PHAR 402, Dr. Lu




Reading Assignments:


Foye, W.O. (1995) Principles of Medicinal Chemistry, Chapter 17. Williams & Wilkins , Philadelphia, pp. 321-344.

                 Goodman & Gilman’s The Pharmacological Basis of Therapeutics, 9th ed., McGraw Hill, 1996, New York, pp. 115-118.


Other References:

 (1)     Cooper, J.R., Bloom, R.E., Roth, R.H. The Biochemical Basis of Neuropharmacology, 5th ed., Oxford University Press, New York, 1986, Chapter 5.

(2)     Nogrady, Thomas, Medicinal Chemistry- A Biochemical Approach, 2nd ed., Oxford University Press, New York, 1988, pp. 138-141, 150-157.


Educational Objectives and Study Guidelines


A student should be able to:

1.         Identify the sites at the cholinergic synapses which can be regulated by drugs and explain how a given drug molecule may act to modify the cholinergic-receptor.

2.                   Know the chemical structure of acetylcholine and explain how it binds to the cholinergic receptor.

3.                    List the locations and types of cholinergic receptors in major organ systems.

4.                    Explain how acetylcholine is biosynthesized, stored, released and inactivated?

5.                    Describe the major chemical and biochemical differences between the nicotinic and muscarinic receptors.

6.                    Know definitions of the following: rate-limiting enzyme, competitive uptake inhibitor, false neurotransmitter, active/inactive receptor conformations, high/low affinity receptor bindings, and active receptor-bound conformation of acetylcholine.

7.                    Relate the structural features of a compound to their cholinergic receptor selectivity.

8.                    Relate the structural features of a compound to physicochemical properties, which may have a major effect on its cholinergic activity.

9.                    Explain how choline esters might be used for the treatment of attention and memory disorders in the elderly.

PHAR 402, Dr. Lu

Handout #1


Sites of Drugs Action at the Cholinergic Synapses



            Site 1:    Uptake of choline (active transport, potential site for the design of cognition enhancers)

            Site 2:    Biosynthesis of acetylcholine (ACh)

Site 3:    Release of ACh, this process is promoted by b-Bungarotoxin, black widow spider venom, and La3+.  ACh release is blocked by butulinum           toxin, cytocholasin B, collagenase pretreatment, and Mg2+. (Note: This is not a good site for the design of cholinergic drugs).

Site 4:    Postsynaptic receptors (main target for drug action).

Site 5:    Metabolism of ACh by acetylcholinesterase (AchE). (This degradative enzyme is inhibited by anticholinesterases and organophosphate nerve       gases).

            Site 6:    Presynaptic receptor (Autoreceptor)

PHAR 402, Dr. Lu

Handout #2

Site 1:  Uptake of Choline (the rate-limiting step)

Competitive Uptake Inhibitor



False Neurotransmitter


Triethylcholine, in addition to competing with choline for uptake into the presynaptic nerve terminal it also enters the nerve membrane and competes with choline for the biosynthesis of acetylcholine.  However, the resulting product, triethylacetylcholine, acts as a false neurotransmitter because it lacks Ach activity.


Study Question:


1.           Delecit was recently marketed in Italy (1990) for the treatment of attention and memory disorders in the elderly.  Explain how it works.

2.     Both of the following compounds are choline analogues.  Explain why compound A has stimulatory activity while compound B exhibits inhibitory action at the cholinergic synapses?



PHAR 402, Dr. Lu

Handout #3

Site 2:  Biosynthesis of Acetylcholine


This step can be blocked by styryl pyridine derivatives such as NVP [4-(naphthyvinyl)pyridine]


 Study Questions:

1.       Why is acetylenic analogue more active than NVP? [Hint: consider binding to the enzyme, ChAT].

2.      What might be the possible reason(s) for the lack of inhibitory activity of compound C?

3.      If the biosynthesis of ACh is not rate-limiting, explain why this is not a desirable step for drug intervention?

PHAR 402, Dr. Lu

Handout #4


Site 4:  Postsynaptic Cholinergic Receptors (Cholinoceptive sites)


Acetylcholine (ACh) is the chemical neurotransmitter for both peripheral and central cholinergic receptors.  These cholinoceptive sites are further divided into nicotinic and muscarinic ACh receptors because of its relative specificity of action toward cholinomimetic [L(+) muscarine, S(-) nicotine] and cholinergic blocking agents (d-tubocurarine, atropine).  Nicotinic ACh receptors are selectively stimulated by nicotine and blocked by curare, C6 and C10.  Muscarinic ACh receptors are activated by L (+) muscarine and blocked by atropine.  It should be noted that nicotine acts at nicotinic sites to produce stimulation in low doses blockade in high doses.




PHAR 402, Dr. Lu

Handout # 5                            Muscarinic Receptor Subtypes


1.        Muscarinic receptors can be classified into at least three pharmacologically well-defined subtypes: M1 (neuronal type); M2 (cardiac type, also known as M2a or M2A); and M3 (glandular/smooth muscle type, also known as M2b or M2B).


2.        Receptor cloning studies have yielded five subtypes m1, m2, m3, m4, and m5 where m1, m2, and m3 correspond to the pharmacologically defined M1, M2, and M3 receptors.  Furthermore, m1, m3, m5 are known to couple to the stimulation of PI metabolism while m2 and m4 are linked to the inhibition of adenylate cyclase (Review your cellular and molecular biology on the signal transduction pathway, i.e., PHAR 331).  


3.        The following radioligands are used for the biochemical and pharmacological characterization of the muscarinic receptors.  They are non-selective muscarinic antagonists, which possess high affinity for the muscarinic Ach receptors (mAChRs).  Can you see how they work chemically?


4.        The following are selective muscarinic antagonists used for receptor binding studies.  They are used to differentiate muscarinic receptor subtypes.


Phar402, Dr. Lu

Handout # 6   


 Muscarinic Receptor Binding of Agonists and Antagonists


Drugs                           3H-QNB Binding                                  Functional studies

                                    (IC50 or PKi, M)                      (Smooth muscle contraction, M)                      



QNB                            2 - 3 x 10-10                                                      5 x 10-10

Scopolamine                 2 - 3 x 10-10                                                      3 x 10-10

Atropine                       3 - 4 x 10-9                                                             1 x 10-9

Methylatropine             1 - 2 x 10-10                                                      5 x 10-10




Oxotremorine               5 - 8 x10-7                                                        - *

Pilocarpine                   7 - 9 x 10-7                                                         2 - 9 x 10-6

Arecoline                      3 - 4 x 10-6                                                       -*

ACh                             2 - 4 x 10-6                                                       1 - 2 x 10-6

Carbachol                    2 - 3 x 10-5                                                         2 x 10-5

Methacholine                2 - 3 x 10-6                                                             3 x 10-6


*Oxotremorine and arecoline are M1 agonists, thus not tested on smooth muscle receptors (M3).  There are excellent correlations between receptor binding studies and functional studies using isolated tissue preparations.

  The relative lower affinity of muscarinic agonists for the muscarinic receptors may be explained by the following biochemical rationale:

Why is this biochemical theory important? Can you see the reason why muscarinic antagonists are used for the biochemical characterization of the muscarinic receptors?

PHAR 402 Dr. Lu

Handout # 7                Conformational Aspects of Acetylcholine

Structure and functional groups of the acetylcholine molecule

Conformational isomers of ACh derived from rotation around the -O-C-C-N axis and their nomenclatures [please note that the receptor-bound conformation of ACh is (+) ac]



                    Description of Steric Relationship Across Single Bonds (From Klyne and Prelog)


                                Torsion angle, 0o                                        Designation


                                      0                30                  Synperiplanar ( sp)

                                   +30       to    + 90                 + Synclinal (+sc)

                                   +90       to  + 150                 + Anticlinal (+ac)

                                 +150       to  + 180                 + Antiperiplanar (+ap)

                                  -150       to   - 180                 - Antiperiplanar (-ap)

                                   - 90       to   - 150                 - Anticlinal (-ac)

                                   - 30       to     - 90                 - Synclinal (-sc)

PHAR 402, Dr. Lu

Handout # 8                Analogues of Acetylcholine                   


a.         Modification of the quaternary ammonium group [please compare the value of the observed equipotent molar ratio (EPMR)]


                   -N(CH3)3            -NH(CH3)2            -NH3           -N(C2H5)(CH3)2                              -N(C2H5)2(CH3)

                     ACh (1)                   (50)                     (2000)                  (3.3)                                        (400)


                  -NH2(C2H5)        -N(C2H5)3            C(CH3)3               -S(CH3)2                       -P(CH3)3

                    (500)                      (inactive)            (inactive)                  (1)                        (12)                       


Study question: 

      1).   What evidence(s) can you find which allow us to conclude that a positively charged functional group is important for binding and thus for the observed agonist activity?

     2).   Is there an overall effective size of this functional group for binding?       


b.          Modification of the ethylene bridge [please note the stereochemistry and the substitution pattern].

             Study questions:

            3).    Explain, with your knowledge of drug-receptor interactions, why a methyl group at the b carbon produces a muscarinic agonist without nicotinic activity while an a methyl substitution provides a compound with only nicotinic activity?

            4).    Provide a reason why a and b dimethyl substitutions destroy cholinergic action at both nicotinic and muscarinic receptors?

 5).    What conclusion can you make regarding muscarinic receptor binding since S (+) b-methacholine is more potent than its R (-) enantiomer?

 Handout # 8 continued

 c.         Replacement of acetyl function gives rise to agonist, partial agonist, or antagonist [please see the size and the nature of the functional group replacement].



            Study question:     Why does carbamoyl function confer oral activity? (Review your organic chemistry)


d.         Conformationally restricted or rigid analogues of ACh [these molecules provide valuable insight regarding the Ach-receptor bound conformation].    

Study question: 

 Can you figure out what conformation of ACh does each of the above conformationally-restricted or rigid compounds mimic? [Hint: please use the Newman projections as illustrated on Handout # 7]


PHAR 402, Dr. Lu

Handout #9


                      Hypothetical Model for the Muscarinic Receptor-Cholinergic Drug interaction                         





 Practice problems:

 1.         Based on Chothia’s model for the binding of cholinergic drugs, explain why 5-methyl L(+)-muscarine lacks both muscarinic and nicotinic activity. 

 2.         The naturally occurring bicyclic alkaloid, (+) anatoxin, is a potent agonist of the nicotinic acetylcholine receptors (i.e., >100 times as compared to carbachol).  With the given structural formula of (+) anatoxin, state whether or not this compound would have any muscarinic agonist activity. Which one of the conformational structure of (+) anatoxin do you believe is involved in binding to the nicotinic and muscarinic receptors?  Provide a chemical rationale to explain why (+) anatoxin is more than 100 times more active than carbachol using the Chothia’s model discussed above.          


 PHAR 402, Dr. Lu

Handout #10               Cholinomimetic Alkaloids


1.         Natural products leads - Can you identify the essential pharmacophore (i.e., ACh-like  moeity) for these molecules?


2.         Synthetic modifications of the above lead molecules - What SAR can be obtained from these analogues?

   a.       Synthetic analogues of L(+) muscarine (non-selective mAChRs agonists)

    b.       Synthetic analogues of arecoline (all of these compounds are relatively more M1-selective)

PHAR 402, Dr. Lu

Handout #11

                               Oxotremoline Analogues


         Oxotremorine, the pharmacologically active metabolite of the synthetic agent tremorine, is a specific muscarinic agonist equipotent to Ach. Oxotremorine is relatively selective as M1-agonist probably due to a favorable distribution to the brain (i.e., a cyclic amide with a basic tertiary amine function).  Our interest in oxotremorine and analogues is due to their potential in the therapy of Alzheimer and related dementias.  Unfortunately, the severe side effects experienced with oxotremorine preclude its clinical use. 


1.      Modification of basic amine group - What SAR may be concluded from the following structures?



2.      Modification of the cyclic lactam function - Please note the drastic changes of its activity



PHAR 402, Dr. Lu

Handout #11 continued


3.         Newer oxotremorine analogues


            “Lead compound”

            To solve this problem, the following strategy was designed:

Note:   The parent compound is sufficiently lipophilic in nature and thus it will enter the brain before it is cyclized to give the quaternary form, which should have greater receptor affinity for the M1-receptors. (Can you see the reason why the researchers did not use bromine or iodine as the leaving group?  Why can we have two carbons between the terminal nitrogen and the chlorine atom?)

PHAR 402, Dr. Lu

Handout #12

Atropine and Solanaceous Alkaloids


Belladonna alkaloids


 1.        What is the pharmacophore for atropine-like molecules?

 2.        Why is the stereochemistry at the benzylic carbon important for antimuscarinic activity?

 3.        What role(s) does the hydroxy group play in enhancing the antagonist potency?

Antimuscarinic activity of tropyl tropates




            Configuration                                        Anticholinergic activity

                                                                          pA2                  log K

              a, S(-) (Hyoscyamine)                         -                      9.4                  

            a, R(+)                                                   -                      6.9

            a, RS ()Atropine                                  9.79                 9.0

            b, RS ( )                                               6.55                 -


PHAR 402, Dr. Lu

Handout #13

Structural Formula of Some Clinically used Antimuscarinic Agents


Quaternary Ammonium Compounds - These compounds are generally not well absorbed from the GI because of their ionic character.  They also lack CNS activity.



Agents Used in Ophthalmology - These compound typically have shorter duration of action.


Agents for Obstructive Pulmonary Disease - Potent quaternary ammonium compounds to provide localized action.

PHAR 402, Dr. Lu

Handout #13, continued

 Agents for Parkinson’s Disease - Tertiary amino ethers or alcohols that have sufficient lipophilicity to allow it to cross blood-brain barrier.


Summary Structure-activity Relationships of Muscarinic Antagonists


1.         Substituent R1 should be carbocyclic or heterocyclic ring for maximal antagonist potency.

2.                   Substituent R2 should be a hydrogen atom, a hydroxy group, a hydroxymethyl group, or a methyl group.

3.                   The nature of the group X only effect the duration of action, the physicochemical properties or the side effect profiles of the drug molecule but not its ability to bind the receptor.

4.                   There is a bulk limitation for the N substitution.  Optimal potency is associated with 2-3 ethyl groups.

5.                   The stereochemistry at the benzylic carbon is critical for muscarinic antagonist potency.  Any compound that can place the phenyl group in the same absolute configuration as depicted in the general formula above will have potent muscarinic antagonist activity.

6.                   The phenyl ring cannot tolerate any substituent other than F on the p-position without losing its antagonist activity.

PHAR 402, Dr. Lu

Handout #14


Hypothetical models for the binding of muscarinic antagonist to the muscarinic receptor


A.        Classical view of binding of lachesine to the muscarinic receptor (by Stubbins et al., 1980)


B.        Proposed receptor-bound conformation of atropine and its interaction with mAChR.


Study Question:

1.     Propiverine hydrochloride (Mictonorm), an agent that treats urinary incontinence, is marketed in Germany (1992) where it is reported to exhibit potent antimuscarinic activity.


Using the proposed hypothetical model for the binding of atropine and superimpose each of the above functional groups onto these receptor binding sites. Explain, based on your knowledge of how atropine-like compounds bind to the muscarinic receptors, why this molecule must undergo metabolic activation before exerting its muscarinic antagonist action? Review your drug metabolism lectures and explain how and where propiverine is bioactivated and by what enzyme(s)?


2.        Based on your understanding of how atropine binds, redraw each of the following known muscarinic antagonists and explain why only one enantiomer is active as a muscarinic antagonist.


3.     In a recent article [Imagination, 3, 99-105 (1997)], a unique chemical substance, Cramnesiaic Acid, has been identified in the brain of college students who attempted to memorize an entire semester of material by pulling an all-nighter to prepare for the final exam.  The effect of this bizarre chemical is to suppress the brain center that controls reasoning.  Its action is further augmented when it reaches the presynaptic cholinergic nerve terminal and combines with choline to form a substance known as Amnesiacholine that causes the student to experience short-term memory impairment.  Explain the above observations with your knowledge of cholinergic nerve transmission in the brain


                                Newer Muscarinic Antagonists