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Medicinal Chemistry-I 2.

16 Physico-Chemical Parameters & Drug Action

Thus, non-critical part of a drug molecule is not involved in drug receptor interactions
but is involved in passive transport of the drug.
While any change or modification of critical part of the drug molecule will result in the
change of its biological activity, only those groups having similar steric, electronic and
solubility characteristics can be interchanged. The study of such groups (bio-isosters) and
their application in medicinal chemistry is known as Bio-isosterism.
More recently Burger classified and subdivided bio-isosters as:
(1) Classical bio-isosters:
(a) Monovalent atoms and groups, e.g. CH2, NH2, OH and SH.
(b) Divalent atoms and groups, e.g. R–O–R', R–NH–R', R–CH2–R' and R–Si–R'
(c) Trivalent atoms and groups, e.g. R – N = R', and R – CH = R'
r r
(d) Tetrasubstituted atom, e.g., = C =, = N =, and = P =
(e) Ring equivalents, e.g. – CH = CH –, – S –, – O – , – NH and – CH2 –
(2) Non-classical bio-isosters:
These non-classical bio-isosters do not rigidly fit the steric and electronic rules of the
classic bio-isosters. These are further subdivided into,
(a) Exchangeable groups
(b) Rings versus non-cyclic structures.
BIO-ISOSTERIC APPLICATIONS
(1) An important compound of catecholamine series is phenylephrine.
OH

CH — CH2 — NHCH3

HO
Phenylephrine
An alkylsulphonamido group may be substituted for the phenolic hydroxyl group. Some
of the resulting compounds have agonist activity whereas others are antagonist.
OH

CH — CH2 — NH2

RO2SNH
Alkylsulphonamidophenylethanolamine
While a classic example of rings versus non-cyclic structures is diethylstilbestrol and
estradiol.
OH
OH

HO HO
Diethyl stilbestrol Estradiol
Medicinal Chemistry-I 2.17 Physico-Chemical Parameters & Drug Action

Diethylstilbestrol has about the same potency as the naturally occurring estradiol. The
central double bond of diethyl stilbestrol is highly important for the correct orientation of
the phenolic and ethyl groups (trans) at the receptor site. Table 2.6 contains a variety of bio-
isosters including classical and non-classical examples, incorporating marketed drugs as well
as interesting experimental compounds.
Applications of bio-isosterism were also found in the designing of histamine -1- receptor
antagonists and anticholinergics (antispasmodics) by replacing benzene by thiophene,

CH – by N –, – CH2 by O or S and so on.

The first application of classical isosterism may be found in ring equivalents. Examples
include pyridine and thiazole, benzene and thiophene.
The sulphur atom of the phenothiazine ring system of neuroleptic agents was replaced
by – CH = CH – or – CH2CH2 – leading to the azepine ring analogues that opened up the
field of tricyclic antidepressants. In imipramine by isosteric exchange of N – with C =,
amitriptyline was obtained.
The purpose of molecular modification is usually to seek subtle change in the compound
that should not alter some properties but change others in order to improve potency,
selectivity, duration of action and reduce toxicity. Bioisosterism makes it possible to limit
some of these changes. All aspects considered, retention of overall molecular shape is the
overriding condition for analogy of action.
In the design of bio-isosters an appreciation of the biochemical mode of action may play
an important role e.g., aspirin acetylates prostaglandin synthetase and thereby deactivates
this enzyme which ordinarily catalyses the biosynthesis of nociceptive prostaglandins.
Isosters of aspirin in which the phenolic oxygen atom (X) has been replaced by 'classical'
isosteric groups or atoms are inactive because they cannot release the acetyl group at all
(X = CH2) or at an adequate rate (when X = S, NH).
OH OH

CH — CH2 — NHCH3 CH — CH2 — NHCH3

O N
H RO2S H

Receptor Receptor
Medicinal Chemistry-I 3.2 Drug Metabolism

Some drugs are considered biochemically inert as they have been excreted unchanged
i.e. without any metabolic transformation e.g., barbitone, diethyl ether, only because they
cannot easily penetrate the lipoprotein membrane of tissues and were rapidly excreted by
active transport mechanisms of kidney.

3.2 METABOLIC BIOTRANS-FORMATION OF DRUGS: PHASE I REACTIONS


On chemical basis, it is classified into reactions which are fundamentally oxidation,
reduction and hydrolysis.
Most of these reactions occur in the liver while hydrolytic reactions of esters and amides
occur in the gut wall, plasma and the lung.
Depending upon the nature and localisation of the enzymes which catalyse these
reactions, it is further classified as:
(a) Metabolic transformations which are catalysed by enzymes of endoplasmic reticulum
of the liver and the other tissues or the microsomal drug metabolising enzymes.
(b) Reactions catalysed by non-microsomal mammalian enzymes i.e. enzymes present in
the mitochondria, lysosomes or cytoplasm of the tissues or in the blood plasma.
(c) Reactions catalysed by intestinal microflora.
(a) Microsomal drug metabolising enzymes:
Among the many enzymes associated with the endoplasmic reticulum, are a group of
enzymes known as 'drug metabolising enzymes'.
These include:
(1) Mixed function oxidases (2) Reductases (3) Esterases.
Very often a drug is subjected to several competing pathways simultaneously and the
extent of formation of the various metabolites depends on the relative rates of the various
interactions. While simple metabolic reactions are followed by conjugation e.g., an alkyl side
chain of a drug may be oxidised to an alcohol which then forms a conjugate with glucuronic
acid or an ester may be hydrolysed to its acid form which then, is coupled with glycine.
Examples:
[A] Oxidation:
Oxidation mainly occurs at aromatic rings, terminal positions of alkyl chains, N-methyl
groups, alcoholic groups and exposed corners of alicyclic rings.
(1) Ethanol, in mammals, is rapidly oxidised by liver alcohol dehydrogenases to a toxic
intermediate acetaldehyde. This is a reversible reaction.
The latter is rapidly oxidised to acetic acid by acetaldehyde oxidase and other enzymes.
This reaction is irreversible and proceeds faster than the former. Acetic acid, then may enter
the tricarboxylic acid cycle and reaches to final stage of oxidation to CO2.
Medicinal Chemistry-I 3.3 Drug Metabolism

Metabolic conversions
Storage
sites Oxidases
reductases Polar drug
hydroxylases metabolite
Lipophilic
drug

Free lipophilic
drug
Conjugation

Elimination
Kidney Liver Lungs

Urine Bile Vapours

Fig. 3.2

(2) Chloral hydrate is transformed to trichloro-acetic acid by aldehyde dehydrogenase.

Cl OH Cl

Cl — C — CHOH Cl — C — COOH

Cl Cl
Chlorol hydrate Trichloroacetic acid
(3)

(4)
Medicinal Chemistry-I 3.4 Drug Metabolism

(5)

(6)

(7) Ketone oxidation: e.g.

(8)

(9)
Medicinal Chemistry-I 3.5 Drug Metabolism

(10) Sulphoxidation:

Other oxidative reactions: These include sulphur atom replacement reaction and ring
formation. Former is an important and significant metabolic reaction for thiobarbiturates
and for phosphorothionate insecticides. Parathion through the latter reaction, is transformed
to a very toxic compound, paraxon (an active cholinesterase inhibitor in mammals) by the
liver microsomes as shown in Fig. 3.3.

O O
C2H5 C2H5
HN HN CH3
CH(CH3)CH2CH2CH3 CH
(O) C 3H 7
S N O O N O
H H
Thiopental Pentobarbital

OC2H5 S OC2H5
O
O2N O—P=S O2N O—P O2N O—P=O
(O)
OC2H5 OC2H5 OC2H5 OC2H5
Parathion Paraxon
Low antichloine-estrase activity High anticholine-estrase activity

CH3 CH3 CH3


N N OH N O
Microsomal
dehydrogenase
Cl N Cl N Cl N

Medazepam 2-hydroxymedazepam Diazepam

Fig. 3.3
Medicinal Chemistry-I 3.6 Drug Metabolism

[B] Reduction:
These processes play an important role in the metabolism of many compounds
containing carbonyl, nitro and azo groups. Bioreduction of carbonyl compounds generates
alcohol derivatives, while nitro and azo reduction leads to amino derivative. Since, the
hydroxyl and amino groups are much more susceptible to conjugation than the functional
groups of the parent compounds, reductive processes facilitate the drug elimination.
Examples:
(1)
OH
– H2O
Cl3C — C — OH Cl3C — CH2OH

H
Chloral hydrate Trichloroethanol
(2)
NO2 NH2

+H
Nitroreductase

HOCH O HOCH O
CH—NH—C—CHCl2 CH—NH—C—CHCl2

CH2OH CH2OH

Chloramphenicol Arylamine
(3)
NH2 NH2
NH2
+H
H2N N=N SO2NH2 +
Azoreductase
NH2
SO2NH2 NH2
Prontosil Sulphanilamide
(4)
N N
O
CH3 (1) Bis-N-de-methylation
CHCH2CH2N CHCH2C — H
CH3 (2) Oxidative deamination
Cl Cl

N
Chlorpheniramine
Reduction
CHCH2CH2OH
Cl
Medicinal Chemistry-I 3.7 Drug Metabolism

(5)
O
C OH
OH CH2 CH3 OH CH2 — CH — CH3
CHC6H5 CHC6H5
+ [H]

O O O O

R (+) Warfarin

(6)
CH2 — CH — CH3 CH2 — C — CH3 CH2 — CH — CH3
[+ H]
NH2 O OH

Amphetamine Phenylacetone 1-Phenyl-2 propanol

(7)
H H
N O N O

O2N N H2N N
Cl Cl

Clonazepam

[C] Hydrolytic Reactions:

The metabolism of ester and amide linkages in many drugs is catalysed by hydrolytic
enzymes present in liver, kidney, intestine, blood and other tissues. The metabolic products
formed, namely carboxylic acids, alcohols, phenols and amines generally are polar and
functionally more susceptible to conjugation and excretion than the parent ester and
amides.
Amide hydrolysis appears to be mediated by liver microsomal amidases, esterases and
deacylases.
COOH COOH
OCOCH3 OH O
(1) + HO — C — CH3

Aspirin Salicyclic acid Acetic acid


Medicinal Chemistry-I 3.8 Drug Metabolism

CH3 CH3
(2)
Cl O — C — COOC2H5 Cl O — C — COOH
CH3 CH3
Clofibrate P-chlorophenoxyisobutyric acid

(3)

N N
H
C=O
H2N Metabolite
Carbamazepine

COOH
(4)
CH3O CH2COOH CH3O CH2COOH
+
N CH3 N CH3
Cl
C=O H

Cl

Indomethacin

CH3 CH3
(5) O
C2H5 C2H5
NH — C — CH2N NH2 + HOOC — CH2N
C2H5 C2H5
CH3 CH3

Lidocaine

(6) NH2 NH2

+ HO — CH2CH2N(C2H5)2

COOCH2CH2N(C2H5)2 COOH
Procaine Para-amino benzoic acid Diethylaminoethanol
Medicinal Chemistry-I 3.9 Drug Metabolism

[D] Other Important Reactions:

(1) N-dealkylation:
CH3 H
N C C CH3 N C C CH3
4 3
CH3 CH3
2
C N C5 1 N
O N CH3 O N CH3

Aminopyrine Antipyrine

(2) O-dealkylation:
NHCOCH3 NHCOCH3

H5C2O HO
Acetophenetidine p-Hydroxyacetanilide

N — CH3 N — CH3

H3CO O OH HO O OH
Codeine Morphine

(3) Aromatization:
COOH COOH

Cyclohexane carboxylic acid Benzoic acid

3.3 CONJUGATION REACTIONS OR PHASE Il REACTIONS


Metabolic transformations or phase - I reactions, do not always produce hydrophilic
(more polar and water soluble) or pharmacologically inactive metabolites. Drugs exhibiting
increased activity or activity different from the parent drug, generally undergo further
metabolism through conjugation or phase II reactions, resulting in deactivation and
excretion of the inactive, highly polar conjugates. All the phase II reactions do not increase
the polarity. Methylation and acetylation, for example, decrease the polarity of drug
metabolite.
Medicinal Chemistry-I 3.10 Drug Metabolism

Phase II reactions are classified mainly into:


(a) Methylation and acetylation which do not generally increase water solubility but
serve mainly to terminate the pharmacological activity.
(b) Attachment of small, polar and ionisable endogenous molecules such as glucuronic
acid, sulphate, glycine and glutamine to the phase I metabolite.
(c) Of minor importance, are the other conjugative pathways e.g. conjugation with
glycosides, phosphate, other amino acids and conversion of cyanide to thiocyanate
Table 3.1: Phase II or conjugation reactions
Conjugation Site Conjugating Functional groups
agent
Glucuronidation Microsomes UDPGA — OH, — COOH, — NH2, — SH
Sulphatation Cytosol PAPS — OH, — NH2
Acetylation Cytosol Acetyl-CoA — COOH
Glutathion Cytosol Glutathion Epoxides, arene oxides
Methylation Cytosol SAM — OH, —NH2
Amino acid Cytosol Glycine
— COOH
UDPGA: Uridine diphosphoglucuronic acid, PAPS: 3'-phosphoadenosine-5'-
phosphosulphate
SAM: S-adenosylmethionine
Thus, phase II reactions include -
(I) Glucuronic acid conjugation.
(II) Sulphate conjugation.
(III) Conjugation with glycine, glutamine and other amino acids.
(IV) Glutathione or mercapturic acid conjugation.
(V) Acetylation.
(VI) Methylation.
(VII) Nucleoside and nucleotide formation.
(I) Conjugation wixh Glucuronic Acid:
The reaction involves the condensation of the drug or its metabolite with the activated
form of a readily available glucuronic acid, [i.e. uridine diphosphate glucuronic acid
(UDPGA)], which is synthesised from glucose-1-phosphate. Glucuronic acid conjugation
proceeds in two steps.
(1) Formation of the activated form from glucose 1- phosphate.
Medicinal Chemistry-I 3.11 Drug Metabolism

(2) Subsequent transfer of the glucuronic group from UDPGA to an appropriate


substrate.
Uridine-5'-diphospho-α-D-glucuronic Acid (UDPGA)
UDP - glucuronyl  Substrate drug

transferase 
β-glucuronide of the substrate
The transfer step is catalysed by microsomal enzymes called UDP-glucuronyltransferases
present mostly in the liver but also occur in many other tissues like kidney, intestine, skin,
lung and brain.
Types of compounds forming glucuronides:
(1) Alcohols and phenols form ether type glucuronides.
(2) Aromatic and some aliphatic carboxylic acids form ester type glucuronides.
(3) Aromatic amines form N-glucuronides and
(4) Sulphhydryl compounds form S-glucuronides.
Phenols, alcohols, amines and amides all form O- or N- glucuronides. Many endogenous
substances, like steroids, are also excreted in this way. Glucuronides are normally non-toxic,
highly water soluble and excreted in the urine or bile.
Glucuronidation of one functional group is usually sufficient to effect excretion,
diglucuronide conjugates usually do not occur.
(a) Compounds containing hydroxyl group:
N — CH3 O
NHC — CH3
OH O
O2N CH — CH — NH — C — CHCl2
CH2 OH
HO O OH OH

Morphine Acetaminophen Chloramphenicol

(b) Compounds containing carboxyl group:

COOH CH3 CH3

OH CH — COOH CH — COOH

CH3O
OC6H5
Salicylic acid Naproxen Fenoprofen
Medicinal Chemistry-I 3.12 Drug Metabolism

(c) Aromatic amines:


O
N
CH2O — C — NH2
CH3
C — CH3 NCH2CH2 — N
C6H5CH2 CH3
CH3CH2CH2 CH2O — C — NH2
CH2CH2CH2 NH CH3
O
Desipramine Meprobamate Tripelennamine

(d) Sulphhydryl compounds: (e) C - Glucuronide:

(II) Sulphate Conjugation:

Sulphate conjugates are formed by the reactions of phenolic and aliphatic hydroxyl
group and of certain amino groups with an activated form of sulphate through an ether
linkage. Hence, they are also termed as "etheral sulphates". Sulphate conjugation generally
results into highly polar compounds that are readily excreted in the urine. The soluble
fraction of liver contains the enzymes that catalyse the sulphur activation and transfer of
sulphate to the substrate.
The sulphate moiety is present in activated state in 3' - phosphoadenosine - 5' -
phosphosulphate (PAPS). The sulphotransferase enzyme then catalyses the transfer of
sulphate group to the phenolic acceptor. The sulphotransferase enzymes are structure
specific. Hence for different substrates, specific sulphotransferase enzymes catalyse the
reactions.
R
O O
Adenine
+ HO — S — O — P — O — CH2 R
O OH O
OH
O O
HO — P — OH O — S — OH
O O
Sulfate conjugate
Medicinal Chemistry-I 3.13 Drug Metabolism

(III) Conjugation with Glycine, Glutamine and other Amino Acids:


The amino acids, glycine and glutamine are utilized by mammalian systems to conjugate
carboxylic acids. In contrast to glucuronic acid, glycine and glutamine are not converted to
activated form. Instead the carboxylic acid substrate is activated with ATP to acetyl CoA
complex. This complex then reacts with glycine and glutamine to form conjugate and free-
acetyl Co-enzyme group.
O OH O

F C—CH2CH2CH2—N F COOH F C—NHCH2COOH

Haloperidol Para flurobenzoic acid Glycine conjugate

O Cl O O O

C — NHNH2 C — OH C — NHCH2C — OH

Hydrolysis

N N N
Isoniazid Isonicotinic acid Glycine conjugate

(IV) Glutathione or Mercapturic Acid Conjugates:


The glutathione conjugation is important in the elimination of polycyclic phenols and
halides. The metabolically generated reactive electrophilic species manifest their toxicity
(e.g., tissue necrosis, carcinogenicity, mutagenicity, teratogenicity) by combining covalently
with nucleophilic groups present in vital cellular proteins and nucleic acids. The tripeptide,
glutathione (cysteine glycine-glutamate) may be coupled via its sulfhydryl group to various
compounds possessing an electrophilic centre.
The sulfhydryl group (-SH), of glutathione reacts with these electrophilic species to form
S-substituted glutathione adducts and thus protects the vital cellular constituents by
effective disposal of electrophiles (i.e., reactive epoxides).
Paracetamol, busulphan and azathioprine are other examples of drugs conjugated by
this pathway. Glutathione conjugates are polar and of high molecular weight and are
eliminated as such in the bile. The glutathione portion of the conjugate may further be
metabolised via the peptide bond to mercapturic acid that are the normal urinary products
of this conjugation pathway.
(1) C H CH3 C6H5 C6H5 O COOH
6 5
CHOCH2CH2N CHO — CH2 CHO — CH2C — O — SCH2 — CH
C6H5 CH3 C6H5 COOH C6H5 H3C — CONH

Diphenhydramine Mercapturic acid conjugate


Medicinal Chemistry-I 3.14 Drug Metabolism

O O O O
(2) C C C C
NH CH3 N CH3 NH CH3 NH CH3

GSH CH
d+ S — CH2 COOH
OH O OH

Acetaminophen Mercapturic acid conjugate


NH — COCH3
(3)
+
CH2Cl CH2 CH2 — S — CH2 — CH — COOH

Benzyl chloride Mercapturic acid conjugate


(4) C2H5OOC COOC2H5 C2H5OOC COOC2H5

C=C C—C
H H H2
S — CH2 — CH — COOH

NHCOCH3
Diethylmaleate Mercapturic acid conjugate

HS
NH2
H 2C H
HC N H 2C CH — COOH
C H2C
O=C
O
N CH2 or HS — G
H
COOH

Glutathione
Medicinal Chemistry-I 3.15 Drug Metabolism

(V) Acetylation Reactions:


Acetylation reactions serve as an important metabolic route for drugs containing
primary amino groups, sulphonamides, hydrazines and hydrazides, which upon conjugation
get converted to their corresponding amide derivatives which are generally inactive and
non-toxic. The transfer of acetyl group is catalysed by N-acetyltransferase enzymes present
mainly in hepatic reticuloendothelial cells, which display broad substrate specificity. Thus,
aromatic primary amines (e.g., sulphonamides) and hydrazine derivatives (e.g., isoniazid) are
acetylated, utilizing acetyl coenzyme A. The acetyl transferase appears to be located in the
soluble fraction of reticuloendothelial cells present in the liver and kidney. Because of a
lowered solubility at acid pH, there is the danger of injury to the kidney resulting from
precipitation of the conjugated sulphonamide in the renal tubular fluid as the kidney
concentrates urine and lowers its pH.

(1) Aliphatic 1º Amines:


Medicinal Chemistry-I 3.16 Drug Metabolism

(2) Aromatic Amines:


NH2 NHCOCH3
N-Acetyltransferase

CH2 N(C2H5)2 CH2 N(C3H5)2

O = C — NH CH2 O = C — NH CH2
Procainamide
NH2 NHCOCH3

N-Acetyltransferase

SO2 NH2 SO2 NH2

Dapsone

(3) Sulphonamides:
NH2 NHCOCH3

N-Acetyltransferase
N N
O2SNH O2SNH

Sulphapyridine N-Acetyl Sulphapyridine

(4) Hydrazines and hydrazides:


CH2CH2NHNH2 CH2CH2NHNHCOCH3

N-Acetyltransferase

Phenelzine
CONHNH2 CONHNHCOCH3
N-Acetyltransferase

N N
Isoniazid
HO HO

Phenyl ethanolamine
HO CH — CH2 — NH2 HO CH — CH2 — NHCH3
N-methyl transferase
OH OH
Norepinephrine Epinephrine
Medicinal Chemistry-I 3.17 Drug Metabolism

(VI) Methylation Reactions:


Methylation differs from other conjugation processes in that,
(1) It is of greater significance in the metabolism of endogenous compounds.
(2) In some cases, it results in the products having greater pharmacological activity than
the parent molecules.
In these reactions, methionine transfers its methyl group via the activated intermediate,
S-adenosylmethionine to the substrate under the influence of methyl transferase enzymes.
Drugs or their metabolites containing primary aliphatic amine, phenolic or sulfhydryl group
may be N-, O or S-methylated respectively. These enzymes are as follows:
(1) Catechol-O-methyl transferases (COMT) They catalyse O-methylation of
catecholamines.
(2) Hydroxyindole-O-methyl transfer-ases. They catalyse O-methylation of substrate
other than catecholamines.
(3) N-methylation is catalysed by substrate specific enzymes. e.g., Phenyl ethanol
amine-N-methyl transferase, Imidazole-N- methyl transferase.
(4) S-methyl transferases: They catalyse S-methylation.

(1) O-methylation:

I I

Hydroxy indole-o-methyl
HO COOH CH3O COOH
transferase

I I
4-hydroxy-3,5-diiodo benzoic acid

HO CH3O

Catechol-o-methyl transferase
HO COOH HO COOH

3, 4-dihydroxy-benzoic acid

(2) N-methylation:

HO HO

Phenyl ethanolamine
HO CH — CH2 — NH2 HO CH — CH2 — NHCH3
N-methyl transferase
OH OH
Norepinephrine Epinephrine
Medicinal Chemistry-I 4.8 Drugs Acting on Autonomic Nervous System

Acetylcholine is biosynthesised by the acetylation of choline molecule. Choline itself has


a weak parasympathomimetic activity and upon injection, causes a fall in blood pressure.
Acetylcholine is about 10,000 times more active than choline molecule.
Acetylcholine is biosynthesised in the nerve terminals as shown in Fig. 4.5.
Active transport mechanisms are involved in picking up choline molecules from the
extrasynaptic fluid into the exoplasm. This transport is dependent upon the intracellular
concentration of Na+ and K+ ions. The choline molecule is acetylated in the cytoplasm by
acetyl coenzyme A which is biosynthesised in the mitochondria present in the nerve
terminal. The acetylation of choline is catalysed by choline acetyl transferase enzyme. The
enzyme is synthesised within the perikaryon and has a molecular weight of about 68,000. In
peripheral cholinergic nerves, it is usually present in higher concentrations. As soon as
acetylcholine is synthesised, it is sequestered within the synaptic vesicles.
Heart
Blood vessel
Smooth muscle
Glands
Autonomic ganglia
CNS

SYNTHESIS STORAGE Muscarinic


receptors
M
Choline RELEASE
CHOLINE + ACETYL – CoA ACh ACh
Acetylase
Glucose +
N
Coenzyme A Nicotinic
Pyruvate
receptors
UPTAKE
Skeletal muscle
DISPOSAL Autonomic ganglia
Choline + Acetic acid Adrenal medulla
Cholinesterase CNS

Fig. 4.5: Biosynthesis of acetylcholine

Site 1: Acetylcholine synthesis can be blocked by styryl pyridine derivatives such as NVP.
Site 2: Acetylcholine transport into vesicles is blocked by vesamicol (AH 5183). (±) Vesamicol
is a potent inhibitor of vesicular ACh storage with L (-)-Vesamicol being more potent
than D(+) - Vesamicol.
Release is promoted by  -bungarotoxin, black widow spider venom, and Ca . Release
++
Site 3:
++
is blocked by botulinum toxin, cytochalasin B, collagenase pre-treatment, and Mg .
Site 4: Postsynaptic receptors are activated by cholinomimetic drugs and anticholinesterases.
Nicotinic receptors, at least in the peripheral nervous system, are blocked by rabies
virus, curare hex amethonium, or dihydro-β-erythroidine; n-methylcarbamylcholine and
dimethylphenyl piperazinium are nicotinic agonists. Muscarinic receptors are blocked by
atropine, pirenzepine, and quinuclidinyl benzilate.
Medicinal Chemistry-I 4.9 Drugs Acting on Autonomic Nervous System

Site 5: Presynaptic muscarinic receptors may be blocked by AFDX - 116 (an M2 - antagonist),
atropine or quinuclidinyl benzilate. Muscarinic agonists (e.g. oxotremorine) will inhibit
the evoked release of acetylcholine by acting on these receptors.
Site 6: Acetylcholinesterase is inhibited reversibly by physostigmine (eserine) or irreversibly by
DFP.
Site 7: Choline uptake competitive blockers include hemicholinium 3, troxypyrrolium tosylate.

The biosynthesised acetylcholine is stored within the synaptic vesicles immediately inside
the membrane of the nerve terminal. Each vesicle is expected to contain about 5,000 -
10,000 molecules of acetylcholine. The number of such vesicles present in the nerve terminal
varies in different organs. For example, a motor nerve terminal may contain 300,000 or more
synaptic vesicles.
When an impulse reaches to nerve terminal depolarisation causes an activation of
calcium ionophore. It allows an influx of extracellular calcium ions which is an essential step
for the rupturing of storage vesicles of almost all neurotransmitters. The extracellular
calcium then leads to the release of acetylcholine from the vesicles. Four calcium ions are
taken up for each molecule of acetylcholine released. The ruptured synaptic vesicles again
re-shape to store fresh neurotransmitter.
The vesicular release of acetylcholine is reported to be inhibited by excess of magnesium
ions. The released acetylcholine along with extracellular calcium ions then mobilise intra-
cellular calcium ions from the sacs present on sarcoplasmic reticulum. The increase in the
concentration of free intracellular calcium ions then activates calmodulin dependent myosin
light chain kinase and phosphorylation of myosin, in turn, creates the conditions that initiate
muscle contraction. In general, minimum concentration of calcium ions needed to evoke
muscle contraction is estimated to be 10–6 mol/litre.
The free acetylcholine present in blood and other tissues, gets quickly hydrolysed by
either e-cholinesterase (present in erythrocytes) or s-cholinesterase (present in serum). Upon
hydrolysis, acetylcholine is converted into acetic acid and choline molecule.
The cholinesterase enzyme is present in high concentration in the synapses of both,
cholinergic and somatic nerves and striated muscle. Hydrolysis occurs in the immediate
vicinity of the nerve ending. At the neuromuscular junction, hydrolysis occurs at the end
plate region after acetylcholine has initiated the muscle twitch. In the autonomic ganglia,
cholinesterase is usually present in the preganglionic fibre. While serum esterase is present
in glial cells, plasma, liver and at other sites.
Cholinesterase enzymes are present in two different forms.
(i) Simple oligomers of a 70,000 dalton catalytic subunit, and
(ii) Elongated forms of complex structure.
The cholinesterases are not very selective enzymes. Both these types hydrolyse a large
number of esters, both of, choline and other carboxylic acids. Cholinesterase is one of the
most efficient enzyme present in the body. It can hydrolyse about 3 × 105 acetylcholine
molecules per mole per minute.

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