The Organic Chemistry of
`Drug Design and Drug Action
`
`Richard B. Silverman
`Department of Chemistry
`Northwestern University
`Evanston, Illinois
`
`Ap
`
`Academic Press, Inc.
`Harcourt Brace Jovanovich,Publishers
`San Diego New York Boston London Sydney Tokyo Toronto
`
`MYLAN EXHIBIT - 1050
`Mylan Pharmaceuticals, Inc. v. Bausch Health Ireland, Ltd. - IPR2022-00722
`
`

`

`This book is printed on acid-free paper. 0
`
`Copyright © 1992 by ACADEMIC PRESS, INC.
`All Rights Reserved.
`No part of this publication may be reproduced or transmitted in any form or by any
`means, electronic or mechanical, including photocopy, recording, or any information
`storage and retrieval system, without permission in writing from the publisher.
`
`Academic Press, Inc.
`1250 Sixth Avenge, San Diego, California 92101-4311
`
`United Kingdom Edition published by
`Academic Press Limited
`24-28 Oval Road, London NW1 7DX
`
`Library of Congress Cataloging-in-Publication Data
`
`Silverman, Richard B.
`The organic chemistry of drug design and drug action / Richard B.
`Silverman.
`p. cm.
`Includes index.
`ISBN 0-12-643730-0 (hardcover)
`1. Pharmaceutical chemistry. 2. Bioorganic chemistry.
`I. Title.
`3. Molecular pharmacology. 4. Drugs--Design.
`[DNLM: 1. Chemistry, Organic. 2. Chemistry, Pharmaceutical.
`3. Drug Design. 4. Phannacokinetics. QV 744 S587o]
`RS403.S55 1992
`615'.19--dc20
`DNLM/DLC
`for Library of Congress
`
`PRINTED IN THE UNITED STATES OF AMERICA
`9 8 7 6 5 4 3 2 1
`HA
`92 93 94 95 96 97
`
`91-47041
`CIP
`
`

`

`To Mom and the memory of Dad,
`for their warmth, their humor, their ethics, their inspiration,
`but mostly for their genes.
`
`

`

`CHAPTER 3
`Receptors
`
`53
`
`54
`
`52
`I. Introduction
`II. Receptor Structure
`A. Historical
`53
`54
`B. What Is a Receptor?
`III. Drug—Receptor Interactions
`A. General Considerations
`54
`55
`B. Forces Involved in the Drug—Receptor Complex
`1. Covalent Bonds, 56 • 2. Ionic (or Electrostatic) Interactions, 56 • 3. Ion-Dipole
`and Dipole-Dipole Interactions, 56 • 4. Hydrogen Bonds, 57 • 5. Charge-Transfer
`Complexes, 60 • 6. Hydrophobic Interactions, 60 • 7. Van der Waals or London
`Dispersion Forces, 61 • 8. Conclusion, 62
`C. Ionization
`62
`D. Determination of Drug—Receptor Interactions
`71
`E. Drug—Receptor Theories
`1. Occupancy Theory, 71 • 2. Rate Theory, 72 • 3. Induced-Fit Theory, 72 •
`4. Macromolecular Perturbation Theory, 73 • 5. Activation—Aggregation Theory, 74
`74
`F. Topographical and Stereochemical Considerations
`1. Spatial Arrangement of Atoms, 75 • 2. Drug and Receptor Chirality, 76 •
`3. Geometric Isomers, 82 • 4. Conformational Isomers, 83 • 5. Ring Topology, 86
`G. Ion Channel Blockers
`87
`H. Example of Rational Drug Design of a Receptor Antagonist:
`88
`Cimetidine
`
`63
`
`95
`References
`General References
`
`97
`
`I. Introduction
`
`Up to this point in our discussion it appears that a drug is taken, and by some
`kind of magic it travels through the body and elicits a pharmaceutical effect.
`Pharmacokinetics (absorption, distribution, metabolism, and excretion) was
`mentioned in Chapter 2, but no discussion was presented regarding what
`produces the pharmaceutical effect. The site of drug action, which is ulti-
`mately responsible for the pharmaceutical effect, is called a receptor. The
`interaction of the drug with the receptor constitutes pharmacodynamics. In
`this chapter the emphasis is placed on pharmacodynamics of general noncata-
`lytic receptors, in Chapter 4 a special class of receptors that have catalytic
`
`52
`
`

`

`II. Receptor Structure
`
`53
`
`properties, called enzymes, will be discussed, and in Chapter 6 another recep-
`tor, DNA, will be the topic of discussion. The drug—receptor properties de-
`scribed in this chapter also apply to drug-enzyme and drug-DNA complexes.
`
`II. Receptor Structure
`A. Historical
`
`In 1878 John N. Langley,' a physiology student at Cambridge University,
`while studying the mutually antagonistic action of the alkaloids atropine (3.1;
`now used as an antisecretory agent) and pilocarpine (3.2; used in the treat-
`ment of glaucoma, but causes sweating and salivation) on cat salivary flow,
`suggested that both of these chemicals interacted with some substance in the
`nerve endings of the gland cells. Langley, however, did not follow up this
`notion for over 25 years.
`
`, CH 3
`
`CH2OH
`
`0
`
`3.1
`
`C2Hf
`
`"CH
`
`CH3
`
`3.2
`
`Paul Ehrlich2 suggested his side chain theory in 1897. According to this
`hypothesis, cells have side chains attached to them that contain specific
`groups capable of combining with a particular group of a toxin. Ehrlich termed
`these side chains receptors. Another facet of this hypothesis was that when
`toxins combined with the side chains, excess side chains were produced and
`released into the bloodstream. In today's biochemical vernacular these excess
`side chains would be called antibodies, and they combine with toxins stoi-
`chiometrically.
`In 1905 and 1906 Langley' studied the antagonistic effects of curare (a
`generic term for a variety of South American quaternary alkaloid poisons that
`cause muscular paralysis) on nicotine stimulation of skeletal muscle. He con-
`cluded that there was a receptive substance that received the stimulus and, by
`transmitting it, caused muscle contraction. This was really the first time that
`attention was drawn to the two fundamental characteristics of a receptor,
`namely, a recognition capacity for specific ligands and an amplification com-
`ponent, the ability of the ligand—receptor complex to initiate a biological
`response.
`
`

`

`54
`
`B. What Is a Receptor?
`
`3. Receptors
`
`In general, receptors are integral proteins (i.e., polypeptide macromolecules)
`that are embedded in the phospholipid bilayer of cell membranes (see Fig.
`2.3). They, typically, function in the membrane environment; consequently,
`their properties and mechanisms of action depend on the phospholipid milieu.
`Vigorous treatment of cells with detergents is required to dissociate these
`proteins from the membrane. Once they become dissociated, however, they
`can lose their integrity. Since they generally exist in minute quantities and can
`be unstable, few receptors have been purified, and little structural information
`is known about them. Advances in molecular biology more recently have
`permitted the isolation, cloning, and sequencing of receptors,4 and this is
`leading to further approaches to molecular characterization of these proteins.
`However, these receptors, unlike many enzymes, are still typically character-
`ized in terms of their function rather than by their structural properties. The
`two functional components of receptors, the recognition component and the
`amplification component, may represent the same or different sites on the
`same protein. Various hypotheses regarding the mechanism by which drugs
`may initiate a biological response are discussed in Section III,E.
`
`III. Drug—Receptor Interactions
`A. General Considerations
`
`In order to appreciate mechanisms of drug action it is important to understand
`the forces of interaction that bind drugs to their receptors. Because of the low
`concentration of drugs and receptors in the bloodstream and other biological
`fluids, the law of mass action alone cannot account for the ability of small
`doses of structurally specific drugs to elicit a total response by combination
`with all, or practically all, of the appropriate receptors. One of my all-time
`favorite calculations, shown below, supports the notion that something more
`than mass action is required to get the desired drug—receptor interaction.5
`One mole of a drug contains 6.02 x 1023 molecules (Avogadro's number). If
`the molecular weight of an average drug is 200 g/mol, then 1 mg (often an
`effective dose) will contain 6.02 x 1023(10-3)/200 = 3 x 1018 molecules of
`drug. The human organism is composed of about 3 x 1013 cells. Therefore,
`each cell will be acted upon by 3 x 1018/3 x 1013 = 105 drug molecules. One
`erythrocyte cell contains about 1010 molecules. On the assumption that the
`same number of molecules is found in all cells, then for each drug molecule,
`there are 101°/105 = 105 molecules of the human body! With this ratio of
`human molecules to drug molecules, Le Chatelier would have a difficult time
`explaining how the drug could interact and form a stable complex with the
`desired receptor.
`
`

`

`III. Drug—Receptor Interactions
`
`55
`
`The driving force for the drug—receptor interaction can be considered as a
`low-energy state of the drug—receptor complex [Eq. (3.1)], where kon is the
`rate constant for formation of the drug-receptor complex, which depends on
`the concentrations of the drug and the receptor, and /coif is the rate constant for
`breakdown of the complex, which depends on the concentration of the drug—
`receptor complex as well as other forces. The biological activity of a drug is
`related to its affinity for the receptor, which is measured by its KD , the
`dissociation constant at equilibrium [Eq. (3.2)]. Note that KD is a dissociation
`constant, so that the smaller the KD , the larger the concentration of the drug—
`receptor complex, and the greater is the affinity of the drug for the receptor.
`
`Drug + receptor
`
`Icon
`
`kofr
`
`drug—receptor complex
`
`KD
`
`[drug][receptor]
`[drug—receptor complex]
`
`(3.1)
`
`(3.2)
`
`B. Forces Involved in the Drug-Receptor Complex
`
`The forces involved in the drug—receptor complex are the same forces experi-
`enced by all interacting organic molecules and include covalent bonding, ionic
`(electrostatic) interactions, ion-dipole and dipole-dipole interactions, hydro-
`gen bonding, charge-transfer interactions, hydrophobic interactions, and van
`der Waals interactions. Weak interactions usually are possible only when
`molecular surfaces are close and complementary, that is, bond strength is
`distance dependent. The spontaneous formation of a bond between atoms
`occurs with a decrease in free energy, that is, AG is negative. The change in
`free energy is related to the binding equilibrium constant (Keq) by Eq. (3.3).
`Therefore, at physiological temperature (37°C) changes in free energy of —2 to
`—3 kcal/mol can have a major effect on the establishment of good secondary
`interactions. In fact, a decrease in AG° of —2.7 kcal/mol changes the binding
`equilibrium constant from 1 to 100. If the Keq were only 0.01 (i.e., 1% of the
`equilibrium mixture in the form of the drug—receptor complex), then a AG° of
`interaction of —5.45 kcal/mol would shift the binding equilibrium constant to
`100 (i.e., 99% in the form of the drug—receptor complex).
`(3.3)
`AG° —RT In Keq
`In general, the bonds formed between a drug and a receptor are weak
`noncovalent interactions; consequently, the effects produced are reversible.
`Because of this, a drug becomes inactive as soon as its concentration in the
`extracellular fluids decreases. Often it is desirable for the drug effect to last
`only a limited time so that the pharmacological action can be terminated. In
`the case of CNS stimulants and depressants, for example, a prolonged action
`
`

`

`56
`
`3. Receptors
`
`could be harmful. Sometimes, however, the effect produced by a drug should
`persist, and even be irreversible. For example, it is most desirable for a
`chemotherapeutic agent, a drug that acts selectively on a foreign organism or
`tumor cell, to form an irreversible complex with its receptor so that the drug
`can exert its toxic action for a prolonged period.6 In this case, a covalent bond
`would be desirable.
`In the following subsections the various types of possible drug—receptor
`interactions are discussed briefly. These interactions are applicable to all
`types of receptors, including enzymes and DNA, that are described in this
`book.
`
`1. Covalent Bonds
`
`The covalent bond is the strongest bond, generally worth anywhere from —40
`to —110 kcal/mol in stability. It is seldom formed by a drug—receptor interac-
`tion, except with enzymes and DNA. These bonds will be discussed further in
`Chapters 5 and 6.
`
`2. Ionic (or Electrostatic) Interactions
`
`For protein receptors at physiological pH (generally taken to mean pH 7.4),
`basic groups such as the amino side chains of arginine, lysine, and, to a much
`lesser extent, histidine are protonated and, therefore, provide a cationic envi-
`ronment. Acidic groups, such as the carboxylic acid side chains of aspartic
`acid and glutamic acid, are deprotonated to give anionic groups.
`Drug and receptor groups will be mutually attracted provided they have
`opposite charges. This ionic interaction can be effective at distances farther
`than those required for other types of interactions, and they can persist
`longer. A simple ionic interaction can provide a AG° = —5 kcal/mol which
`declines by the square of the distance between the charges. If this interaction
`is reinforced by other simultaneous interactions, the ionic interaction be-
`comes stronger (AG° = —10 kcal/mol) and persists longer. Acetylcholine is
`used as an example of a molecule that can undergo an ionic interaction (Fig.
`3.1).
`
`3. Ion—Dipole and Dipole—Dipole Interactions
`
`As a result of the greater electronegativity of atoms such as oxygen, nitrogen,
`sulfur, and halogens relative to that of carbon, C—X bonds in drugs and
`receptors, where X is an electronegative atom, will have an asymmetric distri-
`bution of electrons; this produces electronic dipoles. The dipoles in a drug
`molecule can be attracted by ions (ion—dipole interaction) or by other dipoles
`(dipole—dipole interaction) in the receptor, provided charges of opposite sign
`are properly aligned. Since the charge of a dipole is less than that of an ion, a
`
`

`

`III. Drug—Receptor Interactions
`
`57
`
`0
`-o
`+
`II
`CH3COCH2NMe3
`
`Figure 3.1. Example of a simple ionic interaction. The wavy line represents the receptor
`surface.
`
`dipole-dipole interaction is weaker than an ion—dipole interaction. In Fig. 3.2
`acetylcholine is used to demonstrate these interactions, which can provide a
`AG° of —1 to —7 kcal/mol.
`
`4. Hydrogen Bonds
`Hydrogen bonds are a type of dipole—dipole interaction formed between the
`proton of a group X—H, where X is an electronegative atom, and other
`electronegative atoms (Y) containing a pair of nonbonded electrons. The only
`significant hydrogen bonds occur in molecules where X and Y are N, 0, or F.
`X removes electron density from the hydrogen so it has a partial positive
`charge, which is strongly attracted to nonbonded electrons of Y. The interac-
`tion is denoted as a dotted line,
`to indicate that a covalent
`bond between X and H still exists, but that an interaction between H and Y
`also occurs. When X and Y are equivalent in electronegativity and degree of
`ionization, the proton can be shared equally between the two groups, that is,
`
`The hydrogen bond is unique to hydrogen because it is the only atom that
`can carry a positive charge at physiological pH while remaining covalently
`bonded in a molecule, and hydrogen also is small enough to allow close
`approach of a second electronegative atom. The strength of the hydrogen
`bond is related to the Hammett a- constants.'
`There are intramolecular and intermolecular hydrogen bonds; the former
`are stronger (see Fig. 3.3). Hydrogen bonding can be quite important for
`biological activity. For example, methyl salicylate (3.3), an active ingredient
`
`ion-dipole
`
`O8
`
`NH3
`
`+
`I I
`CH3COCH2NMe3
`8+
`45OH
`
`8+
`
`0
`5
`
`5+
`
`dipole-dipole
`
`Figure 3.2. Examples of ion—dipole and dipole—dipole interactions. The wavy line repre-
`sents the receptor surface.
`
`

`

`58
`
`3. Receptors
`
`intramolecular
`
`intermolecular
`
`Figure 3.3. Examples of hydrogen bonds. The wavy lines represents the receptor surface.
`
`in many muscle pain remedies and at least one antiseptic, is a weak antibacte-
`rial agent. The corresponding para isomer, methyl p-hydroxybenzoate (3.4),
`however, is considerably more active as an antibacterial agent and is used as a
`food preservative. It is believed that the antibacterial activity of 3.4 is derived
`from the phenolic hydroxyl group. In 3.3 this group is masked by intramolecu-
`lar hydrogen bonding'
`
`O
`
`OCH3
`3.3
`
`CH3O
`
`\ /
`
`O
`
`3.4
`
`Hydrogen bonds are essential in maintaining the structural integrity of «-
`helix and /3-sheet conformations of peptides and proteins (3.5)1 and the double
`helix of DNA (3.6).2 As discussed in Chapter 6, many antitumor agents act by
`intercalation into the DNA base pairs or by alkylation of the DNA bases,
`thereby preventing hydrogen bonding. This disrupts the double helix and
`destroys the DNA.
`Another instance where hydrogen bonding is suggested to be important
`arises when the potency of various oxygen-containing drugs becomes reduced
`by substitution of a sulfur atom for the oxygen atom in the drug. Sulfur, which
`is very poor at hydrogen bonding relative to oxygen, presumably cannot
`interact with the receptor group that hydrogen bonds to the oxygen, and
`drug—receptor complex stability becomes diminished.
`The AG° for hydrogen bonding can be between —1 and —7 kcal/mol but
`usually is in the range of —3 to —5 kcal/mol.
`
`From B. Alberts, D. Bray, J. Lewis, M. Raff, K. Roberts, and J. D. Watson, "Molecular
`Biology of the Cell," 2nd Ed., pp. 110 and 109, respectively. Garland Publishing, New York,
`1989, with permission. Copyright © 1989 Garland Publishing.
`2 From B. Alberts, D. Bray, J. Lewis, M. Raff, K. Roberts, and J. D. Watson, "Molecular
`Biology of the Cell," 2nd Ed., p. 99. Garland Publishing, New York, 1989, with permission.
`Copyright © 1989 Garland Publishing.
`
`

`

`0 sheet
`
`NN
`
`1141
`
`nO
`
`2.
`
`CO
`
`a
`
`tT
`O
`a .
`
`A
`
`iv
`D
`3
`
`.y4
`
`3
`fa
`
`CD
`
`a helix
`
`bond
`
`right-handed
`helix
`
`3.5
`
`sugar phosphate
`backbone
`
`1.6 nm
`
`5'
`
`/
`
`V
`
`A
`
`base •
`
`1 hei.cal !urn -- 3-4 nm •
`
`3.6
`
`hydrogen
`bond
`
`

`

`60
`
`5. Charge-Transfer Complexes
`
`3. Receptors
`
`When a molecule (or group) that is a good electron donor comes into contact
`with a molecule (or group) that is a good electron acceptor, the donor may
`transfer some of its charge to the acceptor. This forms a charge-transfer
`complex, which, in effect, is a molecular dipole—dipole interaction. The po-
`tential energy of this interaction is proportional to the difference between the
`ionization potential of the donor and the electron affinity of the acceptor.
`Electron donors contain 77-electrons, for example, alkenes, alkynes, and
`aromatic moieties with electron-donating substituents, or groups that have a
`pair of nonbonded electrons, such as oxygen, nitrogen, and sulfur moieties.
`Acceptor groups contain electron-deficient 7F orbitals, for example, alkenes,
`alkynes, and aromatic moieties having electron-withdrawing substituents, or
`weakly acidic protons. There are groups on receptors that can act as electron
`donors, such as the aromatic ring of tyrosine or the carboxylate group of
`aspartate, as electron acceptors, such as cysteine, and electron donors and
`acceptors, such as histidine, tryptophan, and asparagine.
`Charge-transfer interactions are believed to provide the energy for interca-
`lation of certain planar aromatic antimalarial drugs, such as chloroquine (3.7),
`into parasitic DNA (see Chapter 6). The fungicide, chlorothalonil, is shown in
`Fig. 3.4 as a hypothetical example for a charge-transfer interaction with a
`tyrosine.
`
`Cl
`
`NH-CH(CH2)3N(C2115)2
`CH3
`
`3.7
`The AG° for charge-transfer interactions also can range from —1 to —7
`kcal/mol.
`
`6. Hydrophobic Interactions
`In the presence of a nonpolar molecule or region of a molecule, the surround-
`ing water molecules orient themselves and, therefore, are in a higher energy
`
`CN
`
`CI
`
`CI
`
`CI
`
`CN
`
`OH
`
`Figure 3.4. Example of a charge-transfer interaction. The wavy line is the receptor surface.
`
`

`

`III. Drug—Receptor Interactions
`
`,6\7
`C761/ 4 16,7
`VA767
`7676''c7/447A v,S AAv
`'S,ii
`e`e''>'34 \ 7 7 e.667
`ov,, /
`,, ,,e1v4,
`\7
`.6, AV:n:7
`V:A ////A7A7
`CZAG2FA
`
`i,
`
`/
`
`,
`
`/
`
`61
`
`G. p
`
`:/;43/:///:N/I'''7°//;:/:,:‘,1: ;
`/.-
`v,727:1:6,1:Y. 4,
`/ADA767,4C7
`4'7,6,yAV ,q zs <
`zs,
`D
`D .A
`
`,
`
`b.
`
`Figure 3.5. Formation of hydrophobic interactions. (Reprinted with permission of John
`Wiley & Sons, Inc. from Korolkovas, A. 1970. "Essentials of Molecular Pharmacology," p.
`172. Wiley, New York. Copyright © 1970. John Wiley & Sons, Inc. and by permission of
`Kopple, K. D. 1966."Peptides and Amino Acids." Addison-Wesley, Reading, Massachusetts.)
`
`state than when only other water molecules are around. When two nonpolar
`groups, such as a lipophilic group on a drug and a nonpolar receptor group,
`each surrounded by ordered water molecules, approach each other, these
`water molecules become disordered in an attempt to associate with each
`other. This increase in entropy, therefore, results in a decrease in the free
`energy that stabilizes the drug—receptor complex. This stabilization is known
`as a hydrophobic interaction (see Fig. 3.5). Consequently, this is not an at-
`tractive force of two nonpolar groups "dissolving" in one another but, rather,
`is the decreased free energy of the nonpolar group because of the increased
`entropy of the surrounding water molecules. Jencks9 has suggested that hy-
`drophobic forces may be the most important single factor responsible for
`noncovalent intermolecular interactions in aqueous solution. Hildebrand,19 on
`the other hand, is convinced that hydrophobic effects do not exist. Every
`methylene-methylene interaction (which actually may be a van der Waals
`interaction; see Section III,B,7) liberates 0.7 kcal/mol of free energy. In Fig.
`3.6 the topical anesthetic butamben is depicted in a hypothetical hydrophobic
`interaction with an isoleucine group.
`
`7. Van der Waals or London Dispersion Forces
`
`Atoms in nonpolar molecules may have a temporary nonsymmetrical distribu-
`tion of electron density which results in the generation of a temporary dipole.
`
`NH2
`
`\
`
`/
`
`Figure 3.6. Example of hydrophobic interactions. The wavy line represents the receptor
`surface.
`
`

`

`62
`
`3. Receptors
`
`As atoms from different molecules (such as a drug and a receptor) approach
`each other, the temporary dipoles of one molecule induce opposite dipoles in
`the approaching molecule. Consequently, an intermolecular attraction,
`known as van der Waals forces, results. These weak universal forces only
`become significant when there is a close surface contact of the atoms; how-
`ever, when there is molecular complementarity, numerous atomic interac-
`tions (each contributing about —0.5 kcal/mol to the AG') result, which can add
`up to a significant overall drug—receptor binding component.
`
`8. Conclusion
`
`Since noncovalent interactions are generally weak, cooperativity by several
`types of interactions is critical. To a first approximation, enthalpy terms will
`be additive. Once the first interaction has taken place, translational entropy is
`lost. This results in a much lower entropy loss in the formation of the second
`interaction. The effect of this cooperativity is that several rather weak interac-
`tions may combine to produce a strong interaction. Since several different
`types of interactions are involved, selectivity in drug—receptor interactions
`can result. In Fig. 3.7 the local anesthetic dibucaine is used as an example to
`show the variety of interactions that are possible.
`
`C. Ionization
`
`At physiological pH (pH 7.4), even mildly acidic groups, such as carboxylic
`acid groups, will be essentially completely in the carboxylate anionic form;
`phenolic hydroxyl groups may be partially ionized. Likewise, basic groups,
`such as amines, will be partially or completely protonated to give the cationic
`form. The ionization state of a drug will have a profound effect not only on its
`drug—receptor interaction, but also on its partition coefficient (log P; see
`Section II,E,2,b of Chapter 2).
`
`/ydrophobic
`
`hydrogen bond
`
`charge transfer
`
`1-1""
`
`\
`
`ionic or ion-dipole
`
`:N
`\
`
`CH3CH2CH2CH2O
`
`H
`I+
`N
`NCH2CH2—IrCH2CH3
`CH2CH3
`
`hydrophobic
`
`dipole-dipole
`
`hydrophobic
`
`Figure 3.7. Examples of potential multiple drug—receptor interactions. The van der Waals
`interactions are excluded.
`
`

`

`III. Drug—Receptor Interactions
`
`63
`
`The importance of ionization was recognized in 1924 when Steam and
`Stearn11 suggested that the antibacterial activity of stabilized triphenyl-
`methane cationic dyes was related to an interaction of the cation with some
`anionic group in the bacterium. Increasing the pH of the medium also in-
`creased the antibacterial effect, presumably by increasing the ionization of the
`receptors in the bacterium, Albert and co-workers12 made the first rigorous
`proof that a correlation between ionization and biological activity existed. A
`series of 101 aminoacridines, including the antibacterial drug, 9-aminoacridine
`or aminacrine (3.8), all having a variety of pKa values, was tested against 22
`species of bacteria. A direct correlation was observed between ionization
`(formation of the cation) of the aminoacridines and antibacterial activity.
`However, at lower pH values, protons can compete with these cations for the
`receptor, and antibacterial activity is diminished. When this was realized,
`Albert13 notes, the Australian Army during World War II was advised to
`pretreat wounds with sodium bicarbonate to neutralize any acidity prior to
`treatment with aminacrine. This, apparently, was quite effective in increasing
`the potency of the drug. The mechanism of action of aminoacridines is dis-
`cussed in Chapter 6.
`
`NH2
`
`3.8
`
`The great majority of alkaloids which act as neuroleptics, local anesthetics,
`and barbiturates have pKa values between 6 and 8; consequently both neutral
`and cationic forms are present at physiological pH.13 This may allow them to
`penetrate membranes in the neutral form and exert their biological action in
`the ionic form. Antihistamines and antidepressants tend to have pKa values of
`about 9. The uricosuric (increases urinary excretion of uric acid) drug phenyl-
`butazone [3.9, R = (CH2)3CH31 has a pKa of 4.5 and is active as the anion (the
`OH proton is acidic). However, since the pH of urine is 4.8 or higher, subopti-
`mal concentrations of the anion were found in the urinary system. Sulfinpyra-
`zone (3.9, R = CH2CH2SOPh) has a lower pKa of 2.8 and is about 20 times
`more potent than phenylbutazone; the anionic form blocks reabsorption of
`uric acid by renal tubule cells.14
`
`HO
`
`Ph
`N.
`
`3.9
`
`-Ph
`
`O
`
`

`

`64
`
`3. Receptors
`
`The antimalarial drug pyrimethamine (3.10) has a pKa of 7.2 and is best
`absorbed from solutions of sufficient alkalinity that it has a high proportion of
`molecules in the neutral form (to cross membranes). Its mode of action, the
`inhibition of the parasitic enzyme dihydrofolate reductase, however, requires
`that it be in the protonated cationic form.
`
`CI
`
`NH2
`
`C2H5
`
`N
`
`NH2
`
`3.10
`
`Similarly, there are drugs such as the anti-inflammatory agent indomethacin
`(2.18) and the antibacterial agent sulfamethoxazole (3.11) whose pharmaco-
`kinetics (migration to site of action) depend on their nonionized form, but
`whose pharmacodynamics (interaction with the receptor) depend on the an-
`ionic form (carboxylate and sulfonamido ions, respectively). In a cell-free
`system the antibacterial activity of 3.11 and other sulfonamides was directly
`proportional to the degree of ionization, but in intact cells, where the drug
`must cross a membrane to get to the site of action, the antibacterial activity
`also was dependent on lipophilicity (the neutral form).15
`
`NH2
`
`/ \
`
`CH3
`
`3.11
`Up to this point only the ionization of the drug has been considered. As
`indicated in Section III,B,2, there are a variety of acidic and basic groups on
`receptors. Anionic groups in DNA include phosphoric acid groups (pKa 1.5 or
`6.5) and purines and pyrimidines (pKa —9); anionic groups in proteins are
`carboxylic acids (aspartic and glutamic acids; pKa 3.5-5), phenols (tyrosine;
`pKa 9.5-11), sulfhydryls (cysteine; pKa 8.5), and hydroxyls (serine and
`threonine; pKa —13.5). Cationic groups in DNA include amines (adenine and
`cytidine; pKa 3.5-4) and in proteins include imidazole (histidine, pKa 6.5-7),
`amino (lysine, pKa —10), and guanidino (arginine, pKa —13) groups. There-
`fore, the structure and function of a receptor can be strongly dependent on the
`pH of the medium, especially if an in vitro assay is being used. The pKa values
`of various groups embedded in a receptor, however, can be quite variable,
`and will depend on the microenvironment. If a carboxyl group is in a nonpolar
`region, its pKa will be raised because the anionic form is destabilized. Gluta-
`mate-35 in lysozyme and the lysozyme—glycolchitin complex has a pKa of 6.5
`and 8.2, respectively.16 If the carboxylate forms a salt bridge, it will be stabi-
`
`

`

`III. Drug—Receptor Interactions
`
`65
`
`lized and its pKa will be lower. Likewise, an amino group buried in a nonpolar
`microenvironment will have a lower pKa because protonation will be disfa-
`vored; the s-amino group of the active site lysine residue in acetoacetate
`decarboxylase has a pKa of 5.9.17 If the ammonium group forms a salt bridge,
`it will be stabilized, deprotonation will be inhibited, and the pKa will be raised.
`Now that the importance of drug—receptor interactions has been empha-
`sized, we turn our attention to the principal method for the determination of
`these interactions.
`
`D. Determination of Drug-Receptor Interactions
`
`Hormones and neurotransmitters are important natural compounds that are
`responsible for the regulation of a myriad of physiological functions. These
`molecules interact with a specific receptor in a tissue and elicit a specific
`characteristic response. For example, the activation of a muscle by the cen-
`tral nervous system is mediated by release of the neurotransmitter acetylcho-
`line (ACh; the molecule in Figs. 3.1 and 3.2). If a plot is made of the logarithm
`of the concentration of the acetylcholine added to a muscle tissue preparation
`versus the percentage of total muscle contraction, the graph shown in Fig. 3.8
`may result. This is known as a dose—response or concentration—response
`curve. The low concentration part of the curve results from too few neuro-
`transmitter molecules available for collision with the receptor. As the concen-
`tration increases, it reaches a point where a linear relationship is observed
`between the logarithm of the neurotransmitter concentration and the biologi-
`cal response. As most of the receptors become occupied, the probability of a
`drug and receptor molecule interacting diminishes, and the curve deviates
`
`i
`i
`I
`I
`I
`I
`8 7 6 5 4
`10 9
`-log [ACh] M
`
`100%-
`
`50 -
`
`0-
`
`% Muscle Contraction
`
`Figure 3.8. Effect of increasing the concentration of a neurotransmitter on muscle contrac-
`tion.
`
`

`

`3. Receptors
`
`100%
`
`50
`
`0
`
`66
`
`% Muscle Contraction
`
`10 9 8 7 6 5
`
`4
`
`-log [X] M
`
`Figure 3.9. Dose—response curve for an agonist.
`
`from linearity (the high concentration end). Dose—response curves are a
`means of measuring drug—receptor interactions and are the standard method
`for comparing the potencies of various compounds that interact with a partic-
`ular receptor. Any measure of a response can be plotted on the ordinate, such
`as LD50 , ED50 , or percentage of a physiological effect.
`If another compound (X) is added in increasing amounts to the same tissue
`preparation and the curve shown in Fig. 3.9 results, the compound, which
`produces the same maximal response as the neurotransmitter, is called an
`agonist. A second compound (Y) added to the tissue preparation shows no
`response at all (Fig. 3.10A); however, if it is added to the neurotransmitter,
`the effect of the neurotransmitter is blocked until a higher concentration of the
`neurotransmitter is added (Fig. 3.10B). Compound Y is called a competitive
`antagonist. There are two general types of antagonists, competitive antago-
`nists and noncompetitive antagonists. The former, which is the larger cate-
`gory, is one in which the degree of antagonism is dependent on the relative
`concentrations of the agonist and the antagonist; both bind to the same site on
`the receptor, or, at least, the antagonist directly interferes with the binding of
`the agonist. The degree of blocking of a noncompetitive antagonist (Y') is
`independent of the amount of agonist present; two different binding sites may
`be involved (Fig. 3.10C). Only competitive antagonists will be discussed fur-
`ther in this text.
`If a compound Z is added to the tissue preparation and some response is
`elicited, but not a full response, regardless of how high the concentration of Z
`used, then Z is called a partial agonist (see Fig. 3.11A). A partial