which has an active site
• There are three branches with disulphide bridges that lead
off from the trunk, each of which holds the
acetylcholinesterase enzyme above the surface of the
membrane
• The enzyme itself is made up of four protein subunits, each
of which has an active site.
– Therefore, each enzyme tree has 12 active sites.
• The trees are rooted immediately next to the cholinergic
receptors such that they efficiently capture acetylcholine as
it departs the receptor. In fact, the acetylcholinesterase
enzyme is one of the most efficient enzymes known.
• A soluble cholinesterase enzyme called
butyrylcholinesterase is also present in various tissues
and plasma.
• This enzyme has a broader substrate specificity than
acetylcholinesterase and can hydrolyse a varity of esters
• Design of anticholinesterases
– Its design is depend on
• The shape of the enzyme active site,
• The binding interactions involved with acetylcholine, and
• The mechanism of hydrolysis.
• Binding interactions at the active site
– There are two important areas to be considered
– The anionic binding site and the ester binding site
• Note that:
– Acetylcholine binds to the cholinesterase enzyme by
• (a) Ionic bonding to an Asp or Glu residue,
• (b) Hydrogen bonding to a tyrosine residue
• Crucial amino acids within the active site
– The important amino acids within the active site are
• those which bind acetylcholine, as well as those
involved in the mechanism of hydrolysis.
– As far as binding is concerned, several amino acids are
thought to be involved, but a key interaction is the
interaction between a tryptophan residue and the
quaternary nitrogen atom
– The key amino acid residues involved in the catalytic
mechanism are
• serine, histidine, and glutamate
• Mechanism of hydrolysis
– The histidine residue acts as an acid/base catalyst
throughout the mechanism
– Serine plays the part of a nucleophile (but is poor
nucleophile)
– Activity
• Compare and contrast the speed of enzymatic
hydrolysis and chemical hydrolysis of Ach!!!
• Anticholinesterase drugs
– inhibit the cholinesterase enzyme from breaking down
Ach
• increasing both the level and duration of the
neurotransmitter action
– According to the mode of action, AChE inhibitors can be
divided into two groups:
• irreversible and reversible.
 Reversible anticholinestrase
• Carbamates
– are are organic compounds derived from carbamic acid
(NH2COOH).

– where X can be oxygen or sulphur (thiocarbamate),

– R1 and R2 are usually organic or alkyl substituents, but R1 or
R2 may also be hydrogen, and
– R3 is mostly an organic substituent or sometimes a metal.
– In addition to their use as therapeutic drugs in human medicine
(AD, myasthenia gravis, glaucoma, Lewy bodies, Parkinson’s
disease),
• these reversible AChE inhibitors have been applied as
pesticides, then as parasiticides in veterinary medicine, and in
prophylaxis of organophosphorus compounds (OPs) poisoning
• The carbamates
• Physiostigmine
 it was a natural product that provided the lead for
the carbamate inhibitors.  Was discovered in 1864 as a
product of the poisonous calabar beans from West Africa The
structure was established in 1925

• Structure-activity relationships (SAR)

•  The carbamate group is essential to activity
•  The benzene ring is important
•  The pyrrolidine nitrogen (which is ionized at blood pH) is
important
The overall rate of hydrolysis of physostigmine is 40 × 10 6
times slower than that of acetylcholine.
As a result, the cholinesterase active site becomes blocked and
is unable to react with acetylcholine.
• Physostigmine's antagonistic…
•  The first three stages proceed as normal with the above
steps that we have seen for acetylcholine. However; the
next stage turns out to be extremely slow.

• Why is this final stage so slow?

– The carbamoyl/enzyme intermediate is stabilized because the
nitrogen can feed a lone pair of electrons into the carbonyl group
• Analogues of physostigmine
– Physostigmine has limited medicinal use because of
serious side effect
– only been used in the treatment of glaucoma or as an
antidote for atropine poisoning.
– Simpler analogues have been made and have been used
in the treatment of myasthenia gravis and as an
antidote to curare.
– The analogues should conatian all the structural
requirements of physostigmine
• Miotine
– It is active as antagonist to the enzyme
– it suffers from the following disadvantages:
• susceptible to chemical hydrolysis
• CNS side effect
• Neostigmine and pyridostigmine
– Were designed to deal with both these problems.
– Firstly, a quaternary nitrogen atom is present and so
there is no chance of the free base being formed.
• As the molecule is permanently charged, it cannot
cross the blood–brain barrier and so the drug is free of
CNS side effects.
• Increased stability is achieved by using a
dimethylcarbamate group rather than a
methylcarbamate group.
• Two further points to note about neostigmine
– The quaternary nitrogen is 4.7 A away from the ester
– The direct bonding of the quaternary centre to the
aromatic ring reduces the number of conformations
which the molecule can take up.
• This is an advantage if the active conformation is
retained because the molecule is more likely to be
in the active conformation when it approaches the
active site.
• Both neostigmine and pyridostigmine are in use today.
• They are given intravenously to reverse the actions of
neuromuscular blockers or used orally in the treatment
of myasthenia gravis
• Irreversible Acetylcholinesterase Inhibitors
– AChE is inhibited irreversibly by a group of phosphate
esters that are highly toxic.
– These chemicals are nerve poisons and have been used in
warfare, in bioterrorism, and as agricultural insecticides.
• A general formula of such compounds:

• Where R1 = alkoxy R2 = alkoxy, alkyl, or tertiary amine X =

good leaving group (e.g., F, CN, thiomalate, p-nitrophenyl
• When A is sulfur, bioactivation is required before the
compound becomes effective as an inhibitor of
cholinesterase.
• Inhibition of AChE by organophosphorus compounds takes
place in two steps
– Association of enzyme and inhibitor and Phosphorylation
step, completely analogous to acylation by the substrate.
– Although insecticides and nerve gases are irreversible
inhibitors of cholinesterases by forming a phosphorylated
serine at the esteratic site of the enzyme, it is possible to
reactivate the enzyme if action is taken soon after
exposure to these poisons.
• Several compounds can provide a nucleophilic attack on the
phosphorylated enzyme and cause regeneration of the free
enzyme.
• Nerve gas
– The nerve gases dyflos and sarin were discovered
– Dyflos was developed as a nerve gas in the Second World
War.
– It inhibits AChE by irreversibly phosphorylating the serine
residue at the active site.
• Medicine
– Echothiophate were designed to fit the active site more
accurately by including a quaternary amine to bind with
the anionic region.
– This means that lower doses would be more effective.
– Echothiopate is used medicinally in the form of eye drops
for the treatment of glaucoma and has advantages over
dyflos.
– Unlike dyflos, echothiopate slowly hydrolyses from the
enzyme over a matter of days.
• Insecticides
– Parathion and malathion are relatively non-toxic with respect to
nerve gases.
– The phosphorus-sulfur double bond prevents these molecules
from antagonizing the active site on the acetylcholinesterase
enzyme.
– The equivalent compounds containing a phosphorus-oxygen
double bond are, on the other hand, lethal compounds.
– There are no metabolic pathways in mammals, which can
convert the phosphorus -sulfur double bond to a phosphorus-
oxygen double bond.
– Such pathway does, however, exist in insects
– In the latter species, parathion and malathion act as prodrugs.
• They are metabolized by oxidative desulfuration to give the
anticholinesterases, which irreversibly bind to the insects’
acetylcholinesterases enzymes and lead to death.
• In mammals, the same compounds are metabolized in a
differently way to give inactive compounds which are then
excreted
• Mechanism of Action
– The chemical logic involved in the development of
effective AChEIs was to synthesize compounds that would
be substrates for AChE and
– Result in an acylated enzyme more stable to hydrolysis
than a carboxylate ester.
– Phosphate esters are very stable to hydrolysis, being even
more stable than many amides.
– Application of this chemical property to the design of
AChEI compounds led to derivatives of phosphoric,
pyrophosphoric and phosphonic acids that are effective
inhibitors of AChE.
– These act as inhibitors by the same mechanism as the
carbamate inhibitors except that they leave the enzyme
esterified as the phosphate esters slowly
• The rate of hydrolytic regeneration of the phosphorylated
enzyme is much slower than that of the carbamylated
enzyme and its rate is measured in hours.
• Because duration of action of these compounds is much
longer than that of the carbamate esters, they are referred
to as irreversible inhibitors of AChE.
• An important difference between irreversibly phosphoester
derived AChEIs and reversible AChEIs is that the
phosphorylated AChE can undergo a process known as
aging.
• Aging is the result of cleavage of one or more of the
phosphoester bonds while the AChE is phosphorylated.
• This reaction affords an anionic phosphate that possesses a
phosphorus atom which is much less electrophilic and
therefore, much less likely to undergo hydrolytic
regeneration than the original phosphoester.
• Thus, the aged phoshorylated enzyme does not undergo
nucleophilic attack and regeneration by antidote for
phosphate ester AChEIs.
• This aging process occurs over a period of time, which
depends on the rate of the P-O bond cleavage reaction;
during this time, the antidotes to phosphate ester poisoning
may be effective

Mechanism of AChE inhibition induced by OPs; reactivation, spontaneous hydrolysis, and

aging of the phosphorylated enzyme.
• Cholinesterase Activators (Antidote for Irreversible AChEIs)
– Water is a nucleophile capable of hydrolyzing acetylated AChE rapidly
and regenerating the active enzyme.
– Phosphorylated AChE (irreversibly inhibited), however, was known to
involve a phosphate ester of serine.
– The rate of hydrolysis is much slower for organic phosphate esters
than carboxylate esters, and significantly stronger nucleophile than
water would be required for efficient hydrolysis of phosphate esters.
• Hydroxylamine (NH2-OH) is a strong nucleophilic
compound that efficiently cleaves phosphate esters and
increases significantly the rate of hydrolysis of
phosphorylated AChE but only at toxic concentration.
• Development of structural analog of hydroxylamine
without toxicity
– Logical approach is to design compound with
• a high degree of selectivity and strong binding
affinity for AChE and
• carry a hydroxylamine-like nucleophile into close
proximity to the phosphorylated serine residue.
• Approach: synthesis of hydroxylamine derivatives of
organic compounds possessing a functional group
bearing a positive charge.
• The reaction of hydroxylamine with aldehydes or
ketones affords oximes, which possess the desired
nucleophilic oxygen atom.
• There are three readily available positional isomers of
pyridine aldehyde that can be converted easily to oximes.
– Of the three isomeric pyridine aldoxime methiodides,
the most effective is the isomer derived from 2-
pyridinylaldehyde (2-pyridine aldoxime methyl Iodide
(Pralidoxime, 2-PAM).
– Finally, the nitrogen atom of the pyridine ring can be
converted to a quaternary ammonium salt by treatment
with methyl iodide.
• This cationic charge increases affinity of the
compound for the “anionic binding site” of the
phosphorylated AChE.
– Proposed mechanism for regeneration of AChE
• Binding of the quaternary ammonium nitrogen of 2-
PAM to the “anionic binding site” of phosphorylate
AChE, which places nucleophilic oxygen of 2-PAM in
close proximity to the electrophilic phosphorum atom
• Nucleophilic attack of the oxime oxygen results in
breaking of the ester bond between the serine oxygen
atom and the phosphorum atom, resulting in
regenerated active form of AChE and phosphoryalted 2-
PAM
– Pralidoxime must be given within a short period of time,
– After aging has occurred, 2-PAM will not regenerate the
enzyme.
• Pralidoxime showing a potency as an antidote 106 times greater
than hydroxylamine.
• Because pralidoxime has a quaternary nitrogen, it is
fully charged and cannot pass through the blood–brain
barrier into the CNS.
• This means that the antidote cannot work on any
enzymes that have been inhibited in the brain.
• Pro-2-PAM is a prodrug of pralidoxime which avoids this
problem.
• As a tertiary amine it can pass through the blood–brain
barrier and is oxidized to pralidoxime once it has
entered the CNS.
• Muscarinic antagonists
– Antagonists of the cholinergic receptor are drugs which
bind to the receptor but do not ‘switch it on’.
– By binding to the receptor, an antagonist acts like a plug
at the receptor binding site and prevents acetylcholine
from binding
– The overall effect on the body is the same as if there was
a lack of acetylcholine.
– Therefore, antagonists have the opposite clinical effect
from agonists.
• The clinical effects of muscarinic antagonists are:
– reduced saliva and gastric secretions;
– reduced motility of the GIT and urinary tract by relaxation
of smooth muscle;
– dilatation of eye pupils;
– CNS effects
 The clinical uses are

– shutting down the GIT and urinary tract during

surgery;
– ophthalmic examinations;
– relief of peptic ulcers;
– treatment of Parkinson's disease;
– treatment of anticholinesterase poisoning;
– treatment of motion sickness;
– a potential use for M2 antagonists is in the treatment
of Alzheimer's disease.
• The first antagonists were natural products and in particular
alkaloids.
• Atropine
– belladonna (deadly nightshade) and is included in a root
extract which was once used by Italian women to dilate
their eye pupils.
– Clinically, atropine has been used to decrease
• gastrointestinal motility and
• to counteract anticholinesterase poisoning.
• Atropine has an asymmetric centre but exists as a racemate
• Usually, natural products exist exclusively as one
enantiomer.
• This is also true for atropine, which is present in the plants of
the genus Solanaceae as a single enantiomer called (-)
hyoscyamine .
• As soon as the natural product is extracted into solution,
however, racemization takes place.
– The asymmetric centre in atropine is easily racemized as
• (-) Hyoscine (or scopolamine) is obtained from the thorn
apple ( Datura stramonium )
– it is very similar in structure to atropine.
– It has been used in the treatment of motion sickness.
• These two compounds bind to the cholinergic receptor, but,
at first sight, they do not look anything like acetylcholine.
• If we look more closely though, we can see that a basic
nitrogen and an ester group are present, and if we
superimpose the acetylcholine skeleton on to the atropine
skeleton, the distance between the ester and the nitrogen
groups is similar in both molecules
– There is, of course, the problem that the nitrogen in
atropine is uncharged, whereas the nitrogen in
acetylcholine has a full positive charge.
• This implies that the nitrogen atom in atropine must be
protonated and charged when it binds to the cholinergic
receptor
• Therefore, atropine has two important binding features shared wit
acetylcholine—
– a charged nitrogen when protonated and an ester group.
• It is able to bind to the receptor, but why is it unable to switch it on?
– Because atropine is a larger molecule than acetylcholine, it is
capable of binding to other binding regions within the binding site
which are not used by acetylcholine itself.
• As a result, it interacts differently with the receptor and does
not induce the same conformational changes (induced fit) as
acetylcholine.
– This means that the receptor is not activated.
• As both atropine and hyoscine are tertiary amines rather than
quaternary salts, they are able to cross the blood–brain barrier as
the free base.
• Once they are in the brain, they can become protonated and
antagonize muscarinic receptors which causes CNS effects;
• for example hallucinogenic activity is brought on with high doses,
and both hyoscine and atropine were used by witches in past
centuries to produce that very effect.
• Other CNS effects observed in atropine poisoning are restlessness,
agitation, and hyperactivity.
 Structural analogues based on atropine

• In order to reduce CNS side effects, quaternary salts of

atropine and atropine analogues are used clinically
• For example, ipratropium is used as a bronchodilator in
chronic obstructive pulmonary disease.
• Atropine methonitrate acts at the intestine to relieve spasm.
• A large number of different analogues of atropine were
synthesized to investigate the SAR of atropine, revealing the
importance of
– the aromatic ring, the ester group, and the basic nitrogen (which is
ionized).
• It was further discovered that the complex ring system was
not necessary for antagonist activity, so simplification could
be carried out.
• For example, amprotropine is active and has an ester
group separated from an amine by three carbon atoms.
• Chain contraction to two carbon atoms can be carried out
• These studies came up with the
following generalizations:
– the alkyl groups (R) on
nitrogen can be larger than
methyl (in contrast to
agonists);
– the nitrogen can be tertiary or
quaternary, whereas agonists
must have a quaternary
nitrogen.
– Note, however, that the
tertiary nitrogen is probably
charged when it interacts with
the receptor;
– very large acyl groups are
allowed (R 1 and R 2 =
aromatic or heteroaromatic
rings).
– This is in contrast to agonists
where only the acetyl group is
• Substituents R1 and R2 should be carbocyclic or heterocyclic
rings for maximal antagonist potency.
– The rings may be identical, but the more potent compounds have
different rings.
• Generally, one ring is aromatic and the other saturated or
possessing only one olefinic bond.
• Substituents R1 and R2, however, may be combined into a
fused aromatic tricyclic ring system, such as that found in
propantheline
• The size of these substituents is limited. For example,
substitution of naphthalene rings for R1 and R2 affords
compounds that are inactive, apparently because of steric
hindrance of the binding of these compounds to the
muscarinic receptor.
• The R3 substituent may be a hydrogen atom, a hydroxyl
group, a hydroxymethyl group, or a carboxamide, or i t may
• The X substituent in the most potent anticholinergic agents
is an ester, but an ester functional group is not an absolute
necessity for muscarinic antagonist activity.
– This substituent may be an ether oxygen, or i t may be absent
completely.
• The N substituent is a quaternary ammonium salt in the
most potent anticholinergic agents.
• This is not a requirement , however, because tertiary amines
also possess antagonist activity, presumably by binding to
the receptor in the cationic (conjugate acid) form.
• The alkyl substituents usually are methyl , ethyl , propyl , or
isopropyl .
• The distance between the ring-substituted carbon and the
amine nitrogen apparently is not critical ; the length of the
alkyl chain connecting these may be from two to four
carbons.
• The most potent anticholinergic agents have two methylene
units in this chain.
• Nicotinic antagonists
– Curare (1516) and tubocurarine
• Curare was first identified when Spanish soldiers in
South America found themselves the unwilling victims
of poisoned arrows.
• It was discovered that the Indians were putting a
poison on to the tips of their arrows.
• This poison was a crude, dried extract from a plant
called Chondrodendron tomentosum and caused
paralysis as well as stopping the heart.
• We now know that curare is a mixture of compounds.
• The active principle, however, is an antagonist of
acetylcholine which blocks nerve transmissions from
nerve to muscle.
• Can be used medically if they are taken at the right
dose levels and under proper control.
• The main application is in the relaxation of abdominal
muscles in preparation for surgery. (This allows the
surgeon to use lower levels of general anesthetic)
• Important question to be
raised here
– Why should tubocurarine
bind to and block the
cholinergic receptor with
out having ester functional
group?
– The answer lies in the fact
that the molecule has two
positively charged nitrogen
atoms (one tertiary which is
protonated, and one
quaternary).
• There are three assumptions
– Tubocurarine forms a bridge between two adjacent
cholinergic receptors.
• But latter it was proved that the distance between the
two receptor proteins dimmer was larger than (9-10
nm) the distance between the two positively charged
nitrogen centers (1.4nm).
– The tubocurarine molecule bridges two acetylcholine
binding sites within the one protein complex. Since there
are two such sites within the complex, this appears an
attractive alternative theory.
• However, the two sites are further apart than 1.4 nm
and so this too seems unlikely
– One of the positively charged nitrogens on tubocurarine
binds to the anionic binding site of the acetylcholine
receptor in the protein complex, while the other nitrogen
binds to a nearby cysteine residue 0.9-1.2 nm away
• Anyways despite the above uncertainties, the interaction is
extremely strong and would more than make up for the lack
of the ester binding interaction.
• It is also clear that the distance between the two positively
charged nitrogen atoms is crucial to activity.
• Therefore, analogues which retain this distance should also
be good antagonists.
• Decamethonium and suxamethonium
– Decamethonium is a simple analogue of tubocurarine.
– It is a straight-chain molecule and as such is capable of a
large number of conformations.
– The fully extended conformation would position the
nitrogen centres 1.35 nm apart, which compares well with
the equivalent distance in tubocurarine (1.4 nm).
– The drug binds strongly to cholinergic receptors and has
proved a useful clinical agent.
• Disadvantages;
– Initially it acts as agonist- leads to a brief contraction of
the muscle.
– It binds too strongly and as a result patients take a long
time to recover from its effects.
– Not completely selective for the neuromuscular junction
and has an effect on acetylcholine receptors at the heart.
This leads to an increased heart rate and affect the blood
pressure
• So to overcome such limitations the following approaches
were tried;
• Introduction of instability to the molecule
• Success was first achieved by introducing ester groups into
the chain while retaining the distance between the two
charged nitrogens to give suxamethonium
• The ester groups are susceptible to chemical and enzymatic
hydrolysis.
• Suxamethonium has duration of action of five minutes, but
suffers from other side-effects.(effect on autonomic ganglia)
• Furthermore, about one person in every two thousand lacks
the enzyme which hydrolyses suxamethonium
• The ester groups are susceptible to chemical and enzymatic
hydrolysis.
• Suxamethonium has duration of action of five minutes, but
suffers from other side-effects.(effect on autonomic ganglia)
• Furthermore, about one person in every two thousand lacks
the enzyme which hydrolyses suxamethonium
• Atracurium
• The design of atracurium was based on the structures of
tubocurarine and suxamethonium.
• It is superior to both since it lacks cardiac side-effects and is
rapidly broken down in blood.
• This rapid breakdown allows the drug to be administered as
an intravenous drip.
• The rapid breakdown was designed into the molecule by
incorporating a self destruct mechanism.
• At blood pH (slightly alkaline at 7.4), the molecule can
undergo a Hofmann elimination.
• Once this happens, the compound is inactivated since the
positive charge on the nitrogen is lost.
• It is a particularly clever example of drug design in that the
very element responsible for the molecule's biological
activity promotes its deactivation
• The important features of atracurium are:
• The spacer.
– This is the 13-atom connecting chain which connects the
two quaternary centres and separates the two centres.
• The blocking units;
– The cyclic structures at either end of the molecule block
the receptor site from acetylcholine.
• The quaternary centres.
– These are essential for receptor binding. If one is lost
through Hofmann elimination, the binding interaction is
too weak and the antagonist leaves the binding site.