Presented by: Moein M. Mustafa M.Sc. Pharmaceutical chemistry

Hawler Medical University College of Pharmacy

Department of Pharmaceutical chemistry

Medicinal Chemistry

Fourth stage

Cholinergic System 2

Cholinesterase Inhibitors

• There are two types of cholinesterases in humans, AChE and butyrylcholinesterase (BuChE).

• The cholinesterases differ in their location in the body and their substrate specificity.

• AChE is associated with the outside surface of glial cells in the synapse and catalyzes the hydrolysis of ACh to choline and acetic acid.

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• Inhibition of AChE prolongs the duration of the neurotransmitter in the junction and produces pharmacological effects similar to those observed when Ach is administered.

• These inhibitors are indirect-acting cholinergic agonists.

• AChE inhibitors have been used in the treatment of myasthenia gravis, atony in the GI tract, and glaucoma.

• They have also been used as agricultural insecticides and nerve gases.

• They have received attention as symptomatic treatments for patients with Alzheimer disease.

• BuChE (pseudocholinesterase) is located in

human plasma. Although its biological function is

not clear, it has catalytic properties similar to those

of AChE.

• The substrate specificity is broader, and may

hydrolyze dietary esters and drug molecules in

the blood.

• Three different chemical groupings, acetyl,

carbamyl, and phosphoryl, may react with the

esteratic site of AChE.

• Although the chemical reactions are similar, the

kinetic parameters for each type of substrate

differ and result in differences between toxicity

and usefulness.

Reversible Inhibitors Physostigmine

• Physostigmine is an alkaloid obtained from the dried ripe seed of Physostigma venenosum.

• Solutions are most stable at pH = 6.

• Physostigmine is a relatively poor covalent inhibitor of AChE and is often considered a reversible inhibitor of the enzyme.

• Its cholinesterase-inhibiting properties vary with the

pH of the medium.

• Inhibition of cholinesterase is greater in acid media,

suggesting that the protonated form makes a

contribution to the inhibitory activity well as its

carbamylation of the enzyme.

• Physostigmine was used first as a topical application in

the treatment of glaucoma.

• Its lipid solubility properties permit adequate

absorption from ointment bases.

• It is used systemically as an antidote for atropine

poisoning and other anticholinergic drugs by increasing

the duration of action of ACh at cholinergic sites

through inhibition of AChE.

• Physostigmine has been studied for use in the treatment

of Alzheimer disease.

• Cholinomimetics that are currently used or which have

been recently evaluated in the treatment of Alzheimer

disease include donepezil, galantamine, metrifonate,

rivastigmine, and tacrine.

physostigmine salicylate

Neostigmine Bromide• Neostigmine bromide or Prostigmin bromide, is used as

an antidote to neuromuscular blocking drugs and in the

treatment of myasthenia gravis.

• Use of physostigmine, as a prototype of an indirect-

acting parasympathomimetic drug, facilitated the

development of stigmine, in which a trimethylamine

group was placed para to a dimethylcarbamate group in

benzene.

Neostigmine Bromide

• Better inhibition of cholinesterase was observed when

these groups were placed meta to each other as in

neostigmine, a more active and useful agent.

• Although physostigmine contains a methylcarbamate

functional group, greater chemical stability toward

hydrolysis was obtained with the dimethylcarbamyl

group in neostigmine.

• Of the neostigmine that reaches the liver, 98% is m e t a b o l i z e d i n 1 0 m i n u t e s t o 3 -hydroxyphenyltrimethyl ammonium, which has activity similar to, but weaker than neostigmine.

• A certain amount is hydrolyzed slowly by plasma cholinesterase.

• Neostigmine has a mechanism of action quite similar to that of physostigmine, but its activity does not vary with pH.

• The uses of neostigmine are similar to those of

physostigmine but differ in exhibiting greater miotic activity, fewer and less unpleasant local and systemic

manifestations, and greater chemical stability.

• The most frequent application of neostigmine is to

prevent atony of the intestinal, skeletal, and bladder

musculature.

• An important use is in the treatment of myasthenia

gravis.

Pyridostigmine Bromide• Pyridostigmine bromide is about one fifth as toxic as

neostigmine.

• It appears to function in a manner similar to that of n e o s t i g m i n e a n d i s t h e m o s t w i d e l y u s e d anticholinesterase agent for treating myasthenia gravis.

• The liver enzymes and plasma cholinesterase metabolize the drug.

Pyridostigmine Bromide

Ambenonium Chloride• Ambenonium chloride (Mytelase chloride), is used for

the treatment of myasthenia gravis in patients who do not respond satisfactorily to neostigmine or pyridostigmine.

• This drug acts by suppressing the activity of AChE.

• It possesses a relatively prolonged duration of action and causes fewer side effects in the GI tract than the other anticholinesterase agents.

• Because of its quaternary ammonium structure, ambenonium chloride is absorbed poorly from the GI tract.

• In moderate doses, the drug does not cross the blood-brain barrier.

• Ambenonium chloride is not hydrolyzed by cholinesterases.

Demecarium Bromide• Demecarium bromide (Humorsol), its efficacy and

toxicity are comparable to those of other potent anticholinesterase inhibitor drugs.

• It is a long-acting miotic used to treat wide-angle glaucoma and accommodative esotropia.

• Maximal effect occurs hours after administration, and the effect may persist for days.

Rivastigmine• Rivastigmine (Exelon) is a noncompetitive carbamate

inhibitor of AChE.

• Because of the slow dissociation of the drug from the

enzyme it has long duration of action.

• The Food and Drug Administration (FDA) approved its

use in mild-to-moderate Alzheimer disease in 2000.

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• In 2007, rivastigmine was granted approval for use in

managing mild-to-moderate dementia associated with

Parkinson disease.

Irreversible Inhibitors• Both AChE and BuChE are inhibited irreversibly by a group

of phosphate esters (organophosphate) that are highly toxic.

• These chemicals are nerve poisons and have been used in

warfare, in bioterrorism, and as agricultural insecticides.

They permit ACh to accumulate at nerve endings and

exacerbate ACh-like actions.

• The compounds belong to a class of organophosphorous

esters.

• A is usually oxygen or sulfur but may also be selenium.

• When A is other than oxygen, biological activation is required

before the compound becomes effective as an inhibitor of

cholinesterases.

• Phosphorothionates [R1R2P(S)X] have much poorer electrophilic character than their oxygen analogs and are much weaker

hydrogen bond-forming molecules because of the sulfur atom.

• Their anticholinesterase activity is 105 fold weaker than their oxygen analogs.

• X is the leaving group when the molecule reacts with the enzyme.

• Typical leaving groups include fluoride, nitrile, and p-nitrophenoxy.

• The R groups may be alkyl, alkoxy, aryl, aryloxy, or amino.

• The R moiety imparts lipophilicity to the molecule and

contributes to its absorption through the skin.

• Inhibition of AChE by organophosphorous compounds takes

place in two steps, association of enzyme with inhibitor and

the phosphorylation step.

• Stereospecificity is mainly caused by interactions of

enzyme and inhibitor at the esteratic site.

• The serine residue at the esteratic site forms a stable

phosphoryl ester with the organophosphorous inhibitors.

• 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.

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• Several compounds can provide a nucleophilic attack

on the phosphorylated enzyme and cause

regeneration of the free enzyme.

• Substances such as choline, hydroxylamine, and

hydroxamic acid have led to the development of more

effective cholinesterase reactivators, such as nicotinic

hydroxamic acid.

• Chol ines te rases tha t have been exposed to phosphorylating agents (e.g., sarin) become refractory to reactivation by cholinesterase reactivators.

• The process is called aging and occurs both in vivo and in vitro with AChE and BuChE.

• Aging occurs by part ial hydrolysis of the phosphorylated moiety that is attached to the serine residue at the esteratic site of the enzyme.

sarin

• Symptoms of toxicity are nausea, vomiting, excessive

sweating, salivation, miosis, bradycardia, low blood

pressure, and respiratory difficulty, which is the usual cause

of death.

• The organophosphate insecticides of low toxicity, such as

malathion, generally cause poisoning only by ingestion of

relatively large doses.

• Parathion or methylparathion, however, cause poisoning

by inhalation or dermal absorption.

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• Because these compounds are so long acting,

cumulative and serious toxic manifestations may

result after several small exposures.

• Phosphate esters used as insecticidal agents are toxic

and must be handled with extreme caution.

Isofluorphate• Contact with eyes, nose, mouth, and even skin should

be avoided because it can be absorbed readily through

intact epidermis and more so through mucous tissues.

• Because isofluorphate irreversibly inhibits

cholinesterase, its activity lasts for days or even weeks.

• During this period, new cholinesterase may be

synthesized in plasma, erythrocytes, and other cells.

• A combination of atropine sulfate and magnesium sulfate

protects rabbits against the toxic effects of isofluorphate.

• Atropine sulfate counteracts the muscarinic effect, and

magnesium sulfate counteracts the nicotinic effect of the

drug.

• Isofluorphate has been used in the treatment of glaucoma.

Isofluorphate

Malathion• Malathion, has been used as an agricultural insecticide.

• Malathion is a poor inhibitor of cholinesterases.

• Microsomal oxidation, which causes desulfuration,

occurs slowly to form the phosphothioate (malaoxon),

which is 10,000 times more active than the

phosphodithioate (malathion) as a cholinesterase

inhibitor.

• Insects detoxify the phosphothioate by a phosphatase,

forming dimethyl phosphorothioate, which is inactive

as an inhibitor.

• Humans, however, can rapidly hydrolyze malathion by

a carboxyesterase enzyme, yielding malathion acid, a

still poorer inhibitor of AChE.

• Phosphatases and carboxyesterases further metabolize

malathion acid to dimethylphosphothioate.

Parathion• Parathion is used as an agricultural insecticide.

• It is a relatively weak inhibitor of cholinesterase;

however, enzymes present in liver microsomes and

insect tissues convert parathion to paraoxon, a more

potent inhibitor of cholinesterase.

• Parathion is also metabolized by liver microsomes to

yield p-nitrophenol and diethylphosphate; the latter is

inactive as an irreversible cholinesterase inhibitor.

CHOLINERGIC BLOCKING AGENTS• A wide variety of tissues respond to ACh released by the

neuron or exogenously administered chemicals to mimic this

neurotransmitter’s action.

• Peripheral chol inergic receptors are located a t

parasympathetic postganglionic nerve endings in smooth

muscle, sympathetic and parasympathetic ganglia, and

neuromuscular junctions in skeletal muscle.

• Although ACh activates these receptors, there are antagonists

that are selective for each.

• Atropine is an effective blocking agent at parasympathetic postganglionic terminals.

• Like most classic blocking agents, it acts on all muscarinic receptor subtypes.

• d-Tubocurarine blocks the effect of ACh on skeletal muscle, which is activated by N1 nicotinic receptors.

• Hexamethonium blocks transition at N2 nicotinic

receptors located in autonomic ganglia.

• Anticholinergic action by drugs and chemicals

apparently depends on their ability to reduce the

number of free receptors that can interact with Ach.

Structure–Activity Relationships• A wide variety of compounds possess anticholinergic

activity.

• The development of such compounds has been largely

empiric and based principally on atropine as the prototype.

• Nevertheless, structural permutations have resulted in

compounds that do not have obvious relationships to the

parent molecule.

There are several ways in which the structure–activity

relationship could be considered, in general, the

considerations of Long et al., who based their

postulations on the 1-hyoscyamine molecule being one of

the most active anticholinergics and, therefore, having an

optimal arrangement of groups.

1-hyoscyamine

• Anticholinergic compounds may be considered chemicals that

have some similarity to ACh but contain additional

substituents that enhance their binding to the cholinergic

receptor.

• An anticholinergic agent may contain a quaternary

ammonium function or a tertiary amine that is protonated in

the biophase to form a cationic species.

• The nitrogen is separated from a pivotal carbon atom by a chain

that may include an ester, ether, or hydrocarbon moiety.

• The substituent groups A and B contain at least one aromatic moiety capable of van der Waals interactions to the receptor surface and one cycloaliphatic or other hydrocarbon moiety for hydrophobic bonding interactions.

• C may be hydroxyl or carboxamide to undergo hydrogen bonding with the receptor.

THE CATIONIC HEAD• It is generally considered that the anticholinergic

molecules have a primary point of attachment to

cholinergic sites through the cationic head (i.e., the

positively charged nitrogen).

• For quaternary ammonium compounds, there is no

question of what is implied, but for tertiary amines, one

assumes, with good reason, that the cationic head is

achieved by protonation of the amine at physiological

pH.

THE HYDROXYL GROUP• Although not requisite for activity, a suitably placed

alcoholic hydroxyl group enhances antimuscarinic activity.

• The position of the hydroxyl group relative to the nitrogen

appears to be fairly critical, with the diameter of the

receptive area estimated to be about 2 to 3 Å.

• It is assumed that the hydroxyl group contributes to the

strength of binding, probably by hydrogen bonding to an

electron-rich portion of the receptor surface.

THE ESTERATIC GROUP• Many of the highly potent antimuscarinic compounds

possess an ester grouping, and this may be a contributing

feature for effective binding.

• This is reasonable because the agonist (i.e., ACh) possesses a

similar function for binding to the same site.

• An esteratic function is not necessary for activity, because

several types of compounds do not possess such a group

(e.g., ethers, aminoalcohols).

CYCLIC SUBSTITUTION• Examination of the active compounds reveals that at

least one cyclic substituent (phenyl, thienyl, or other) is

a common feature in almost all anticholinergic

molecules.

• Aromatic substitution is often used in connection with

the acidic moiety of the ester function.

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• Use of aromatic acids leads to low activity of these

compounds as anticholinergics but potential activity

as local anesthetics.

• In connection with the apparent need for a cyclic group, it

has been pointed out that the “mimetic” molecules, richly

endowed with polar groups, undoubtedly require a

complementary polar receptor area for effective binding.

• As a consequence, it is implied that a relatively

nonpolar area surrounds such sites.

• Thus, increasing the binding of the molecule in this

peripheral area by introducing flat, nonpolar groups

(e.g., aromatic rings) should achieve compounds with

excellent affinity but without intrinsic activity.

• This postulate is consistent with most antimuscarinic

drugs, whether they possess an ester group or not.