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This dissertation has been microfilmed exactly as received 69-16,652

GHOURI, Mohammad Sarfraz Khan, 1940·­ CHEMISTRY AND PHARMACOLOGY OF BLOCKING AGENTS.

University of Hawaii, Ph.D., 1969 Pharmacology

University Microfilms, Inc., Ann Arbor, Michigan CHEMISTRY AND PHARMACOLOGY OF

ADRENERGIC BLOCKING AGENTS

A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

IN PHARMACOLOGY

JANUARY 1969

By 1-- r ((I. f f LO 11 Mohammad S~ K!' Ghour1.

Dissertation Committee:

Thomas J. Haley, Chairman Louis J. Casarett Daniel D. Palmer Lawrence H. Piette Martin D. Rayner ACKNOWLEDGEMENT

The only wisdom I have displayed in this study was in the choice of my professors who tmpressed upon me the importance of human relations in molding a career. It is with deep sense of gratitude that I wish to express my appreciation to the members of the Dissertation Committee for their guidance and valuable suggestions.

I am deeply indebted to the East-West Center for financial support and for providing a unique opportunity to participate in an international program for Cultural and Technical Interchange.

Appreciation are expressed to many others in the Department of

Pharmacology. I am particularly grateful to Mrs. Kathryn Yamashiro for her skill in converting a palimpsest to a legible typescript, and to

Mr. Nathan Komesu for his cooperation.

My final thanks are due to the following companies for the generous supply of the drugs used in this investigation~ Ciba Pharmaceuticals;

Merck, Sharp and Dohme; Eli Lilly and Company; U. S. Vitamins and

Pharmaceutical Corporation; Burroughs Wellcome and Company; Warner­

Lambert, Wyeth Laboratories Inc.; McNeil Laboratories; Sterting­

Winthrop, and Smith, Kline and French Laboratories. CHEMISTRY AND PHARMACOLOGY OF ADRENERGIC

BLOCKING AGENTS

A dissertation submitted to the Graduate Division of the University

of Hawaii in partial fulfillment of the requirements for the

degree of Doctor of Philosophy.

ABSTRAGr

Studies on the isolated rabbit ileum indicate that this preparation reacts differently to stimulation or blockade of the alpha or beta adrenergic receptors, and thus may be used as a differentiation test object to ascertain the type of involved in the response obtained. This preparation can also be used to differentiate between the drugs that produce their effects by releasing . This tissue is also suitable for demonstrating nonspecific musculotropic actions of drugs. The treated rabbit uterus had alpha and beta adrenergic receptors, and stimulation of the former results in contraction and the latter in relaxation.

During the course of study on sympathomimetic drugs, a peculiar observation was made that effectively blocked isoproterenol-induced inhibition on the ileal and the uterine preparations. Cyclopentamine-induced beta receptor blockade was not to be expected since the structure of this drug had none of the characteristic features of the known beta receptor blocking agents.

This finding not only points out that the current hypotheses regarding the structure-activity relationships of adrenergic drugs are inadequate but also makes it imperative that any further speculations on the ~ structure-activity relationships in adrenergic drugs and thereby on the chemical nature of adrenergic receptors ~t consider the dynamic nature of the structures of drug, receptor and the receptor environment.

MOreover, it may be stressed that the drug molecule as a whole determines the nature of the biological activity of the drug, and the receptor environment should not be considered irrelevant in any receptor-dependent pharmacological activity. It seems that

cyclopentamine has the ability to produce beta receptor blockade by

directly acting at beta as well as by causing a

change in the degree of influence of the receptor environment on the beta receptor activity. Cyclopentamine also provides an indirect

evidence that the beta receptor activity is susceptible to the dynamic

changes occurring in the vicinity of the receptor. The beta receptor

blocking action of cyclopentamine, therefore, suggests a new approach

to adrenergic mechanisms, and provides a new basic structure for the

development of beta receptor blocking drugs.

The presence of an isopropyl group on the amino nitrogen and the

catechol hydroxyl groups were found to increase specificity with

resultant increase in the intrinsic activity at beta receptor. But

these groups do not seem to define absolute structural requirements for

activity at beta receptors. However, the presence of the beta hydroxyl

group in beta receptor is essential.

The only essential requirement for intrinsic activity at alpha

adrenergic receptor is the presence of the ethylamine side chain.

Substitution in the ethylamine moiety invariably decreases alpha

receptor stimulating activity. With large groups alpha v receptor blocking property is obtained. It appears that an aromatic ring is required for alpha adrenergic receptor blocking action since no aliphatic was found to have alpha receptor blocking activity.

Substitution in the ethyl moiety decreases affinity for alpha receptor,

but produces sympathomimetic that act by releasing

norepinephrine. TABLE OF CONTENTS

PAGE

ACi

4. Structure-Activity relationships of beta-receptor blocking agents • 56 5. Stereochemical aspects • ·· · 72 6. Effects of adrenergic receptors· · · · blocking agents on the nervous system · • 75 7. Antiarrhythmic properties· ·· ·· · ·• 79 8. Metabolic effects of adrenergic · ·· receptors blocking agents 85 B. Statement of the Problem · • · • · · 93 C. Objectives of the Studies · · ·· 97 vii TABLE OF CmlTENTS (Cont'd)

PAGE

CHAPTER II. METHODS AND MATERIALS •• . . . · .. . · ... 96 1. Introduction ••••••••••••• 96 2. Selection of tissue preparations ••••• 96

(a) Isolated ileal preparation 99 (b) Isolated uterine preparation •• 99

3. Experimental details 101

(a) Determination of alpha receptor stimulating property •••••• 103 (b) Determination of alpha receptor blocking property •••••• 104 (c) Determination of beta receptor stimulating property ••••• 105 (d) Determination of beta receptor blocking property ••••••••• 105 4. Analysis of results •• 106 5. Drugs and solutions •• 111

CHAPTER III. RESULTS, DISCUSSIONS AND CONCLUSIONS 115

1. Basis for interpretation and integration data . • · 115 2. Alkylethylamine series · ·· · · 118 3. Phenylalkylamine series · · . . · · ·· · 123 4. Phenylalkanolamine series· · · .. · 129 5. Heterocyclic compounds • · · · · 132 6. Congeners of dichloroisoproterenol • · · • 137 CHAPTER IV. SUMMARY ...... 163 APPENDIX ...... 168 BIBLIOGRAPHY ...... ·. 173 LIST OF TABLES

TABLE PAGE

I COMPARISON OF ACTIONS OF NOREPINEPHRmE AND ISOPROTERENOL AND THE TYPES OF ADRENERGIC RECEPTORS IN VARIOUS TISSUES ••••••••••• 3

II ADRENERGIC BLOCKING DRUGS ••• 11

III STRUCTURE-ACTIVITY RELATIONSHIPS m PHENYLALKANOLAMINES ••• ••• 57

IV STRUCTURE-ACTIVITY RELATIONSHIPS IN SULFONAMIDO SUBSTITUTED PHENYLALKANOLAMINES . . ... 65

V COMPARISON OF POTENCY OF ALPHA-RECEPTOR BLOCKING AGENTS AND THEIR ISOMERS •••• ...... 74 VI COMPARISON OF ALPHA-RECEPI:OR STIMULATING PROPERTIES •••••••••••• ...... 119 VII POTENCY COMPARISON OF ALPHA-RECEPTOR STD1ULANTS••••••••••• 120

VIII STRUCTURE-ACTIVITY RELATIONSHIPS m ALKYLETHYLAMmE SERIES ••••••• 122

IX COMPARISON OF ALPHA-RECEPI:OR BLOCKING PROPERTIES ON ISOLATED RABBIT ILEUM •• •••••••••••••• 124

X COMPARISON OF ALPHA RECEPTOR BLOCKING PROPERTIES ON ISOLATED RABBIT UTERUS •••••••••••••••• 125

XI POTENCY COMPARISON OF ALPHA-RECEPTOR BLOCKING AGEN'TS •••••••••••• ••••• 126

XII STRUCTURE-ACTIVITY RELATIONSHIPS m PHENYLALKYLAMINE SERIES •••• 127

XIII STRUCTURE-ACTIVITY RELATIONSHIPS IN PHENYLALKANOLAMINE SERIES •••• •• ...... 131

XIV COMPARISON OF SLOPE RATIOS AND SLOPE FACTORS FOR. ALPHA-RECEPTOR ...... 138 xv COMPARISON OF SLOPE RATIOS AND SLOPE FACTORS FOR ALPHA-RECEPTOR BLOCKING AGENTS ON ISOLATED ILEUM ••••• 139 ix

LIST OF '!ABLES (Cont'd)

'!ABLE PAGE

XVI COMPARISON OF SLOPE RATIOS AND SLOPE FACTORS FOR ALPHA-RECEPTOR BLOCKING AGENTS ON ISOLATED UTERUS ••••• ...... 140 XVII COMPARISON OF BETA RECEPTOR BLOCKING PROPERTIES •••• · ...... 143 XVIII POTENCY COMPARISON OF BETA RECEPTOR BLOCKING AGENTS •• · ...... 144 XIX COMPARISON OF BETA RECEPTOR STIMULATING PROPERTIES 148

xx POTENCY COMPARISON OF BETA RECEPTOR STIMULANTS ••• ·...... 149 XXI STRUCTURE-ACTIVITY RELATIONSHIPS OF CONGENERS OF DICHLOkOISOPROTERENOL 151

XXII COMPARISON OF SLOPE RATIOS AND SLOPE FACTORS FOR BETA RECEPTOR STIMULANTS 155

XXIII COMPARISON OF SLOPE RATIOS AND SLOPE FACTORS FOR BETA RECEPTOR BLOCKING AGENTS ...... 156 LIST OF ILLUSTRATIONS

FIGURE PAGE

1 .ALKYLA.TION MECHANISM BY BETA HALOALKYLAM!NES ••••••• 24

2 CATALYZED METABOLIC PATHWAYS •••••••••••• 87

3 LOG DOSE-RESPONSE CURVES FOR NOREPINEPHRINE AND AGAmSt PHENTOIAMmE ON ISOLA.!ED ILEUM OF RABBIT ••••••••• · ... . 108

4 LOG DOSE-RESPONSE CURVES FOR PHENYLEPHRINE AGAmSt PHENtO:.AMLNE AND M.J 1999 ON ISOLA.!ED UTERUS OF RABBIT •••••••• · . .. . 110

5 LOG DOSE-RESPONSE CURVES FOR PHE~'YLEPHRINE AGAmST VARIOUS CONCENtRATIONS OF ON ISOLAtED ILEUM OF RABBIT • · .. .. 141

6 LOG DOSE-RESPONSE CURVES FOR ISOPROTERENOL AGAmST CYCLOPENTAMINE AND M.J 1999 ON ISOLATED ILEUM OF RABBIT •••• •• ·. . . . 146

7 BETA ADRENERGIC BLOCKmG AGrIVITY OF CYCLOPENTAMINE ON ISOLA.!ED ILEUM OF RABBIT 147 CHAPTER I

INTRODUCTION

A. REVIEW OF THE LITERATURE1

Drugs that owe their biological activity to their chemical constitution have contributed enormously to the development of the drug-receptor interaction concept that provides, sometimes, the only simple explanation of many drug effects. Subtle changes in such a molecule may result in profound alteration in the activity of the compound. Studies based on structure-activity relationship find their justification in the compounds belonging to this group. Falling in this category are the adrenergic receptor blocking agents, the new chemical compounds which have become valuable pharmacological tools and effective therapeutic agents. This review aims at discussing the literature on adrenergic drugs mainly from the chemical and the pharmacological point of view.

Langley (1) postulated a "receptor" substance to account for the antagonistic behavior of pi.1ocarpine and . Dale (2) found that epinephrine exerted two types of actions in the spinal cat, a pressor action and a depressor action demonstrable after pretreatment with ergotoxine. Cannon and Bacq (3), Cannon (4) and Cannon and

Rosenb1ueth (5) suggested that excitatory and inhibitory transmitters released from sympathetic nerves combined with a receptive substance to form sympathin E (excitatory) and sympathin I (inhibitory) respectively.

1 A part of this review has been accepted for publication in the Journal of Pharmaceutical Sciences. 2 Since norepinephrine has been found to be the transmitter for all adrenergic nerves irrespective of the nature of response, it became apparent that the effector cell deterPJined the excitatory or inhibitory nature of the response. Ahlquist (6) tested a number of closely related on different tissues and came to the conclusion that only two sets of structure-activity relationship could exist in these amines.

The relative potencies of catecholamines in producing contraction of smooth muscle were epinephrine norepinephrine a-methylnorepinephrine isoproterenol and, in producing relaxation, the order of potency was isoproterenol epinephrine norepinephrine a-methylnorepinephrine.

This led him to distinguish two types of receptors, alpha receptors that subserve excitatory responses in most tissues, the intestinal alpha receptors being inhibitory; and beta receptors that subserve the inhibitory responses except in the where the response is excita­ tory. All the excitatory effects of the alpha receptors can be viewed as membrane effects brought about by depolarization, and all the inhibitory effects of beta receptors can be interpreted as linked to repo1arization phenomena (6-8). The distribution as well as the responses obtained by the stimulation of alpha and beta receptors in various tissues are summarized in Table I (9, 10).

The true chemical nature of the adrenergic receptor is still a matter of speculation although working hypothetical models have been constructed (11, 12). Since virtually nothing is known about the precise nature of these receptors, some physico-chemical character­ istics of compounds found to be active at these receptor sites have been studied to indicate the kinds of interaction which might be 3

TABLE 1. COMPARISON OF ACTIONS OF NOREPrnEPHRrnE AND ISOPROTERENOL AND THE TYPES OF ADRENERGIC RECEPTORS IN VARIOUS TISSUES

Action of Action of Tissues Norepinephrine Isoproterenol Receptors (N) (I)

Heart Increased rate Increased rate (I> N) beta Increased force Increased force (I> N) beta

Blood vessels Constriction Dilatation alpha, beta

Bronchial smooth muscle Relaxation Relaxation (I > N) beta

Intestinal smooth Relaxation Relaxation alpha, muscle (I > N) beta

Vas deferens Contraction Inhibition of alpha, contractions beta

Pilomotor muscles Contraction No contraction alpha

Uterus, rabbit and Contraction Relaxation alpha, human (I > N) beta

Uterus, rat and non­ Relaxation Relaxation alpha, pregnant cat (I > N) beta

Dilator Pupillae Contraction No contraction alpha

Nictitating membrane Contraction Inhibition of alpha, contractions beta

Muscle Glycolysis Glycolysis (I > N) beta

Liver (Potassium (No potassium ( release ( release alpha, (Glycolysis (Glycolysis beta

Adipos~ tissue Fatty Fatty acids alpha, released released beta

Bowman ~ al. (86) 4 incorporated in such receptor models (13). Receptors are characterized not by their composition, shape, size or location, but by the chemical molecules that somehow manage to bring about the physiological response that has been identified with the activity of the receptor.

The specificity with which a receptive substance selects or differentiates between chemical entities is its most fundamental property. The optical isomers of epinephrine and norepinephrine provide a good example, the levo isomer being 10-100 times more active than the dextro isomer. The absolute configuration of these isomers have been worked out, the levo form having L or R configuration (14).

In order to explain the difference in potency between the optical isomers a three-point attachment to the receptor was envisaged. Belleau

(12) has presented a series of sketches to show what an adrenergic receptor should look like in different stages of its activity with agonists and antagonists. The characteristic feature of this illustration is the incorporation of Ca++ as an integral part of the primary receptor site. Calcium plays a key role in a multitude of biochemical processes in the body and has been shown to participate in adrenergic mechanisms (12). Though stimulating, these models ignore the possibility that the relative disposition of structures in the vicinity of the receptor site may influence the physico-chemical characteristics of the receptor itself. The structures surrounding a receptor site may also undergo similar changes themselves by virtue of their being in a dynamic state coincident with the activity of the cell as a whole.

Thus the structural groups forming a receptor site may be constantly undergoing conformational Changes, some conformations occurring more 5 frequently than others, depending upon the extrareceptor activity and the relative stability of the conformational transitions. To conceive of a rigid receptor structure with its functionally active groups oriented in a rigid conformational pattern seems no longer justiable in view of the fact that molecules differing in their structures can elicit response at one and the same receptor and a single chemical molecule can be active on different types of receptors (9). This means that for a given molecule of a drug, the capability to interact with the receptor will depend upon: 1) the intrinsic nature of the receptor, i.e. the nature and arrangement of the active chemical groups forming it, 2) its ability to acquire different conformations and their relative frequency of occurrence depending upon their relative stability, 3) the nature of the receptor environment and the degree of its influence on the receptor substance, both factors being controlled by the activity of the whole cell, and 4) the nature of the drug molecule.

The failure to isolate a receptor substance has been an intractable problem in pharmacology possibly because the controlling forces that help induce certain conformational patterns in the chemical groups which form a receptor are lost the moment the receptor environments are disturbed. Such effects will be intermolecular in origin and may be called the "environmental factor." This factor involves two main problems, namely the stereochemistry of molecules in different stages of aggregation, and the effect of temperature on the molecules in a given state of aggregation. It will be clear that the environmental factor depends on the operation and magnitude of intermolecular forces.

A change in its surroundings may cause a sufficient alteration of the 6 behavior of a receptor as to make it desirable to postulate many sub-types of the receptor in different organ systems responding differently to a single chemical entity (15, 16, 17). Table I compares the relative actions of norepinephrine and isoproterenol on different organs of various species. It has been emphasized that this classification of the adrenergic receptors into two types is in better agreement with the antagonists of catecholamines than with the amines themselves (18). The element of uncertainty that lies in fitting metabolic, intestinal, cardiac and CNS effects of catecholamines into this scheme is a challenge in itself, and there is no clear evidence as to whether differences between alpha and beta receptors are related to basic structural features, to flexible conformational variants or to the relative accessibility of the receptors (19).

The evolution of alpha- and beta-receptor blocking agents, a term first used by Moran and Perkins (20), in the last two decades is the immediate consequence of Ahlquist's classification. The many symposis and excellent reviews are indicative of the remarkable progress that has been made in this field (21-30). Various methods are in use for the screening of adrenergic antagonists. A brief review of some of those commonly employed will be given here.

Preparations with Alpha-Receptors

Isolated vas deferens of the guinea-pig without the nerve supply has been extensively used (31, 32, 33). The same tissue with an intact nerve supply has also been described (34, 35). The sensitivity of the preparation decreases in the order norepinephrine epinephrine . Stone and Loew (36) used the isolated 7 seminal vesicles (If guinea pig in a similar manner. The isolated rat seminal vesicle preparation has also been used (36A). The nictitating membrane of the anaesthetized cat in ~ or in vitro is very sensitive to epinephrine-induced contraction (36, 37). Another preparation described by Lewis and Koess1er (39) consists of strips of rabbit sorta. This is extremely sensitive to very low concentrations of epinephrine and norepinephrine (41). The sensitivity of this preparation decreases in the order norepinephrine histamine acetylcholine. Pissemski (41) and Sch10ssmann (42) observed the vasoconstrictor action of catecholamine on perfused blood vessels.

Fastier and Smirk (43) and Burn (44) described the use of perfused rabbit ear and perfused rat bindquarter. The sphinctor pupillae muscles have been used to a limited extent (45).

The activity of compounds on alpha receptors can be assessed in experiments on the of spinal or anaesthetized animals and in man and the factors involved have been discussed in detail (46,

47). Contraction of the sympathetically innervated sphinctoer pupillae produces dilation of the pupil, and can be used to measure activity at the receptor. To increase the sensitivity, the superior cer~ical ganglion can be removed sometime before the experiment (48, 49).

However, this preparation is not very well suited to quantitative determination in vivo since the dose range is too narrow to study drug effects. A general method described by Levy and Ahlquist (50) consists in recording arterial pressure, heart rate, intestinal contraction and contraction of the retractor penis in the anaesthetized dog. Four test amines are used. These are epinephrine and ethylnorepinephrine 8 (alpha, beta activators), phenylephrine (alpha activator) and isoproterenol (beta activator). In human subjects change in blood pressure and change in blood volume passing through the limb are the criteria employed to assess activity at the alpha receptor. Both these changes can be brought about by action at either type of receptor and it is not feasible to block only the beta receptor in man (26).

Preparations with Beta Receptors

Among the isolated preparations, rabbit uterus has been the test object of choice (30) although cat uterus (51) and pregnant uterus of the rat (52) have also been used. The relative sensitivity of alpha and beta receptors in hormone-dominated rabbit uterus has been studied

(53, 54). Miller (55) has thoroughly reviewed the work pertaining to the types of adrenergic receptors in the myometrium. It seems that all mammalian uteri contain both alpha and beta receptors. Stimulation of alpha receptors results in contraction and stimulation of beta receptors in inhibition. The rat uterus has been considered a different type of receptor (56). It differs from the beta receptors in the heart since it is blocked by . Not all alpha blocking agents block the rat uterus response, and it is not certain whether inhibition is due to both alpha and beta responses. Castillo and De Beer (57) described isolated tracheal chain of the guinea pig. The whole bronchial tree including the lungs has also been used by many workers

(58, 59, 60). The isolated intestine of the rabbit has been used extensively and since it has high spontaneous activity, it is not necessary to use any spasmogen (61). Sympathetic stimulation or 9 addition of catecholamines produces relaxation of the muscles. This preparation is also useful for distinguishing adrenergic antagonists and those drugs which prevent the release of sympathetic transmitter from nerve ending. The isolated preparation of the muscles of the fundus of the rat's stomach also relaxes in response to catecholamine

(62, 63). It is uncertain whether this preparation has only beta type receptors. Evidence has been presented to show the presence of both types of receptors in the canine ileum (64).

A modification of the epinephrine "reversal" test (65) that consists in blocking the responses at the alpha receptor thereby unmasking those at beta receptor has been devised (66, 67). Dornhorst and Herxheimer (68) made use of the effects of isoproterenol on the passage of air through the lungs in anaesthetized animals and found that these effects can also be determined in conscious man. Another method consists in reducing or blocking the positive chronotropic and inotropic effects of isoproterenol on the heart (69). The spontaneously beating auricles of the guinea pig or rabbit have been used, as have perfused whole (70, 71, 72). Bohr (73) has discussed the type of adrenergic receptor in coronary arteries. Small coronary arteries possess almost entirely beta type receptors whereas large coronary arteries contain both.

SAR of Alpha-Adrenergic Blocking Agents

Drugs that antagonize the effects of sympathomimetic amines are classified into alpha- and beta-receptor blocking agents depending upon whether the type of action blocked is associated with alpha or the beta 10 receptor stimulation. Drugs which exert effects within the

cerebrospinal axis, at the autonomic ganglion or along the postganglionic fibers, and interfere with the transmitter release at

the sympathetic nerve endings are described as adrenergic neuron blocking drugs. They will not be discussed here. Each of these

classes of adrenergic blocking drugs is further divided on the basis of their chemical structure. Compounds that block the alpha-receptor do not show a great deal of structure selectivity and various groups of

compounds derived from structures unrelated to one another have been

found to possess potent antagonistic properties.

Table II gives the types of chemical structures and the names of

the representative drugs that have been in medical use.

Ergot alkaloids: - Historically, these are the most important compounds.

During investigations of methods for the bioassay of tissue extracts,

Dale (2) discovered that ergot alkaloids (ergotoxine) possessed anti­ adrenergic activity. Stoll and Hofmann (74) showed that "ergotoxine"

was a mixture of three alkaloids: ergocristine, ergokryptine, and

ergocornine. The ergot alkaloids are derivatives of lysergic

amide. There are twelve naturally occurring alkaloids consisting of six

isometric pairs; the optical activity depends on the lysergamide group,

the levo isomer, in each pair, being more active pharmacologically

than the dextro isomer (which is also less soluble). The levo isomers

are said to be derived from lysergic acid and the dextro isomers from

isolysergic acid. These acids differ only in the arrangement of the

carboxyl group and atom at the 8-position (I). The chemistry

and pharmacology of the ergot alkaloids have been discussed (75-83). 11

TABLE II. ADRENERGIC BLOCKING DRUGS

Type of Main Compounds Type of receptor chemical structure in medical use blocked

1- Lysergic acid amides Ergot alkaloids alpha (some beta)

2. Yohimbine-like alkaloids None alpha

3. Benzodioxanes Piperoxane alpha 4. Phenoxyalkylamines Gravitol alpha

5. Haloalkylamines alpha

6. Imidazolines Phentolamine alpha 7. Dibenzazepines None alpha

8. Pyrrolidines None alpha

9. Aminotetrazoles None alpha

10. Analogues of isoproterenol beta 12

CO-NH-R

There are three main actions of ergot alkaloids - they cause the plain muscles of uterus to contract; cause. an intense , and block alpha-type of adrenergic receptors. The vasoconstriction is very long lasting and it has been suggested that this might be a action on adrenergic receptors (84). , first isolated by Stoll (85), has both adrenolytic and properties. It has only a weak oxytocic action. The saturation of the double bond in ergotamine produces dihyroergotamine (DEE) with much less vasoconstrictor and oxytocic activity, but a greater anti­ adrenergic activity. DEE is the most potent epinephrine antagonist in the ergot group, and combines reversibly with alpha-receptors. Its action was considered to be competitive (86). Ergonovine (2­ aminopropan-lol amide of lysergic acid) is the simplest and the most powerful oxytocic amongst the ergot alkaloids. Methyl substitution in the amino propanol chain of ergonovine does not increase adrenergic blocking activity. The relative activities of ergot alkaloids and 13 their dihydroderivatives in antagonizing the effects of epinephrine on the rabbit uterus and on the guinea pig seminal vesicles indicate that the activity is greatest with large on the tricyclic polypeptide residue and that the activity is considerably increased by hydrogenation of the double bond (83). DEE prevented the inhibitory response of epinephrine in the isolated rabbit intestine (87). Evidence to support the alpha-blocking action of DEE is sufficient, but there is no convincing proof of its ability to block beta-receptor although it has been suggested that DEE has a slight beta-blocking activity (88, 89).

Ariens (90), however, has recently pointed out that the terms alpha- and beta-adrenergic block are not applicable to such drugs as DHE, since he

could not agree that specific or competitive blockade had been demonstrated. Barlow (26), on the other hand, has some doubt as to the real significance of the term competitive blockade. Recently Van Rossum

(91) has discussed the merits and limitations of the mathematical

approach to drug receptor interaction at molecular levels. De Bonnevaux

~ al. (92) observed that DEE depressed central sympathetic action

following compression of both carotids in chloralosed dogs, and that

this action preceded the peripheral adrenergic blockade.

Ergot alkaloids inhibit the increase in metabolic rate and blood

sugar produced by epinephrine (93, 94). Other effects include a fall in

systolic pressure rather than a rise produced by epinephrine in

hypertensive subjects after large parenteral doses of ergot alkaloids,

reduction in the incidence of ventricular filbri11ation and ectopic

beats caused by cyclopropane; relief from renal ischemia caused by

epinephrine, asphyxia and electrical stimulation of splanchnic nerves 14

(95). Ergot alkaloids do not antagonize epinephrine-induced vasodilatation in skeletal muscles of isolated limbs and the positive

inotropic effect of epinephrine on the isolated heart in situ (96).

DEE produces sympatholytic effects in lower doses than are required to

produce adrenolytic effects. The adrenergic blockade develops slowly with these compounds which led to criticism (26) of the ability of

ergot alkaloids to produce competitive blockade (97).

Yohimbine and Related Alkaloids: Yohimbine (II) and a number of other naturally occurring alkaloids and their semisynthetic derivatives have

been shown to posses sympatholytic and adrenelytic properties.

Chemically they resemble the ergot alkaloids in containing an indole

grouping as part of a complex molecule, yet their alpha-receptor

blocking activity is not very high. The yohimbine molecule is

remarkably flat (77), the ester group being equatorial. Changing che

configuration of the ester group from equatorial to axial (as in

) decreases the ability to antagonize the effects of

epinephrine on the blood pressure (88). Unsaturation of the ring

bearing the alcoholic hydroyxl and the ester groups (99) as well as the

removal of any of these groups reduces activity; but the ester and the

hydroxyl are not the essential requirements for activity, since

desoxyyohimbol (III) is not inactive (100). Yohimbine has many other

pharmacological actions such as vasodilatation, ADH release and local

anesthesia.

Benzodioxans: Many of these compounds were synthesized by Fourneau and

the first synthetic compounds with potent adrenergic blocking activity, 15

III II (F933) (IV) and prosympal (V), were described by Fourneau and

Bovet (101).

IV v

The most potent and the most toxic among the basically substituted benzodioxans was the diethylaminomethylbenzodioxan also known as prosympal or F883 (102, 103). It is both adrenolytic and sympatholytic.

However, F883 and F933 did not reduce the toxicity of epinephrine in mice (104). The levo isomer of prosympal was found to be about six ttme~ more active than the dextro form in blocking the pressor action of epinephrine in cats (105, 106). The toxicity of the secondary amines increases with molecular weight, but the epinephrine antagonism reaches 16 a maximum between 2 and 3 atoms. substitution diminishes sympatholytic property whereas anti-epinephrine activity is maintained. Piperoxan is the most active of the piperidine-substituted benzodioxans.

The most recent addition to this group is dibozane (VI). O'Leary

(107) showed that it lowered the mean blood pressure in anaesthetized dogs and was 5 - 20 times more active than piperoxan in antagonizing the hypertensive response to carotid occlusion, and about equally potent in antagonizing the hypertensive effect of epinephrine.

VI

Ahlquist and Levy (108) used dibozane as the typical alpha-blocking agent for production of epinephrine "reversal" and the prevention of the inhibition of intestinal motility in anaesthetized dogs. Rapela and

Green (1961) tested dibozane, azapetine and phenoxybenzamine for their ability to block the adrenergic receptor in the skeletal muscles of the dog. They found that dibozane and phenoxybenzamine were equipotent in antagonizing the effects of norepinephrine and epinephrine whereas

azapetine was much less active. The effectiveness of these agents

increased progressively with increasing dose of showing that a

noncompetitive blockade was produced. A persistent blockade produced 17 by the largest dose of dibozane was not observed with the largest dose of any of the other two compounds. In man, dibozane does not produce consistent effects (111).

Phenoxyethylamines: These compounds have not been found useful therapeutically because of their side effects (112). They bear a close chemical resemblance to different groups of drugs including adrenergic neuron blocking agents (e.g. xylocholine), antihistaminic compounds

(e.g. thymoxyethyldiethylamines, F929) and local anaesthetics (e.g. procaine). Eichholtz (113) and Schmidt and Scholl (114) described oxytoxic action of gravitol (VII). Many reports have shown that these compounds also possess the ability to antagonize some of the effects of epinephrine (112, 115-119).

The simplest member of this group, phenoxyethyldiethylamine (F-928)

(VIII) is active in blocking the pressor action of epinephrine. It is also the starting point in the development of antihistaminic compounds

VII VIII belonging to this series. The parent compound phenoxyethylamine (IX) is sympathomimetic. In all other phenolic ethers of tertiary alkylamines, the position of the phenolic OR groups has a profound effect on the adrenergic blocking activity of these compounds. 18

IX

Substitution in the O-position is consistent with anti-adrenergic activity, whereas compounds substituted in the meta- and p-positions have, respectively, pressor and -like activities. Replacement of phenolic-OR group by a tertiary alkyl group in O-position produced the antihistaminic compound (F929) (X). Bulky substituetns on the nitrogen atoms yielded compounds with depressor activity resembling isoproterenol.

In general, secondary and tertiary phenoxyethy1amines exert an adrenergic blocking action which is relatively weak and of short duration. A quaternary derivative (XI) has been shown to interfere with the norepinephrine synthesis from and to deplete amine stores

(120) •

x XI

Beta-Ra1oa1ky1amines: Nickerson and Goodman (121) were the first to observe that N,N-dibenyzl-2-ch1oroethy1amine (XII) antagonized the 19

XII actions of injected epinephrine on the blood pressure of the cat.

This soon followed by a considerable interest in this series of compounds and hundreds of compounds related to it were tested for their hypotensive property (112, 122-124). Like phenoxyethy1amines, these compounds have not proved useful as therapeutic agents, but have served as valuable tools in the investigati.on of the pharmacology of drug receptor interaction.

In contrast to other classes of adrenergic blocking agents that produce competitive, reversible blockade, beta-haloalky1amine blockade has been designated by Nickerson (126) as "nonequilibrium antagonism" by Gaddum (127) as "unsurmountable antagonism;" and by Furchgott (125) as "irreversible competitive antagonism." The persistent effect of beta-haloalkylamines, (128), even after supposedly complete removal of the drug from the tissue, was designated as complete blockade occurring only in the second state due to covalent bond formation between the receptor and the drug; first stage of this reaction is reversible, competitive and surmountable (129).

The original explanation by Nickerson ~!!. (130) that ethyleneiminium ion (El ion) might be involved in this persistent 20 blocade now seems to be true. Many experimental factors endorsing the view that these substances owe their activity to the ring closer followed by El ion formation are: (1) among the halogens; chlorine, bromine and iodine produced active beta-haloalkylamines whereas fluorine did not (131); (2) the rates of formation of halide ions, of hydrogen ions, and of El ions determined by means of thiosulfate titra­ tion for a number of compounds indicate that there is a close correlation between the degree of adrenergic blocking activity and El ion concentra­ tion (132, 134); and (3) compounds in the series RR'Nch2Ch2X where R and R' are kept constant and only the halogen atom X is altered, have the same duration and intensity of adrenergic blockade showing that the same El ion is produced (132, 134, 135) although they differ in the rate of onset of blockade depending upon the ease of El ion formation.

Harvey and Nickerson (133), however, observed that N,N-dicyc1ohexyl­ beta-ch1oroethylamine was devoid of activity. This indicates that the ease of formation of E1 ions, its stability, its activity at the receptors, and the stability of the complex formed with the receptors, all contribute to the observed activity.

Not only the rate of formation and concentration of El ions but also the structure of this ion is important in order that a stable drug receptor complex could be formed. Belleau (136) examined a number of dibenamine analogues in which the nitrogen is part of a ring, and concluded that the stability of various 2-ch1oroethylamines to produce

El ions was dependent on the size of the ring and therefore on conformational factors. He showed that whereas compound (XIII) could give rise to El ion (XIlla), compound (XIV) was too rigid to produce its 21 corresponding El ion (XIVa). On the basis of the doses of dibenamine and its analogs required to block the pressure effect of epinephrine in anaesthetized cats it appears that only 3,4-dihydroxyphenylisopropyl grouping gives rise to a better compound than dibenamine with a 4 - 10 fold increase in potency (37). Similarly, the introduction of methyl or methoxy substituents into the aromatic rings produces little change in potency; ortho- and para-methylphenyl isomers have decreased rate of onset of action. Addition of Cl or an alkyl group bigger than methyl proved detrimental to activity except m-chlorophenyl which still retained some activity (138).

XIII

XIV XIVa

Graham (139) studied the effect of halogen substitution in the phenyl

ring in a series of compounds having the structure (XV) and showed that

the position of halogen in the phenyl ring was important for antagonism, 22

xv

with 0- and meta-substitution being more active than para. Bromo- and iodo-compounds were more active than chloro-compounds in their ability to release El ions. A similar situation occurs with dibenzyl-2-halo- ethylamines; the bromo-compound being five times more effective than the chloro-compound and having a rapid action with greater toxicity

(140) •

The degree of activity of the substituted beta-haloalkylamines belonging to the series of N,N-dimethyl-2-chlorophenethylamine (DMEA)

(XVI) is dependent upon the closeness of the structure to that of epinephrine (XVII). Chapman (141) and Graham and James (14·2) studied

XVI XVII

this series of compounds and showed that (XVIII) and (XIX) were 10 -

20,000 times more active than dibenamine. The structural requirements 23

Br

XVIII

XIX for activity include (a) an aromatic ring, (b) a beta-ha1ogenoethy1 group and (c) a secondary or tertiary amino group. Para substitution in the phenyl ring favors activity. Here again, bromo- and iodo- compounds have greater activity than ch1oro-compounds. They are more rapidly acting and have shorter duration of activity than dibenamine and much more toxicity. It was proposed that these compounds a1ky1ate the alpha-receptor by way of carbonium ion information, yet their site of action is not different from that of dibenamine (143, 144). This mechanism can be represented diagrammatically as follows (Fig. 1): 24

>

Receptor

FIGURE 1. ALKYLATION MECHANISM BY BETA-HALOALKYLAMmES

DMEA (XV) has been shown to possess muscarinic and nicotinic properties (145). Replacement of by a hydrogen atom and a methyl group on nitrogen by ethyl (XX) or chloroethyl group (XXI) changes the compound into a cholinesterase inhibitor (146). 25

xx

CH 3 HCI· CI-CH2 -CH 2 -N

In general, the only conclusion that can be drawn from the structure action relationship of beta-ha10a1ky1amines is to quote the results of Nickerson and Gump (147) that a compound could act like dibenamine if it has (1) a tertiary or quaternary nitrogen atom, which is attached at least to one beta-ha10a1ky1 group capable of forming an

E1 ion, (2) has an unsaturated ring structure attached to the nitrogen atom to stabilize the intermediate by resonance, and (3) has all the substituents on the ring lying in its plane.

Mechanisms of Action of Beta-Ha10a1ky1amines

Several detailed reviews on the proposed mechanism of action of beta-ha10a1ky1amine are available (148, 123, 149, 124). The only certainty is the ability of beta-ha10a1ky1amine to affect covalent bond formation via E1 ion at or around the receptor site (150, 150), a 26 property shared by nitrogen mustards (152). Recently, Belleau (12) proposed an interesting explanation as to how alkylation is brought about. This beautiful, step-by-step diagrammatic treatment is intellectually satisfactory enough to make one feel comfortable momentarily, yet how far this oversimplication leads in the direction of reality still remains uncertain. Belleau visualized the El ion of dibenamine alkylate as a carboxylate ion at the accessory site of the alpha receptor; the carboxylate ion being normally an acceptor of a phosphoryl group in a phosphoryl transfer process linked with the Ca++ release into the cell. In their dynamic receptor concept, Bloom and

Goldman (153) preferred a phosphate radical to represent the nucleophilic site at the alpha-receptor. However, current evidence lends support to Belleau's choice of the carboxylate ion.

Graham and Al Katib (154) carried out an interesting experiment on the isolated vas deferens of the guinea pig using various hydrolases, viz, trysin, alpha-chymotrypsin, alkaline and acid phosphatase, phosphodiesterase, Naja venom and papain, and determined their effects on the stimulatns: epinephrine, norepinephrine, dopamine, histamine, acetylcholine, bradykinin, K+ and three beta-haloalkylamines

(dibenamine, SY28 and L2). Only trypsin reversed the blockade. The ease of reversal is in the order L2 > SY28 > dibenamine. There was a linear relationship between the concentration of trypsin and the degree of blockade produced. From this, they suggested that trypsin may catalyse the recovery of alkylated alpha receptors. This recovery would occur by action at an ester linkage on L-arginine or L-lysine, implying that the anionic acceptor site in the alpha receptor is a 27 free carboxyl rather than a phosphate, and that the receptor is in part an chain containing arginine or lysine or both. Chymotrypsin or higher concentrations of trypsin desensitizes the receptors to all agonists possibly by rupturing bonds. However, another report from Moran ~ al. (155) does not confirm the role played by trypsin in the regeneration of adrenergic receptors. Using tritium labelled N­

(2-bromoethyl)-N-ethyl-l-naphthylamine (SY28), these investigators blocked norepinephrine responses on the isolated vas deferens of rabbit and incubated this preparation with trypsin. They noticed a 20 per cent increase in the response to norepinephrine of both the treated and the control preparations. From this, they inferred that the recovery of response following trypsin is probably unrelated to alpha receptor regeneration. Moreover, H3-SY28 stays longer on the receptor.

Trypsin action is dependent upon the pH of the solution; alkaline pH destroys the enzyme. Since none of these reports has indicated the pH of the solution, it is not at all certain that both the experiments were carried out under identical optimum conditions for enzyme activity. A more convincing proof of the influence of trypsin on this regeneration process of the alpha receptor would be provided by blocking the £-amino group of arginine and lysine with amine reagents and

showing that in that case, trypsin could not affect alpha receptor

activity. What has been said before for other series of beta­ halogenoalkylamines also applied to dibenamine analogues in which

N-benzyl group is replaced by a naphthyl-methyl moiety (XXII) where R may be an alkyl or an aryl group and X a halogen atom. These compounds 28

XXII

have the dual property of blocking certain effects of both histamine and epinephrine (156-158). the most effective compounds were N-ethyl-

N-2-chloroethyl~1-naphthylmethylamine(SY 14) and N-ethyl-2-bromomethyl- l-naphthy1-methy1amine (SY 28). the point of attachment of the naphthalene group to the rest of the molecule is also important.

Graham and Lewis (1959) discovered that 1-naphthylmethy1 derivatives

(XXII) were more effective than 2-naphthylmethy1 derivatives (XXIII).

CH2-N-CH2-CH2-X ·HX oo-I I. R XXIII

As usual, the nature of the halogen in the ha10ethyl group was important: Br-derivatives were more active than cl-derivatives,

F-derivatives being inactive. the compounds in which R = Ph, Me, Et 29 and X = F had no anti-epinephrine and only slight antihistaminic activity. On oral administration to mice N-alkyl-N-(2-chloroethyl)- benzhydrylamines (XXIV), which are devoid of antihistaminic properties,

XXIV

exerted a moderate degree of blockade of the excitatory responses to epinephrine and sympathetic nerve stimulation (160). Benzhydryl- derivatives, like naphthylmethyl derivatives of beta-haloalkylamine, are devoid of properties. On the other hand, N-ethyl-

N-(2-chloroethyl)-9-fluorenamine (SY 21) (XXV) potentiated acetylcholine spasm on isolated seminal vesicles of the guinea pig (36). Increasing

xxv 30 the size of the alkyl group on the nitrogen in f1uoreny1 compounds progressively decreased antiadrenergic activity. the ethyl group produces the optimum effect (161). In all these different series of compounds related to beta-ha1oa1ky1amine no parallelism could be estab- between the relative order of potency with respect to antagonism to epinephrine, histamine, and acetylcholine although the underlying mechanism in all these instances is based on the ability of beta- ha1oa1ky1amines to behave as a1ky1ating agents.

Kerwin et al. (162, 163) examined a munber of beta-ha1oa1ky1ammonium compounds (XXVI) for antiadrenergic activity. these compounds were devoid of any such activity indicating that E1 ion formation was not possible in these compounds. In contrast to this observation, salts of

XXVI

bensy1-N,N-dimethy1-N-ethy1ammonium (XXVII) and its orthobromo and 2,4- dibromo derivatives exhibit considerable activity in decreasing arterial pressure in animals and blocking contraction of the nictitating membrane in response to electrical stimulation of preganglionic and 31

-HX

R· 0 -Sr 0, p-dl Sr XXVII

postganglionic sympathetic nerves (164). Hence these compounds block transmission of impulses on the level of presynaptic adrenergic structures. In addition it appears that the position of Br substitution in the phenyl ring is cricical; in the O-position Br exhibits a weak sympathomimetic activity, and in the para-position it produces a nicotine-stimulating and adrenolytic activity. The sympatholytic activity is probably based on the inhibition of spontaneous norepinephrine release.

Phenoxybenzamines: The attempt to combine the phenoxyethyl grouping with the beta-haloalkylamine has been fruitful, and resulted in the

evolution of compounds with enhanced activity. Many of these compounds are considerably more active, more specific, and more rapid in action.

Phenoxybenzamine (XXVIII) is highly effective, both orally and

intravenously, in doses of about one-tenth of those of dibenamine. the

structure action relationship of these compounds has been discussed in 32 detail by Nickerson and Nomaguchi (165) and by Gump and Nikawitz (138) and only salient features need be mentioned here.

XXVIII·

(1) Phenoxyethyl-alkyl-beta-haloalkylamines are somewhat active, but

diphenoxyethyl and phenoxyethyl-benzyl-beta-haloalkylamines are very potent adrenergic blocking agents.

(2) Ortho-methyl substitution of the aromatic ring of phenoexyethyl-

amines produces a considerable increase in activity while meta

and para substitutions significantly reduce activity. Increasing

the size of the alkyl substituent from methyl to isopropyl

produces a progressive increase in activity, but n-amyl decreases

activity. Compounds with an alpha-methyl substituent on the

oxyethyl chain are an exception to this rule in that unsubstituted

or methyl substituted derivatives exhibit maximal activity.

(3) A definite potency relationship exists within each series of

similarly substituted phenoxyethylamines, the phenoxyethyl-alkyl-

diphenoxyethyl and phenoxyethyl-benzyl derivatives increasing in 33 activity in that order.

(4) Replacement of a phenoxyethyl grouping by a phenylthioethyl

causes a reduction in activity.

Kerwin ~ al. (163) investigated the effects of the introduction of an alkyl and halogen group into N-(Aryloxyisopropyl)-beta- haloethylamines (XXIX) and came to the conclusion that a methyl group

CH 3

ftOCH2- 6H- ~-CH2-CH 2 -CI· HCI

R CH 2 CS HG

XXIX

at position 1 was necessary for oral activity and a methyl or an isopropyl group at position 2 would increase intravenous activity in the cat. The bromo analog was not active orally, as its greater chemical activity makes it more susceptibel to destruction in the gastrointestinal tract. Decreased activity also results from substitution in the phenoxy ring. However, Lindenstruth and Vander

Werf (166) reported that a fluoro- or a trifluoromethyl group in the para-position of the phenoxy ring produced an active compound, whereas similarly substituted meta and para compounds were one-fifth or one-half as effective. 34 Loew and Micetich (167) observed the antagonism produced by

2-bipheny1 derivatives of the type (XXX) to epinephrine, histamine and acetylcholine in dog and mice and showed that this series of compounds were only moderately active against epinephrine and diminished the

xxx

depressor action of histamine in dog. Their anti-acetylcholine action was the weakest among the beta-ha1oa1ky1amines. The effects of some compounds derived from

were tested for their ability to antagonize the blood pressure responses to epinephrine in cats and dogs (168). All these compounds in which R = Me, Et, benzyl, or napthylmethy1, and R' = l,4-benzodioxan- 2-y1 and X = C1 or B were weak adrenergic antagonists except one (N-benzy1-N-(2-bromomethy1)-2-aminoethy1-1,4-benzodioxan-2-ylmethy1 35 ether.Hbr) which was 2 - 3 times more active than dibenamine. Recently,

the thymol ether derivatives of beta-haloalkylamines (XXXI) have been described (169). 1-(4-chlorothymoxy)-N-(2-chloroethyl)-N-ethyl-2-

propylamine (WV0062) is superior to phenoxybenzamine, has good enteric

XXXI

absorption and has a good therapeutic index. Its analog without a chlorine atom at the 4-position of the thymoxy ring (WV0080, Rl = H) the same activity but three times more toxicity. On the other hand, changing the chloroethyl group to bromoethyl and changing the isopropyl to an ethyl group (R3 = H; R4 = CH2CH2Br) in WV0062 yielded WV823 LN-(2-bromoethyl)-1-(4-chlorothymoxy)-diethylamin~1 which has the same therapeutic index but only one-fourth the enteral activity of the parent compound. The adrenolytic effects are not improved if the Cl in

4-position is replaced by Br, I, F, NH2' N02' or CH3CO; nor are they 36 improved if the Cl or the ethyl group in the side chain is replaced by another halogen or hydroxyl group, or if the non-halogenated ethyl moiety is replaced by a methyl or a phenyl group.

Another recent inclusion in the phenoxybenzamine series is the introduction of phenoxathiinium compounds. Shriver and Rudzik (170) tested two such compounds MPT (10- methylphenoxathiinium tetrafluoro borate) and its 2-chloro derivatives (ClMPT). ClMPT is ten times more active than MPT or phenoxybenzamine in antagonizing the cardiovascular effects of epinephrine in dogs; it produces a diphasic blockade not observed with phenoxybenzamine. On the cat nictitating membrane, ClMPT and phenoxybenzamine have approximately the same activity whereas MPT was about half as active.

Mechanism of action of phenoxybenzamines: It is now clear that phenoxy­

'ethylamines and phenoxybenzamines do not affect adrenergic blockade by a common mechanism. A number of observations suggest that it is of some importance to consider the electrical properties of these compounds in relation to their adrenergic blocking activity. For example, it has been reported that the electron withdrawing groups in the molecule that decrease the availability of electrons in the benzene ring would also decrease the stability of El ion (147). Conversely, electron releasing groups, by increasing the electron density in the ring would contribute to the stability of El ion. Furthermore, it has been demon­ strated that the substituents that produce +1 or +E effects would enhance activity unless they interfere with binding at the para- and meta positions of the ring (148). Not only are the formation and stability of the El ions important, but the ability of these ions to 37 to quickly adsorb and rapidly alkylate the receptor site involves stereochemical considerations as one of the prerequisites of the activity. Belleau (171) has also discussed these factors in detail.

The fact that replacement of the ethereal in

PhOCH2CH2NRCH2CH2X by a methylene group to get PhCH2CH2CH2NRCH2CH2X2 results in complete loss of activity (122) led Belleau (172, 173) to speculate on the conformational characteristics of the alkylating El ions. He supposed that in order to alkylate the anionic site, the distance relationship between the carbonium ion and the benzene ring must be such as to reproduce the distance relationship characteristics of the pattern (XXXII). Lately, Belleau (12) has

XXXII

abandoned the role of the carbonium ion derived from the N,N-dimethyl-

2-phenyl-aziridinium ion (XXXIII) in the alkylation process. To differentiate from dibenamine and to account for the lack of specificity of the receptor towards the optical isomers of aziridinium ion, he has assumed a two point attachment, simultaneously pushing the 38 e1ectrophi1ic benzylic carbon atom out of the plane of the receptor surface.

XXXIII

In view of their ability to a1ky1ate tissue, it should not be

surprising that these compounds have antihistaminic, anti- and

anti-muscarinic properties. They inhibit cholinesterase and have the

ability to release some of the norepinephrine from the storage site

and prevent its uptake, potentiating the amine action on the heart and

on the beta receptor. They do not, however, affect the release of the

sympathetic transmitter by nerve impulses or by acetylcholine acting at the nerve endings (86). Recently, Boul1in --et a1. (174) have shown that blockade of adrenergic receptors by phenoxybenzamine causes overflow of norepinephrine in cat1s colon after nerve stimulation, and suggested

that phenoxybenzamine prevents the reincorporation of nerve-liberated norepinephrine by preventing the transmitter from combining with the

adrenergic receptors rather than by a direct action on the nerve ending.

This action makes phenoxybenzamine an invaluable tool for studies of

the sympathetic nerve ending since the amount of norepinephrine

escaping into the blood may represent the amount of transmitter 39 actually released and bound to receptors. During repetitive stimulation,

the efflux of norepinephrine progressively declines and in 15 minutes

practically ceases although less than 10 per cent of the amine stores have been released. This indicates that in the presence of phenoxybenzamine, the vesicles are selectively depleted of the

transmitter by repetitive stimulation.

Many authors have maintained the view that higher concentrations of an adrenergic blocking agent are needed for the sympatholytic effect than are required for adrenergic blockade (175-178). However, DHE and prosympal have been known to cause sympatholysis at lower doses, a property that has also been claimed for phenoxybenzamine. Levin and

Beck (179) examined the ability of alpha and beta blocking agents to modify neurogenically and humorally induced constriction in the perfused extremity of the dog. Phenoxybenzamine reduced the vasoconstriction

produced by pre- and post-ganglionic sympathetic nerve stimulation

significantly more than the constriction response produced by

intraarterially injected norepinephrine. Phentolamine blocked the

responses to norepinephrine and nerve stimulation to the same extent.

Consistent with this is another observation of the ability of

phentolamine to suppress pressor action in smaller doses than was

required to block the direct hypertensive effect of epinephrine (180).

It seems reasonable to ascribe the greater sympatholytic activity of

alpha-adrenergic blockers to their central effects. Supporting

evidence has been afforded by Boissier ~ ale (181) who noted that

phenoxybenzamine, dibenamine, phentolamine, yohimbine, DHE, ,

and propranolol reduce spontaneous locomotor activity of the mouse and 40 potentiated an infra hypnotic dose of pentobarbital. A definite answer can not be provided at the moment as to the nature of the factor that determines the relative efficacy of these drugs on nerves and on the receptor. Nevertheless, a compromise between the classical view and the experimental results reviewed here can be obtained by assuming that a qualitative change in the receptor decides whether or not the receptor responds more vigorously or becomes abnormally more sensitive to nervous stimulation. Any agent that depresses nervous activity should, therefore, produce a greater blockade of this abnormal receptor response. Consistent with this view are the results of Varma (182) that sympathetic denervation of the cat nictitating membrane specifically affects the antagonistic effect of adrenergic blocking agents, and that hypertensive patients showed exaggerated blood pressure responses to pressor stimuli and drugs when compared to normotensive controls (183, 184).

Dibenzazepines With failure to obtain a clinically useful compound from a vast group of beta-haloalkylamines, search was directed to develop molecules without a chlorine atom since it became apparent that the untoward effects of dibenamine series were the manifestations of the presence of the chlorine atom in the molecules. A new series of adrenergic blockers were synthesized on the framework of 6,7-dihyro-sH-dibenz

~c,~/-azepines.

First described by Wenner (185), these compounds were studied by

Randall and Smith (186). The most active member of this series is

azapetine, L6-allyl-6,7-dihydro-sH-dibenzLc,~azepinephosphat~ 41 (Ro-2-3248) (XXXIV) which has many properties in common with benzylimidazolines and resembles closely in its short duration of action. It has a sympatholytic as well as a direct action

2 CH ) N - CH 2 - CH =CH 2 • H 3 P0 4

CH 2 XXXIV

on the blood vessels (187). Slightly higher doses are needed to cause sympatholytic effects than those which produce adrenergic blockade.

Azepetine also exerts a papaverine-like action on coronary arteries in animals (186). It prevents hyperglycemia resulting from stimulation of the sympathetic nerves (183) and mesentric vasoconstriction caused by norepinephrine and epinephrine (189). '!hese compounds have no appreciable activity on acetylcholine and histamine receptors. 'Ihe fact that a substitution larger than propyl on the nitrogen atom destroyed all adrenergic blocking activity and that the most active compound Ro-2-3248 has an allyl group indicates that the stereoelec- tronic properties of the molecule are of initial significance, a group that increases the polarisability of the nitrogen will result in enhanced activity. Pyrrolidines: derivatives are just one example in which the N-CH2 part of beta-haloalkylamine group is transformed into a 42 component of the heterocyclic ring. Schipper ~ ale (190) first described a series of such compounds. The most effective compound,

1-benzyl-2,S-bischloromethy1 pyrro1idine (ERL-49l) (XXXV) inhibits the pressor response to epinephrine in anaesthetized dog and compared to dibenamine, is several hundred times more effective with a considerably more favorable therapeutic index.

xxxv

In contrast to beta-ha10alkylamines, substitution of the benzyl group of ERL-491 by I-naphthyl, and 4-methylbenzyl led to a moderate loss in activity, and by 4-methoxybenzy1 to a marked loss in activity.

The 4-ch10robenzy1 analog exhibited a low order of adrenolytic activity whereas the 2-bromobenzyl analog was approximately as active as ERL-49l.

The addition of a phenoxya1kyl group, however, ~id not improve the activity; 1-(2-phenoxyethy1)-2,S-bisch10romethylpyrrolidine and its

2-phenoxyisopropyl analogues were about as active as ERL-491. The mechanism proposed by the authors takes into consideration the ability of the E1 ions derived from the 2,S-bisch10romethy1pyrrolidine to conform to Belleau's celebrated phenethy1amine pattern; and the steric factors that contribute to the stability of the El ion and its fixation at the receptor surface. 43 A shift from an adrenolytic to a sedative property occurs when the

ethyl group in 3-phenyl-N-ethylpyrrolidine (XXXVI) is replaced by a

homoveratryl group (XXXVII) (191).

XXXVI XXXVII

Unlike (XXXVI) in which the adrenergic blocking property is due to the presence of phenylethylamine group in the heterocyclic structure, the presence of a phenmethylamine grouping in 2-phenylpyrrolidine

(XXXVIII) and in 2-phenylpiperidine derivatives (XXXIX) (192) is

compatible with adrenergic blocking activity.

Piperazines: This class of compounds is unique insofar as its very varied pharmacological characteristics are concerned. Adrenolytic,

Q Ph Q Ph I I R R

XXXVIII XXXIX 44 and antihistaminic properties of l-

(XL) were first mentioned by Bovet and Bovet-Nitti (98). Numerous papers have since appeared (193-199). In l-arylpiperazine series, hypotensive, vasodilating and neuroleptic effects have also been emphasized.

Among the 1,4-disubstituted piperazines (XL1), a great variety of substituents have been tried and it is only occasionally worth while to discuss structure activity relationship in this series of compounds.

r--\ I R-N N-R LJ

XL XLI

For example, only one compound, l-methyl-4-, has a potent adrenergic blocking activity whereas other piperazines with one alkyl and one aryl substituent groups showed only a trace activity

(196). This compound possesses weak anticholinergic, weak anti- histaminic, and a potent local anaesthetic activity comparable to p~ocaine. It appears that the adrenolytic but not the hypotensive property is greatly susceptible to structural changes. A change in the following compound (XLII) from R = (CH2) 30CH3 to R = H results in a marked loss of adrenergic blocking activity without affecting hypotensive activity (200). There is no correlation between the degree of effectiveness of a compound as anti-adrenergic, antihypertensive and antihistaminic, as 45 each property varies independently with an alteration in the molecule.

Substituted 1,4 diary1 piperazines (XLIII) exhibit maximum activity

202). In anaesthetized cats and dogs, these compounds produce both

--:or',- 0 CH 3 " ·---N N-R O \.....J XLII XLIII hypo- and hypertensive responses depending upon the dose;

3,4-dimethoxybenzy1piperazine has a direct musculotropic action as strong as that of papaverine. 1-L3-ethoxy-3-(p-to1y1)-propyl/-4­

(0-to1y1)- dihydroch1oride (SU-12080) (XLIV) has been shown to possess strong antiadrenergic, weak antihistaminic, weak anti- cholinergic, and potent direct spasmolytic activity (203-205).

Similarly, the compound 1,4-bis(1,4-benzodiozan-2-y1methy1)piperazine

OC 2 H5 . CH 3 I r-'\ o-CH-CH2 -CH 2 -N\...-IN-o

XLIV 46 (McN-18l) was more active than dibozane both in its sympatholytic and adrenolytic activity and was without any hypotensive effect (206).

The ring structure of piperazine is not essential for activity although it increases the activity over that found in open chain compounds. An example is the N,N'-disubstitution of ethylenediamines

(XLV) and piperazines with benzodioxanylmethyl and phenoxyethyl groups.

XLV

Symmetrical secondary amines with two benzodioxanylmethyl or phenoxyethyl groups are active but comparable derivatives of piperazines are even more active. In both the series, replacement of either of these substituents with phenyl or carbethoxy groups decreases activity, and a greater loss in activity occurs if both the substituents are replaced by phenyl, benzyl, phenoxypropyl, or ethoxyethyl groups.

Substitution on benzodioxane ring and on carbon atoms of piperazine ring also produces compounds with decreased activity (207). R = dioxanylmethyl or phenoxyethyl. Phenylpiperazine esters (XLVI) and carbamates (XLVII) possess moderate antiadrenergic activity, and on intravenous injection in dog bring about a sustained hypotensive effect. As expected, these are also sedatives (208). Surprisingly, the amides (XLVIII) have no 47

2HCI

XLVI

o r-"\ . \I Ar-N N-(CH2)n-O-C-NH-R \...J

, XLVII antiadrenergic activity, but they are potent sedatives as well as hypotensive agents (209).

XLVIII

Substituted indo1y1a1ky1pheny1piperazines form a group of tranquilizers (210A) and a member of this group 1 L5,6 dimethyoxy-2­ methy1(3-indo1y1)-ethyY-4-pheny1piperazine (XLIX) has been shown to possess norepinephrine depleting action on sympathetic nerves, adrenolytic, and antiarrhythmic properties (211). 48

XLIX

Replacing the substituted indolyl group by 3-substituted 2,4- quinazolinediones (L) results in complete disappearance of anti- adrenergic activity whereas sedative and hypotensive properties become more pronounced (212). Recently, triazine substitution has yielded a

L compound: 2-amino-4-methoxy-6-£2-(4-phenylpiperazine-1-yl)-ethYl/-s- triazine (LI), capable of producing long lasting alpha receptor blockade with central, peripheral and direct dilator effects (213). Lastly, an

OCH;, N~N

NH 2 llN..J- (CH 2)2- N=N ()

LI 49 attempt to convert piperazine moiety into one of diazabicyclooctane has not been successful. Boissier ~ ale (214) investigated a number of

3-aralkyl, and 3-acyl substituted 8-methyl-3,8-diazabicycloL3.2.!7 octanes (LII) and noticed antagonistic activity of 3-methyltropyl derivative to acetylcholine showing that these compounds correspond well to derivatives and can not be considered as N-methylpiperazine analogues. Obviously, a modification of the size and shape of the ring would affect the molecule as a whole resulting in loss of affinity for the receptor. On the other hand, tropyl ester of 2,3-diphenylpropionic acid (LIII) is sympatholytic (215).

LII L III

Tetrazoles

Since the earlier reports by Gross ~ ale (216-220) on the

convulsive, analeptic, and sedative properties of aminoalkyl- and

aminophenyltetrazoles, only a few papers have discussed the 50 antiadrenergic action of this series of compounds. Search for potential antihypertensive agents in all sorts of compounds has led to the discovery of potent tetrazo1e derivatives that antagonize the adrenergic response. None of these compounds, however, has brought a significant advance over those that are already existing but they do provide a new basic structure on which new compounds can be designed with better and selective effects. Recently, Hayao ~ a1. (221) have reported that most of the aminoa1ky1tetrazo1es possess potent alpha adrenergic blocking activity, the 5-/2-(4-ary1-1-piperaziny1)eth~/- tetrazoles being the most active series. This is to be anticipated since it was shown in the preceding section that pheny1-, phenylmethy1- and dtmethoxyindo1y1piperazines constitute a large series of compounds with a wide range of pharmacological activity, including potent sympatholytic and adrenolytic effects. Rodriquez ~ a1. (222) examined a whole series of phenylpiperazinetetrazole derivatives and recognized a high degree of adrenergic blocking activity, in compounds of the formula (LIV) where Rl ,R2 are alkyl, aryl, aralkyl and substituted pheny1a1ky1piperazy1 groups. Their results indicate that

5,2-(4-pheny1-1-piperazy1)ethy1tetrazo1e dihydroch1oride (NA 1277) (LV)

LIV LV 51 is outstanding in its antiadrenergic action. It blocked aortic strip

and nictitating membrane responses to epinephrine, antagonized the vasoconstrictor responses to norepinephrine, and reversed the blood

pressure response to epinephrine. It did not, however, block

resulting from reflex mechanisms and/or sympathetic nerve

stimulation. It resembles DEE in its ability to exhibit greater in vivo than in vitro activity, but, unlike DEE, it does not cause

stimulation of smooth muscle. The compound has a rapid rate of onset

of action and produces an effect that lasts longer than that of

phentolamine and azapetine. Comparable adrenergic blocking activity

is also exhibited by a non-tetrazoline derivative of phenylpiperazine

(MA 1211) (LVI) which resembles phentolamine in its mode of action.

LVI

A recent paper (223) has described some pharmacological effects of

w-substituted alkylamino-3-aminopyridines. Aminopyridines have long

been known for their CNS and sympathomimetic effects (224-226). All

attempts at evolving a selective and potent adrenergic blocker have

resulted in the formation of and pressor agents. Exchange of substituted 3-aminopyridine group with aminoquinolines, and

corresponding imidazo and triazolo groups; has not yielded any 52 substantial gain. A new compound, 4-(3,4 dihydroxyphenethyl)amino-3­ aminopyridine (LVII) has been claimed to be capable of blocking both alpha, beta receptors and to produce persistent at a high dose and hypertension at a low dose.

LVII

Imidazolines

Hartmann and Isler (227) studied the effects of 2-substituted imidazoline on blood vessels. Among the alkyl substituted imidazoline derivatives vasodilator and vasodepressor activity could be found if the alkyl group was from 6 to 8 atoms in length. Tolazoline, 2-benzyl- imidazoline (LVIII) exhibits a wide range of pharmacological activity including a powerful hypotensive, relatively feeble adrenolytic, sympatholytic, cholinergic and histaminic properties (228). Alpha adrenergic blockade produced by tolazoline appears to be competitive

LVIII 53 (21, 97). Introduction of phenolic or methoxy groups into the benzene ring (e.g. tr~ethyoxybenzyl, phedracin), or the exchange of benzyl for alpha-naphtyl (naphazolin) or an indolyl group reverses the activity from depressor to pressor.

Phentolamine (LIX) presents an almost entirely different structure with greatly enhanced adrenolytic activity (229). Phentolamine is

5 - 7 t~es more active than tolazaline on the alpha receptors in dog's

HO

LIX

hind l~b, and is even more effective in rabbit aortic strips and perfused rabbit ear (230). In addition to its antiadrenergic action, phentolamine acts directly on tissues and affects cholinergic receptor sites.

Beta-Receptor Blocking Agents Until 1957, Ahlquist's concept of alpha and beta adrenergic receptors had only the status of a hypothesis. The synthesis of dichloroisoproterenol (DCI) (LX) by Mills (231) and the evaluation of its pharmacological properties by Powell and Slater (232) not only brought a fitting recognition to Ahlquist's classification, but also marked the beginning of a renewed interest in adrenergic drugs. 54 Adrenergic beta receptor blocking drugs, in fact, have attained a

position which was once considered to be the sole prerogative of ergot

alkaloids. The search for greater specificity for beta receptor

blockade has led to the discovery of new kinds of pharmacological

activity unrelated to adrenergic blockade as a by-product.

OH ~ I / CH 3 ~ Cl o-'I CH-CH 2 -NH-CH CI - .\ CH 3 LX

Following the fortunate discovery of DCI, several other potent compounds were introduced in quick succession: in 1962 by Black

~ a1. (233); in 1964, propranolol by Black ~ a1. (234); MJ 1999 by

Larsen and Lish (235); analogs by Burns ~ al. (236); in

1965 KO - 592 by Engelhardt (237), INPEA by Somani and Lum (238); and

in 1966 H 56/28 by Brandstrom ~ a1. (239). Pronethalol, the first beta-blocking agent thought to be useful clinically, had to be withdrawn from clinical trials when it was shown to possess a carcino-

genic property in the mouse (240). The only other compound to offer

some promise of safe and potent action is propano101 which is being

used in Europe while the search for an ideal beta-blocking drug

continues. The subject of beta-adrenergic blocking drugs has been thoroughly

covered in a conference of the New York Academy of Sciences (29). An

excellent review on the beta-blocking drugs has been presented by Moran 55 (241) that covers all essential points. For structure-action relation­ ship, papers by Ariens (242) and by Biel and Lum (24) may be consulted.

A very recent review by Ahlquist (30) is concerned with the pharmacodynamics of these compounds. The following discussion is intended to deal with the important aspects of recent progress in this area.

Adrenergic beta receptor blocking agents that have been introduced so far bear a remarkable structural resemblance to isoproterenol. This makes it possible to work out a definite SAR among these blocking agents.

In synthesizing the new beta-blocking agents, the chemical structure of isoproterenol has been attacked at critical points deemed necessary for beta-adrenergic activity. For a molecule to behave as a potent beta receptor agonist it was considered necessary to have a phenylethylamine structure with dihydroxy substitution at 3,4-positions of the benzene ring (243-245), with a hydroxyl group at beta-carbon atom of ethylamine side chain (246-251) and an isopropyl group at the terminal nitrogen end (252, 253). The importance of these structural characteristics in affecting a drug-receptor interaction has been postulated on the basis of stereochemical considerations (11) the characteristics themselves have been subjected to frequent revision (12). Whereas many alternatives can be thought of to describe the union of drug molecule with the receptor, the present lack of knowledge of the true chemical nature of the adrenergic receptor indicates that a solution can not be achieved without recourse to more practical approaches. 56 Structure-Activity Relationships of Beta-Receptor Blocking Agents

The search for a compound with a selective effect free from the stimulatory effects of isoproterenol on cardiac muscles led to the discovery that the manipulation of the phenethanolamine structure of isoproterenol could give rise to structures with varying degrees of stUnulating and inhibitory action on bronchial smooth muscles and the muscles of the heart. Among the variously substituted phenyalkanolamines (Table III) it can be concluded:

(1) Halogen substitution in the ring of isoproterenol produces analogs

which are more potent beta adrenergic receptor blocking agents

than those of epinephrine (DCE) which, in turn, are stronger than

those of norepinephrine (DCNE). DCI first stimulates and then

depresses the rabbit heart, where as both DCE and DCNE have only a

depressant action (20, 254). Replacement of the beta-hydroxyl

group by a chlorine atom produces alpha-receptor blockade (50).

The 2-chlorophenyl analog is a potent bronchodilator with prolonged

action in the perfused rabbit lung (255) and is effective orally

in man (256). All other halophenyl analogs of isoproterenol

produce blockade of bronchial relaxation (257-258). Unlike DCI,

the 2,4-dichloro analog is devoid of positive chronotropic effect

on the heart. Bringing 4-methyl and 3,4-dimethyl groups to

positions other than 3 and 4 results in weak antiadrenergic, weak

antiarrhythmic, and strong negative inotropic activity.

(2) 3,4-dimethyl- and 4-methylphenyl analogs of DCI are potent blocking

agents with considerable stimulating effect on the heart rate

(259). H-35-25 (LXI) appears to be capable of producing a TABLE III. STRUCTURE-ACTIVITY REIATIONSHIPS IN PHENYLALKANOLAMINES

OH R3 ~H RI--=O-/- - bH- NHR 4 • Hel R2

Beta Broncho- receptor Chronotropic Rl R2 R3 R4 Antiarrhythmic dilator blocking and Inotropic - potency effect effect effect DC1 4-C1 3-C1 H CH( CH3)2 enhanced block 1 +

Netha1ide naphthyl H CH(CH3)2 .1 1.2 ?

4-CH3 3-CH3 H CH(CH3)2 1 1.2 + 4-CH3 3-CH3 CH3 CH(CH3)2 .2 .2 4-C1 3-C1 CH3 CH(CH3)2 .OS .08 weak 4-CH3 3-CH3 CH3 n-C4H9 .2 .1 4-CH3 3-CH3 CH3 CH(C2HS)2 .1 .06 4-C2HS 3-C2HS H CH(CH3)2 .1 .08 ? 4-CH3 3-H H CH(CH3)2 .2 1.2 + 4-C1 3-Cl C2HS CH(CH3)2 .OS .1S S-CH3 2-CH3 CH3 CH( CH3)2 .2 .06 4-C2HS H CH3 CH(CH3)2 .04 .2 4-CH3 H CH3 CH(CH3)2 .1 .2 H 3-CH3 CH3 CH(CH3)2 .2 .2 4-C1 3-CH3 H CH(CH3)2 1.0 .....\J1 TABLE III. (Continued) STRUCTURE-ACTIVITY RELATIONSHIPS IN PHENYLALKANOLAMINES

Beta Broncho- receptor Chronotropic Antiarrhythmic dilator blocking and Inotropic Rl RZ R3 R4 potency effect effect effect

4-CZHS HH CH( CH3)Z .15 + 4-CH~ 3-CH3 H CHCHZCHZ .08 4-(CHZ ZCH3 HH CH( CH3)Z .1 4-CH3 3-C1 H CH( CH3)Z 1.0 4-CH3 Z-CH3 CH3 CH(CH3)Z .03 H 3-CH3 CH3 CH( CH3)Z .04 .05 4-C1 3-C1 HH .1 4-C1 3-C1 H CH3 .5 H 3-C1 H CH( CH3)Z moderate 4-C1 H H CH( CH3)Z block 4-C1 Z-C1 H CH( CH3)Z block 4-CH3 Z-C1 H CH( CH3)Z block 1 S-C1 Z-C1 H CH(CH3)Z block H Z-C1 H CH( CH3)Z dilator 4-CH30 3-CH30 H CH(CH3)Z 4-CZHSO 3-CZHSO H CH( CH3)Z 4-CH30 3-CH3O CH3 CH(CH3)Z S-CH30 Z-CH3O CH3 CH(CH3)2 enhanced weak S-CH30 2-CH3O CH3 t-but C H1S S-CH3O 2-CH3O CH3 CH enhanced CH3

\J1 00 TABLE III. (Continued) STRUCTURE-ACl:IVITY RELATIONSHIPS IN PHENYLALKANOLAMINES

Beta Broncho- receptor Chronotropic Rl R2 R3 R4 Antiarrhythemic dilator blocking and Inotropic - potency effect effect effect 4-NHS02CH3 H H CH( CH3) 2 1 decrease 4-tolyl H H CH( CH3)2 .5 Tetralin H CH( CH3) 2 1 4-N02 3H H a+(CH3)2 1

V1 \0 60 selective blockade of beta receptors in femoral vascular structures

in a dose range that has little blocking action on cardiac beta- receptors (259A).

OH CH 3 \ I / CH 3 f' CH-CH-NH-CH 0-- \ CH 3 LXI

2-Ch10ro-4-methy1 substitution has no sympathomimetic effect (260).

The introduction of an alpha-methyl group into the beta-

phenethano1amine side-chain of DCI results in diminution of the

blocking of positive inotropic effects whereas the vasodilator

blocking effect is maintained (261, 262). Substitution of the

alpha-alkyl group abolishes the positive inotropic effect of DCI

with a concomitant decrease in beta-receptor blocking ability

(259). This relationship is maintained in alpha-alkyl substituted

3,4-dimethy1 analogs of DCI. p-To1y1 compounds are analogous

to 3,4 dimethyl compounds, but tetra1in analogs lack the

sympathomimetic activity of 3,4-dimethy1 derivatives without

change in the blocking activity (260).

(3) The presence of the N-isopropy1 group is crucial irrespective of

whether the drug is a blocker or an agonist. Cyc1ization of this

group into cyc10propy1 eliminates all of the activity (259).

(4) The "methoxamine" series has created another example of receptor

selectivity. Methoxamine (LXII) caused a long-lasting 61 vascoconstriction and rise in blood pressure in doses about 100 times those of epinephrine and norepinephrine (263), and restored the epinephrine pressor response after the response had been reversed by phenoxybenzamine and dibenamine (264).

OH CH 3 OCH 3 \ \ o-CH-CH-NHz

OCH 3

LXII

Karim (264), on the other hand, observed the antagonizing effect of methoxamine on the action of epinephrine, norepinephrine, and isoproterenol in the cat and the rat's perfused heart, and the isolated rabbit atrial preparation.

These demonstrations lead one to believe that methoxamine has alpha-stimulating and beta-blocking property, a conclusion not acceptable to Ahlquist (30).

Isoproterenol itself exhibits this dualistic behavior: the l-isomer acts as an alpha- while the d-isomer acts as alpha-adrenergic blocking agent (251, 266, 267).

Isoproterenol in large doses has been shown to block its own action on beta-receptors of vascular smooth muscles in the cat

(268) and of the isolated rat uterus (269). The classical 62 structure-activity hypothesis can not be considered to provide an

adequate explanation for these seemingly anomalous phenomena, and

it must, of necessity, give way to newer interpretations based on

a broadened outlook. Individual reactions can be blocked or

elicited by different mechanisms, but a single fundamental mechanism must be common to all the reactions controlled by one receptor. An agonist by triggering this mechanism must produce all receptor effects, and a true antagonist (as defined by

Ahlquist) must antagonize this fundamental mechanism and thus be equally effective against all agonist actions. Characteristically, a beta-receptor blocking agent will produce the following responses

(a) decrease in heart rate unless there is no significant ongoing adrenergic influence on the heart; (b) some decrease in force of myocardial contraction unless there is no significant ongoing

adrenergic influence; (c) a possible increase in intestinal and myometrial activity and (d) a possible depressor response in

intact animals due to the effects on the heart (30).

N-isopropylmethoxamine (IMA) and methoxamine have been denied

the status of typical beta-blocking agent since they failed to

block significantly the increase in femoral flow, intestinal

inhibitory actions, and positive chronotropic effects produced

by isoproterenol in anaesthetized dogs (270). They do, however,

produce ethylnorepinephrine and isoproterenol "reversal" which

can be prevented by dibenamine (254). The reversal is thought to

be brought about by a vasoconstrictor action of these agents not

involving beta-receptors. Their ability to block inhibitory 63 responses in the rat uterus (56) and to block metabolic effects of catecholamine, as does DCI (271-273) leads to the proposal that either the drugs have a great degree of tissue specificity or the beta-receptors are not alike in all tissues (274).

A more probable explanation might involve the role played by the "environmental factor." N-tert-butylmethoxamine (TMA) exerts its blocking effect on the vasodilator responses to isoproterenol and ethylnorepinephrine in the anaesthetized dog and shares this activity with DCI, pronethalol, IMA and methoxamine in the isolated rat uterus. But like IMA and methoxamine, TMA does not have a broad spectrum beta-blocking activity. TMA differs from IMA in many respects: it does not produce bradycardia and a gradual increase in mean arterial pressure, does not have an appreciable effect on blood pressure or on heart rate, and it reduces the femoral flow response to intraarterial isoproterenol (274).

A new addition to this class of selective beta-receptor blocking agents is dimethyl-isopropyl-methoxamine which closely resembles TMA (275). The selective beta-blockade produced by the alpha-substituted DCI analogs, methoxamine, IMA and TMA indicates that not only alpha-alkyl substitution is important in segregating beta-blocking effects, but almost any change in the molecule affects the molecule as a whole in a way which may be presumed to account for the altered physiological behavior.

The emerging situation is critical. The appearance of selective beta-receptor blocking agents and the lack of confidence in defining beta-receptor function make it imperative to choose a 64 universally acceptable criterion to which each new compound must

conform fully in order to qualify as a beta-receptor blocking agent.

(5) p-Methysulfonamido substitution into the phenyl ring produced

compounds whose activity ranged from depressor to pressor and

from beta-stimulation to alpha-blockade (276). The most out-

standing compound in this series was MJ 1999 (LXIII) (277-279).

LX III

The results of Larsen and Lish (276) are reproduced in

Table IV.

It will be seen from a study of Table IV that the para

position of the methylsulfonamido group is crucial to beta-

blocking activity; moving it to the meta position of the phenyl

ring completely abolishes all the beta-receptor blocking

property in this series.

(6) Nitro-derivatives of isoproterenol behave in much the same way

as the methylsulfonamido derivatives. The most active compound

of this series is 1-(4-nitrophenyl)-1-hydroxy-2-isopropylamino-

ethane (INPEA) (LXIV). Somani ~ ale (280) examined a series of TABLE IV. STRUCTURE-ACTIVITY RELATIONSHIPS IN SULFONAMIDO SUBSTItUTED PHENYLALKANOLAMINES

OH R3 1 1 R. f' CH - CH - NHR 4 R 0-- 2

Compound R R R R Activity l 2 3 4

MJ 1999 CH3S02NH H H CH( CH3)2 Depressor

MJ 1998 CH S0 NH H CH CH Pressor 3 2 3 3 MJ 1996 H CH3S02NH H CH3 (Pressor and beta (receptor stimulation

MJ 1995 H CH3S02NH H CH( CH3) CHZOC6H5 (beta receptor stimulation, (depressor

MJ 1994 H ArSOzNH H CH(CH3)CH20C6H5 beta receptor depressor

MJ 1993 OH CH3S02NH H CH3 beta-stimulation, pressor

MJ 1992 OH CH3S02NH H CH( CH3)2 beta-stimulation, depressor

MJ 1991 OH CH3S02NH H CH(CH3)CH20C6H5 Depressor, alpha-block

0\ Vt 66 these compounds on the isolated rabbit heart preparation. Their results indicate that substitution with a single N02 group in the para position of the phenyl ring yields the most active compound, activity decreases on moving the N02 group to meta or ortho

LXIV

positions. The adrenergic blocking activity is also decreased by substituting with two N02 groups in 2,4- and 3,5-positions and by substitution with p-NH2 or p-CH3S02 groups in the ring. Bicyclic aromatic alkanolamines have yielded therapeutically active compounds. First introduced by Black and Stephenson (281), pronethalol(2-isopropylamino-1-12-naphthy!~-ethanol)(LXV) decreases the heart rate in intact cats without greatly affecting

OH I I CH 3 I ~ CH-CH -NH-CH ~ 2 W \ C.H 3 LXV 67 positive inotropic effects. This compound, however, is not entirely devoid of sympathomimetic effects although it is much weaker than DCI. It may stimulate or depress the heart rate and the force of contraction depending upon the dose and (238, 282-284). Pronethalol also elicits a fall in blood pressure on intravenous injection due to peripheral vasodilation and mycocardial depression (238, 281, 282, 283).

It has no effect on the vasodilator actions of histamine and acetylcholine (238).

Crowther ~ ale (284, 286) established structure activity relationships and arrived at the following conclusions:

(a) Isopropyl, sec-butyl, and t-butyl substitution on the

nitrogen atom gave potent compounds (287) and cycloalkylamino

moieties yielded inactive compounds (24). Di-substitution

on the nitrogen also resulted in inactivation.

(b) Branching at the alpha-carbon atom was detrimental for beta­

receptor blocking activity (286).

(c) Alteration of the hydroxyl group at the beta-carbon atom

resulted only in decreased activity (286).

(d) Moving the alkanolamine side chain to the alpha-position

of the naphthalene ring increased sympathomimetic activity

without an appreciable effect on blocking activity.

Halogenation or alkoxylation of the remote phenyl ring

decreased both blocking and stimulant action at the beta­

receptor. Replacement of the naphthalene group by

polycyclic ring structures produced less active compounds. 68 Indole derivatives (XVI), however, retained blocking as well as sympathom~etic activity (286).

OH I I CH 3 CH-CH2-NH-CH \ CH O::rlN 3 I R "LXVI

Another heterocyclic compound, Ro3-3528(6,7-d~ethyl­ alpha-Lisopropylamino)-methy!i-2-benzfuranmethanol (LXVII), has recently been shown to have considerable activity on the isolated rabbit atria and the heart in situ (287A).

LXVII

Moderate activity is found in compounds in which the naphthalene ring is separated by a methylene group from the ethanolamine side chain. Changing this side chain from the beta- to alpha-position on the naphthalene ring increases the activity many fold, but a corresponding compound with an ether linkage between the naphthalene and the side chain 69 exhibits a sharp rise in activity as in propranolol

(LXVIII) (288-290).

/ CH 3 - CH z - NH - CH \ CH 3

LXVIII

Propranolol is not only ten times stronger than pronethalol but is also less toxic (234). Exchange of the naptha1ene moiety for a substituted phenyl group does not affect activity; an example is KB-592 Ll-(3-methy1phenoxy)­

3-isopropy1aminopropanoi! (LXIX) which is as active as propranolol (237, 291) although three times less active orally but which unlike propranolol has some sympathomimetic activity (292).

OH

I . / CH 3 OCH z - CH - CH Z - NH - CH, o CH 3

LXIX 70

Lish ~ al. (227) described methanesulfanilides (LXX)

II related to KO-592 which contain the methylsulfonamido radical at para position of phenyl ring. 'Ihese compounds produce

LXX

potent and selective blockade to the effects of isoproterenol on respiratory, uterine, and cardiac muscles, but have no anaesthetic activity.

Ablad ~ al. (293, 294) have extensively studies a new compound, 1-(O-allylphenoxy)-3-isopropylamino-2-propanol

H-56/28 (LXXI) and have shown it to be equally potent to propranolol in antagonizing positive chronotropic and

o LXXI 71 inotropic effects of isoproterenol or electrical

stimulation of the cardiac sympathetic nerves. H-56/28

also exhibits a moderate degree of beta-receptor stimulation

on the heart muscle of reserpinized cat. This stimulatory

effect is blocked by propranolol. Quite interestingly, an

allyloxy derivation of H-56/28 (trasicor) has been shown

recently to possess hypotensive, antiarrhythmic and negative

chronotropic activity in man. Unlike propranolol, which

produces depression, this compound caused stimulation of the

central nervous system. Trasicor (39089-Ba) (LXXII), however,

is slightly less potent than propranolol (295).

OH I OCH 2 -CH-CH 2 -NH- o OCH 2 -CH =CH 2 LXXII

A compound that has no structural resemblance to isoproterenol is iproveratril (LXXIII). Iproveratril was studied by Haas and Hartfelder

(296, 297) and was shown to antagonize the effects of isoproterenol on the heart, blood pressure and phosphorylase activity. Pretreatment with iproveratril reduced the toxic effect of K-strophanthin on the guinea pig heart and decreased heart frequency. Such effects were not 72 affected by or reserpine pretreatment (298). It is a potent

coronary dilator (299).

L XXIII

STEREOCHEMICAL ASPECTS

Ever since Cushny (300) drew attention to the biological relations of optically isomeric substances, it became increasingly a matter of great concern to relate physiological activity to chemical constitution

of biologically active molecules (301). To study the stereochemical relationships of catecho1amines and their antagonists with the receptor

it was necessary to ascertain the absolute configuration of the

catecholamine structures. It has been established that the active

isomers of catecho1amines and the active isomers of the antagonists have D-configuration (14, 302). Isomers with L-configuration are either less active or inactive (303). Optical isomers of beta-receptor blocking agents can be used to dissociate effects that are due to beta-receptor stimulation from other unrelated actions. Levo isomers

of both DCl and pronethalol were forty times more effective than dextroisomers in their ability to block isoproterenol-induced

tachycardia in chloralosed cats and paralleled the bronchodilating 73 action of the corresponding analog of isoproterenol in guinea pigs

(304). Both d- and l-isomers of Del are cardiac stimulants (304) whereas the l-isomer of pronethalol has beta-adrenergic blocking as well as antiarrhythmic properties and the d-isomer is devoid of

beta-blocking action but has antiarrhythmic activity (305). the

dextro and laevo isomers of H-56/28 and propranolol can abolish

ouabain-induced ventricular tachycardia on intravenous injection in

the unanaesthetized dog. But dextro H-56/28 differs from laevo

H-56/28 and propranolol in having a rapid onset and longer duration of

antiarrhythmic action (306). Intraarterially administered racemic

propranolol and racemic H-56/28 were equipotent in their effects on

basal blood flow and on isoproterenol-induced vasodilation whereas

the dextro H-56/28 was a much weaker beta-adrenergic blocking agent

(307). the laevo isomer of INPEA was five times more active than its

racemate (308), which in its tuen was much more potent than the dextro

isomer on the rabbit heart preparation (309). the weak beta-receptor

blocking activity of the dextro isomers of the beta-receptor blocking

agents Unplies that beta-OH in these compounds and beta-OH in

catecholamine bind to the same site on receptor (280).

table V summarizes the relative potencies of beta-receptor blocking

agents on electrically driven rabbit left atria (310), on isolated

guinea pig atria and the atria and papillary muscles of kittens (311),

and on the isolated guinea pig tracheal chain (303, 312). 74

'!ABLE V. COMPARISON OF POmNCY OF BETA-RECEPrOR BLOCKING AGENTS AND THEIR ISOMERS

Rabbit Guinea Pig Guinea Pig Papillary left Tracheal Atria Muscle of atria Chain Kitten P~ P~ PA2 dl-Pronethalol 1 7.3 dl-KO-592" 7.9 8.2 8.2 dl-Propranolol 6.7 8.5 8.5 8.8 Del 6.34 7.8 7.8 l-Pronethalol 2.08 7.1 7.3 l-MJ 1999 1.8 6.8 6.2 6.6 d-Propranolol .76 6.5 6.5 6.8 dl-MJ 1999 .62 d-Pronethalol .14 5.2 dl-MJ 1998 .12 d-MJ 1999 .06 5.15 l-IMA 6.53 5.5 5.5 l-MA 6.25 5.1 5.3 dl-des MA 5.09 dl-des IMA 4.85 d-MA 4.37 d-IMA 3.5 l-TMA 7.2 d-Tma 4.0 dl-Pseudo TMA 4.0 l-INPEA 6.5 6.5 6.5 d-INPEA 4.22 dl-H56/28 6.7 8.5 8.8 75 Effects of Adrenergic Blocking Agents on the Nervous System

Both alpha and beta-blocking agents are potentially capable of

influencing the functional physiology of the entire nervous system

from brain to nerve endings. these actions, for the most part, are

not related to adrenergic receptor blockade, and include a wide

spectrum of pharmacological activity. However, they constitute an

easily accessible means to explore the mechanism of action of many nonadrenergic drugs. For example, the finding that both alpha and beta

receptor blocking agents, except DCl, antagonize the

effect of lends support to the view that catecholamines

are involved in the mechanism of action of acetazolamide which is also

inhibited by amine-depleting agents like reserpine, alpha-methyltyrosine

and several benzoquinolizine derivatives (313). The failure of

adrenergic blocking agents, except dibenzyline, to antagonize the

anticonvulsant effects of diphenylhydantoin and chlordiazepoxide

supports the results of Rudzik and Mennear (314) that catecholamine

depletion is not involved in this case. Propranolol and pronethalol

are CNS depressants whereas INPEA is a CNS stimulant (315, 308).

Vaugham Williams (316) has emphasized the need to demonstrate whether

any apparent effect is truly on the CNS or secondary to some peripheral

action. As observed by Haley and McCormick (317) and Feldberg (318),

catecholamines produce soporific or anaesthetic effect in cats, dogs

and mice following injection into the cerebral ventricles, and as

shown by Dell (319), marked cortical arousal and facilitation of

postural reflexes follow the intravenous injection and the endogenous

release of epinephrine. This dual behavior is best explained by 76 considering that the total effect of a drug is the algebraic sum of possible negative and positive influences at different sites (316).

A study of the effects of beta receptor blocking agents will signify the tmportance of these suggestions.

The activity of beta-blocking agents at sympathetic nerve terminals is one of the factors that determines the extent of intrinsic sympathomtmetic activity, antiarrhythmic activity, and the suitability for experimental and clinical use. There is no necessary correlation between activity at nerve endings and activity at the beta receptor.

Both of these effects may occur in the same dose range as is the case with pronethalol, or they may be sufficiently separated by dose as in

MJ 1999 and MJ 1998 (320). In anyone tissue, the may have one or more of three separate actions: It may (a) occupy a receptor, (b) prevent the uptake of norepinephrine, and (c) lower the norepinephrine content either by prevention of uptake or by interfering with the binding of norepinephrine (299). Thus, pronethalol inhibits accumulation or uptake or norepinephrine into the heart following norepinephrine infusion in the anaesthetized rat. Doses larger than necessary for beta receptor blockade are required to produce a decrease in endogenous levels of norepinephrine (299). In the rat heart propranolol has no such action (299). Verapami1 produces a very weak inhibition of norepinephrine, but does produce a significant decrease in the amine content of rat heart after daily injections for several days (299).

However, propranolol induced a transient vasodilation followed by a sustained vasoconstriction in the denervated auto-perfused hind ltmbs of the dogs. The alpha-receptor blocking agents phentolamine and 77 phenoxybenzamine abolished the pressor response to propranolol. This

pressor response is not obtained in spinal dogs, or in dogs whose

adrenal glands were excluded from the circulation. These results

suggest that the pressor response to propranolol is due to the reflex

release of catecholamine from the adrenal medulla or that a direct CNS

effect of propranolol induces catecholamine release (321). Reflex mechanism, however, seems less significant inview of the fact that

pronethalol is taken up strongly by central nervous tissue (233) and

affects spinal reflexes (316, 322, 323).

DCI and phenoxybenzamine afford an example of tissue selectivity

in their ability to prevent norepinephrine uptake. In the cat, phenoxy­

benzamine prevented uptake of norepinephrine by kidney but not by the

uterus; DCI prevented the uptake of norepinephrine by the uterus but not

by the kidney. In the rat, phenoxybenzamine prevented the uptake of

norepinephrine by the heart, spleen, and uterus, and reduced the uptake

by the duodenum. DCI and phenoxybenzamine lowered the content of

norepinephrine of some tissues of the rat. Farrant et al. (324),

therefore, concluded that the way in which these adrenergic antagonists

affect the storage sites for norepinephrine varies between organs, and

that activity at the receptors bears no apparent relationship with the

activity at the storage site. In another study, Dhalla (325) has

provided evidence that DCI can produce sympathomimetic effects by

directly stimuhting the beta receptor, or indirectly, by releasing

norepinephrine. Pretreatment of rats with reserpine abolished the

phosphorylase activation by DCI in the atria but not in the diaphragm

indicating that both direct and indirect mechanisms were involved. 78

On the electrically driven rat atria, the positive inotropic effect of

DCI and nicotine, but not of or norepinephrine, was inhibited by , , iproniazid, and hexamethonium.

The effects of DCI, tyramine and nicotine were blocked by both propranolol and , whereas the action of norepinephrine was potentiated by cocaine and antagonized by propranolol. On the spontaneously beating rat atria, pentolinium, hemicholinium, phenoxybenzamine and methylxylocholine ether inhibited the positive inotropic and chronotropic actions of DCI and nicotine but did not alter the effects of tyramine and norepinephrine, MJ 1999 antagonized the cardio-stimulant effects of DCI, tyramine, nicotine and norepinephrine whereas atropine and piperoxan were ineffective. When the atria were made tachyphylactic to tyramine; DCI and nicotine, unlike norepinephrine, failed to produce a sympathomimetic action.

Dhalla concluded that unlike tyramine, DCI and nicotine release norepinephrine by depolarization of the sympathetic nerve terminals and the amine then produces its usual positive chronotropic and inotropic effects. It is interesting to note that beta receptor blocking agents not only can affect release of norepinephrine from the nerve endings but also can displace alpha-receptor blocking agents to restore the catecholamine responses. In dogs and in the isolated rabbit aortic strip, propranolol interacts with a previously blocked alpha receptor, in some undefined way, to displace phenoxybenzamine, phentolamine and tolazoline, restoring the normal pressor response to norepinephrine (326). This property is also shared by methoxamine and (327). 79 Antiarrhythmic Properties

Experience has shown that beta-adrenergic blocking agents suppress certain types of arrhythmias (328-333), but this action can not be attributed entirely to beta receptor blockade (238, 305, 334-336).

The extent of the antiarrhythmic action of beta-blocking agents depends on the nature of the agent that produced the arrhythmia, the nature of the beta-blocking agent, the electrical characteristics of the cardiac muscle, and the metabolic state of the cell. The last two factors have been very ably discussed by Trautwein (337). The ability of beta receptor blocking agents to prevent experimental catecholamine­ induced cardiac arrhythmias has been ascribed to specific and competitive beta receptor blockade (338-342). However, the concept of nonspecific antiarrhythmic action arose when it was shown that DCI could reverse ouabain-induced arrhythmias in experimental animals

(334) and that both laevo and dextro isomers were capable of reversing digitalis-induced arrhythmias (343, 344) although the laevo isomer was forty times more potent in its beta blocking action than the dextro isomer (304).

How this nonspecific antiarrhythmic effect manifests itself depends on several factors that are affected to varying degrees by beta receptor blocking agents. These include (a) a direct effect on the rate of the pacemaker potential which is more marked in the heterotropic pacemakers than in the sinus node, (b) a decrease in diastolic excitability, (c) prolongation of the absolute and relative refractory period with a decrease in vulnerability of the heart to stimuli during these periods, and (d) a reduction in conduction velocity 80 in all cardiac fibers (337). Quinidine and procainamide exhibit their antiarrhythmic properties by reducing the increase of Na+ conductance brought about by depolarization, altering the time constant of this effect and by raising the threshold for excitation (337). Hoffman and Singer (345) have demonstrated that pronethalol also affects the cardiac electrical activity probably by a direct action on the cardiac cell membrane. These observations are in agreement with the general view that the antiarrhythmic properties of beta-blocking agents are due to their local anaesthetic action (346-348). Standaert and Roberts (320) determined the neurotoxic effects of pronethalol on the soleus motor nerve terminal preparation and suggested that the capacity of pronethalol to prevent the digitalis-induced ventricular arrhythmia may be related to its nerve terminal depressant action.

Supporting evidence for this view has been provided by many workers.

It consists of the ability of pronethalol to affect equally the adrenergic nerve endings and injected catecholamine (241), the weaker activity of MJ 1999 and MJ 1998 than pronethalol on motor nerve terminals as well as on adrenergic nerves (279), and the greater efficacy of beta­

TM10, procaine and diphenylhydantoin against digitalis-induced ventricular arrhythmias due to their strong depressant action at motor nerve terminals (349-350). MJ 1998 has no antiarrhythmic activity since it produces a very slight depression of the nerve terminal (279).

Pronethalol, propranolol and quinidine equally reduced the maximal rate at which the guinea pig isolated atrium followed an electrical stimulus.

They were equal in their effect in increasing toxic dose of ouabain in guinea pig (351). H 56/58 (293), INPEA (238) and MJ 1999 (347) do not 81 suppress ouabain-induced cardiac arrhythmias or arrhythmias due to coronary artery ligation. As expected, pronethalol is also without effect on arrhythmias due to coronary artery ligation (238). A recent report describes the effectiveness of propranolol in antago­ nizing Ba-induced cardiac arrhythmias in rats, dogs and rabbits (352). Barium causes KT loss from the mycodardium and propranolol app~ars to antagonize this effect through beta receptor blockade and a quinidine­ like action; also Ba-to1erance is increased by atropine whereas reserpine is ineffective. It is not yet certain whether propranolol together with MgS04 and ~ could be used effectively in Ba-induced arrhythmias. Following reversion of ouabain-induced ventricular tachycardia by dextro H 56/28 or propranolol, there was a consistent slowing of the heart rate below the initial value. H 56/28 did not produce a pronounced bradycardia because of its slight intrinsic sympathom~etic activity in contrast to the sympathetic blocking action of propranolol (306).

Bowman and Raper (353) demonstrated the presence of beta receptors in skeletal muscle fibers, and the presence of alpha receptors in the motor nerve endings. Raper and Jowett (354) investigated the antiadrenergic and antifibrillary activities of beta receptor blocking drugs on isolated rabbit suricles and spontaneously fibrillating chronically denervated skeletal muscle. They observed that (a) despite the differences in the species, the tissues and the method of assessment used, there was sufficient similarity in the orders of potency of the range of drugs used to suggest that antifibrillary action in denervated muscle might be the result of the same effect as 82 that responsible for prolongation of the effective refractory period in muscles, (b) local anaesthetics procaine and lignocaine produce similar effects indicating that membrane stabilization may be the basic property underlying both effects. Consistent with this hypothesis is the fact that MJ 1999 which is devoid of local anaesthetic activity

(277) is also without antifibri1lary activity in denervated muscle and, as compared to procainamide, is the least effective quinidine-like drug on cardiac muscle. Another important aspect of their study is the similarity between the action of methoxamine, IMA and IMA that of quinidine, procainamide, procaine and lignocaine on cardiac muscle in that antiadrenergic actions occurred only in doses which also produced some quinidine-like effect. In denervated muscle, for a given degree of antiadrenergic effect, the decrease in background fibrillation was less than that produced by quinidine and the local anaesthetics. Furthermore, antiadrenergic activity outlasted the depressant action on fibrillation.

Denervated muscle therefore provides a useful test preparation for distinguishing antiadrenergic activity from quinidine-like activity

(354).

In contrast to its action in immobilized laboratory animals, propranolol in a dosage of 2.5 to 5 mg/kg intravenous had no effect on the telemetrically recorded heart rate of unrestrained normal dogs.

!he difference is ascribed to emotional reactions in the restrained animals. !he tachycardia occurring in the latter seems to be due chiefly to a decrease in parasympathetic tone (355). Propranolol,

KO-592 and INPEA showed no correlation between the decrease in heart rate and cardiac output on intravenous injection. INPEA in doses of 83 50 mg had no blocking activity whereas propranolol and KO-592 had steeper dose-responses curves. INPEA antagonized the positive chronotropic effects of isoproterenol and its optical isomers were not different in activity (356). (H 56/28) blocked both positive chronotropic and inotropic effects of isoproterenol and electrical stimulation of cardiac sympathetic nerve in doses equal to that of propranolol (293). In reserpinized cats alprenolol caused a moderate increase in cardiac rate and contractile force that could be inhibited by propranolol. Higher doses of both the levo and the dextro isomers of alprenolol, like propranolol, are equally effective in producing direct cardiac depression (293). In human studies, alprenolol and propranolol were equipotent blockers of the cardiovascular effects of isoproterenol, but they influenced the basal hemodynamics differently

(357). Cardiac output decreased after propranolol (22 T 4.2 per cent) but not after H 56/28. Mean arterial blood pressure was not significantly altered by either agent. On the basis of results from animal studies with the two compounds, Forsberg and Johnson (357) concluded that propranolol reduced cardiac output by inhibiting endogenous sympathetic tone on the cardiac beta receptors and H 56/28 inhibited endogenous sympathetic tone on the heart to the same degree as propranolol, but the hemodynamic consequences of this action of

H 56/28 were overcome by a cardiac stimulation due to the slight

"intrinsic" beta receptor stimulating action of this drug. The potency and time-effect relationships of intravenously administered racemic

H 56/28, the levo from H 56/28, and racemic propranolol were studied in man (358) and in the cat (359) by recording heart rate and blood 84 pressure responses to repeated intravenous infusions of isoproterenol.

The ratios of equipotent intravenous doses were found to be about 2:1:2.

All agents had the same time effect relationship and the effect was maximal within 10-15 minutes after administration and still persisted at the end of 2 hours.

The ratio between the smallest (negative) inotropic dose and the smallest beta-adrenolytic dose were determined on the dog heart-lung preparation for propranolol, KO-592," and (360). They were 6.1 for propranolol, 4.9 for KO-592," 6.5 for prenylamine, and

1.3 for Verapamil. This ratio for Verapamil is too small to show a specific beta receptor blocking effect. Unlike propranolol and KO-592," prenylamine had positive inotropic and positive chronotropic effects in a smaller dose range. Even in marked negative inotropic doses, these drugs do not affect the spontaneous frequency of the heart, but a distinct depressant effect readily occurs in reserpine treated isolated hearts. The negative inotropic effect of beta-blockers has been found to depend on extracellular ea++ concentration. At low Ca++ concentration, this effect is decreased to a larger extent than at higher concentration of Ca++. In isolated guinea pig artria, the action of quinidine and the beta receptor blocking effect was the same at all concentration of ea++

(361). Beta-blockade can prevent the usual positive inotropic and chronotropic effects of nicotine that result in hypertension, increased cardiac output, and stroke volume as well as the indirect beta-dilator effect on peripheral vessels. In the presence of beta-blocking drugs, nicotine fails to cause an increase in heart rate indicating that the unopposed alpha-receptor activation by the norepinephrine released from 85 the peripheral stores can result in more uniform blood vessel constriction with sufficient increase in total peripheral resistance to impair stroke volume and cardiac output (362). An example of physiological antagonism on the blood vessels is the isoproterenol vasomotor reversal by sympathomimetic amines in anaesthetized cats and dogs (363, 364). Here, phenylephrine reverses the depressor response to isoproterenol by producing vasoconstriction rather than producing beta receptor blockade (30).

METABOLIC EFFECIS OF ADRENERGIC BLOCKING AGENTS

In recent years, many attempts have been made to classify metabolic actions of catecholamine into those exerted via alpha- or beta-receptors.

It is becoming more and more imperative to treat metabolic effects of catecholamines and their inhibition by adrenergic receptor blocking drugs on separate grounds which may be related to but not soley based on the known actions of the catecholamines and their antagonist on adrenergic receptors (365). A particular metabolic response, for example the hyperglycemic effect of epinephrine, can be the resultant

of several different and independent actions of epinephrine, each of which may participate to a different extent in different species, or

under different conditions in the same species (366). In the absence

of complete understanding of each of these independent metabolic routes,

it is not predictable which of them is blocked by a blocking drug or whether all the different mechanisms are receptor-dependent. It is

also a highly improbable assumption that all the actions of

catecholamines and those of their antagonists are exerted only via 86

adrenergic receptors. Recently, ~-Hagen (366) has admirably dealt with the whole spectrum of metabolic effects of adrenergic drugs in a very comprehensive review.

Conversion of ATP into 3,5 cyclic AMP, Figure 2, under the

influence of adenylcyclase and catecholamine is the most important event

in the wide range metabolic action of the amine that manifests itself

as hyperglycemia, lacticacidemia, and hyperlipidemia (367). Whether

Furchgott's -receptor (15) is indeed adenylcyclase is yet to be

conclusively demonstrated although this suggestion has been made (368).

The inhibition of catecholamines by beta receptor blocking drugs can

cause considerable alterations of substrate concentrations in blood.

Changes of blood levels in glucose, lactate and pyruvate are considered

to be parameters of the metabolic effects of epinephrine. They are

exceeded if expressed in percentage as well as in significance by fat

mobilization and metabolization products. The lipolytic effects of

epinephrine and the inhibitory effects of beta-blockers on adipose

tissues are reflected by changes in the concentrations of plasma fatty

acids and glycerol (369). Propranolol in a dose of 20 ug/kg/min on

intravenous administration in the anaesthetized dog completely prevented

the positive chronotropic, hyperglycemic and plasma free fatty acid

(FFA) elevation produced by isoproterenol administered intravenously at

a rate of .02 ug/kg/min. A dose of 2 ug/kg/min of isoproterenol

surmounted the blocking effect, of propranolol showing that competitive

blockade was produced. Propranolol affects the FFA level more readily

than the glucose level (370). In anaesthetized rats, the calorigenic TRIGLYCERIDES i ) FFA + GLYCEROL

INAOTIVE LIPASE i ) ACTIVE LIPASE

ATP i CYCLIC - 3', 5' - AMP

ADENYLCYLASE i CATECHOLAMINE

PHOSPHORYLASE b ') PHOSPHORYLASE a (INACTIVE ) r (ACTIVE)

PHOSPHORYLASE KINASE

GLYCOGEN 1) GWCOSE-6- PHOSPHATE

FIGURE 2. CATECHOLAMrnE CATALYZED METABOLIC PATHWAYS

00 "-J 88 and the positive chronotropic properties of different doses of epinephrine, norepinephrine and isoproterenol were shown to be (371)

Isoproterenol:Epinephrine:Norepinephrine

1: 5 :8 identical metabolic activity

1: 4 :10 identical heart frequency

The order of potency of epinephrine and norepinephrine in this report is just the reverse of that on adipose tissue (372, 373). Not only catecholamines but also many aromatic amines related in structure to phenethylamine but lacking a catecholamine ring and a beta-OH group in the side chain have been shown to increase lipolysis in adipose tissue

(374, 375) as is true of many polypeptide , ACTH, TSH, glucagon and others (376-378). Lipid mobilizing action of ACTH is blocked by beta receptor blocking agents (379). Recently, Cepalik et a1. (390-392) have undertaken studies on the problem of lack of specificity of the agents affecting lipid mobilization. Interactions of propranolol, with norepinephrine and with ACTH were followed using the release of FFA from rat epididymal adipose tissue in vitro. Propranolol with ACTH produced a purely noncompetitive action (pD2 = 3.83) whereas antagonism to norepinephrine was competitive (pA2 = 5.48). It appears that ACTH does not affect the adrenergic receptor site in adipose tissue; its lipid mobilizing action is due to a different trigger mechanism (373,

380). The dose-response curves of norepinephrine and pheny1-t­ buty1noroxedrine (FtBuNOX) (LXXIV) for affecting the release of FFA from rat epididymal adipose tissue in vitro were studied per se and when interacting with propranolol, FtBuNOX starts to exert an autoinhibitory action prior to reaching its maximum possible effect. Both 89 sympathomimetics differed distinctly in the slope of their dose- response curves. In the case of FtBuNOX the slope corresponded well to

OH CH2 I J OJ -~ P'il, CH-CH2-NH-C-CH HOV I .2 "=I CH 3 LXXIV

the usual presumption of a bimolecular reaction (drug and receptor) and the markedly steeper slope of the norepinephrine curve was in good agreement with the presumption of a trimolecular (2-receptors) reaction

(382). The relative lipid mobilizing potencies of isoproterenol, norepinephrine and epinephrine (2:1:06) on rat epididymal adipose tissue were almost identical with those obtained on FFA release in blood plasma

(0.5: 33: .2) in vivo and in vitro in the same animal (381, 383). Cepalik et ale (380) proposed that adrenergic lipo-mobilization characterizes a certain degree of beta-tropism, and this function could be considered for a specific adrenergic reaction. On the basis of hypothetical models of l-receptor and 2-receptor reactions, Wenke ~ ale (381, 384) showed that whereas FtBuNOX could produce lipid mobilization in a single step, catecholamines exert a 2-step reaction when affecting such mobilization.

This and the dynamic receptor theory of Bloom and Goldman (153) center around a mechanism that involves the catecholamine nucleus and the beta-OR group as the basic structural requirement for lipid mobilizing 90 activity. Unfortunately, the real picture can not be as sUnple as is being assumed. If differences in potency exist among structurally sUnilar catecholamines, why are structurally dissimilar antagonists, pronethalol, DHE, IMA and phenoxybenzamine almost equally effective in blocking the release of FFA in vitro (385) despite the simultaneous interplay of many other variables, e.g., penetration to receptor sites, difference in FFA passage across cell membrane and change in ionic environment of enzyme. Added to this list of vaguely understood phenomena is the fact that differences in activity of epinephrine may be detectable in different seasons; also the ACTH response is minimum in February, March, and April (386). A recent report by Eisenfeld ~ al.

(387) indicates that adrenergic blocking agents can inhibit the extraneuronal metabolism of norepinephrine by preventing its access to the metabolizing enzymes. Specific transport mechanisms exist for the entry of extracellular catecholaimine into sympathetic nerves. This finding lends support to the suggestion by Brooker ~ al. (385) that potency differences among catecholamines are due to their varying susceptibilities to COM! and MAO, and to the proposal by Ellis ~ al.

(388) that the alpha-receptor may also be involved in catecholamine­ induced increase in metabolic activity.

The situation becomes more complex when it is shown that the relative potencies of catecholamine in producing a particular response may vary from one tissue to another in one species, or from one species to another for one tissue (389), and when an analogous picture is presented by alpha- and beta-receptor blocking agents. For example, the ability to activate phosphorylase or to induce glycogen breakdown 91 in the rat liver decreases in the order epinephrine norepinephrine isoproterenol (390, 391) whereas a different order of potency has been shown for the same metabolic effect in the rat heart (390, 392), rat skeletal muscle (390) and dog liver (393). Similarly, the hyperglycemic response to epinephrine is inhibited by beta-adrenergic receptor blocking agents in the dog (394, 395); and this blocking action varies in the rat (390, 392, 397, 398). Moreover, alpha-blocking drugs do not affect epinephrine-induced hyperglycemia in the dog (394, 399) and man

(396), yet they produce variable effects on phosphorylase activation

(390, 392, 398, 400-1) and on glycolysis (402, 403). In direct opposition to their irregular mode of action on epinephrine-induced lipolysis and hepatic glycolysis, and the reported species differences, beta receptor blocking agents consistently block phosphorylase activation by epinephrine in heart and skeletal muscle whereas alpha­ receptor blocking drugs are without this effect (390, 391, 392, 401,

404). One aspect of the regulation of phosphorylase activity in the cell which has received little attention is the effect of ions on the enzymes of the cyclic AMP phosphorylase system (405). At least, a study of this kind would be needed to ensure that the difference in activity of alpha- and beta-receptor blocking agents in heart and skeletal muscle are dependent upon the nature of the receptors present, and are not due to some bioelectrical phenomenon. In the isolated, perfused guinea pig heart, the positive inotropic action and phosphorylase activity of isoproterenol are blocked by levo-IMA but are not affected by dl-IMA. However, the latter does transiently block the stimulation of contractility and phosphorylase activity of 92 norepinephrine. Neither dl-IMA nor l-IMA, however, reduces the phosphorylase activity to a greater extent than the mechanical actions.

On the other hand, KO-592 completely abolishes all the actions of both norepinephrine and isoproterenol (406). Not only is phosphorylase activation by norepinephrine in the heart blocked to varying degrees by optical isomers of blocking drugs, but the inhibition of norepinephrine­ induced lipolysis in isolated fat cells of the rat exhibits varying degrees of susceptibility to the blockade produced by 4-INPEA, 3-INPEA and 2-INPEA - these isomers differing only in the position occupied by the nitro group in the phenyl ring. 4-INPEA was approximately ten times as potent as 2- and 3-INPEA in antagonizing norepinephrine-induced lipolysis (407). During the course of discussion, it has been emphasized that the 1evo isomers of adrenergic as well as adrenergic blocking drugs possess the maximum activity on adrenergic receptors. A recent report by Bjorntorp et a1. (408) showed that the 1evo form of

H 56/28 in lower concentration (5x10-7M) inhibited norepinephrine­ induced increase in lipolysis in rat epididymal fat pad in vitro but in higher concentration (5x10-5M) had an intrinsic norepinephrine-like effect on lipolysis. The dextro form had no intrinsic activity and blocked the norepinephrine effect only in higher concentrations. In anaesthetized dogs, the 1evo form of H 56/28 partially blocked the increase of FFA in plasma by norepinephrine and itself increased FFA concentration. The dextro form, as usual, had only slight blocking activity. Isopropylmethoxamine has often been claimed to possess only the selective metabolic blocking property (236, 273, 409, 410) with 93 consequent doubts as to its being a typical beta-adrenergic blocking agent (30). Isopropylmethoxamine, however, has been shown to possess ability to block isoproterenol-induced relaxation of guinea pig tracheal chain to a moderate degree (pAZIMA = 5.97, pAZDCI = 7.7) (380), and to exert a weak blocking action on the positive inotropic effect of epinephrine on isolated rabbit and turtle hearts (411).

Any explanation, such as the one proposed by Northrop and Parks

(398) that beta-blockers prevent formation of and the alpha-blockers prevent the action of preformed cyclic AMP, might point out the right approach to delineate the difference between the mode of actions of alpha- and beta-blocking agents on the metabolic actions of catecholamines.

The differences among catecholamines and the differences among organs, and those among species in their response to a single catecholamine may lead to the supposition that many types of receptor for catecholamine might exist; these several receptors might simply be different adenylcylases (isoenzymes), and the mixture of adenylcylases present in a tissue would determine the response to a given catecholamine

(366) •

B. STATEMENT OF THE PROBLEM

1. Summary of Background

Over the years attempts have been made to devise general

hypotheses pertaining to structure-activity relationship and

the mechanism of action of adrenergic drugs. Recent efforts

include variations of receptor occupancy hypotheses, rate or 94 kinetic hypotheses, and more chemically descriptive concepts.

In reviewing publications of the past several years that could be considered pertinent to structure-activity relationships, one is impressed by the inconsistency that exists between most synthetic new molecules and the general hypotheses developed.

This lack of agreement might indicate that these hypotheses are sterile or inadequate for either predictive or correlative purposes, and can not serve as vehicles for correlating structure­ activity relationships in adrenergic drugs. MOst of these hypotheses have indeed offered little that is of substantive value from a chemical perspective of devising novel molecules of biological interest. As hypotheses become more chemically descriptive, one can anticipate that there will be a greater consideration of such hypotheses by the designers of new biologically interesting molecules.

Probably the most complex description of a drug receptor to have been constructed to date is that of the adrenergic receptor.

Attempts to design adrenergic antagonists using classical concepts of isosterism on the phenylethylamine nucleus of the major adrenergic amines have been signally unsuccessful and have produced no clues concerning the nature of the adrenergic receptors. In principle, isosterism consists of the substitution in a compound of one atom or group of atoms with another which has a similar electronic and steric configuration in order to produce a second compound which may have similar or antagonistic properties. Drugs synthesized on phenylethylamine pattern show 95 a great deal of variability in their effects on adrenergic receptors. Some specifically act on alpha or beta adrenergic receptors whereas others affect both. The effects of these drugs on adrenergic receptors mayor may not be the same qualitatively, and a compound may stimulate one type of receptor while simultaneously blocking the other. Observations of this kind resulted in the generalization that the pheny1­ ethy1amine nucleus was essential for activity at adrenergic receptors (12); the terminal amino group being responsible for alpha adrenergic activity (8, 12, 90), and the isopropy1amino, catechol ring and the hydroxyl group on the beta carbon atom in the side chain were thought to be absolutely essential for beta receptor activity (8, 12, 90). Bulky substituents on the amino nitrogen atoms were anticipated to produce alpha receptor blockade whereas replacement of the catechol hydroxyl groups by other groups, e.g., chlorine, was regarded necessary for beta adrenergic receptor blocking property (90).

Obviously the notion that there is a clear-cut structural relationship between the beta receptor stimulating drug, isoproterenol, and the various beta receptor blocking agents was considered as an established fact serving as the guiding principle for the syntheses and the discussions on the structure­ activity relationships of drugs acting on beta adrenergic receptors. Despite these suggestions, the potent and the better known alpha receptor blocking agents do not conform to phenylethylamine pattern and catecho1amines with specific and 96 more powerful action than the natural amines could not yet be synthesized. Similarly, an ideal beta receptor blocking drug has not yet been found and no compound has ever improved on isoproterenol.

There is a distinct class of the sympathomimetic amines that produce adrenergic effects by releasing norepinephrine from the storage sites. They are structurally related to epinephrine and norepinephrine in having the same phenylethyl­ amine basic structure. The structural similarity was in itself a temptation to investigate if these drugs had any other adrenergic receptor activity. It was reasonably expected that some of these compounds would have a certain affinity for adrenergic receptors that would be manifest as stimulating or inhibitory action on the receptors. During the preliminary phase of this study, an utterly unprecedented observation was made that an alicyclic sympathomimetic amine, cyclopentamine, could effectively antagonize the beta receptor agonist, isoproterenol, on the isolated rabbit intestine and the uterus.

This finding instigated a more detailed appraisal of this new structural aspect for adrenergic activity by way of comparison with various sympathomimetic drugs selected from different classes of compounds including phenylalkylamines, phenyl­ alkanolamines, aliphatic alkylamines, heterocyclic amines and the congeners of the classical beta receptor blocking agent, dichloroisoproterenol. 97 2. Objectives of the Studies

This study was aimed at determining specifically

(a) whether sympathomimetic drugs that act by releasing

norepinephrine could also directly act on alpha or

beta adrenergic receptors producing stimulation or

blockade;

(b) whether there was a relationship between the structure

and the adrenergic receptor activity of these

indirectly acting drugs;

(c) what was the nature of cyclopentamine-induced beta

receptor blockade, and in what way it was related

to the classical beta receptor blocking agent,

dichloroisoproterenol;

(d) whether the congeners of dichloroisoproterenol that

are supposed to be beta receptor blocking agents

show any activity besides affecting beta adrenergic

receptors; and

(e) whether the data on cyclopentamine reflects the

inadequacy of the current hypotheses on the

structure-activity relationships in adrenergic drugs. CHAPTER. II

METHODS AND MATERIALS

Introduction

The experiments were based on the actions of drugs on pieces of tissues which had been taken from freshly killed animals and were kept alive in physiological salt solution. Delineation of the types of adrenergic receptors involved in the drug response was made on the basis of the qualitative action of drugs and the use of specific adrenergic blocking agents.

Selection of tissue preparations

For assay purposes, the isolated intestine and the uterus of the rabbit were chosen since they offered some practical advantages over other preparations. These preparations continued to give uniform responses for many hours if kept in Locke-Ringer's solution. Since both tissues were isolatee from the same animal, they could be more effectively employed to observe the differences in both the qualitative and quantitative responses of a given drug on the two types of tissues.

Besides, a simultaneous and a more direct assessment of tissue differences could be made under identical experimental conditions.

Both alpha and the beta adrenergic receptors have been demonstrated in the ileum of the rabbit (64) and the rabbit uterus (55), and it was thus feasible to investigate whether a drug stimulated one or both types of receptors, stimulated one and blocked the other, or blocked both. Since alpha receptor stimulation in the intestine produced an inhibitory response and in the uterus, an excitatory response, and since 99 both these qualitatively different effects were quantitatively antagonized by the same alpha receptor blocking agent, phentolamine,

it was possible to demonstrate clearly the effects of drugs affecting

alpha adrenergic receptors, and the possibility of a nonspecific musculotropic action could be readily eliminated.

The isolated ileal preparation. The isolated rabbit intestine reacted

differently to stimulation or blockade of the alpha or beta adrenergic receptors and served as a differentiation test object to ascertain the

type of adrenergic receptor involved in the response obtained.

Phenylephrine primarily activates alpha receptors. The intestinal

inhibitory response to phenylephrine is unaffected by the beta receptor

antagonist, dichloroisoproterenol, but is completely blocked by the

alpha receptor blocking agent, phentolamine. Therefore, by definition,

the intestinal inhibitory response to phenylephrine must involve

activation of alpha receptors. Isoproterenol activates primarily beta

adrenergic receptors. The intestinal inhibitory response to

isoproterenol is unaffected by the specific alpha adrenergic receptor

blocking agent, phentolamine, but is completely blocked by the beta

receptor blocking agent, dichloroisoproterenol. Therefore, the

intestinal inhibitory response to isoproterenol must involve activation

of beta receptors. Epinephrine activates both types of receptors, and

the intestinal inhibitory response to epinephrine is blocked by a

mixture of the alpha and beta adrenergic receptor blocking agents,

phentolamine and dichloroisoproterenol.

The isolated uterine preparation. Uteri of many species have been used

under various experimental conditions for testing activity at adrenergic 100 receptors. Species differences are a paramount factor in pharmacolo­

gical work on the uterus. The other important conditions that deserve

careful consideration are (i) hormonal state of the animal used, (ii)

ionic composition of the physiological solution, and (iii) the type of

response to be measured since it is not practicable to use one and the

same kind of uterine preparation to measure accurately the activity of all uterotropic drugs (412).

Considerable attention has been directed to the classification of mammalian uteri on the basis of the qualitative actions of epinephrine

and norepinephrine. The effects of these agents on the tone or

spontaneous contractions of the uterus have been broadly considered as

inhibitory, stimulatory, or biphasic in which stimulation is followed by inhibition. In contrast to many other types of smooth muscles,

these qualitative actions can vary with the species, with the hormonal

state, or type of the uterine preparation employed for study.

The mechanisms underlying these differences are poorly understood.

Two types of adrenergic receptors have been postulated to be

present in the uterus, one for uterine stimulation and one for

inhibition (55). Uterine stimulation is associated with the interaction

of epinephrine, norepinephrine and phenylephrine with alpha receptors while, uterine inhibition occurs when epinephrine and isoproterenol

combine with beta receptors. It may be necessary to block alpha

receptors with phentolamine in order to unmask the beta receptor

stimulatory effect of epinephrine. The effect which is observed depends

on the extent to which one or the other of these receptors is activated

and predominates. 101

During the last several years, the rat uterus has often been used in in vitro preparation (52), but the suitability of this preparation for inhibitory actions on beta receptors has not been unequivocally established. It has not yet been established whether this preparation has true beta adrenergic receptors (413), although no suggestion has been made (56). The isolated rabbit uterus has proved to be a better preparation for studying inhibitory responses of drugs on the uterus.

To measure inhibitory effects, i.e., the decrease of uterine activity, an evaluation based on isotonic responses of the isolated rabbit uterus is suitable since a lowering of the basal tone is not an important component of the effect and the uterine contractions are of a regular shape.

This preparation is very sensitive to the influence of estrogen and progesterone, and the responsiveness of this tissue to stimuli can be enhanced by prior hormonal treatment. The estrogen treated uterus reacts to stimuli of equal intensity with a steadily increasing tension development, whereas the progesterone treated uterus reacts with a series of contractions the height of which is continuously decreasing

(414). Thus a positive correlation between the dose and the response is seen in estrogen dominance, whereas negative correlation between dose and response can be seen in progesterone dominance. An appropriate hormonal treatment of the rabbit should be expected to impart optimum sensitivity to the tissue (53).

Experimental details Female rabbits, 3 to 4 kg in weight, were primed by giving intra­ peritoneal injections of a 5 p g dose of estradiol daily for three 102 days followed by 1 mg doses of progesterone for three days. Two days

of rest were allowed before killing the animal. Segments of ileum were

obtained from the last ten inch portion of the small intestine. The

lumen of the intestine was flushed several times with Locke-Ringer's

solution. One end of an ileal segment, about 2 to 3 cms in length, was tied to an anchoring glass rod and immersed in a 40 ml bath

containing Locke-Ringer's solution which was aerated with 95 per cent

oxygen and 5 per cent carbon dioxide and maintained at 37.50 C

thermostatically. The other end of the segment was connected by means

of a thread to a lever for recording of isotonic longitudinal muscle

activity. The recordings were obtained by means of an ink writing

lever and a kymograph drum. The preparation was allowed to

equilibrate for 15 minutes. The weight on the writing lever was adjusted

so as to exert a minimal tension on the isolated intestine, thereby

allowing the preparation to develop a certain degree of tone in order

to get significant responses to the drugs applied. The doses were

administered into the bath in a volume of 0.5 to 1 mI. All drugs were

added in molar doses, and all solutions were freshly prepared in Locke­

Ringer's solution. Drugs not soluble in Locke-Ringer's solution were

dissolved in distilled water.

Initially, each drug was tested for its individual effects on

tissues that were not treated with any other drug. If a drug showed an

appreciable effect, a dose that gave a maximum response was determined.

The qualitative nature of the drug effect then was further analyzed

using various antagonists. All drugs were examined for adrenergic

blocking activity in doses that did not affect tissue activity. In 103 cases where the tissue activity was seriously affected by the non­

specific actions of drugs, a spasmogen ( 12 ~ g per liter) was added to the bath fluid to maintain tissue activity near normal as far as possible. The antagonist was allowed to act for one minute before injection of the agonist dose which was then allowed to act for 30

seconds. It was necessary to wash out previous doses of all drugs completely and to obtain a maximum response to the agonist each time before testing a new dose. This step eliminated any cumulative drug effect and indicated the state of tissue sensitivity at regular intervals. The sensitivity of the tissue usually returned to normal after 5 to 10 minutes, but a tissue that showed a deteriorated sensitivity to the drugs was discarded. Even after repeated washings

if the tissue did not produce the same or nearly the same response to a given dose of a drug applied before and after washing then the drug was considered to be "fixed" on the tissue.

Uterine strips were set up in a manner similar to that described

for ileal strips except that the time required to equilibrate the

preparation was longer. Spontaneous activity of the uterine preparation was induced by repeated treatment with phenylephrine and washing.

Phenylephrine treatment decreased equilibration time, and once the

tissue had developed its full activity, no further treatment was necessary.

Determination of alpha receptor stimulating property. When alpha

adrenergic receptor stimulants were given to intestinal preparations,

there was a reduction in the amplitude of rhythmic contraction and in

the tone of the preparation. The inhibitory responses of different 104 doses of a were measured in terms of the percentage reduction in the tone and amplitude of the thytbmic contraction in the presence of a standard dose of phentolamine. The use of phentolamine served two purposes, it showed that a test drug was, in fact, acting at alpha adrenergic receptors and the results were standardized since they were based on the ability of the test drug to antagonize phentolamine rather than on a more variable tissue response. This is discussed in more detail in Chapter III.

On the uterus, alpha receptor stimulants produced contractions with an increase in the amplitude and the tone of the contractile tissue. The percentage increase in the amplitude and the tone of the spontaneously contracting uterus against a standard dose of phentol­ amine was the measure of the alpha receptor stimulating effect of a given dose of the agonist.

Phentolamine in a constant dose of 7x10-3 p Mwas used as a specific alpha receptor blocking agent and phenylephrine in a dose range of 2.4x10-2 to 6x10-2 p Mwas used as a reference for potency comparison of alpha receptor stimulants.

Determination of alpha receptor blocking property. In order to evaluate the alpha receptor blocking property of an antagonist different doses of the antagonist were tested against standard doses of phenylephrine, norepinephrine and epinephrine. The percentage rlecrease in the inhibitory responses of the agonists on the ileum and the decrease in the excitatory responses of the agonists on the uterus was the measure of the alpha receptor blocking property of the given dose of the antagonist. 2 3 Phenylephrine 2.4xlO- )J. M, norepinephrine 4.7x10- }J M and epinephrine 105

4.5xlO-3 p Mwere used as standard alpha adrenergic receptor stimulants, and phentolamine in a dose range 3xlO-3 to 1.5xlO-2 p Mwas used as a reference for potency comparison.

Determination of beta receptor stimulating property. Stimulation of the beta receptors in the intestine and the uterus produced inhibition of the motility of the isolated tissues. In contrast to alpha receptor agonists, beta receptor agonists needed a slightly more prolonged time to cause complete inhibition of the muscle activity. The beta receptor stimulating activity on the ileum was determined against a standard dose of lxlO-2 p M of the specific beta receptor blocking agent, dichloroisoproterenol. Dichloroisoproterenol did not antagonize beta receptor agonists on the uterus because of its intrinsic activity and, therefore, p-methylsulfonamidoisoproterenol (MJ 1999) in a standard dose of 8.5xlO-2 ~ Mwas used on this preparation. Isoproterenol was used as a reference for potency comparison in doses of 8.lxlO-4 ~ M to

1.3xlO-2 p M. The percentage inhibition produced by different doses of the agonist in the presence of a standard dose of the beta receptor blocking agent determined the drug activity on the beta adrenergic receptors. The argument for using beta receptor antagonists in this study is the same as given for the use of phentolamine in the determi­ nation of alpha receptor stimulating activity (See Chapter III).

Determination of beta receptor blocking property. Since beta adrenergic stimulation in both the intestinal and the uterine preparation caused inhibitory responses, the ability of drugs to antagonize the inhibitory response of isoproterenol on these tissues was assessed. The standard dose of isoproterenol used on the intestine was 4.lxlO-3 p M and that on 106 the uterus was 1.lx10-3 p M. p-Methy1su1fonamidoisoprotereno1 in a dose range 3x10-2 to 1.2xlO-2 p Mwas used as reference for potency comparison.

Analysis of results

Biological responses to drugs are, as a rule, graded. They can be measured on a continuous scale, and there is a systematic relationship between the dose (or effective concentration) of drug and the magnitude

(or intensity) of the response it elicits. It is convenient to use the word "dose" in discussing the data, but since the volume of the bath is kept constant, this is essentially the same thing as the concentration of the drug in the bath. In terms of experimental results, isolated tissues afford an advantage over the intact animal since the tissue can be exposed to known concentration of drug for known times, and equilibrium conditions can be established by largely eliminating factors such as the distribution and fate of the drugs in the body and the contribution of many other tissues to the observed effect. These

complicating factors are of great significance once it is realized that the effects are likely to depend on the intervals between the times when the two drugs are applied and the time when the measurement

is made.

It is now generally agreed that an observed biologic effect is a reflection of the combination of the drug molecules with the receptors.

The dose-response relationship is based on the law of mass action and the response is considered proportional to the receptor occupancy (hence the necessity of using molar concentration) (415). If the effect is plotted

against log dose, it is generally possible to fit an approXimately 107 S-shaped curve to the results. From the practical standpoint, the logarithmic dosage scale is preferable since line segments rather than hyperbolic curves are obtained which are much easier to deal with in statistical analysis. Moreover, drugs that produce the same effect by the same mechanism but differ in potency yield parallel line segments, and the constant horizontal separation of the lines is a measure of the potency ratio for the two drugs (416). This is shown in Figure 3 which represents experimental log dose-response curves for norepine­ phrine and phenylephrine tested on the isolated rabbit ileum. It may be noted that the decrease in activity manifests itself as an increase of the dose necessary to obtain an effect, which implies a decrease in affinity. Another practical advantage of the logarithmic dosage scale is that a wide range of doses can be presented readily in a single graph. The log dose-response curve is symmetrical about the point at which 50 per cent of the maximum response is obtained, and its maximum slope and point of inflection occur at this point. The position of a log dose-response curve on the x-axis reflects the affinity of the drug for its receptors (243). Since by convention the scale of dosage increases from left to right, it follows that for a series of congeneric drugs interacting with the same receptors a set of parallel log dose­ response curves is expected. The curves for the most potent drug will be at the left, curves for drugs with poorer affinities for the receptor will lie further to the right. Just as the parallelism of these curves is regarded as consistent with an identical mechanism of action (i.e. interaction with the same receptor site), so in general would a lack of parallelism create the strong presumption that two drugs produce the z o -..... -al 100 r NE =NOREPINEPHRINE -:J: PA =PHENTOLAMINE -Z PE= PHENYLEPHRINE w 50 (!) ~ z w u 0' , c=:' ..... ,...,..... , a::: w -4 -3 -2 -I a. log [AGONIST] (JL M)

FIGURE 3. LOG DOSE-RESPONSE CURVES FOR NOREPINEPHRINE AND PHENYLEPHRINE AGAINST PHENTOLAMINE ON ISOLATED ILEUM OF RABBIT ot-' 00 109 same end effect by different mechanisms. Further evidence that the two combine with the same receptor would be provided if the same maximum response is obtained (243).

The analysis of antagonism in the framework of the log dose­ response curve is straightforward. In the presence of a competitive

antagonist, the curve for an agonist will be shifted to the right, but neither the slope nore the maximum response would be expected to change. The antagonist simply alters the effective affinity of the agonist drug for receptor. It is possible to reverse the blockade by antagonist by increasing the dose of the agonist showing that the blockade is surmountable and competitive (415). The effect of a non­ competitive drug upon the log dose-response curve is different. The agonist curve will again be shifted to the right, but the slope will be decreased and the maximum response will diminish in relation to the degree of noncompetitive blockade established (243). Noncompetitive blockade is not surmountable. Figure 4 represents log dose-response curves for phenylephrine tested on the isolated uterus of the rabbit in the presence of phentolamine and MJ 1999 respectively. It is apparent that phentolamine causes a parallel shift in the curves indicating that this compound acts as a competitive antagonist of phenylephrine.

MJ 1999 on the other hand, causes no parallel shift but produces depression in the maximal height of the curve, which indicates a non­ competitive antagonism between phenylephrine and dichloroisoprotereno1.

A series of such analyses constitute the basis on which the results obtained from the studies on isolated tissues can be interpreted in terms of potency, types and sites of action of adrenergic drugs. The z o -r- u

FIGURE 4. LOG DOSE-RESPONSE CURVES FOR PHENYLEPHRINE AGAINST PHENTOLAMINE AND I-' MJ 1999 ON ISOlATED UTERUS OF RABBIT I-'o 111 results are statistically analyzed using Litchfield-Wilcoxon method

(417). This method of statistical analysis provides many practical advantages over other more complicated methods which can be stated as follows:

(i) This is a graphical method and gives not only the EDSO and

the slope of the curve, but also their confidence limits

(P 0.05).

(ii) This method uses the data in original units throughout.

(iii) Zero and 100 per cent effects are used effectively.

(iv) This method recognizes heterogeneity when present and gives

corrected confidence limits in such cases.

(v) This method facilitates both the comparison of the two

curves for parallelism and the computation of relative

potency with its confidence limits. An example of this

statistical treatment is given in Appendix I.

Drugs and Solutions

Physiological salt solution. The isolated tissue preparations were examined for the intensity of their responses to drugs in different physiological salt solutions. The tissues were found to work best in

Locke-Ringer's solution that was used throughout. The composition of

Locke-Ringer's solution is as follows:

NaCl 9 ~

~l 0.42 ~

MgC12 0.2 ~

CaC12 0.24 ~ 112

NaHC03 0.5 gm

Dextrose 0.5 gm

The ingredients were dissolved in distilled water to make one

liter of the solution.

Drugs. The drugs that were investigated for adrenergic receptor activity were chosen from structurally distinct classes of compounds including aliphatic alkylamines, phenylalkylamines, phenylalkanolamines, congeners of dichloroisoproterenol and heterocyclic compounds. The doses of each of these compounds are mentioned against each.

Aliphatic alkylamines

Methylhexamine 1 to 5 )J. M

Isometheptene mucate 1 to 5 )J. M

Cyclopentamine hydrochloride 1 to 20.8 )J. M

Propylhexedrine 1 to 5 )J. M

Tuaminoheptane sulfate 1 to 10 )J. M

Phenylalkylamines

Hydroxyamphetamine 1 to 5 )J. M

Chlorophentermine hydrobromide 1 to 5 ~ M

Mephentermine sulfate 1 to 5 )J. M

Phenylpropylmethylamine hydrochloride 1 to 5 ~ M

dl-o-Methoxy-N,alpha-dimethylphenethylamine hydrochloride (U-0433)

2.3 to 21.6JlM o-Methoxy-N,alpha,alpha-trimethylphenethylamine hydrochloride (U-0588)

1 to 5 J1 M 113 N-Benzyl-O-methoxy-N,alpha-dimethylphenethylamine N-Oxide

hydrochloride (U-062l7) 1.5xlO-l to 6.2 p M

N-Benzyl-o-methoxy-N,alpha-dimethylphenethylamine hydrochloride (U-0277)

3.5xlO-2 to 7.9xlO-l p M

p-Methoxy-N,alpha-dimethylphenethylamine hydrochloride (U-089l) 1 to

5pM

N-Isopropyl-o-methoxy-alpha-methylphenethylamine hydrochloride (U-0287)

2.lxlO-l to 8.6 p M

Epinine hydrochloride 2xlO-2 to 3.5xlO-l p M

Phenylalkanolamines

l-Phenylephrine hydrochloride 2.4xlO-4 to 6xlO-2 p M

dl- tartrate 4.5xlO-l to 3.5 p M

dl-Ephedrine hydrochloride 1 to 5 p M

Phenylpropanolamine hydrochloride 7.5xlO-l to 11.3p M

Metaraminol bitartrate 3.5xlO-2 to 4.2xlO-l p M

dl-Cobefrine hydrochloride 1.lxlO-2 to 1.2xlO-l p M

Methoxamine hydrochloride 1.5xlO-2 to 10.8xlO-2 ~ M

Isopropylmethoxamine hydrochloride 0.25 to 2.5 u M

Nylidrine hydrochloride 1 to 5 p M

Congeners of dichloroisoproterenol

1-(3,4-dichlorophenyl)-2-homoveratrylaminoethanol hydrochloride

(Compound 21047) 9xlO-3 to l4.5xlO-2 p M

1-(3,4-dichlorophenyl)-2-tert-butylaminoethanol hydrochloride

(Compound 31060) 3.5xlO-l to 4.2 p M 114

1-(3,4-dich1oropheny1)-2-cyc1ohexy1aminoethano1 hydrochloride

(Compound 31075) 1 to 5 ~ M a1pha-1-(3,4-dich1oropheny1)-2-LI-(3,4-methy11enedioxypheny1)-2­

propy1amin£! ethanol hydrochloride (Compound 31470)

6.1x10-2 to 7.5x10-1 p M beta-1-(3,4-dich1oropheny1)-2-/I-(3,4-methy1enedioxy~heny1)-2­

propy1amin£! ethanol hydrochloride (Compound 31471)

1.2x10-1 to 2.4 p M

1-(3,4-dich1oropheny1)-2-(3-penty1amino)ethano1 hydrochloride

(Compound 31447) 1 to 5 p M

Heterocyclic compounds

Methylphenidate hydrochloride 1.2x10-1 to 9.5x10-1 p M

Tetrahydrozo1ine hydrochloride 1.3x10-2 to 9.8x10-2 p M

Xy1ometazo1ine 5x10-3 to 12.5x10-2 p M CHAPTER III

RESULTS, DISCUSSIONS AND CONCLUSIONS

Each class of the compounds studied in this project showed a specific trend in adrenergic receptor activity coincident with alterations in the molecule. This has necessitated a separate discussion of each class of compounds before attempting a comparative description of all the compounds producing the same qualitative response. Compounds belonging to alkylethylamine, phenylalkylamine, phenylalkanolamine and heterocyclic series were selected since they have a common basic structure similar to that of epinephrine, norepinephrine and isoproterenol, and act through a common mechanism involving norepinephrine release from the storage sites. It was anticipated that these compounds would have some activity on adrenergic receptors.

The basis for interpretation and integration of data

There are two ways to gain information about a receptor. The first and only really satisfactory approach is to identify and isolate it.

The second way of obtaining information about receptor is indirect. It has dominated pharmacologic research in the past. The approach is to draw inferences about a receptor from the biologic end results caused by drugs. Even in the relatively simple biological test systems, e.g. isolated tissues, it may not be readily feasible to ascribe the measured response to a particular receptor. The complexity of the structure of the tissue and the multiplicity of its physiologically active components are the complicating factors which make it imperative to employ other 116 criteria in order to differentiate between the specific and non-specific interactions of a drug with cellular components. Two of these criteria, namely the log dose-response curves and competitive antagonism, have found sound theoretical basis and have established their usefulness in determining the specificity of drug action (243, 415). The reliability of these tests has been verified several times in the past and enjoys universal acceptance (26, 243, 415).

In discussing the results that follow a drug will be designated as acting at alpha or beta adrenergic receptors if it demonstrates the properties described below.

Qualitative evaluation: Drugs that stimulate alpha or beta adrenergic receptors in the intestine produce inhibitory action (6, 418). All other drugs acting at cholinergic, histaminic and serotonin receptors produce contractions in this tissue and can be easily differentiated

(243). The inhibitory action of drugs that release norepinephrine may be a possible interfering mechanism but it has been shown that sympathomimetic amines do not release norepinephrine in the isolated rabbit intestine (418). A recent paper demonstrated that endogenous catecholamines are rapidly liberated and autolysed at temperatures above 20 0 C (419). Since in the isolated tissues a rapid turnover of the catecholamine is not feasible, sympathomimetic amines that act by releasing norepinephrine must produce a rapid tachyphlaxis. In fact,

sympathomimetic amines that fail to release norepinephrine have been

shown to behave as alpha receptor blocking agents (418). In the presence

of phentolamine these indirectly acting drugs will be soon rendered

ineffective since phentolamine specifically prevents the uptake of 117 endogenous norepinephrine (420). Thus slow synthesis of norepinephrine, rapid liberation, fast autolysis, and blocked uptake preclude any possible indirect contribution to the measured drug effect on adrenergic receptors. A part from tachyphlaxis and the alpha receptor blocking action of the indirectly acting sympathomimetic amines, the inhibition of the peristaltic movement of the rabbit intestine caused by these drugs should be slow in onset and of a lesser magnitude as compared to the rapid and complete action of the compound that directly act on the alpha adrenergic receptors (418). Additional evidence for the lack of an indirect action is provided by the quantitative evaluation of drug effects as explained below. The same arguments can be applied to the isolated rabbit uterus.

Quantitative evaluations: Using log dose-response curves the following evaluations can be made:

(a) The competitive relationship between an agonist and an

antagonist is shown by a parallelism in the log dose-response

curves as explained in Chapter II. If the slope ration (81/82) is less than the slope factor (fSR) then the two lines are

considered sufficiently parallel. These values are

statistically obtained as described in Appendix I.

(b) The affinity of a drug for its receptor is inversely related

to the dissociation constant for drug - receptor complex. The

affinity constant has the dimensions of (Concentration)-l, and

increases when the activity of a drug increases (415). Thus a

change in affinity manifests itself as a change in the

effective dose of a drug, the ED50' and results in a parallel 118 shift of the dose-response curve along the log-dose axis

(Figure 3 in Chapter II).

(c) The blockade produced by a competitive antagonist is

reversible i.e. an increased concentration of agonist can

break through the blockade.

(d) The ability of the agonist to produce the same max~um

possible response in the presence and in the absence of the

competitive antagonist (Figure 4 in Chapter II).

Alkyethylamine Series

Results: and cyclopentamine were the only two compounds that showed a weak alpha adrenergic receptor activity on the isolated rabbit uterus. Both the compounds were inactive on th~ isolated rabbit intestine in doses as high as 5 u M. Cyclopentamine had about half of the activity of tuaminoheptane on the uterus (Tables VI and VII).

Tuaminoheptane and cyclopentamine exerted no direct musculotropic action and no tissue fixation was observed. Methylhexaneamine, , and were completely inactive on both the tissue preparations in all doses studied.

Cyclopentamine, however, proved to be an outstanding compound for its ability to reversibly block the inhibitory responses of isoproterenol on the isolated ileum and on the uterus. This was a surprise observation since the cyclopentamine structure would not have been expected to be compatible with such an activity. The implications of this peculiar finding will be discussed along with the classical beta receptor blocking drug, dichloroisoproterenol, and its congeners. 3 TABLE VI. COMPARISON OF ALPHA-RECEPTOR STIMULATING PROPERTIES IN THE PRESENCE OF 7x10- J..l M OF PENTOr.AMINE

Ileum Uterus Agonist ED50 and Range* Slope and Range ED50 and Range Slope and Range ~ pM 1-Pheny1ephrine 0.024 2.26 0.036 2.41 (0.015-0.038) (1.14-3.17) (0.019-0.054) (1.91-3.42) 1-Norepinephrine 0.005 2.70 0.004 2.72 (0.002-0.006) (1.53-3.54) (0.003-0.005) (2.15-3.24) d1-Cobefrine 0.040 1.95 0.082 2.33 (0.022-0.065) (1.32-3.1) (0.03 -0.15) (1.62-3.45) Tetrahydrozoline 0.042 2.21 0.045 2.01 (0.023-0.061) (1.8 -3.9) (0.031-0.069) (1.30-4.50) Xy1ometazo1ine 0.05 1.83 No activity (0.025-0.082) (1.12-2.96)

Methoxamine 0.059 1. 75 0.098 2.40 (0.24- 0.097) (1.12-3.46) (0.058-0.20) (1. 73-3.01) 0.15 1.96 0.21 1.87 (0.08- 0.29) (1.31-2.78) (0.12-0.33) (1.13-2.76) Epinine 0.16 2.03 0.26 1.93 (0.09- 0.26) (1.45-3.01) (0.15-0.34) (1.39-3.4) 0.45 1.65 0.91 1.82 (0.22- 0.66) (0.98-3.25) (0.69-1.8) (1.01-4.60) Tuaminoheptane No activity 3.50 1. 74 (1.85-5.32) (1.21-3.67) d1-Synephrine 1.81 2.18 2.51 2.31 (1.22- 2.68) (1.09-3.20) (1.21-4.08) (1.62-3.77) 5.32 2.27 5;80 2.08 (3.2 - 7.6) (1.53-3.98) (2.93-7.98) (1.01-3.79) Cyc1opentamine** No activity 5.92 2.31 (3.69-7.37) (1.58-3.24)

t-' * 95 per cent confidence limit t-' \0 **EDSO and slope for beta receptor blocking activity given in Table XVII 120

TABLE VII. P(Y£ENCY COMPARISON OF ALPHA ADRENERGIC RECEPTOR STIMULANTS

Potency Comparison Agonist Ileum Uterus

1-Pheny1ephrine 1.0 1.0

1-Norephinephrine 5.2 8.5 d1-Cobefrine 0.6 0.45

Tetrahydroz0 line 0.57 0.8

Xy1ometazo1ine 0.48 No activity

Methoxamine 0.4 0.37

Metaraminol 0.16 0.17

Epinine 0.15 0.16

Methylphenidate 0.053 0.04

Tuaminoheptane No activity 0.015

d1-Synephrine 0.013 0.014

Phenylpropanolamine 0.005 0.006

Cyc1opentamine* No activity 0.006

* Potency for beta recep~or blocking activity given in Table XVIII. 121 Discussion: Table VIII summarizes structure-activity relationships in alkylethylamine series. None of these compounds had fewer than seven carbon atoms in the chain. Tuaminoheptane with an open chain structure and without a substituent group on the amino nitrogen was the most active member of this series. Shortening the alkyl chain by one carbon atom without decreasing the total number of carbon atoms as in methylhexaneamine abolished all of the alpha receptor activity. This corresponds with the observations of several authors (421-423) who showed that a weak pressor activity could only be found in long chain aliphatic compounds with seven and eight carbon atoms in the straight alkylamine chain. Isometheptene, however, had eight carbon atoms of which seven were in a straight chain with an ethylenic linkage between the fifth and the sixth carbon atoms and was found inactive. Cycliza­ tion of the chain did not improve on the open chain analog.

Cyclopentamine had the least alpha receptor stimulating property, whereas the cyclohexyl derivative was completely inactive. Cyclo­ pentamine, isometheptene, and propylhexedrine have an assymmetric carbon atom next to the amino nitrogen and exist in dextro and levo forms. But both the cyclic and the simple straight chain aliphatic series have been shown to possess only a moderate degree of stereospecificity (424). This is in contrast to aromatic derivatives of the phenylethylamine series, in which stereospecificity determines the magnitude of activity to a large extent (14). Thus the prerequisite for alpha adrenergic receptor stimulating activity in the alkylamine series are an amino group, and a saturated alkyl chain consisting of seven or eight carbon atoms. 122

TABLE VIII. STRUCTURE-ACIIVITY RELATIONSHIPS IN ALKYLETHYLAMINE SERIES

Structure Activity Drug

Tuaminoheptane H alpha-receptor stimulation

Methy1hexaneamine H inactive

Isometheptene inactive

Cyc1opentamine* cyc1openty1 alpha-receptor stimulation beta-receptor block

Propy1hexedrine cyc1ohexy1 inactive

*beta-receptor blocking property discussed along with congeners of dich1oroisoprotereno1. 123 Phenylalkylamine Series

Results: This series of compounds was characterized by a complete lack of affinity for beta adrenergic receptors. Intrinsic activity at alpha adrenergic receptors was detectable only with epinine, whereas U-0433,

U-062l7, U-0277 and U-0287 produced alpha receptor blockade. Hydro- xyamphetamine, , , phenylpropylmethylamine,

U-0588, and U-089l were inactive. The most active alpha receptor blocking agent, U-0277, was about 17 tUnes less active than the reference drug, phentolamine (Tables IX, X and XI). U-0277 was also different from the rest in having a direct musculotropic action on the rabbit uterus. The contractions produced by this drug in the isolated rabbit uterus were observed in all doses studied. This effect was not antagonized by phentolamine, and it was obvious that the contractions were not due to alpha receptor stUnulation. I Discussion: The influence on the adrenergic receptor activity of various substituent groups on the terminal amino nitrogen, on the alpha carbon atom next to the nitrogen, on the beta carbon atom in the side chain and in the ring varied with the nature and the size of the substituent groups (Table XII). Methylation of the alpha carbon atom with concommitant para hydroxylation in the phenyl ring as in hydroxyamphetamine, dUnethylation of alpha carbon atom as in chlorphentermine, and the addition of a methyl group on the amino nitrogen atom in chlorphentermine to get mephentermine produced inactive compounds. Introduction of a methoxyl group in the ortho position of the phenyl ring of mephentermine gave U-0588 but did not confer activity. Removing a methyl group at alpha carbon atom in TABLE IX. COMPARISON OF ALPHA RECEPTOR BLOCKING PROPERTY ON ISOLATED RABBIT ILEUM

Pheny1ephrine**- Norepinephrine*** Epinephrine**** Antagonist EDSO and range* Slope and EDSO and range Slope and EDSO and range Slope and pM range pM range pM range

Phentolamine 0.007 2.13 0.009 2.3 0.008 2.9 (0.004-0.013) (1.8-3.6) (0.006-0.014) (1.16-3.S) (0.007-0.019) (1.3-3.9)

U-0277 0.13 2.3S 0.138 2.65 0.21 2.9 (0.09-0.23) (1.2-4.2) (O. 08-0.23) (1.S-4.6) (0.12-0.38) (1. 3-S. 2)

U-06217 0.93 1.86 1.2 1.73 loS 1.93 (O. 67-1. 6) (0.6-3.9) (0.7-2.1) (1.1-3.6) (0.9-2.9) (0.9-3.7) U-0287 1.6 1.92 1.96 2.2 2.3 2.7 (0.85-2.5) (1.3-3.S) (1.1-3.7) (1.7-3.9) (1.3-3.S) (1.6-4.6)

U-0433 7.4 2.01 0.1 1.54 3.4 1.96 (S.2-9.6) (1. 7-3.9) (6.S-10.9) (1.2-3.7) (S.1-11.6) (0.9-4.2)

* 95 per ~ent confidence Itmit. ** 2.4x10- p M *** 4.7x10-3 ~ M **** 4.5x10-3 p M

t-' N ~ TABLE X. COMPARISON OF ALPHA ADRENERGIC RECEPTOR BLOCKING PROPERTY ON ISOLATED RABBIT UTERUS

Pheny1ephrine** Norepinephrine*** Epinephrine**** Antagonist EDSO and range* Slope and EDSO and range Slope and EDSO and range Slope and }JM range pM range }.lM range

Phentolamine 0.006 2.01 0.01 2.2 0.003 2.45 (0.004-0.01) (1.2-3.9) (0.007-0.019) (1.1-3.7) (0.005-0.013) (1. 3-4. 2) U-06217 0.73 2.32 0.81 1. 76 0.74 2.42 (0.56-1.2) (1.2-4.2) (0.52-1.3) (0.9-3.8) (0.63-1.2) (1.9-4.9) U-0287 1.91 1.96 2.4 1. 78 2.5 2.1 (1.1-3.2) (1.2-3.6) (1.8-3.7) (1.2-4.5) (1.9-3.4) (1.26-4.3) U-0433 7.3 1.89 7.6 2.06 7.8 1.9 (5.1-11. 2) (0.96-3.7) (4.3-10.1) (1. 3-4. 3) (5.9-10.3) (0.0-3.9)

U-0277 produces persistent contraction at all doses studied.

* 95 per ~ent confidence limit ** 2.4x10- }J M 3 *** 4.7xlO-3 }J M **** 4.Sx10- ~ M

I-' N VI TABLE XI. POTENCY COMPARISON OF ALPHA RECEPTOR BLOCKING AGENTS

Potency Comparison Antagonist Ileum Uterus PE* NE** EP*** PE NE EP

Phentolamine 1.0 1.0 1.0 1.0 1.0 1.0

U-0277 0.06 0.065 0.057 produces persistent contraction

U-0287 0.004 0.005 0.005 0.003 0.004 0.005

U-0433 0.0009 0.001 0.001 0.008 0.001 0.002

U-062l7 0.008 0.008 0.008 0.008 0.012 0.014 - * phenylephrine ** norepinephrine *** epinephrine

I-' N Cl\ TABLE XII. STRUcruRE-ACTIVITY RELATIONSHIPS IN PHENYLALKYLAMINE SERIES

RR4 R5 3 I I /R 7 R2 f' CH -C - N R,0-- I 'Re Rs

Structure Drug Rl R2 R3 R4 RS R6 R7 R8 Activity Hydroxyamphetamine H OH HH H CH3 HH inactive Chlorphentermine HH H H CH3 CH3 H H inactive Mephentermine HHHH CH3 CH3 H CH3 inactive Phenylpropylmethylamine HH H CH3 HHH CH3 inactive U-0433 H H OCH3 H H CH3 H CH3 alpha-receptor block H CH CH H CH inactive U-OS88 HH OCH3 3 3 3 U-06217* H H OCH3 HH CH3 CH2C6HS CH3 alpha-receptor block U-0277 H H OCH3 H H CH3 CHC6HS CH3 alpha-receptor block U-0891 H OCH3 HH H CH3 H CH3 inactive U-0287 H H OCH3 HH CH3 H CH(CH3)2 alpha-receptor block Epinine OH OH HH HHH CH3 alpha-receptor sttmu1tant

* N-oxide of U-0277

I-' .....N 128 U-0588 produced U-0433 with weak alpha receptor blocking activity.

Addition of a benzyl group at amino nitrogen in U-0433 gave rise to

U-0277 with enhanced alpha receptor blocking activity. This observa­ tion is in agreement with the previous reports (9) that bulky groups on the amino nitrogen atom increase alpha receptor blocking activity.

U-06217, an N-oxide of U-0277, exhibited a diminished alpha receptor blocking action. Such compounds have not been tested before. U-0287, the N-isopropy1 analog of U-0433, was more active than U-0433.

Despite the presence of an isopropyl group, U-0287 had no affinity for beta adrenergic receptors since the beta hydroxyl group and the catecholamine nucleus were missing in its structure. Moving the ortho methoxy1 group in U-0433 to the para position as in U-0891 resulted in complete loss of alpha receptor blocking activity. Simultaneous methylation at the amino nitrogen and at the beta carbon atom in the pheny1ethy1amine basic structure as in pheny1propylmethy1amine was not compatible with adrenergic receptor activity, but the elimination of the methyl group on the beta carbon atom and the introduction of two hydroxyl groups in the para and the meta positions in the ring produced the alpha receptor stimulant, epinine.

In general, dimethy1substitution on the alpha carbon atom results in the loss of alpha receptor blocking activity indicating that more complex stereochemical characteristics are involved in this case. It appears that the size and the number of substituent groups on the terminal nitrogen atom determine the degree of the alpha adrenergic receptor blocking activity if the alpha carbon atom next to the nitrogen is not fully substituted. The lack of intrinsic alpha receptor 129 activity in these compounds, endorses the view that substitution on the terminal nitrogen atom and the alpha carbon atom abolishes intrinsic activity on the alpha adrenergic receptors and a change from the stimulant to the blocking action occurs (90). Bulky substituents like benzyl confer enhanced alpha receptor blocking activity which is diminished by a simultaneous addition of an N-oxide linkage. No substituent group on the alpha or the beta carbon atom in the side chain is tolerated with regard to alpha receptor stimulating action, whereas the presence of a single methyl group on the alpha carbon atom with an unsubstituted beta carbon atom results in some affinity for alpha receptor. Substitution at the ortho and the meta position in the phenyl ring maintains affinity for alpha adrenergic receptors; para substitution, on the other hand, inactivates the compounds. The lack of affinity of these compounds for the beta adrenergic receptors was to be expected since the structural requirements for beta receptor activity (24) were not fulfilled.

Phenylalkanolamine Series

Results: Unlike the phenylalkylamine series where predominantly alpha receptor blocking activity was encountered, this series of compounds exhibited a high degree of intrinsic activity at alpha adrenergic receptors (Tables VI and VII). None of the compounds, however, proved to be an alpha , although no activity was traceable with 5 u M doses of ephedrine and nylidrin. Phenylephrine, synephrine, phenylpropanolamine, metaraminol, cobefrine and methoxamine were alpha receptor stimulants and were completely blocked by phentolamine. 130 Isopropylmethoxamine antagonized isoproterenol on the rabbit uterus but had no such activity on the isolated rabbit ileum. It was apparently devoid of affinity for alpha adrenergic receptor.

Discussion: Pheny1a1kano1amines are very closely related to epinephrine and norepinephrine chemically (Table XIII). They differ from pheny1­ a1kamines in having a hydroxyl group at the beta carbon atom next to the phenyl ring. This hydroxyl group is considered to be of paramount importance in facilitating the association of the drug molecule with its receptor. The carbon atom bearing the hydroxyl group is asymmetric and gives rise to optical isomers. The levo isomers are uniformly much more active than the dextroisomers (14).

The nature and the size of the substituent groups on the amino nitrogen was crucial for intrinsic activity at alpha adrenergic receptors. A group larger than methyl was not tolerated. Isopropyl group resulted in the affinity for beta receptor. The ability of alpha and beta receptors to differentiate sharply between chemical entities was clearly exemplified by methoxamine and its isopropyl analog.

Methoxamine with an unsubstituted amino group behaved as an alpha receptor activator whereas the presence of an isopropyl grouping on the amino nitrogen in isopropylmethoxamine was reflected in beta receptor blocking activity. A very distinctive feature was the complete loss of alpha receptor affinity on simultaneous addition of substituted groups on the amino nitrogen and the alpha carbon atom as in ephedrine and nylidrin, whereas affinity for the beta adrenergic receptor could still be seen in isopropylmethoxamine. This would indicate that the spatial arrangement of groups on and in the vicinity of the amino nitrogen are TABLE XIII. STRUcrURE-AcrIVITY RELATIONSHIPS IN PHENYLALKANOLAMINE SERIES

OH R R 4 I II R2 0-' CH-CH-NHR 5 R3 -

Structure Drug R R R R R Activity l Z 3 4 S l-Phenylephrine HH OH H CH3 alpha-receptor stimulant dl-Synephrine H OH H H CH3 alpha-receptor stimulant Ephedrine HHH CH3 CH3 inactive Phenylpropanolamine H H H CH3 H alpha-receptor stimulant Metaraminol HH OH CH3 H alpha-receptor stimulant dl-Cobefrine H OH OH CH3 H alpha-receptor stimulant Methoxamine OCH3 H OCH3 CH3 H alpha-receptor stimulant Isopropylmethoxamine OCH3 H OCH3 CH3 CH( CH3)Z beta-receptor block Nylidrine H OH H CH3 CH3 inactive

CH

(CHZ)Z

I-' C6HS lJJ I-' 132 critical for alpha receptor activity. Among the alpha receptor agaonists, activity decreased with methyl substitution on the alpha

carbon atom, but a hydroxyl group at the meta position of the phenyl ring improved activity. Thus phenylpropanolamine was a weak alpha receptor stimulant, but metaraminol was about 40 times as active as

phenylpropanolamine (Tables VI and VII). Introduction of another hydroxyl group at the para position in the phenyl ring as in cobefrine

further increased the activity four-fold. Synephrine which has a

single hydroxyl group at the para position was only about 3 times as

active as phenylpropanolamine. It seems that the para position is not

significantly important for intrinsic alpha receptor activity.

Heterocyclic Compounds

Results: All the three compounds, methylphenidate, tetrahydrozoline

and , activated alpha adrenergic receptors.

Xylometazoline was not active on the uterine preparation whereas it was

about half as active as phenylephrine on the ileum (Tables VI and VII).

Xylometazoline and tetrahydrozoline were about equally effective and

methylphenidate was almost ten times less potent.

Discussion: These compounds conform to arylalkylamine pattern and are

formed by the incorporation of the amino nitrogen and the alpha carbon

atoms into a heterocyclic ring. Since the relative disposition of the

nitrogen, alpha and beta carbon and the phenyl moiety is not appreciably

altered, these compounds showed affinity for alpha adrenergic receptors.

An analogous situation is seen among the pyrroldine derivatives that

show an alpha receptor blocking property (191). Tetrahydrozoline (LXXV) 133

~NH VV N~

LXXV

has naphthalene moiety with one of the two rings saturated whereas xy1ometazo1ine (LXXVI) has two methyl groups at 2 and 5 positions and a tertiary butyl group at the para position. 'rhese two compounds were not very different in their alpha receptor activity, indicating that substituents in the phenyl ring follow the same course as was observed for pheny1alkano1amine derivatives wherein a greater change in activity

LXXVI 134 appeared only with the meta substitution. Incorporation of the nitrogen atom into pieridine ring as in methylphenidate (LXXVII) resulted in decreased activity.

LXXVII

Conclusion

(a) Alpha Receptor Stimulants: From the foregoing description of the

structure-activity relationships in the various classes of

compounds it is possible to develop a generalized picture as to

the nature of the chemical groups whose presence in the molecule

is essential for alpha receptor activation. These structural

requirements may be summarized as follows:

(i) The introduction of N-alkyl groups of increasing size results

in a decrease in the intrinsic alpha adrenergic activity.

N-alkylation leads to an increase in the doses of these drugs

required to obtain responses from alpha adrenergic receptors.

A gradual decrease in the affinity of such compounds for the

alpha adrenergic receptor is observed and compounds are

obtained which are practically devoid of alpha adrenergic 135

activity. The same type of relationship is found if

N-alkyl and N-aralkyl substitution is applied to para and

to meta hydroxyphenylethanolamines. The amino group is

of special significance for intrinsic activity on the

alpha receptors.

(ii) The introduction of substituents alpha to the nitrogen

atom produces an additional asymmetric center, and reduces

the susceptibility of the molecule to the attack of amine

oxidases (425). The substitution of an alpha methyl

group reduces activity at alpha receptors.

(iii) Compounds that lack the alcoholic hydroxyl group at the

beta carbon atom in the side chain are much less active,

but this alcoholic group is not essential for activity at

alpha adrenergic receptors.

(iv) The meta hydroxy compounds are quite active whereas the

parahydroxy compounds are only feebly active.

(b) Alpha Receptor Blockers: Since in.this study the compounds that

showed alpha adrenergic receptor blocking activity were all

developed on the basic arylalkylamine pattern differing in regard

to the nature and the position of the substituent groups, any

generalization made will be pertinent only to such compounds.

Alpha adrenergic receptor blocking agents are characterized by a

nonspecific nature of their chemical structures, and this lack of

specificity precludes any generalized considerations.

(i) Starting from norepinephrine, substitution of alkyl or aralkyl

groups on the amino nitrogen atom results in a gradual change 136 from sympathomimetic to sympatholytic. The complementary

character of norepinephrine with respect to its specific

receptor is shown by its high affinity for the receptor.

This implies that changes in the structure of norepine­

phrine will result in a decrease of the affinity, because

the highly specific structural requirements are disturbed.

When a planar ring is introduced into the molecule by

substitution of aralkyl groups on the nitrogen, more van

der Waal's forces become active and a strong contribution

to the binding forces or affinities may be predicted, and

lytics with a high potency result. The phenyl group

probably interacts with a receptor surface outside the

surface of interaction for norepinephine. Here, also,

additional receptor parts for the 1ytics are involved.

Thus a decrease in the affinity will be compensated for

as soon as additional binding forces on a newly acquired

receptor surface come into play.

(ii) Mono substitution at the alpha carbon atom is favorable

but disubstitution is not.

(iii) Hydroxyl group on the beta carbon atom in the side chain

and in the ring are detrimental to alpha receptor blocking

activity.

(iv) The presence of a methoxy1 group in the ortho position

contributes to alpha receptor blocking activity wherea a

methyoxyl group at the para position destroys the activity. 137 (v) The presence of an aromatic ring is mandatory since alpha

receptor blocking activity is not found in aliphatic

amines.

The slope ratios and slope factors for alpha receptor stimulants ".' are ,:.g1ven in Table XIV and those for alpha receptor blocking drugs in

Tables XV and XVI. The slope ratios are considerably slower than the corresponding slope factors indicating that the agonists and antagonists behave in a competitive way and the log dose-response curves are parallel. The competitive antagonism is also indicated by the reversible blockade produced. Figure 5 represents log-dose-response curves for phenylephrine in the absence and in the presence of different concentrations of phentolamine on the isolated ileum of rabbit. It is apparent that higher concentrations of the antagonist (phentolamine) necessitate an increase in the dose of the agonist (phenylephrine) without affecting the slope and maximum effect.

Congeners of dichloroisoproterenol

A comparison of the chemical structures of the synthetic beta adrenergic receptor blocking agents in the literature suggests that work in this area presumes that the structure-activity relationships of such drugs have been well established. However, the absolute structural requirements for beta adrenergic receptor activity have not been clearly defined and all possible manipulations of presumed structural features have produced drugs with only a narrow spectrum of potency. Thus the evolution of more specific, potent, and reliable beta receptor blocking agents has been retarded by relucatance to deviate from a unilateral TABLE XIV. COMPARISON OF SLOPE RATIOS AND SLOPE FACTORS FOR ALPHA RECEPTOR STIMULANTS

Ileum Uterus Agonists Slope Ratio Slope Factor Slope Ratio Slope Factor 1-Pheny1ephrine* 1-Norepinephrine 1.19 2.13 1.12 1.87 d1-Cobefrine 1.15 1.86 1.03 1.65 Tetrahydrozo1ine 1.02 2.30 1.19 1.98

Methoxamine 1.29 2.15 1.0 2.02 Metaraminol 1.15 2.40 1.29 2.35

Epinine 1.11 1.86 1.24 2.65

Methylphenidate 1.35 2.05 1.32 2.89 Tuaminoheptane -- -- 1.38 3.25 dl-Synephrine 1.03 2.25 1.04 1.95

Phenylpropanolamine 1.0 1.95 1.15 1.95 Cyclopentamine -- -- 1.04 2.16 Xylometazoline 1.23 2.65 - * Reference drug. w/-' 00 TABLE xv. COMPARISON OF SLOPE RATIOS AND SLOPE FAcrORS FOR ALPHA ADRENERGIC BLOCKING DRUGS ON ISOLATED RABBIT ILEUM

Phenylephrine Norepinephrine Epinephrine

Antagonists Slope ratio slope factor Slope ratio Slope factor Slope ratio Slope factor

Phentolamine*

U-0277 1.01 2.35 1.15 2.54 1.0 1.88

U-062l7 1.14 2.46 1.32 2.68 1.50 1.93

U-0287 1.10 3.15 1.04 2.75 1.07 1.87

U-0433 1.05 2.85 1.49 1.97 1.43 1.89

* Reference drug.

.... UJ \0 TABLE XVI. COMPARISON OF SLOPE RATIOS AND SLOPE FACTORS FOR ALPHA RECEPTOR BLOCKING AGENTS ON ISOLATED RABBTI UTERUS

Phenylephrine Norepinephrine Epinephrine Antagonists Slope Ratio Slope Factor Slope Ratio Slope Factor Slope Ratio Slope Factor

Phentolamine*

U-06217 1.15 2.35 1.25 3.16 1.01 1.81

U-0287 1.02 2.42 1.23 2.25 1.16 1.96 U-0433 1.05 2.32 1.06 1.87 1.28 2.38 U-0277

* Reference drug.

~ z o ~ 100 -m ::c- -z w 50 l- f. _1ft i II f (!) f" ~ ~ ...... ~ Z cv· l.LJ U I .J~~ 0::: 0 W -3 -2 -I Q.

log [1- PHENYLEPHRINE] (fLM )

FIGURE 5. LOG DOSE-RESPONSE CURVES FOR PHENYLEPHRINE AGAINST VARIOUS CONCENTRATIONS ~ OF PHENTOLAMINE ON ISOIATED ILEUM OF RABBIT t-' 142 approach based on the prototype structure of isoproterenol. The chance discovery of dichloroisoproterenol simply served to strengthen the old belief that any powerful beta adrenergic blocking property was only to be found in isoproterenol analogs thereby putting more reliance on the hypothetical models depicting adrenergic mechanisms. According to these models the isopropyl groups, beta hydroxyl group and a phenyl ring substituted especially at para and meta positions constitute a prerequisite for beta receptor blocking activity (12, 24, 242). Using dichloroisoproterenol and its congeners, para-methylsulfonamido­ isoproterenol, and isopropylmethoxamine, an attempt was made to find out whether beta receptor blocking activity was solely dependent on such structural features, and as to how a structurally distinct drug, cyclopentamine, produced a similar effect.

Results: Dichloroisoproterenol and its congeners, except compound­

31075, consistently reduced muscle tone in both the tissues in doses above 0.5 ~ M, and it was necessary to add 12 p g per liter of carbachol to maintain sufficient muscle tone. Of all the drugs tested only dichloroisoproterenol and compound-2l047 showed what has been described as beta adrenergic receptor bocking property under the conditions of

these experiments. Compound-2l047 was about twice as active as

p-methylsulfonamidoisoproterenol but had only one third the activity of dichloroisoproterenol (Tables XVII and XVIII). Dichloroisoproterenol

blocked isoproterenol-induced relaxation in the intestine but consist­

ently relaxed uterine preparation because of its intrinsic

sympathomimetic activity. Similarly, isopropylmethoxamine was completely

inactive on the ileum and produced a weak beta adrenergic receptor TABLE XVII. COMPARISON OF BETA-RECEPrOR BLOCKING PROPERTIES IN THE PRESENCE OF ISOPROTERENOL

Ileum** Uterus***

Antagonist ED50 and range* Slope and range ED50 and range Slope and range pM ..uM p-Methy1su1£onamidoisoproterenol 0.073 2.62 0.082 2.34 (0.034-0.014) (1.37-3.69) (0.035-0.132) (1.09-3.62)

Dichloroisoproterenol 0.011 2.46 Relaxes (0.006-0.029) (1.89-3.65)

Compound-21047 0.033 2.44 0.028 1.86 (0.018-0.072) (1.78-6.12) (0.016-0.065) (1.19-3.71)

Isopropylmethoxamine No activity 0.78 1.78 (0.52-1.38) (0.92-3.81)

Cyc10pentamine 8.9 1.73 12.1 2.32 (6.35-12.46) (1.08-2.77) (6.9-26.2) (1.46-3.59)

* 95 percent confidence limit. ** 4.lxlO-3 p M of isoproterenol used on ileum. *** 1.lxlO-3 p M of isoproterenol used on uterus.

~ VJ 144

TABLE XVIII. PCYrENCY COMPARISON OF BEtA RECEP'I:OR BLOCKWG AGEN'!S

Potency Comparison Antagonist Ileum Uterus

MJ 1999 1.0 1.0

DCl 6.6

Compound-2l047 2.2 2.9

Isopropylmethoxamine 0.105

Cyclopentamine 0.008 0.007 145 blockade only on the uterus. p-Methysu1fonamidoisoprotereno1 had no sympathomimetic activity and antagonized isoproterenol on both the preparations.

An interesting observation was the ability of cyc10pentamine to reversibly block isoproterenol-induced inhibition of the ileal and the uterine smooth muscles. It was about 810 times less potent than dich10roisoprotereno1 on the intestine and about 150 times less so than p-methy1su1fonamidoisoprotereno1 on the uterus (See Figure 6 and Tables

XVII and XVIII). Cyc10pentamine in a dose of 19.8 pM also blocked epinephrine (4.5xlO-3 pM) in the presence of phentolamine (7xlO-3 ~ M), but did not affect the tissue responses to alpha receptor agonists, phenylephrine and norepinephrine (Figure 7, Beta adrenergic receptor blocking activity of cyc10pentamine on rabbit ileum. A. Cyc10pentamine,

19.8 p M; B. Norepinephrine 4.7xlO-3 pM; C. Cyc10pentamine 19.8 ~ M plus norepinephrine 4.7x10-3 pM; D. Phenylephrine 2.4xlO-2 p M;

E. Cyclopentamine 19.8 p M plus phenylephrine 2.4xlO-2 p M;

F. Isoproterenol 4.1x10-3 p M; G. Cyc10pentamine 4.9 p M plus isoproterenol 4.1x10-3 p M; H. Cyc10pentamine 9.8 p M plus isoproterenol

4.lx10-3 » M; I. Cyc10pentamine 14.7 p M plus isoproterenol 4.lx10-3p M;

J. Epinephrine 4.5x10-3 p M, cyc10pentamine 14.7 p M, epinephrine

4.5x10-3 p M, cyc10pentamine 19.8 p M, epinephrine 4.5x10-3 p M, phentolamine 7x10-3 p M, cyc10pentamine 19.8 p M, epinephrine 4.5x10-3 pM respectively).

Compound-31470, compound-3147l and compound-31060 behaved as beta adrenergic receptor stimulants with activity decreasing in that order

(Tables XIX and XX). Compound-31470· had about one fifth the activity z 100 .--o -m p)­ - ~ ::L -....: Z ~o, '-= w 50 ~ (.!) ~~ ~ ~ z (()~ w ~. u "" a:: w a. a ' .,.... .c;::.... , -4 -3 -2

log [ I-ISOPROTERENOL]

FIGURE 6. LOG DOSE-RESPONSE CURVES FOR ISOPROTERENOL AGAINST CYCLOPENTAMINE AND ill 1999 ON ISOlATED ILEUM OF RABBIT .p-.... O' 147

t B , c t t

n11111!t~ ~~~(lI~~II~'l{fJr~qf1t; 111 , t F l 0 E

t , H I t· t

J t "t,ttt

FIGURE 7. BETA ADRENERGIC BLOCKING AcrIVITY OF CYCLOPENTAMINE ON ISOLATED ILEUM OF RABBTI (See text for legend) TABLE XIX. COMPARISON OF BETA RECEPTOR STrnuLATING PROPERTmS

I1elD1l** Uterus*** Agonist EDSO and range* Slope and range EDSO and range Slope and range pM pM

Isoproterenol 0.004 2.42 0.001 2.64 (0.002-0.007) (1.39-3.9) (0.000S-0.002) (0.S8-3.17) Compound-31470 0.19 2.21 0.22 2.1S (0.12-0.36) (1.S1-3.87) (0.12-0.3S) (0.98-4.1S) Compound-31471 O.Sl 2.26 0.3S 1.86 (0.28-0.94) (1.41-3.7) (0.26-0.S3) (0.86-3.71)

Compound-31060 1.007 1.S7 1.24 2.14 (0.64-1.8S) (0.9-2.8) (0.S9-2.1) (1.14-4.80)

* 9S per cent confidence l~it ** in the presence of 1x10- »M of DCI. *** in the presence of 8.Sx10-2 ~ M of MJ 1999.

~ 149

TABLE XX. POTENCY COMPARISON OF BETA RECEPXOR STIMULANTS

Potency Comparison

Agonist Ileum Uterus

Isoproterenol 1.0 1.0

Compound-3l470 0.2 0.005

Compound-3l471 0.008 0.003

Compound-3l060 0.004 0.0008 150 of isoproterenol on the ileal preparation. On the isolated rabbit uterus isoproterenol showed highly exaggerated beta receptor stimu­ lating activity, a property that was not shared by any other beta receptor agonists found in this study. Compound-3l075 and compound­

31447 were inactive. None of the congeners of dichloroisoproterenol showed any affinity for the alpha adrenergic receptor.

Isoproterenol produced an incomplete auto-inhibition of its inhibitory effect on the intestinal tissue at a dose of 1.3xlO-2 p M.

This effect could not be overcome by repeated washings but gradually disappeared within 30 minutes. Successive high doses of isoproterenol produced more nearly complete and longer lasting auto-inhibition of its relaxing effect on ileal strips. Cyclopentamine potentiated the isoproterenol-induced auto-inhibition on the isolated ileal prepara­ tion. No such inhibition was observed on the rabbit uterus.

Discussion: Dichloroisoproterenol and its congeners have been derived from the basic phenylethylamine structure as is evident from Table XXI.

A number of beta adrenergic receptor blocking drugs have been constructed as to preserve isopropylaminoethylamine moiety while allowing changes to occur in the rest of the molecule. Among the

3,4-dichlorophenyl derivatives the presence of the isopropyl group on the amino nitrogen atom produced the most active compound, dichloro­ isoproterenol (232), and the only other grouping that showed any beta receptor blocking activity in this study was homoveratryl, whereas

3-penthylamino group as in compound 31447, and cyclohexyl group as in compound-3l075 produced inactive compounds. The association of beta receptor blocking activity with homoveratryl group is quite interesting TABLE XXI. STRUcrURE-AcrIVITY RELATIONSHIPS OF CONGENERS OF DICHLOROISOPROTERENOL

OH R3 II RIO-' CH-CH-NHR4 R - 2

Structure Drug Activity Rl R2 R3 R4 DCl Cl Cl H CH(CH ) beta-receptor block

MJ 1999 CH SO NH HH CH(CH ) beta-receptor block

Compound-3l447 Cl Cl H CH(C H) Inactive

Compound-3l060 Cl Cl H C(CH ) beta-receptor stimulant

Compound-3l075 Cl Cl H Cyclohexyl Inactive

Compound-21047 Cl Cl H Homoveratryl beta-receptor block

Compound-3l470 Cl Cl H alpha-3,4-methylenedioxyphenylpropyl beta-receptor stimulant

Compound-3l471 Cl Cl H beta-3,4-methylenedioxyphenylpropyl beta-receptor stimulant

I-' V1 I-' 152 since iproveratril (LXXIII) which has no apparent structural bearing on isoproterenol and dichloroisoproterenol still exhibits potent beta receptor blocking activity (296-299). The lack of affinity for the beta adrenergic receptor in compound 31075 that has a cyclohexy1amino moiety lends support to the observation that cycloa1ky1amino moieties yield inactive compounds (24). So far as intrinsic activity on the beta adrenergic receptor is concerned, the isopropyl group gives more active compounds e.g. isoproterenol, but is not an absolute requirement for such an activity since tert-buty1amino group as in compound-31060, and 3,4-methylenedioxyphenylpropy1 group in compound-3l470 and compound-3147l yield active compounds. This indicates that the generally held opinion that the isopropy1amino moiety is essential for any activity at beta adrenergic receptor (259), is not entirely true.

Compound-3l470 and compound-31471 are identical in structure although the attachment of the 3,4-methylenedioxyphenylpropyl group at the terminal nitrogen in the two compounds is conformationally different.

The configuration of 3,4-methy1enedioxypheny1propy1 group is alpha in compound-31470 and is beta in compound-3147l. Both the compounds are beta receptor agonists. Compound-31470 is about 48 times less potent than isoproterenol whereas compound-3147l is even weaker in being about

128 times less effective than isoproterenol. The amino group is the salient structural entity required for adrenergic receptor binding (9).

It has been suggested that alpha and beta receptors represent a single structural entity which will elicit an alpha adrenergic response if attacked first by the amino portion of the catecholamine. In case of a sterica11y hindered amino group such as isopropylamino, the catechol I 153 moiety may attack the receptor first, thereby eliciting a beta

sympathomfmetic response (426, 427). It ha~ also been suggested that

the larger the alkyl substituent on the nitrogen atom of norepinephrine,

the greater will be the affinity of the resulting compound for the beta

adrenergic receptor (428). This would imply that the confi~ration of

groups on the tennina1 nitrogen atom in compound-31470 produce more restricted interaction of this cationic moiety with the anionic receptor

site thus allowing more facilitated approach of the phenyl ring to the

receptor. But the fact that the maximum beta adrenergic receptor

activity is obtained with compounds having the isopropyl group indicates

that this grouping is capable of fonning a sterica1ly hindered amino

cationic group without appreciably distorting the structural set-up

around the receptor area. The larger groups would be able to achieve the

first object more readily than isopropyl but the perturbation in the

receptor vicinity would also be great resulting in diminished affinity

for the receptor, ~~d hence, in less activity. Eventually, this

reasoning may provide a simple explanation as to why the isopropyl group

is so important for activity at beta adrenergic receptors, and why no

other compound has ever improved on isoproterenol.

Replacement of catechol hydroxyl groups in isoproterenol by chlorine

atoms gave rise to dichloroisoproterenol and it was considered that the

para position in the phenyl ring was important for activity at the beta

adrenergic receptor (254-260). Consistent with this is the beta

receptor blocking action of p-methylsulfonamidoisoproterenol and

isopropy~ethoxamine (Tables XVII and XVIII). These drugs had slope 154 ratios that were indicative of specificity in their effects (Tables

XXII and XXIII).

An intriguing situation arises with the incidence of cyclopentamine-induced beta receptor blockade. This drug demonstrates all the characteristic features of a competitive beta receptor blocking agent as described earlier. Cyclopentamine bears no structural resemblance to any of the known drugs that activate or block beta adrenergic receptor and could not have been expected to have affinity for beta adrenergic receptor. Obviously, this anomalous behavior of cyclopentamine falls outside the realm of the current hypotheses, and therefore, warrants a detailed discussion here.

Molecular pharmacology pictures a drug effect as the end result of a series of events. A drug must be structurally so constituted as to be capable, first of all, of interacting with a receptor. The degree of interaction and the nature of the binding forces between drug and receptor will determine the affinity of the drug for the receptor.

Affinity by itself is not enough to produce a response and a drug that stimulates the receptor must have intrinsic activity for the drug­ receptor complex to exert a drug reaction (90). The structural moieties of a drug responsible for high receptor affinity may be different from thos that confer strong intrinsic activity on the compound. The concept of receptor is valid only if a group of drugs can produce a series of observable biological effects all of which can be specifically antagonized by selectively acting blocking agents. Both criteria (drug effect and specific drug antagonism) must be met before a receptor can be fully identified, and a series of drugs which elicit TABLE XXII. COMPARISON OF SLOPE RATIOS AND SLOPE FAGrORS FOR BETA RECEPTOR STIMULANTS

Ileum Uterus

Agonists Slope Ratio Slope Factor Slope Ratio Slope Factor

Isoproterenol* Compound-3l470 1.09 2.68 1.22 3.16

Compound-3l471 1.07 2.78 1.42 2.26 Compound-31060 1.54 2.32 1.23 2.23

* Reference drug.

~ \J1 VI All

TABLE XXIII. COMPARISON OF SLOPE RATIOS AND SLOPE FACTORS FOR BETA RECEPTOR BLOCKING AGEN.I:S

Ileum Uterus

Antagonists Slope Ratio Slope Factor Slope Ratio Slope Factor - MJ 1999*

Dichloroisoproterenol 1.06 1.95 1.25 2.2

Compound-2l047 1.07 2.10 Isopropylmethoxamine -- -- 1.31 1.85 Cyclopentamine 1.51 2.3 1.01 2.5

* Reference drug.

t-' U1 0'1 157 qualitatively similar biologic responses can thus be grouped together as one class with a cammon mechanism of action. Only then does it become feasible to analyze the different structural features of such drugs and speculate on the salient moieties necessary for the drug­ receptor interaction which presumably triggers a series of biological events culminating in an observable response.

It might seem that the concept of "molecular dissection", that is, the attempt to determine the essential structural features of a complex molecule by the synthesis of analogs containing differing segments of the parent molecule, is an essentially false and misleading one.

Substitution of a variety of inert non-reactive groupings into a molecule may have a profound effect on the spatial distribution of the groups within that molecule, as it must be considered as a three­ dimensional entity. When any molecule possesses a structural component that confers a degree of flexibility, then the molecule must be considered as a consortium of different structures; this is not an infinite number, as the older conceptions of free rotation about single bonds have it, but a restricted number, definable within limits. the effects of minor changes in the chemical structure on the relative importance of these different conformations must also be considered when relating chemical structure to biological activity, as well as their effects on the other, well known, modulators of biological activity, such as tissue distribution and metabolic disposal. Each member of a series of molecules might then be treated as an individual case, and comparisons between different compounds must be based on an extensive analysis of its properties and not rely simply on the 158 demonstrations of similarities of pattern in their formal structures.

The study of structure-activity relationships is at best a very indirect way of studying receptors, and is beset with many difficulties of interpretation unconnected with the basic problem of the drug receptor interaction. Nevertheless, it is a technique that is potentially capable of yielding much valuable information about the receptor if the results are consistently interpreted in terms of a limited number of identifiable chemical structures. A consensus of such studies could then possibly give information on the sort of chemical groups most likely to occur in receptors.

The forces that lead to interaction between drugs and receptors are very weak, with the exception of the strictly limited number of cases where covalent bonds are formed. The analysis of these forces have shown that the correlation between the drug and the receptor must be good for the interaction energy to be great enough to overcome thermal motion of the drug and allow a reasonable duration of binding. The necessity for multiple group interaction, the importance of the correct relative orientations of the different groups within the molecule, the consequences of the alterations in the bulk or shape of a molecule are all understandable, and in principle, have implications beyond the demonstrations that many of the phenomena revealed by the study of structure-activity relationships are explicable in terms of known forces.

There is a great deal of evidence to suggest that the association of small molecules with proteins leads to structural changes. A change in receptor conformation accompanying the formation of a drug-receptor complex would be expected by analogy with enzyme-substrate combination. 159 The difference between the enzyme and the receptor would then be that

in the case of the enzyme the induced structural change in the protein

leads to a change in the substrate, in the case of the receptor the

induced change would lead to a change in the properties of the matrix

of proteins of which the receptor protein is a part. The idea of a

rearrangement of a membrane protein produced by stimulant drugs could

also provide a reasonable explanation for the initiation of the series

of events that leads to the observed response. Combination of a

stimulant drug with the receptor induces a structural change; this

structural change may alter the configuration of the groups which lead

to the binding of the stimulant and therefore a necessary consequence

of the protein rearrangement would be dissociation of the stimulant­

receptor complex. Combination with a drug which does not lead to a

rearrangement would result in a stable binding of the drug and a

blocking action would be observed. Association of a molecule with a

protein need not lead to a disorganization of the protein; the drug­

protein complex could stabilize the protein.

Drug-receptor interaction characterizes a direct and specific

response. This is distinguishable from all other effects that are due

to the action of the drug molecules with structures other than a

receptor. These definitions have been helpful in classifying drugs;

but the drug molecule, receptor site, drug-receptor complex, and the

extra-receptor structures have been assumed to be too rigidly oriented

in space to represent a real picture and sometimes this creates a

difficulty in reaching a solution for observations that tend to deviate

from the presumably established stereochemical patterns. Neither the 160

drug nor the receptor, nor the receptor invironment is static. The

environmental factor is one of the important factors that affects the

receptor activity at any given instance, and a drug molecule that

drastically alters the degree of the influence of the environmental

factor on the receptor activity should be expected to bring about a measurable change in the response of the receptors, for the receptor

conformation would have been changed by a disturbance in its

association with its neighbors.

The inability of isopropylmethoxamine, tuaminoheptane and

cyclopentamine to stimulate adrenergic receptors in the intestine, the

lack of activity of xylometazoline on the uterus, and the auto­

inhibition produced by isoproterenol in the intestine but not in the

uterus are examples of a vaguely understood phenomenon of tissue

selectivity. Pertinent in this discussion is the striking difference

in the individual behavior of drugs on one type of tissue while showing

a similar mode of action on the other (324). If drug-receptor inter­

action is based on complementary structural characteristics, then the

two entities must adopt certain specific conformations in order to

achieve this objective. The induction of favorable conformation would

not only depend upon the mutual moulding of the drug and the receptor

but would also be dependent on the state of activity of the receptor

environment. Thus the degree and the chances of the drug-receptor

interaction would be determined by the resultant of the overall mutual

impact of drug, receptor and receptor environment. It is conceivable

that such interactions would be greatly influenced by the hormonal and

the metabolic state of the tissue. A reasonable conclusion is that the 161 assumption of a specific conformation by the receptor and the receptor accessibility to a drug molecule are conditioned by the degree of the influence of such environmental factors. Evidence to support this concept is provided by many adrenergic blocking drugs. As for instance, on the isolated rabbit aortic strip propranolol interacts with a previously blocked alpha receptor to displace phenoxybenzamine, phentol­ amine and to1azo1ine (326), a property that is also shared by methoxamine and ephedrine (327). Furthermore, the beta receptor blocking action of isopropy~ethoxamineis prevented by dibenamine (254).

Since it has been generally agreed that the adrenergic receptor is an enzyme protein, it is evident that any discussion on drugs affecting the adrenergic receptor must be based on the known three dimensional organization of the protein. A polypeptide chain can assume an infinite number of conformations but tends to take on a conformation in which hydrogen bonding is at a maximum; the major force in maintaining the shape or conformation of protein is the hydrophobic bond (429). this means that intramolecular distances and the presence of ionic groups in a drug molecule determine whether or not the hydrophobic bond in the protein is affected by an approaching drug molecule with accompanying change in the pharmacological response. this view draws its support from the fact that the inter-atomic distances of the 0-CH2-CH2-N occurring in the side chains of local anaesthetics, adrenergic blocking, cholinergic, antispasmodics and histaminic drugs has been calculated to be 5.0~ (430). It is also knowa that many adrenergic blocking agents affect more than one type of receptors. One possible conclusion that can be drawn is that the drug molecule can actually make its first 162 impact on the protein helical structure between any two turns or o pitch since the difference between two turns is 5.4A (431).

The foregoing discussion leads to two possible mechanisms of action of cyclopentamine at the molecular level. The first involves occupancy of beta adrenergic receptors and the second mechanism may incorporate allosteric sites.

Conclusion: The structural requirements for stimulating activity at beta adrenergic receptor are relatively more pr.ecise than those for activity at alpha adrenergic receptors. This allows somewhat more reliable predictions on the change in activity consequent upon a change in structure. The presence of isopropylamino group, beta hydroxyl group, and catechol hydroxyl groups seem to be essential for maximum activity, but, except the beta hydroxyl group, replacement of the isopropyl and catechol hydroxyl groups by other substituents still produces beta receptor agonists. In analogy with isoproterenol, the structural features of dichloroisoproterenol are suitable for beta receptor blocking activity, but do not necessarily constitute the only basic framework for developing naw beta receptor blocking drugs.

Cyclopentamine provides a new insight into the adrenergic mechanisms and may prove an alternative basic structure for the synthesis of new beta receptor blocking drugs. CHAP'!ER IV

SUMMARY

Initially, this study was undertaken to determine the activity of sympathomimetic drugs on alpha and beta adrenergic receptors. The drugs used were known to act by an indirect mechanism involving the release of norepinephrine from the storage sites. Almost all of the sympathomimetic drugs have been synthesized on the basic pheny1ethy1­ amine pattern, and even in the alicyclic and heterocyclic sympathomimetic drugs the ethy1amine portion of the pheny1ethy1amine structure is invariab1y maintained. In view of the structural similarity between these amines and the major catecho1amines, epinephrine and norepinephrine, it was anticipated that these drugs would have certain affinity for adrenergic receptors. The drugs used were representatives of aliphatic a1ky1ethy1amine, ary1a1k1yamine, ary1a1kano1amine and heterocyclic series.

The adrenergic receptor activities of these compounds were evaluated on the isolated rabbit ileum and the hormone-treated rabbit uterus. Studies on the isolated rabbit ileum indicated that this tissue reacts differently to stimulation or blockade of the alpha or beta adrenergic receptor, and thus may be used as a differentiation test object to ascertain the type of receptor involved in the response obtained. Also direct or indirect effects of the drug may be determined.

The isolated rabbit uterus was found to be a useful preparation capable of providing a rapid means of confirmation of the results obtained on the ileum, and had the added advantage of being isolated and used 164 s~u1taneous1y to provide a more accurate study of tissue differences under strictly identical experimental conditions. It was experienced that the hormonal state of the animal had a great influence on the responsiveness of the uterus to adrenergic drugs. By instituting an appropriate hormonal treatment before killing the animal, it was possible to enhance tissue sensitivity and to induce reproducibility of its responses to drugs.

The alpha receptor activity was found in compounds that were derived from aliphatic alkylamine, pheny1a1ky1amine, pheny1a1kano1amine, and from heterocyclic compounds in which ethy1amine moiety had been transferred into a heterocyclic ring. The terminal amino group played an essential role in determining the affinity of the drug for alpha receptor. Substitution at this amino group was critical for alpha receptor stimulating activity, and activity was lost with any substituent larger than methyl whereas much larger groups ultimately resulted in alpha receptor blocking activity. Substitution at alpha carbon atom in the side chain had many important effects, it decreased the intrinsic activity of the drug at alpha adrenergic receptor while increasing alpha receptor blocking activity in the presence of a bulky substituent on the amino nitrogen atom. If no substitution at the terminal nitrogen had occurred, then the methyl substitution at alpha carbon atom made the drug less susceptible to enzymatic degradation, and showed only norepinephrine releasing property. D~ethy1 substitution, however, resulted in complete loss of affinity for alpha receptor whereas norepinephrine releasing action was maintained. The presence of the hydroxyl group at the beta carbon atom was not crucial 165 for alpha receptor affinity but was detrimental to alpha receptor blocking property. No compound with beta hydroxyl group had any alpha receptor blocking activity but this group showed a marked rise in the intrinsic activity at alpha adrenergic receptors. Methyl substitution at the beta carbon decreased alpha receptor activity affecting norepinephrine releasing property. All of the aliphatic amines were devoid of alpha receptor blocking activity. The presence of the aromatic ring was essential for a drug to behave as an alpha receptor blocking agent. Substitution at the ortho and the meta position of the phenyl ring was compatible with alpha receptor stUnulating or blocking activity whereas para substitution decreased both. Substituents at the meta position were more favorable than at the ortho position.

The presence of an isopropyl group at the terminal amine nitrogen was not found critical for beta receptor affinity but it seemd to impose a limiting stereochemical factor. This is contrary to the generally held view that isopropyl group is absolutely essential for activity at beta adrenergic receptors. The absolute structural features for beta receptor stimulating and beta receptor blocking activity are not essentially analogous, the presence of a para and a meta substituted phenyl ring and of a beta hydroxyl group in the side chain is essential for stimulating activity, but is not required for beta receptor blocking activity.

Cyc10pentamine which is known to act indirectly by releasing norepinephrine was found to stimulate the alpha adreaergic receptor.

Contrary to expectation, this drug also showed a weak beta receptor blocking activity. This finding necessitated a re-check of a number of 166 sympathomimetic amines to see whether they had any affinity for the beta adrenergic receptor. No such activity was discernible in any other compound. A series of dich1oroisoprotereno1 derivatives was examined to compare and contrast the modes of action of these compounds with that of cyc1opentamine. The congeners of dich1oro­ isoproterenol had affinity for and moderate intrinsic activity at the beta adrenergic receptors. The suitability of the dich1oroisoprotereno1 structure for developing potent and specific beta receptor blocking agent is biased by the high incidence of beta receptor stimulating activity in these compounds. It is, therefore, not surprising that during the last ten years none of the congeners of dich1oroisoprotereno1 has drawn much attention. These compounds possess a direct, dose­ dependent musculotropic action in moderate doses, and the dose range for the evaluation of their adrenergic receptor activity on the isolated tissues is restricted. This difficulty can be overcome by using carbachol as a spasmogen. Dich1oroisoprotereno1 and its congeners were found to have no affinity for alpha adrenergic receptor.

Cyc1opentamine, on the other hand, provides a new, basic structure which seems to be potentially capable of yielding powerful beta adrenergic receptor blocking drugs. A very significant aspect of cyc10pentamine structure is the complete lack of structural features that confer beta receptor stimulating activity on the molecule, and thus, carefully designed cyc10pentamine analogs may be expected to produce a pure beta receptor blockade.

Isoproterenol was ~ound to inhibit its own inhibitory effect on the isolated rabbit ileum but not on the isolated rabbit uterus. 167 tuaminoheptane, cyclopentamine and isopropylmethoxamine were active on the hormone-treated uterus but were completely inactive on the

isolated rabbit ileum. Similarly, xylometazoline and dichloro­

isoproterenol were active on the ileal strips and had no activity on

the isolated rabbit uterus. the ability of the compounds to be

selectively affect one type of tissue at the exclusion of others, and

the unexpected beta receptor blocking action of cyclopentamine exposed hitherto unrecognized aspect of drug-receptor interaction that neither

the drug nor the receptor nor the receptor environment is static. the

environmental factor is one of the important factors that modifies the

receptor activity at any given instance. this concept also implies

that a drug molecule as a whole is responsible for the nature and the

degree of observable response that it produces. APPENDIX I

TREATMENr OF THE RAW DATA

For statistical analysis of the results, the Litchfield-Wilcoxon Method (417) was used.

Procedure

A. The Data and Graph

1. The actual doses and the percentage blocking effects were

listed.

2. The doses were plotted against percentage effects on

logarithmic-probability paper leaving space for but omitting

any 0 or 100 per cent effects.

3. With a transparent straight edge, a straight line was drawn

through the points, particularly those in the region of 40 to

60 per cent effect.

B. Plotting 0 or 100 per cent Effects.

1. The expected per cent effect, as indicated by the line drawn,

was read and listed for each dose tested.

2. Using the expected per cent effect a corrected value for each o or 100 per cent effect listed was obtained from the table given in the method. These values were recorded and plotted

on the graph.

C. The (Chi)2 Test.

1. The difference between each observed (or corrected) per cent

effect and the corresponding expected per cent effect was

listed. 169

2. Using each difference and the corresponding expected per cent

effect the contribution to (Chi)2 as read from nomograph was listed.

3. The contributions to (Chi)2 were added together and multiplied

by the average number of tissue segments per dose, i.e., the

total number of animals/K, the number of doses. This gave the

(Chi)2 of the line. The degrees of freedom were two less than the number of doses plotted, i.e., n = K = 2. 4. If the (Chi)2 of the line was less than the value of (Chi)2

given in the table for n degrees of freedom, the data were

considered not to be significantly heterogenous, i.e., the

line was a good fit.

D. The Median Effective Dose (EDSO) and fEDSO 1. The doses for 16 per cent, SO per cent and 84 per cent effects

(ED16 , EDSO and ED84) were read from the line on the graph. 2. The slope function, S, was calculated as:

ED84/EDSO+EDSO/ED16 S = 2

3. From the data tabulation, the total number of animals tested at

those doses whose expected effects were between 16 and 84 per cent was obtained. It was designated as liN ." 4. The factor for EDSO was calculated as 2.77/ N

fEDSO = S

s. The confidence limits of the EDSO were calculated as: 170 EDSO x fEDSO = upper) ) limit for 19/20 probability EDSO / fEDSO = lower)

E. The slope (5) and slope factor (fs)

1. The dosage range was calculated as a ratio

R = largest/smallest dose plotted

2. Using this value of R and that of 5, a complex number, A,

was read from nomograph.

3. The factor for slope was obtained as

fs = A10(K-l)K N

4. The confidence limits of 8 were calculated as

8 x fs = upper) ) limit for k9/20 probability 5 1 fs = lower)

F. The Test for Parallelism of Two Lines

1. The slope function ration, 8. R, was calculated as

8. R. - 51/82 where 81 is the larger value

2. Using fs, and fs2, fs.R was read from nomograph. 3. If the value of 8. R was less than fs.R then the curves

were considered to be parallel within the experimental

error.

The following example illustrates the use of the method. 171 SOLUTION OF THE DOSE-EFFEcr CURVE OF CYCLOPENTAMINE AGAINST ISOPROTERENOL

Dose Tissue Observed Expected Observed Contribu~ion No. (u M) Segments %block %block minus to (Chi) Expected

1 4 5 9 7 2 0.0060

2 8 5 38 42 4 0.0065

3 12 S 74 71 3 0.0040

4 16 5 84.5 86.5 2 0.0030

5 20 5 88.5 93.5 5 0.0400

Total segements = 25 Total 0.0595 Ntunber of doses = 5 (Chi) = 0.0595x5 = 0.2975 Segements/Dose = 25/5 = 5 Degree of Freedom, n = K-2=3

(Chi)2 from Table for n of 3 = 7.82. 0.2975 is less than 7.82, therefore, the data are not significantly heterogenous.

ED84 = 15.6 u M

ED 8.9 u M 50 = ED16 = 5.2 u M

= 15.6/8.9+8.9/5.2 = 1 73 S 2 •

N = 20

fED50 = S2.77/ N = SO.61 = 1.730•61 = 1.4

EDSO x fED50 = 8.9 x 1.4 = 12.46 u M

EDSO ; fED50 = 8.9 ; 1.4 = 6.35 u M

ED50 and 19/20 confidence 1tmists:8.9(6.35 to 12.46) u M 172 R = largest/smallest dose = 20/4 = 5

A = 1.3 from Nomograph, using 8 = 1.73 and R = 5

fs = (A)10(K-1)/K N = 1.310 (5-1)/5 20 = 1.31•79 = 1.6

8 x fs = 1.73 x 1.6 - 2.768

S ; fs = 1.73 ~ 1.6 = 1.08

8 and 19/20 confidence limits:

1.73(1.08 to 2.768)

The slope ration 8 /8 2.62/1.73 1.51 = 1 2 = =

81 = 2.62 (MJ 1999) 8 1.73 (Cyc1opentamine) 2 =

fs.R = 2.3 (from Nomograph using the two fs values of MJ 1999 and cyclopentamine).

8.R of 1.51 is less than fs.R of 2.3, therefore, the deviation from parallelism is not significant. BIBLIOGRAPHY

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