Ref- Foye's Principle of Medicinal chemistry and journals and my own thoughts

1. Cholinergics and
Anticholinesterases
Acetylchlonine (Ach)

2. Our Nervous System
Because of its wide and important involvement,
understanding Nervous system is important to treat
many diseases
Functions –
• To transmit signals to and from
body organs or cells to carry out
o Heartbeat, Respiration
o Digestion, Hormone secretion
o Movement, body pressure
• To process sensory information
• Logic, Decision and Memory

3. Drugs based on Nervous System treat
various clinical conditions
Cholinergic NS NS based on other
Neurotransmitters
Impaired or excessive
gastric/secretion
Epilepsy – irregular neuronal
activity leading to muscle spasm
Glaucoma – increased
pressure on eye
Bradycardia – slow heart beat
Anxiety – feeling irrational fear
Relief from pain
Alzheimer’s and –loss of
memory
Parkinson disease – loss of
movement control
Myasthenia gravis – muscle
weakness
Cause unconsciousness during
surgery

4. First lesson in Medicinal chemistry
• Medicinal Chemistry aims to cure diseases. But
to do that we need to know what biological
factor is causing disease. Then we create drugs
that interact with that particular factor to either
suppress it or stimulate it.
• These biological factors arise from alteration in
the process of normal body functioning. Thus to
find and understand what goes wrong during
illness, we must first study the normal
functioning of body. This allows us to recognize
a central biological factor that is most involved
in causing disease. Then we make drugs to act
on it.

5. Neurons
• Neurons are individual cells of the Nervous
System that process and transmit signals by
electrical and chemical process.
• Adjacent neurons are physically separated by
the each other. The gap region is called
synapse.

6. Fig: Neurotransmitters moving through
Synapse between two neurons
Pre-synaptic
Neuron
(sends signal)
Post-synaptic
Neuron
(receives signal)

7. • Neurotransmitters (NT) are endogenous
(produced by body) chemicals that transmit
signals across a synapse from sending presynaptic
neuron to the target postsynaptic neuron
• They are synthesized and stored in neuron itself
• There are many NTs eg Acetylcholine, Adrenaline,
serotonin, dopamine, GABA
• The process of transmission of signal along a
neuron and over the synapse is called
neurotransmission. Signal can pass over the
synapse by either chemically or electrically
• One neurons interacts with many other neurons
in all possible directions.

8. Our Nervous system

9. Types of peripheral NS
• Somatic NS-
– controls voluntary muscle Movement
– Transmits sensory information to brain
• Autonomic NS
– Controls involuntary body functions such as Heart
beat, secretion (GI acid/insulin), fight or flight
responses

10. Two types of Autonomic NS
Parasympathetic NS
Uses Acetylcholine
Sympathetic NS
Uses Adrenaline
Makes body
ready for fight
or flight
Makes body
ready for rest

11. Did you note the mono-directionality ?
• Instead of a single light switch that you can
turn on/off or a single volume knob you can
turn high/low…..
• In this case you have two independent control
system for doing opposing things, eg
sympathetic increases heart beat while
parasympathetic is required to slow heart
beat, there is no way neither NS can reverse or
undo it’s action by itself.

12. Introduction
• Cholinergics refer to the part of Nervous
system that utilize Acetylchlonine (Ach) as a
neurotransmitter. It is key NT in the
parasympathetic NS
• A unique feature of Ach is that the same
molecule can bind with two different
receptors (muscarinic and nicotinic receptor)
using different conformation.
Acetylcholine
Acetyl Choline

13. Physio-Chemical property of Ach
• It is ester of acetic acid and choline
• It is soluble in water due to salt form at Nitrogen
• In solid form it is stable but in solution, the ester group gets
hydrolyzed (ie ester group turns into acid and alcohol).
• If acid or base is present, as in stomach, then rate of hydrolysis is so
fast that it prevents oral dosing of Ach
• Even if we prepare it’s solution in neutral water and inject in blood
so as to bypass the acidic stomach, an ester hydrolyzing enzyme
called butrylcholinesterase significantly degrades it s that the
pharmacological response is very weak
• Even if it was administered in stable form, It’s ionic ammonium
group prevents good penetration across the lipophillic cell wall
• This chemical property makes it a weak agonist plus since it is non-
selective agonist of Muscarinic and Nicotinic
• (Thus having no ester group and no ionic amine is an approach tp
making more stable and strong Ach agonist. However selectivity is a
different issue)

14. Based on above info we can make simple
changes to Ach hopefully design derivatives to
improve stability and cell penetration.
Ach
Conceptual Agonist drugs
Drug design is a conceptual thing. We don’t really know if drug will really work as
expected until we synthesize and test them. To improve chances of success the design part
should not be random but have some rational

15. Muscarinic receptor
• They are agonized by a poisonous mushroom
derived compound called muscarine
• They occur primarily in the CNS and in Autonomic
NS, and are part of a large family of G-protein-
coupled receptors
• Physiological functions include heart rate and
force, contraction of smooth muscles and the
release of other neurotransmitters.
• There are five subtypes of muscarinic AChRs
based on pharmacological activity: M1-M5

16. Nicotinic receptor
• They are agonized by nicotine
• They also occur in the CNS and Autonomic NS plus are
exclusive in neuromuscular junction, and are part of a
ligand gated ion channel receptors
• Physiological functions depend upon muscle-type or
neuronal-type
• Muscle-type nicotinic AChRs are localized at
neuromuscular junctions and allow muscle contraction
and maintain muscle tone; (thus these are targets for
muscle relaxants)
• Neuronal type are involved in cognitive function,
learning and memory, arousal, reward, motor control
and analgesia.

17. H3C O
O
CH2 CH2 N(CH3)3
Quaternary
Ammonum group
Ethylene
group
Acyloxy
group
SAR of cholinergics as Muscarinic agonist
• Cholinergic drugs mimic action of Ach on
Muscarinic or nicotinic receptors and produce
the same effect as Ach but in greater
magnitude
• A general strategy of making an agonist is to
use the original compound, in this case Ach,
as a framework

18. 1) Modification of quaternary Ammonium group
a) Presence of nitrogen in quaternary ionic form is
important for agonist activity. Replacement of
Nitrogen with other elements such as Sulphur,
Arsenic and Phosphorous reduces activity
b) Presence of three methyl group is needed for
agonist activity. Changing the three methyl
group by higher alkanes or Hydrogen also causes
loss of activity
H3C O
O
CH2 CH2 N(CH3)3
Quaternary
Ammonum group
Ethylene
group
Acyloxy
group

19. Replacement with Arsenic or Phosphorous maintains + charge but still
reduces activity
Replacement with sulphur removes the + charge and causes
reduced activity
Only this has good potency
Conclusion
1) positive charge needed 2) Positive charge should be on Nitrogen only
3) Charge on Nitrogen only possible when it bonded to 4 atoms ie quaternary form needed
Valency
S = 4
P = 3
Ar = 3
N =3

20. • If R = methyl,(CH3) --> active
• If R = ethyl (C2H5) --> antagonist!
• If R = propyl (C3H9) and higher alkyls --> inactive
• If only one of the R = ethyl or propyl  active
but less potent than Ach
• If any or all R = H  activity goes on decreasing

21. 2) Change in the ethylene group
c) A “rule of five” idea states that there should
be no more than 5 atoms between the
Nitrogen and the terminal Hydrogen
As the chain length increased from two, activity
is rapidly lost.
C O
N
O
H
H
H
1
2
3
4
5
This tells us
about the
relative size of
binding site

22. d) Inclusion of methyl group in the ethylene
carbons can alter selectivity
Methyl inclusion in β carbon relative to N
retains potency of Ach and more selective to
muscarinic receptor. This compound is called
methacholine and used clinically
Methyl inclusion in α carbon relative to N
reduces potency but makes more selective to
nicotinic receptor. This is not used clinically

23. •Methyl group in Beta carbon
•As potent as Ach
•Selective to Muscarinic receptor
•Used clinically
•Methyl group in alpha carbon
•Not As potent as Ach
•Selective to Nicotinic receptor
•Not Used clinically
Methacholine
H3C O
O
CH2 CH N(CH3)3
CH3
H3C O
O
CH CH2 N(CH3)3
CH3

24. 3) Modifications to the Acetoxy group
e) Substituting the Acetyl with higher
homologous group such as propionyl or butyryl
Reduces activity.
R O
O
CH2 CH2 N(CH3)3
If R = propionyl (C3H7), butyrl (C4H9) or
higher than activity is reduced

25. f) The ester group isn’t mandatory as
quanternary amine group but an oxygen
atom is required in this region
g) Since ester group makes it liable to hydrolysis,
alternate groups were included and found that
replacing the ester with carbamate, ether or
ketone function resists hydrolysis while
maintaining activity

26. H3C O CH2 CH2 N(CH3)3
H3C CH2 CH2 N(CH3)3
O
H2N O
O
CH2 CH2 N(CH3)3
carbamates
Ethers
Ketone
(Carbamates are resistant enough
to Gastric acid to be given orally)
Modification to
reduce hydrolysis

27. SAR of
cholinergics/Muscarinic
agonist
1. Presence of nitrogen in quaternary ionic form is
important for agonist activity
2. Presence of three methyl group in Nitrogen is needed for
agonist activity
3. A “rule of five” idea states that there should be no more
than 5 atoms between the Nitrogen and the terminal
Hydrogen
4. Inclusion of methyl group in beta carbon to N makes
muscarinic selective in alpha carbon to N makes nicotinic
seelctive
5. The ester group isn’t mandatory as quanternary amine
group but an oxygen atom is required in this region
6. Replacing the ester with carbamate, ether or ketone
function resists hydrolysis while maintaining activity
H3C O
O
CH2 CH2 N(CH3)3
Quaternary
Ammonum group
Ethylene
group
Acyloxy
group

28. Designing a better drug by using SAR
• Problem with Ach was it’s instability and
unselective activity
• Combine point 4 and point 6 into a new
structure and see what you get?
• How does it compare with Ach?

29. Pharmacological action of Ach
A) Through the muscarinic receptor
Cardiac effects
• Bradycardia,
• decrease of atrioventricular conduction.
• decrease of the strength of atrium
contractions.
Blood vessels
• Acetylcholine injection causes release of nitric
oxide (NO) which dilates blood veins

30. • Effects on smooth muscles
• intestine: an increase in tone with sometimes an
increase in the peristaltic contractions. This can lead to
Nausea and vomiting.
• ureters: increase in tone.
• bronchi: bronchoconstriction. (An aerosol of
acetylcholine can cause an attack of asthma)
• Effects on secretions
• Acetylcholine increases digestive (abundant saliva),
bronchial, cutaneous (sweat) and lacrimal (tears)
secretions.
• Effects on the eye
• Acetylcholine induces a decrease of iris diameter or
miosis which can lower the intra-ocular pressure

31. • Through the nicotinic receptor
• In Autonomic NS, Ach allows nerve transmission
• In neuromuscular junctions
– At low dose Ach allow skeletal muscle movement
(important for breathing)
– At high does, it causes muscle paralysis!
• In Brain, cholinergic deficiency causes Alzheimer
disease

32. Bio-synthesis of Ach

33. Specific Muscarinic agonist
Drugs other than Ach that produce the same effects
As Ach at Muscarinic receptor but for longer time
and greater intensity
– Methacholone Chloride
– Carbachol Chloride
– Bethanecol Chloride
– Pilocarpine Hydrochloride
Mechanism of Action (MOA) : They act directly by
binding to muscarinic receptor as a agonist and
produce the same effects as Ach

34. Methacholone
Chloride
• It is a muscarinic selective cholinergic agonist.
• It’s S enantiomer is 240 times more potent than R
enantiomer. Howeever, R iosmer is a weak
inhibitor of Ach degrading enzyme called
Acetylcholinesterases.
• Thus this is given as a racemic mixture
• Use – It is used to induce bronchospam in asthma
patients for purpose of verifying the diagnosing
asthma
• MOA

35. Carbachol
Chloride
• It is carbamate analog of Ach. This feature
makes it very resistant to hydrolysis by both GI
acid and Acetylcholinesterase such that it can
be given orally. But it is not selective to
muscarinic or nicotinic receptor.
• Uses: It’s use is limited to treatment of
glaucoma and constrict the pupils during eye
surgery.
• MOA

36. Bethanecol
Chloride
• It is an carbamate derivative of Ach which
contains a methyl group in beta carbon to
Nitrogen. This makes the molecule very stable to
hydrolysis and selective to muscarine too.
• Uses:
– treat urinary retention resulting from general
anesthetic
– treat gastrointestinal atony (muscles lose their
peristalic ability)
• MOA

37. Pilocarpine
Chloride
• It is a plant derived alkaloid whose structure does not
match the established SAR but still acts like an
cholinomimetic
• It is not selective to muscarine. Unlike other muscarine
agonists, it can penetrate the eye well following topical
application
• Uses:
– Treat dryness of mouth caused by radiation therapy in the
head or neck
– Glaucoma
– constrict the pupils during cataract surgery
• MOA

38. • Tell why carbamates resist AchE

39. Anticholinesterase
• Acetylcholinesterase (AchE) is a enzyme that
hydrolyzes Ach into Acetic acid and Choline
• MOA: Anticholinesterase drugs work by inhibiting the
enzyme Acetylcholinesterase which prevents hydrolysis
of Ach thus increasing their concentration in the
synapse which promotes more Ach action. Since they
promote Ach activity without binding to any receptor
they are also called indirectly acting cholinergic
agonists
• Note: There is enough Acetylcholinesterase in the
synapse to hydrolyze 3 X 108 molecules of Ach in 1
millisecond (10-3sec). Normally only 3 X 106 Ach are
released into synapse

40. Applications
• Improve muscle strength in Myasthenia gravis
• Glaucoma
• Alzheimer's
• Insecticides
• Chemical weapon (serine gas)
• There are two types: Reversible and Irreversible

41. Theory of AchE inhibitors
If instead of acetyl group there is carbamate group then
hydrolysis will be resisted. The AchE which is not
hydrolyzed cannot be used again. Thus goal of AchE
inhibitor is to provide such hydrolysis resistant functional
group such as carbamates or phosphate ester
During hydrolysis of Ach, the AchE gets acylated. It needs to
be hydrolyzed by water to be regenerated in free from or
else it can’t function again.

42. Reversible Anticholinesterase
• These are compounds can act by two ways:
• A) They bind but don’t react with AchE with
greater affinity than Ach like Ach does or
• B)these compounds that bind and react with
AchE to form acylate AchE which is mores table
form but still capable of being easily hydrolyzed
• Reversible means that they inhibit AchE for short
time (only few mins)
• These compounds have more therapeutic uses
than irreversible ones eg Physostigmine and
Neostigmine

43. Physostigmine
• It is an alkaloid type anticholinesterase obtained from
the seeds of calabar beans
• It has no charged amine and is more lippohillic and can
thus penetrate the blood brain barrier
• It has very great affinity for AchE but that can only in
charged form. Thus there is pH limitation in it’s activity.
• Uses
– Glaucoma
– Counter CNS poisoning by atropine and tricyclic
depressents
• MOA – It inhibits AchE by binding and reacting to it and
carbamylating it. This carbamylated enzyme is slow to
hydrolysis

44. Neostigmine
• It is a synthetic anticholinesterase based on Physostigmine.
• It resembles the aromatic features of physostigmine and
also the distance between the ester and ammonium is
same
• But since it has charge on Nitrogen, it cannot cross the CNS
like physostigmine does
• Also its half life is shorter than physostigmine
• Uses
– Myasthenia gravis
– To counter Urinary retention
• MOA – It inhibits AchE by binding and reacting to it and
carbamylating it. This carbamylated enzyme is slow to
hydrolysis

45. Irreversible Anticholinesterase
• These compounds act by only one way: that bind and
react with AchE to AchE with greater affinity than Ach
to form acylated enzyme which strongly resist
hydrolysis
• Irreversible means that they inhibit AchE for very long
time(many hours)
• These compounds have less therapeutic uses than
reversible ones.
• Serine gas and organophosphate insecticides are based
on this concept.
• They cause cholinergic crisis
• Their common structural feature is the presence of
Phosphate ester bond which strongly resist hydrolysis

46. Cholinergic crisis
• A cholinergic crisis is an over-stimulation at a
neuromuscular junction due to an excess of
acetylcholine (ACh). This happens due to
inactivity (perhaps even inhibition) of the AChE
enzyme, which normally breaks down
acetylcholine. This is a consequence of some
types of nerve gas, (e.g. sarin gas) or insecticides.
• It causes muscle paralysis and respiratory failure

47. Aging
Irreversible Anticholinesterase containing Phosphate ester
resist hydrolysis very strongly. They also undergo a feature
called aging which increases this resistance even more.
When AchE becomes acylated for a long time with, one of
the ester bond is broken. This creates a negative charge that
oppose nucleophillic attack on phosphorous and in this
state it resists hydrolysis even more. At this stage antidotes
against Irreversible anticholinesterase such as PAM doesn’t work.
Thus regeneration of AchE is blocked for even longer periods
leading to cholinergic crisis

48. Organophosphate poisoning
Most insecticides use concept of irreversible AchE
inhibition to kill pesticides. These compounds
contain Phosphate ester bond that strongly resist
hydrolysis. They are very lipophillic and volatile
also. They can quickly enter the blood stream and
inhibit AchE for a many hours. This promotes Ach
activity in the synapse which leads to ‘cholinergic
crisis’. When this happens muscles stop responding
to Ach causing paralysis and respiratory failure
(death)

49. Antidote to insecticides
Because insecticides can lead to chloinergic
crisis, antidotes were designed to hydrolyze the
acylated AchE. A successful antidote was PAM
It has a quaternary ammonium and a very
strong nucleophile called oxime group and both
work together to free AchE from the phosphate
ester compounds
Pralidoxime
(PAM)

50. How PAM reverses poisoning by
organophosphate or serine gas ?
There are two sites in the binding pocket of AchE. One is a anionic site
which is empty and other is the Esteric site where the phosphate
compound sits. 3 event follows
• First the charged ammonium of PAM binds to the anionic site.
• From there PAM’s strong nucleophillic oxime group can be in close
distance to attack the ester bond between Phosphorous and serine
amino acid of AchE
• This form free AchE and phosphorylated PAM
Limitation: For PAM to work, it must be used immediately following
exposure to insecticides or serine gas or no more than 36 hrs of
exposure or else aging will occur and it wont be able to dislocate the
phosphate form the receptor
The search is on for stronger nucleophilic molecules that effectively displaces phosphate even
in aged form. This is a problem entirely of pure chemistry. No receptor/enzyme consideration
But enzymes are very powerful in catalyzing any reaction. What if we could engineer a protein
to specifically cleave this bond? Protein engineering is a established science!

52. Thank You

53. Revision
Autonomic NS/
Parasympathetic

54. Anatomical Location

55. Subtype and Anatomical location

56. Subtype

57. Muscarinic receptor Nicotinic receptor
Named after it’s agonist Muscarine Named after it’s agonist Nicotine
It is a GPCR It is a Ligand gated ion channel
Subtypes : M1-M5 Muscle type or neuronal type
Location CNS, Autonomic NS Location : CNS, Autonomic,
Neuromuscular junctions
Allow Smooth Muscle contraction Allow Skeletal muscle contraction

58. Acetylcholine
2 problems prevent it from being a good drug
– Unselective
– Unstable (Easy to hydrolyze)
Solution
– Ethylene group control selectivity
– Acyloxy group controls stability
H3C O
O
CH2 CH2 N(CH3)3
Quaternary
Ammonum group
Ethylene
group
Acyloxy
group
(maintains
activity)
(controls
selectivity)
(modify
stability)

59. 2 ways of promoting Ach activity
• 1) Directly: Agonize muscarinic receptor
• 2) Indirectly : Inhibit Acetylcholinesterase
(AchE) ---> Ach not hydrolyzed ---> more Ach
to bind to receptors for longer time
(But This also promotes nicotinic activity!)

60. • During AchE mediated hydrolysis of Ach, AchE
gets gets attached with acyl group
• This acylated AchE needs to be hydrolyzed by
water to be free and function again
• Anticholinesterase inhibitor, both reversible
and irrversible contain functional groups
which makes this step very difficult for water
since water is not a strong nucloephile

61. • Order of Resistance to hydrolysis
• AchE-Phosphate ester>>>>>> AchE-carbamate
>> AchE-ester
• Reversivle Anticholinesterase block AchE for
few min----> thus have therapeutic value
• Irreversivle Anticholinesterase block AchE for
many hours---> thus have toxic effect -->
cholinergic crisis --> respiratory failure

62. Q) How Ach gives only specific Muc or Nic
response if it can bind to both of them?

63. Design criteria of good AchE inhibitor
• All AchE inhibitor based insecticide below
Phosphorylate the enzyme and have a peculiar
group in the right side of compound. Figure out
the organic chemistry.
• Remember: Rational drug design is not limited by
not having software to look at binding site or do
any fancy docking, QSAR. In this case the fact that
acylated AchE needs to be hydrolyzed and and
hydrolysis is a nucleophilic attack that is affected
by leaving group is all that you need

65. • But there is no rationality for making an
irreversible AchE inhibitor unless you are
making a chemical weapon.
• Our focus should be on making a better PAM
alternative. All existing drugs are based on
oximes because it is a powerful nucleophile.
Do you know any stronger nucleophilies? How
about sulphur based compounds?

66. All these are useless in aging and the charge on Nitrogen
makes it difficult to reverse poisoning in CNS.
We need a antidote that
• Has stronger nucleophilic group than oxime
• Enough lipophilicity to penetrate into brain
•You will later see that charge in nitrogen isn’t mandatory

67. First lesson in Drug design
• When we say a drug or endogenous ligand interacts
with its target receptor or enzymes we mean that there
is formation of chemical bond such as
– H-Bond
– Dipole bond
– Ionic bond
– Covalent bond
– Van der wall bond
– Hydrophobic
– Pi-pi bond, pi-cation bond (you won’t find this in books but
this is important too as you will see in next slide)

68. Introduction to Structure based drug design (SBDD)
Implementation of knowledge of chemical bonding and
binding site to design drugs

69. Notice how enzmye catalysis works.
1) In AchE binding site, Hydrogen from a Histidine
protonates O of Ach and OH of erine attacks as a
nucleophile, just like what happens in lab: first C=O
protonation by some acidic catalyst then OH of
water attacks as a nucleophillic. Difference is in
enzyme both functionality is built in by various
amino acids, one acidic type (Histidine – it is basic
in neutral form but because of it’s basicity it mostly
remains in conjugated acid form) and other
nucleophilic type (Serine)

70. Amino Acids grouped by chemical property

71. 2) The charged Nitrogen is used to anchor Ach into
binding site, where it forms not the expected
ionic bond (cation of Ach and anion in enzyme)
but cation-pi bond. The pi refers to aromatic rings
provided by tryptophan and phenylalanine of the
enzyme.
What this implies is that positive charge on PAM,
which makes brain penetration difficult, is not
really required. Instead we can include functional
groups that do pi-pi bonding with the enzyme ie
aromatic rings. This increases lipophilicity too!

72. Thus, In theory………
N
OH
this should also work
N
OH
this should also work and
penetrate brain more
We made specific changes in PAM in response to knowledge of how
Ach chemically binds to AchE. This change has advantage of
penetration brain better. Because we used the binding site as a
reference, this is called Structure based Drug Design and is one of
the principle of Rational drug design.
Assignment: Watch this video and learn about the Structure based
Drug Design of the first Anti-influenza drug, Zamanavir and bring
written report
Youtube/How protein crystallography changed drug design
N
N
OH
instead of this

73. How chemical interaction dictates conformation?
Ach When bound to nicotine
receptor Ach When in aqueous solution
trans form Gauche form
Ref : Conformation of acetylcholine bound to the nicotinic acetylcholine
receptor. Proc. Natl. Acad. Sci. USA 85 (1988)
Q)Normally, gauche is more stable than trans form in solution. However when bound to nicotine
Ach readily adopts such Trans conformation. Why?
Ans) At this trans form the total entropy ie chemical interaction with receptor overcomes
any entropy loss to finally result a large change in gibbs free energy ie thermodynamics of
binding matters