University Microfilms, a XEROX Company, Ann Arbor, Michigan

Home , Adrenaline, Methylphenidate, Norepinephrine, Pseudotropine

I I

71-22 ,*+69

EFFLAND, Richard Charles, 1942- SYNTHETIC AND PHARMACOLOGICAL STUDIES OF AGENTS AFFECTING PERIPHERAL ADRENERGIC ACTIVITY.

The Ohio State University, Ph.D., 1971 Chemistry, pharmaceutical

University Microfilms, A XEROX Company, Ann Arbor, Michigan

THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED SYNTHETIC AND PHARMACOLOGICAL STUDIES OF AGENTS

AFFECTING PERIPHERAL ADRENERGIC ACTIVITY

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

U r Richard CC^Effland, B .S .

$ $ $ $ $ $

The Ohio State University 1971

Approved by

/ Adviser Oollege of Pharmacy ACKNOWLEDGMENTS

I wish to express my sincere appreciation to

Professor Jules B. LaPidus, for his guidance and support as adviser throughout this study

Professor Popat N. Patil, for his patience, time, and assis tance in carrying out the pharmacological studies

Professor Donald T. Witiak, for his encouragement and assis tance during and prior to these studies

My Mother and Grandmother, for their many sacrifices that have made this possible

My wife, Mary Louise, for her encouragement, understanding, and confidence, and to whom, along with my son DuWayne, I dedicate this work. VITA

August 26, 1942 ...... Born—Peoria, Illinois

1965...... B. S. Pharmacy, The University of Iowa, Iowa City, Iowa

1965-196 6...... Teaching Assistant, College of Pharmacy, The Ohio State University, Columbus, Ohio

1966-197 0...... NIH Pre-doctoral Fellow, College of Pharmacy, The Ohio State University, Co lumbus, Ohio

1970-1971 ...... Research Assistant, College of Pharmacy, The Ohio State University, Columbus, Ohio

FIELDS OF STUDY

Major Field: Medicinal Chemistry TABLE OF CONTENTS

ACKNOWLEDGMENTS...... ii

VITA...... iii

LIST OF TABLES...... v

LIST OF FIGURES...... vi

INTRODUCTION...... 1

RESULTS AND DISCUSSION

A. SYNTHETIC...... 24

B. BIOLOGICAL...... 66

EXPERIMENTAL

A. SYNTHETIC...... 85

B. BIOLOGICAL...... 102

SUMMARY...... 106

BIBLIOGRAPHY. .j ...... 109

iv LIST OF TABLES

Table Page

1. Chemical shifts and coupling constants for the 1- benzylic proton (H^) and 2-methyl protons of the diastereomeric l-hydroxy-2-methyl-2,3,4,5-tetrahydro- 3-benzazepines ...... 45

2. Proton resonance characteristics of substituted tetrahydro-3-benzazepines ...... 52

3. Relative activities of isomeric cocaines and related compounds on response of rat vas deferens to (-)- norepinephrine ...... 67

4. Norepinephrine potentiating effects of isomeric cocaines and trojJacocaines on rat vas deferens ...... 70

5. Relative activities of isomeric cocaines and related compounds on norepinephrine induced contractions of rat vas deferens ...... 72

6. Relative activities for alpha-adrenergic blockade by atropine related compounds ...... 76

v LIST OF FIGURES

Figure Page

1. The nrar spectrum of l-hydroxy-2-methyl-2,3,4,5- tetrahydro-3-benzazepine (A) ...... 43

2. The nmr spectrum of l-hydroxy-2-methyl-2,3,4,5- tetrahydro-3-benzazepine (B) ...... 44

3. The nmr spectrum of l-hydroxy-2,3,4,5-tetrahydro- 3-benzazepine ...... 55

4. Log dose-response curves for the norepinephrine potentiating effects of (-)-cocaine, (±)-allops eudococaine, and pseudococaine ...... 69

5. Dose-response curves for (-)-norepinephrine and pseudoecgonine methyl ester ...... 75

6. Log dose-response curves for alpha-adrenergic blocking activity of homatropine hydrobromide...... 77 INTRODUCTION

While the pharmacological effects of many adrenergic agents have long been known, the mechanism of action of these substances at the receptor level has remained the object of intensive research. Adrenergic drugs are those chemical compounds that exert an effect on the adrenergic nervous system, or that part of the peripheral autonomic nervous system mediated by the neurochemical hormone norepinephrine. The many pharmacological actions of a number of epinephrine related substances led Ahlquist^ to suggest the I presence of two different types of adrenergic receptors. A series of closely related catecholamines were ranked in order of potency on a variety of tissues and vascular systems. His studies revealed two distinct orders of potencies for the sympathomimetic agents. In one, epinephrine was the most potent and isoproterenol the least. In the other, isoproterenol was the most potent.

However, it was also found that these receptors could produce both inhibitory or excitatory action depending upon the tissue. Therefore, classification of the two receptor types as inhibitory or excitatory seemed inappropriate.

Ahlquist instead proposed that those receptors toward which epinephrine and norepinephrine were the most active be classified as alpha receptors and those toward which isoproterenol was the most active be classified as beta

1 2

receptors. Alpha receptors were associated primarily with excitatory acti vities except in the intestine where inhibition resulted from stimulation of the

alpha receptors. Similarly, stimulation of the beta receptors produced pri marily inhibition, except for myocardial tissue where excitation resulted.

Many attempts have been made to define the nature of the adrenergic

receptors at the molecular level, and several theories of drug-receptor in teraction have been proposed, such as the ’’Conformational Perturbation 2 Theory’’ of Bernard Belleau. As a basic postulate, it is proposed that the key drug induced change which is significant with respect to a given response is a conformational perturbation of the protein-like molecule which acts as the receptor. This event would serve to transform the receptor from a latent catalytic unit to an active species capable of catalyzing the chemical modifica tion of a substrate molecule forming an integral part of the chemical machi nery underlying contractile processes. In most cases of enzyme activation, it is thought that the interaction of small molecules with regulatory sites in duces specific conformational changes which alter the catalytic efficiency of the enzyme. A similar idea, an "induced fit theory", has been advanced by 3,4 5,6 Koshland. Belleau has suggested that the agonist effect produced by a catecholamine at excitatory adrenergic alpha receptors results from ion-pair formation between the protonated amine head of the agonist and an anionic site on the receptor surface, of which ATP is an integral part. The contractile response is presumed to result from complete charge neutralization at this 3

site. Bloom and Goldman have modified Belleau's adrenergic receptor hy

pothesis, and offered an alternative proposal, the "Dynamic Receptor 7 Hypothesis." They envisioned the adrenergic receptors as enzyme-sub-

strate complexes, consisting of a phosphorlytic enzyme and adenosine tri

phosphate (ATP). Catecholamine agonists interact with the nucleotide sub-

strate-phosphorolytic enzyme complex in such a manner as to stimulate the

rate of phosphorolysis or phosphoryl group transfer reactions of the terminal

phosphate group of ATP, with adenosine 5'-diphosphate (ADP) produced. The

production of a response by this interaction involves receptor (Enzyme-

Substrate) destruction, with a concommitant release of calcium ion, which

in turn initiates muscle response. The compatibility of the dynamic receptor

hypothesis with existing theories of drug action such as the occupancy 8,9 10 theory, Belleau's conformational perturbation theory, and Paton's rate 11 7 1! theory is also discussed by Bloom and Goldman. More recently, Belleau 13 has revised his original proposal as well as incorporating and rejecting 7 various aspects of Bloom and Goldman's hypothesis. Belleau maintains

charge neutralization of the terminal phosphate group of ATP by the agonist

amine as a starting point, followed by transfer of the terminal phosphate to

a nearby carboxylase anion. Specific ligand formation between calcium and

the functional groups involved in the transfer process would underlie activa tion of ATP cleavage. In addition he postulates a resting state, prior to acti vation by the agonist amine, involving an accessory binding site of the alpha- 4

receptor occupied by the quaternary moiety of lecithin. It is suggested that

an imidazole cation would counteract an anionic charge of the terminal phos

phate of ATP, while the quaternary cation of lecithin would pair with the

carboxylate anion acting as the acceptor in the phosphoryl transfer process.

With respect to the beta-receptor, a growing body of evidence indicates

that the stimulation of the enzyme adenyl cyclase, which converts ATP to

cyclic 3', 5'-adenosine monophosphate (AMP), resembles most closely those

effects which have been described as being mediated by beta adrenergic re- 14 ceptors. The beta-adrenergic agonist is postulated to facilitate intramole

cular attack by the 3’-hydroxyl group of ribose on the innermost phosphorus

atom of ATP to form cyclic 3', 5'-AMP which then serves as the chemical mediator for a multitude of beta-effects. The nature of the amine is con sidered the primary determinant of whether alpha or beta agonism capacity resides in a given compound, and the benzylic hydroxyl group is relegated to a secondary role. The cationic amine group neutralizes the anionic charge on the phosphate oxygen, thereby lowering the energy barrier to approach of the attacking ribose 3'-hydroxy nucleophile. This charge neutralization by the cationic amine is considered to be a size-insensitive role, as compared with the size-sensitive role of facilitating the conversion: ATP -* ADP. There- 7 fore, bulky groups on nitrogen preclude alpha activity but not beta. Cyclic

3',5'-AMP is currently the subject of extensive investigation, and evidence is very strong that most of the metabolic effects of the catecholamines are 5

mediated by cyclic AMP, and that in most and perhaps all tissues the beta

receptor and adenyl cyclase are the same. In addition, it has been suggested

that the alpha receptor may also be related to adenyl cyclase, that the alpha

and beta adrenergic receptors might in reality represent different sites for

hormone-enzyme interaction on the adenyl cyclase molecule.

More recently a somewhat different approach concerning adrenergic 15 interactions between drugs and receptors has been employed. Emphasis

has been placed oni the nature of the catecholamine chemical species at the

adrenergic receptors. It has been proposed that the characteristic species

for a beta adrenergic agonist is a quinone methide, while for the alpha

adrenergic agonists the characteristic catecholamine seems to be an aziri-

dine, an internally cyclized quinone methide derivative. The relative ability of a particular catecholamine to form either a quinone or aziridine interme diate is reflected in the ratio between alpha and beta adrenergic agonist pro pensities.

Structure-activity relationships of the beta-phenethylamine moiety, which can be regarded as the parent nucleus of adrenergic amines, have been 16 17 extensively studied. ’ The alpha-amino group, the beta-hydroxyl group and the aromatic ring are considered to be the key functional groups involved in the interaction of adrenergic drugs with their receptor sites. While the amine function is regarded as an indispensable moiety for adrenergic acti vity, the phenolic hydroxyl groups of the catechol system and the beta- 6

hydroxyl group are considered important but non-essential groups for acti

vity. Maximum adrenergic activity at both the alpha- and beta-receptor

systems depends however, upon the presence of these hydroxyl functions.

The phenolic and beta-hydroxyl groups presumably enhance binding and op timum orientation required for maximum catalysis by the adrenergic agonist.

The uniformly lower agonist activity on non-catechol structures is attributed 7 to an increased frequency of unproductive interactions. Some compounds

not possessing the catechol portion may have a longer duration of action

since, unlike catecholamines, they cannot be metabolized by catechol-O- 18 methyl-transferase, which gives less active O-methylated derivatives.

In any analysis of structural or stereochemical requirements for ac tivity, it is important to keep in mind that drugs which are closely related structurally, such as the phenethylamines, may produce pharmacologically identical effects by different mechanisms. Adrenergic agonists represent such a case. Sympathomimetic agents may act predominantly directly, that is at the effector site; predominantly indirectly, by releasing endogenous nor epinephrine; or by a combination of these processes. There is evidence that the mechanism of action of indirect-acting sympathomimetic amines is more complex, and that many indirect acting agents may block the uptake 19 of norepinephrine as well as cause its release. Many sympathetic amines and related agents inhibit the uptake of norepinephrine into the sympathetic nerve endings. This implies that a greater concentration of norepinephrine 7 will then be available for activation of pharmacologic receptors, with result ing potentiation of norepinephrine effects. A systematic pharmacologic ap-

! 20,21 proach has been used by Swamy and colleagues to define some stereo chemical characteristics of uptake sites, with the assumption that potentia tion of exogenous norepinephrine reflects the ability of molecules to inhibit the uptake of norepinephrine. Other studies of chemical structure of sym pathomimetic amines and its relationship to inhibition of uptake have been 22,23 24 performed. In a recent review, Patil et aL make the following gen eralizations regarding steric structure-action relationships with respect to inhibition of uptake:

(a) In phenolic amines, deoxy derivatives are more potent than their corresponding (-)-isomers which are in turn more potent than (+)-isomers, (b) In nonphenolic amines such as ephedrine and norephedrine, deoxy derivatives are also more potent than (-)-isomers. But in the latter case the situation is complicated by two asymmetric carbons, (c) (+)-Deoxynorephedrine is about twenty times more potent in inhibiting norepinephrine up take than that of (-)-deoxynorephedrine. (d) (+)-Norpseudo- ephedrine is more potent than either (-)-norephedrine or (+)- deoxyno rephedrine.

Recent studies have indicated that tissue accumulation of nonphenolic amines may occur by a different mechanism than that involved with catechol amines, since the accumulation of labeled amphetamine, norephedrine and 25,26 phenylethanolamine was not influenced by cocaine.

Often very subtle stereochemical differences may determine whether a compound possesses primarily direct sympathomimetic activity or indirect 27 activity. In 1933, Easson and Stedman postulated for an asymmetric mole

cule like R-(-) -epinephrine (1), a three point attachment with the receptor,

involving: (a) the basic nitrogen; (b) the aromatic group (with m- and p-

hydroxyl groups which determine the intensity of attachment); and (c) the

alcoholic hydroxyl group. In the less active S-(+)-isomer (2), since the alco

holic hydroxyl group is oriented in the wrong position, only two-point interac

tion is expected, as if the hydroxyl group were missing. This is in agreement

with the fact that the agonist effects of (+)-epinephrine and the deoxy deriva- 28 tive epinine (3) are equal.

HO HO OH

HO C-CHo-NHCH

HO CH9CH9-NHCH

There were, however, examples where the activity of deoxy derivatives 29 and (+)-isomers was not in harmony with the Easson-Stedman hypothesis. 30,31 In order to test the Easson-Stedman hypothesis, Patil and colleagues selected a series of (-)- and (+)-isomeric sympathomimetic amines and their 9 deoxy derivatives. Using vas deferens from normal as well as reserpine- pretreated rats, they were able to show that with respect to direct activity the (+)-isomer and the deoxy derivatives were equiactive, but for those agents acting indirectly the deoxy derivative was generally more active than the (+)-isomer. Thus in the reserpine treated catecholamine depleted tissues, where only direct activity is observed, results were in agreement with the Easson-Stedman hypothesis. The hypothesis apparently holds for adrenergic amines which exert their effect by direct action on the adrenergic receptors, but is not applicable to those amines whose action are mainly of an indirect nature. The greater indirect activity of the deoxy derivatives over the (+)-isomers was attributed to a higher rate of neuronal uptake and/or - increase in the basic release mechanism by the deoxy derivative. In (+)- isomers the OH-group may be incorrectly oriented with respect to transport mechanisms, and the rate of transport, therefore, is slower than that of the corresponding deoxy derivative where such a group is absent. Since exo genous norepinephrine is equally potentiated by (•+)-isomers and their corres ponding deoxy derivatives, the inhibition of reuptake of endogenously liberated neurohormone may be the same for both agents. This would suggest that the higher activity of deoxy derivatives is not due to a greater ability to inhibit reuptake, but rather due to either faster uptake and/or faster displacement of norepinephrine. This type of study reveals the critical importance of distin guishing between, the direct and indirect components of action before conclu- 10

32 sions concerning a structure-activity relationship are drawn.

A wide variety of sympathomimetic amines and related compounds have been studied to determine steric structure activity relationships for both in direct activity and direct activity. Among nonphenolic amines, the ephedrine isomers present a unique opportunity to study steric structure-activity rela- 24 tionships. There are four stereoisomers of ephedrine, with sympathomi metic activity ranging from direct to indirect. The pattern of the pharmaco logic activity of all four ephedrine isomers in the rat vas deferens appears as 1R,2S-D(-)-ephedrine (4) > IS, 2R-L(+)-ephedrine (5) 1S,2S-L(+)- pseudoephedrine (6) » 1R, 2R-D(-)-pseudoephedrine (7). The pattern of the

or >NHCH3 CHij ^NHCH3 c h 3 ^n h c h 3

potentiation of exogenous norepinephrine by these agents in the reserpine- 24 pretreated tissues also appears to be the same. Patil and co-workers have indicated a relationship between the absolute stereochemistry at the benzylic carbon and the direct action of peripherally acting sympathomimetic 11

30 31 33 amines. * ’ D-configuration of the beta-carbon favors direct action,

while there is relatively more indirect component in amines where

the beta-carbon has the L-configuration. 34 While in general the R-configuration favors direct action, these

generalizations must be viewed with caution when considering adrenergic

agents with two asymmetric centers. For example, 1R, 2S-D(-)-ephedrine

(4) and 1R, 2R-D(-)-pseudoephedrine (7) differ only in absolute configuration

about the alpha-carbon, yet their effects, both in vivo on blood pressure and 35 heart rate, and in vitro on vas deferens, are quite different. D(-)-pseudo-

ephedrine can reduce or block the pressor effects of D(-) -ephedrine in

anesthetized dogs. On the isolated rat vas deferens D(-)-pseudoephedrine

did not show any intrinsic effects, but markedly antagonized contractions due

to tyramine and D(-)-ephedrine, as well as potentiating the effects of nor

epinephrine. Thus D(-)-pseudoephedrine appears to act at both the catechol

amine uptake site and the alpha-adrenergic site, with only affinity but very 36 little or no intrinsic activity at the latter site. LaPidus et al. pointed out

the stereochemical similarities between (-)-ephedrine and (-)-pseudoephedrine.

In both of these molecules, the functional groups, the phenyl ring, beta-

hydroxyl group, and the amino group could fit the same three points on the

hypothetical receptor. 34 In a stereochemical study of the ephedrine isomers, Portoghese em ployed nuclear magnetic resonance spectroscopy to predict the preferred solu- 12 tion conformations of the free bases and salts. From a comparison of the preferred conformations of the protonated forms, it was suggested that the C-methyl group in 1R, 2R-pseudoephedrine may hinder effective interac tion with the receptor. Both D-(-)-ephedrine and D-(-)-pseudoephedrine have the 1R configuration, but the C-methyl group of the former projects above the plane of the phenethylamine moiety, while in the latter it is below the plane. Only D-(-)-ephedrine possesses some direct activity.

H. H H\ v H H H v 'O Ph = Phv . 0 ' ' 0 Ph

H H H H + / Me'^ MeiiljySNH H"'"j\NH H » r Me Me j[/[e Me H Me Me

D-(-) D - ( - ) - i | r L-(■*■) (1R,2S) (1R, 2R) (IS, 2R) (IS, 2S)

37 Tye and co-workers studied the beta adrenergic effects of ephedrine isomers on guinea pig trachea. Both D-(-)-ephedrine and D-(-)-pseudoephedrine (1R isomers) appeared to be essentially direct acting, and were nearly equipotent. This equipotency on a predominantly beta tissue, in contrast to results obtained on other tissues, suggests less stringent steric requirements for the 2-methyl group. The L-(+) -isomers (IS) possessed an indirect com ponent in their action.

I 13

Information concerning stereochemical requirements of a given recep tor site is obtained primarily by altering the structure of an agonist molecule and observing the resulting effect on biological response or activity. Since it is difficult to obtain direct information concerning the nature of the receptor site, indirect information is obtained in this manner. The more detailed our knowledge of the structure of the reacting agonist at the time of receptor- agonist interaction, indicated by a pharmacological response, the more we can learn of the stereochemical requirements of the receptor site. This study of the relations between the chemical structure of drugs and their pharmaco logical activity can possibly provide information of the receptor surface be cause of the complementarity between drug and receptor.

Adrenergic agents such as epinephrine and ephedrine have free rotation about the central carbon-carbon bond. While studies can provide reliable in formation concerning preferred ground state conformations in solution, there is no guarantee that this is the conformation that reacts with the receptor to produce the biological response. A number of studies have been carried out 34 38 39 on the preferred solution conformations of the ephedrines. * ’ However, as a result of this free rotation and relatively low energy barrier, a thermo dynamically less stable conformation could be the biologically active species.

To eliminate this uncertainty and to attempt to determine the biologically ac tive conformation of a molecule, rigid analogs of many drugs have been pre- 14 pared. In discussing structure-activity relations and receptor surface, 40 Ariens states:

The more rigid the structure of a drug molecule, the more it tells us about the properties of specific receptors, and the smaller the chance that it will fit different types of receptors.

In a rigid system the stereochemistry of the functional groups is maintained the same in solution or during receptor interaction.

A number of systems have been used in preparing ephedrine-like molecules with a rigid or semi-rigid structure. In some, the functional groups are incorporated into a heterocyclic ring, such as cis-2-amino-4- 41 methyl-5-phenyl-2-oxazoline (8). In others, the functional groups are

CHq c 6h 5

N O

NH2 13 substituents on a npn-heterocyclic ring system. The semi-rigid isomeric 42 2-aminotetralols, prepared as norephedrine homologs, are examples.

There are four possible isomers of 2-aminotetralol: (+),(-)-cis-form (9) and (+), (-)-trans-form (10). Only the cis-form was effective as a pressor agent. (+) -cis-2-aminotetralol was about five times as active as (-1-cis-

2-aminotetralol. The arrangement of functional groups in (+)-cis-2-amino tetralol was claimed to be like that in the most active form of norephedrine. 24 Patil et al. point out that the higher pressor potency of these two agents may

reflect their possible catecholamine-releasing effects.

As part of a conformational study of beta-phenethanolamine receptor 43 sites, Smissman and Gastrock prepared conformationally rigid derivatives

of the isomeric norephedrines, utilizing the trans-decalin system. These 3- amino-2-phenyl-terns-2-decalols were tested as racemic mixtures, with compounds 11 and 12 representing two conformers of erythro configuration and compounds l£and 1£ representing conformers of threo configuration. OH Ph

11 12

Ph n u

■Ph

13 14 16

In pharmacological testing these compounds were reported to be about l/lOOth as active as norepinephrine in rat vas deferens preparations. This adrenergic activity was attributed to possibly both direct and indirect activity, although no attempt to distinguish the mode of action was reported.

The synthesis of the conformationally rigid racemic 3-pheny 1 -3 -hydroxy- 44 trans-decahydroquinolines was also reported by Smissman and Chappell.

The demethyl analogs 15 and 16 were prepared.

15 16

Pharmacological testing on the isolated rat vas deferens revealed that -4 there were no intrinsic effects from these agents. However, in 10 M con centrations, effects of exogenous norepinephrine were potentiated. This po tentiation indicates that these agents possibly interact with uptake sites. 45 Nelson et aL , using a similar approach, prepared the isomeric 9- hydroxy-10-amino-l, 2 ,3 ,4a, 9,10,10a-(trans-4a, 10a)-octahydrophenanthrenes,

P7, 18. 19 and 20. Two of the compounds 17 and 20 are representative of two 17 OH

17 18

NH2 •n h 2

19 20

conformations of norephedrine possessing the threo configuration while the other two compounds l£and lj) represent conformers of the erythro confor mation. None of the compounds tested in vas deferens showed intrinsic ac tivity of greater than 10% of (-)-norepinephrine. Compounds 17 and 20^ showed adrenergic blocking activity, the latter demonstrating both competitive and noncompetitive antagonism. Compound 18 exhibited potentiation at 1 x 10 M while a noncompetitive type interaction was evident at higher concentration.

The synthesis and biological testing of butane analogs of ephedrine has 46 recently been reported. The dl-erythro isomer of 3-amino-2-phenyl-2- butanol (21) showed direct agonist effect in that it was equally effective on -4 the normal and reserpinized rat vas deferens in 10 M concentration. The dl -threo isomer 22 was identical in qualitative responses with the erythro 18

compound but much less effective. These results are similar to those obser

ved with erythro ephedrine and threo -ephedrine racemates.

CH3,

HoN OH OH

21 22

The N-isopropyl derivatives of the trans-decalins 11, 12, 13 and 1£

as well as the N-isopropyl derivatives of norephedrine and norpseudoephe- 47 drine, were recently prepared and tested for biological activity. Neither

dl -erythro-N-isopropylnorephedrine nor dl -threo-N-isopropylnorpseudo-

phedrine (10-4M) showed any effect on the normal or reserpinized rat vas

deferens. Both gave slight potentiation of norepinephrine. Among the trans- -4 decalins, the N-isopropyl derivative of 14, in 10 M concentration, poten

tiated the response of norepinephrine, increasing the pDg of norepinephrine

from 6.4 to 7.4. The N-isopropyl derivatives of IT and 12 were less effec

tive, with JL3 the least active.

The relatively low activity exhibited by many of these rigid analogs is generally attributed to the large excessive bulky groups used as a framework for the key functional groups. Although these compounds provide a rigid arrangement of the functional groups, they nevertheless impart a large degree of lipophillic character and steric bulk to the molecule not normally present in the non rigid parent molecule. This extra lipophillicity and steric hin drance could possibly alter the compound’s ability to penetrate membranes or hinder its ability to occupy either the alpha-receptor, uptake site, or effect release of endogenous transmitter.

Thus it was desired to prepare analogs of ephedrine and phenylethanolamine that would provide a degree of restricted mobility and rotation, yet with a minimum of extra bulk being introduced into the molecule. The tetrahydro derivative 23^ of 3-lH-benzazepine (24) appeared to be a suitable system to use as a cyclic derivative or analog of the parent phenethylamine moiety.

The compounds tetrahydro-l-hydroxy-3-benzazepine (25) and tetrahydro-1- hydroxy-2-methyl-3-benzazepine (26) represent the cyclic analogs of phenylethanolamine (27), and ephedrine (28), respectively. Part of this dissertation concerns the preparation, biological evaluation, and conformational and con

figurational analysis of these compounds.

The attempted synthesis of a related compound 1,5-epoxytetrahydro-3-

benzazepine (29) utilizing several possible synthetic approaches, is also

N-H O N-H

29 30

discussed. This compound was of interest not only because of its relation to the above benzazepines but also because it represents a cyclized derivative of the phenylmorpholine system found in phenmetrazine (30), a drug possessing central activity.

The ability of certain amines to potentiate norepinephrine responses in adrenergic innervated organs was mentioned previously. Normal physiological I inactivation of released neuronal norepinephrine occurs either by metabolism 21

by the enzyme catechol-O-methyl transferase (COMT) and/or by re-uptake into the nerve terminal. The enzyme monoamine oxidase (MAO), once thought to be a primary source of inactivation of released norepinephrine, is located primarily intraneuronally where it metabolizes cytoplasmic norepine phrine outside the storage granules. Inactivation by re-uptake into the pre-

synaphtic nerve terminal is now generally considered the primary route of inactivation of norepinephrine.

Numerous agents, including methylphenidate, tricyclic antidepressants, and cocaine, have been reported to inhibit the uptake of norepinephrine and 48 other amines into adrenergic neurons. The potentiation of the response of an effector organ to norepinephrine by cocaine has been attributed to an in hibition of this uptake process. Such inhibition would presumably result in an increased concentration of norepinephrine at the receptors and therefore 49 an enhanced response. Some reports, however, indicate that the poten- 50 tiating action of cocaine may be more complex. Maxwell et al., from studies on rabbit aortic strips, suggested that cocaine may have an action in the smooth muscle cells which may account in part for the supersensitivity 51 effect. Bevan and Verity studied the potentiating effect of cocaine on nerve free vascular muscle and concluded that the action of cocaine was due to both a presynaptic action and a direct action at the adrenergic receptor. From 52 studies on cat spleen strips, Reiffenstein similarly concluded that blockade of uptake is insufficient to explain potentiation by cocaine in vitro, and sug 22 gested an "allosteric activation" whereby cocaine may deform adrenergic re ceptors to alter receptor kinetics to allow increased receptor utilization. In 53 addition, Trendelenburg has reported that in guinea pig atria, cocaine also has a "tyramine-like" effect, causing release of norepinephrine. A number of other reports have provided support for the concept that the potentiating effects of cocaine may be due to some mechanism other than inhibition of 54 uptake.

The ability of cocaine to stereospecifically potentiate the response of 55 isomeric amines has been examined. The results indicate that cocaine- induced supersensitivity is indeed stereospecific. Cocaine has been found to potentiate the response to (-)-norepinephrine more than (+)-norepinephrine.

This was intially attributed to a greater inhibition of uptake of (-)-norepine- 56 phrine than (+)-norepinephrine at the neuronal membrane. However, other reports indicate that the uptake of norepinephrine through the neuronal mem brane has little or no stereospecificity, and that the lack of potentiation of

(+)-norepinephrine by cocaine is due to the low potency of this isomer which exerts pharmacological effects only in concentrations which saturate the up take mechanism. The effect of cocaine would then become negligible when 57 58 uptake is saturated. Malmfors has suggested that stereospecificity of uptake resides in the mechanism of retention rather than that of uptake. It is possible that both retention and neuronal uptake may exhibit stereospeci ficity, but that the rate of uptake into the nerve is much faster than into the

I 23 I 59 granule. Von Euler and Lishajako have studied uptake into granules and found uptake to be stereoselective. Rates of metabolism by MA.0 may also be a factor, as well as experimental methods and tissue used. Selectivity of uptake and storage mechanism has been discussed in a review by Patil , 24 et al.

In addition to potentiating the direct action of various amines, cocaine antagonizes the indirect action of amines that release norepinephrine from 60 the nerve terminals.

Considerable study has been made relating the ability of cocaine to selectively potentiate the response of isomeric amines as well as amines differing structurally or in substitution. However, very little study has been done on the potentiating abilities of isomeric cocaines or of structurally sim i- 61 lar tropane derivatives. Therefore, the norepinephrine potentiating ability of the isomeric cocaines, (+)-pseudococaine and (±)-allopseudococaine, as well as various intermediates, was examined using isolated rat vas deferens as the test tissue. Derivatives of tropine and pseudotropine, as well as atropine related compounds, were also tested. RESULTS AND DISCUSSION

A. SYNTHETIC

The synthesis of l-hydroxy-2,3,4,5-tetrahydro-3-benzazepine (25) was achieved by way of the intermediate ketone tosylate 38_. Phenethylamine tosylate (33), prepared from phenethylamine (31) and p-toluenesulfonyl chlo ride (32), was refluxed with ethyl chloroacetate in acetone in the presence of potassium carbonate to yield the ethyl ester of N-phenethyl-N-toluene-p- sulfonylglycine (34). Hydrolysis afforded the free acid 35 in 83 per cent 62 yield. The ester is usually obtained as an oil and hydrolyzed directly.

It will solidify however if subjected to high vacuum to remove all ethyl chloro acetate, and on recrystallization from 95 per cent ethanol give a pure white solid in 85 per cent yield.

pyridine H2 -CH 2 -NH 2 + CHg so2ci

31 32

k 2 c o 3 H 2 CH2 -N -T s ClCHoCOoEtf

H CH2 33 34 C02Et TS — p—CH g-0—SOg-

I 25

H2 -CH 2 -N -T s 1) NaOH 2) HC1/H20 ^ CH2C02H

63 Previous work on cyclization of (3-arylethylglycines had shown that

under the commonly employed Friedel-Crafts conditions, unless the aroma

tic ring is activated with methoxy groups, only tetrahydroisoquinolines are 64 obtained. Rehman and Proctor, however, reported the successful prepa

ration of azabenzocycloheptenones utilizing low temperature and aluminum

chloride. Using this method and the conditions reported, 3£was refluxed

with thionyl chloride in dry benzene to form the corresponding acid chloride

36. The acid chloride was added to a mixture of an equivalent amount of an

hydrous aluminum chloride in dry methylene dichloride at -70°C. The pre-

sci’ibed work up of the reaction afforded no expected benzazepinone. Instead,

a yellow oily solid was obtained which, when recrystallized from methanol, yielded a white crystalline compound exhibiting no carbonyl peak in the infra

red. Nmr and melting point confirmed this to be N-tosyl tetrahydroisoquino- 65 line (37). It was later found that the desired benzazepinone 38_ could be ob- I

1 eg.

soci 2 A1C13 35 > o ~ ° h 2 - c h 2 - n - t s benzene c h 2 c i 2 -70° CH 36 2 A0C1 Ts 37

tained in 82 per cent yield by employing three quivalents of aluminum chlo

ride instead of one. Reduction of 38 with sodium borohydride afforded the

3 eg.

AlClc NaBH 4 ^ 36 N -T s N"Ts “ 95% ^ c h 2 c i 2 EtOH -70°

38 39

corresponding alcohol 39^ in 92 per cent yield.

Previous attempts by Proctor to detosylate the sulfonamide 3£by base 64 catalysed elimination of the toluene-p-sulfonyl group failed. Of the methods commonly used to cleave sulfonamides, reductive cleavage with dissolving metals has probably been utilized most extensively. The mixture of sodium and liquid ammonia or sodium and alcohol has often been employed in peptide 66 chemistry to cleave tosyl protecting groups. The successful use of sodium 67 and alcohol on a system similar to 39 had been reported, but proved un- 27 successful when applied to 39. Better results were obtained using sodium and ammonia.

Sodium metal was added to liquid ammonia, which was cooled in dry ice and acetone and flushed with nitrogen, until a deep blue color persisted.

The sulfonamide 39 was added, followed by sufficient sodium to maintain the blue color for fifteen or twenty minutes. Ammonium acetate was added until the blue color disappeared, and then the ammonia evaporated. Examination of the isolated product revealed the presence of a free N-H peak as well as a large amount of unreacted starting material. Since the sulfonamide 39 was relatively insoluble in liquid ammonia, it was dissolved in ether, and the ether solution added to the sodium-ammonia mixture. Upon neutraliza tion with ammonium chloride and work up of the residue following removal of the ammonia, the free amine 25 was obtained in 50-65 per cent yield.

39 Na/NH 3

Similar results could be obtained by using dimethoxyethane as co-solvent and lithium metal instead of sodium. Column chromatography of the crude reac tion products yielded another compound that spectroscopic data and melting point indicated to be p-thiocresol. 28

The preparation of the diastereomericl-hydroxy-2-methyl-2,3,4,5- tetrahydro-3-benzazepines (48) and (47) was carried out in a similar man- 64 ner. Phenethylamine tosylate (33) was condensed with ethyl cv-bromopropionate by refluxing in dry acetone in the presence of potassium carbonate

for 133 hours. Removal of excess ethyl a-bromopropionate under high

vacuum and hydrolysis of the ester 40 afforded the acid 41 in 82.7 per cent yield. The acid chloride 42 was obtained from 41 by refluxing with thionyl

(3Ho-CHo-N~Ts 1) NaOH/H20 MeOH 2I 2 ) H3^3 CHCHg

Br a 40 COgEt

Ho-CHo-N-Ts ^ CH2 -Cfa 2 -N -T s

CHCHo CHCHg I c o 2h COC1

41 42

chloride in benzene; cyclization of 42 with aluminum chloride in methylene

dichloride at -70° afforded the tetrahydrobenzazepinone 4 j3 in 20-40 per cent yield. Reduction of 4£ with sodium borohydride gave a mixture of cis and trans diastereomers, 44 and 45, in 80-92.5 per cent yield. Since sodium or lithium and ammonia had been used successfully in

cleaving the tosyl group on the structurally similar 1-hydroxy-2,3,4,5-tetra-

hydro-3-benzazepine (25), the same procedure was followed for the mixture

of 44 and 45. However, all attempts at detosylation by this method failed to

yield any isolatable free amine, regardless of the metal or co-solvent used.

Thus it was felt that separation of the diastereomeric alcohols prior to de tosylation would be necessary, rather than after as previously planned.

Analysis by gas-liquid partition chromatography (glpc) revealed an approximately equal mixture of the two isomers, which could not be separated by fractional crystallization. Separation was achieved first by thin layer chromatography on Silica Gel GF (for thin layer) using ether-Skelly C (2:1) as the solvent system. The two isomers were then obtained by column chro matography using Silica Gel G (for columns) and ether-Skelly C (2:1) as 30 I i eluent. The compound with the higher Rf value (. 45) was called A and the one with the lower Rf (. 28) called B. The purity could be checked with thin layer, using fluorescent Silica Gel GF and U. V. light, or iodine as a means of detection. It is interesting to note that the compound with higher Rf value on TLC (A) has a longer retention time in glpc.

The separated isomeric alcohols were again subjected to detosylation attempts with metal-ammonia and metal-alcohol but again no detosylated amine could be isolated from either A or B. Thus other means of detosyla- 68 tion were investigated. Ji et ed. reported the cleavage of a number of sulfonamides in excellent yield by sodium naphthalene. The sulfonamide 39 was used as a test system since it had already been successfully detosylated by an alternate method. A solution of the sulfonamide in 1,2-dimethoxyethane

was added to a solution of the sodium naphthalene anion radical in 1 , 2 -d i- methoxyethane and stirred for one hour at room temperature. Addition of water and subsequent work up yielded only a yellow oil or gummy solid from which no amine could be isolated. Repeated attempts gave the same results.

A mixture of red phosphorous and hydroiodic acid has been used as a 69 means of detosylation of amines, but due to the presence of a benzylic hydroxyl group, the use of this method was not attempted.

The use of lithium aluminum hydride as a means of cleaving sulfon amides appears to have received little attention. There have been however, a number of reports on both successful and unsuccessful attempts to cleave 31

70 sulfonamides with this reagent. The sulfonamide 39^ was again used as a test system, adding a solution of the sulfonamide in dry tetrahydrofuran to a stirred suspension of lithium aluminum hydride. After refluxing for 24-48 hours the reaction mixture was worked up to yield 2£in 55 per cent yield.

The product was identical in all respects to that obtained by the sodium- ammonia method. It was also found that the ketone 38^ could be reduced di rectly to the desired detosylated amino alcohol 25.

THF THF T s-N LiAlH 4 CO LAH

39 25 38

The isomeric alcohols (A) and (B), subjected to the same procedure

using lithium aluminum hydride, afforded the corresponding l-hydroxy- 2 - methyl-2,3,4,5-tetrahydro-3-benzazepines. The diastereomeric 1-hydroxy-

OH CH CH THF N-Ts N-H LAH

(A) (A) (B) (B) 32

2-methyl-2,3,4,5-tetrahydro-3-benzazepines will hereby be referred to as

A and B, and the corresponding tosylates as A tosylate and B tosylate.

A number of possible synthetic approaches to l,5-epoxy-2,3,4,5- tetrahydro-3-benzazepine (29) were investigated. It was felt that the l s 5- diol 46 might be a useful precursor to the desired epoxy derivative 29. There-

N-H

fore, initial attempts were directed toward the preparation of this compound.

Cyclization of the bis-o-dichloroacetylbenzene 47_ appeared to be a possible route to 46. The analogous para isomer had been prepared by treating ter-

O II

C-CH -C l

71 phthaloyl chloride with diazomethane and hydrochloric acid. However, similar treatment of phthaloyl chloride resulted only in a complex mixture 33 from which no major component could be isolated. The para isomer had also 72 been prepared by chlorination of p-diacetylbenzene. The preparation of o-diacetylbenzene by reaction of methyl magnesium bromide with o-phthal- 73 aldehyde, followed by permanganate oxidation, had been reported. How ever preliminary investigations of this procedure gave poor results.

Reductive cyclization of o-xylylene dinitriles (48) has been used as a fairly general method for the preparation of saturated imines and tetrahydro- 74 3-benzazepines (49). A method for the preparation of adrenaline involving

the reduction of the cyanohydrin 50 to the corresponding amine 5JL has been 75 reported. However the exact method of reduction, conditions, or other references were not given. HONs^»XT v cH(OH)CN —* H„XJ Q ^ . CH(OH)CH2 NH2 50 51 34

The preparation of the bis cyanohydrin .53 was accomplished by treat

ment of o-phthalaldehyde (52) with potassium cyanide in glacial acetic acid.

OH o c - s * o i " i OH 52 53

76 Attempts to prepare 53 using acetone cyanohydrin or liquid HCN with po tassium cyanide were unsuccessful. Reductive cyclization of 53 was attempted

using platinum oxide or 5% rhodium on alumina as catalyst. The latter has been used to reduce the aromatic ring of mandelic acid without hydrogenoly- 77 sis of the benzylic hydroxyl group, and to effect low pressure reduction of nitriles in the presence of benzyl substituted amines, without concurrent hy- 78 drogenolysis of the benzyl groups. Nevertheless hydrogenolysis of the benzylic hydroxyl group occurred more readily than reduction of the nitrile with either catalyst. This could be observed from the infrared spectra. As the reaction time increased the OH peak became smaller and the cyano peak more intense. It has been observed that the presence of an electronegative group such as a hydroxyl on the same carbon as a nitrile results in a de- 79 creased absorbance of the nitrile. 35

A method of preparing arylamino alcohols from aryl-aldehydes through 80 an oxirane intermediate was reported by Duncan, et al.

/ \ RoNH I ArCHO > ArCH—CH„ — - > ArCHCH0 -NR,

Using similar conditions, a solution of o-phthalaldehyde (52) was added to a solution of sodium hydride and trimethylsulfonium iodide in DMSO and

THF. Work up of the resulting reaction mixture left a viscous brown intract able oil that could not be purified by crystallization or by distillation, even at high temperature and vacuum. Infrared indicated the presence of carbonyl and hydroxyl absorptions.

The cyclization of cis-2 ,5-bis (hydroxymethyl)-tetrahydrofuran deriva tives (54) to the 8-oxa-3-azabicyclo (3.2.1) octane ring system (56) had been 81 reported by Cope and Anderson. It appeared that a similar approach to 29

CH0NHR

L ____ /

54 55 56 36 from a suitable bis substituted phthalan, such as 58, might be possible.

Treatment of o-phthalaldehyde (52) with nitromethane afforded the hemi- 82 acetal 57. Numerous attempts were made to obtain the transformation of

CHoNO

OH 57

57 to either 58 or an intermediate diol. However all attempts proved un successful.

Another possible synthetic sequence to ^involving a cis - bis substi tuted phthalan is shown below. Ozonolysis of 1,4-dihydronaphthalene-l, 4- 37

o 2h 59 60

LiAlH4 NH o 61 29

endoxide (59) followed by oxidative work up of the ozonolysis product appeared to be a means of acquiring the desired cis-dicarboxylicphthalan 60. Esteri- fication of this product followed by cyclization with ammonia would give the imide J31, which could be reduced to the desired 2 ,3 ,4 ,5-tetrahydro-3-benzazepine-1,5-endoxide (29).

The 1,4-dihydronaphthalene-l, 4-endoxide (59) was readily prepared by reaction of furan with a solution of benzyne generated in situ from anthranilic 83 acid. Ozonolysis of 59 followed by oxidative work up with formic acid and

isoam yl ■> 59 nitrite 38 peroxide yielded an acidic product identical in all respects with phthalic acid.

Nmr Study of cis and trans 1 -Hydroxy-2-methyl-2,3,4,5- tetrahydro -3 -benzazepines.

The use and limitations of nuclear magnetic resonance spectroscopy

(nmr) and other physical methods for configurational assignment and confor mational study of A and B will now be discussed.

The dependency of vicinal proton coupling constants upon the dihedral angle between the vicinal protons enables nuclear magnetic resonance spec troscopy (nmr) to be used as a method of determining the relative configura tion of vicinal substituents. The relationship between the vicinal coupling 84 constant Jh-C-C-H' the dihedral angle 0 was shown by Karplus to be

0 2 w _ o . o J cos 0 - C (0 £ 0 £ 90 ) JHHf J1 8 0 cos 2 0 - C (90° £ 0 £ 180°)

0 180 where J =8.5 Hz, J = 9.5 Hz and C = -0.3 Hz. For six-membered rings in the chair conformation the coupling constants for protons on adjacent saturated carbon atoms are generally 8-13 Hz for J and 2-6 Hz for SIX} SIX

. In comparable systems J0„ is usually slightly smaller (~ 1 Hz) clX} 6u vi| than Jax.eq* 85 39

For rigid systems where the dihedral angles are fixed, vicinal coupling constants are a reliable means of assigning relative configurations. This method has been employed in assigning or confirming relative configurations of rigid and nearly rigid ephedrine and norephedrine analogs mentioned pre- 43,44,45 viously. Vicinal proton coupling constants have been used to deter- 86,87 mine preferred conformations of flexible ring systems as well as freely 34 38 rotating systems as ephedrine and pseudoephedrine ’ , where J . repre sents a weighted average of all conformations.

Assignment of threo and erythro configurations in conformationally mobile diastereoisomers has also been made on the basis of vicinal proton 85,88 coupling constants. A discussion of the application of this approach for assigning the relative configuration (erythro or threo) of the diastereomeric tetrahydro-3-benzazepines A and B follows.

The nmr spectra of the diastereomeric 1-hydroxy-2-methyl-2,3,4,5- tetrahydro-3-benzazepines A (Fig. 1) and B (Fig. 2) could be divided into

four distinct parts: ( 1 ) the aromatic region, ( 2 ) the benzylic proton region,

(3) the methylene and methine region, and (4) the methyl region. The OH and NH protons were covered in the methylene envelope. The coupling con stants of the vicinal protons on Cj (H^) and Cg (H^-) were obtained by ex amining the benzylic proton (Hx ) signal, as.H^ was obscured by the methy lene envelope. Since Hx represents the X portion of an AgMX type system, 89 a first order analysis is possible. The three methyl protons represent the 40

Ag portion, and the Cg methine proton the M portion.

The seven-membered ring of both cis-1 -hydroxy-2-methyl-2.3 ,4 ,5 - tetrahydro-3-benzazepine (62) and trans-l-hydroxy-2-methyl-2.3,4,5-tetrahydro-3-benzazepine (63) can theoretically assume two separate chair con formations and two separate boat conformations, as well as twist boat forms.

62 63

The interconvertible conformational chair and boat forms are shown below:

(a) 62

PH3

Lx "M I

41 OH

N-H

-H HO

(a) 63 (b)

-H CH CH

(c) (d)

Conformational analyses have been performed on cycloheptene and 87,90,91,92 benzocycloheptanes. The presence of a double bond in the seven- membered ring affords a rigidity which prohibits the pseudorotation that complicates the analysis of cycloheptane. Cycloheptene and benzocycloheptane can therefore exist in the classical boat and chair conformations. In vestigations have shown that benzocycloheptane exists predominantly in the 92 chair form (95%), with 13 kcal/mole activation energy for ring inversion.

In the boat form a severe repulsion exists between the near hydrogen at the

’'prow" of the boat and the double bond of cycloheptene or the aromatic ring 91 of benzocycloheptane. While the presence of a nitrogen atom may lower somewhat the energy difference between the chair and boat forms, it is still 42

90 likely that the chair form will be preferred. As with other systems, the

presence of substituents will no doubt alter these energy differences, either

increasing or decreasing the relative stability of a given conformation.

Examination of Dreiding models of 62^ and 63 revealed that the dihedral

angle between Hx and in the cis isomer 62 could vary only from 0 to 90°,

while in the trans isomer 6j3 this angle could vary from 0 to 180°. It was

also apparent from the model of the cis isomer 6 £ that the preferred confor

mation on a steric basis should be the chair conformation 62b. This is also

the conformation most favorable for intramolecular hydrogen bonding between

the OH and N. In either chair form, the dihedral angle between Hx and

appears to be approximately 75-90°. Therefore, the coupling constant

Jh^Hj^ w0 “ld ke expected to be quite small.

For the trans isomer, in the chair form 63a, the OH and Me are trans diequatorial and in the chair form 63b are trans diaxial. However, in this conformation intramolecular hydrogen bonding can occur. The dihedral angle for HXHM in 63a appears to be about 150-165°, while in 63b it appears to be o o close to 35-40 . Only in the boat form 63d can the angle approach 90 . In the other boat form 63c and in all twist boat forms, this dihedral angle is large. Therefore, the coupling constant j % hm would be expected to be lar

ger for the trans (erythro) isomer 6 £ than for the cis (threo) isomer 62.

The nmr spectra of A (Fig. 1) and B (Fig. 2) showed f°r compound I A to be essentially zero, and for compound B, 6-7 Hz (Table 1). This would 3.0 3.0 5.04.0 6.0 • . 0

1 000

4 0 0 3 0 0 200 100

-fc*~4 »» yi»4>41»+iX srf"

S. 0 7.06.0 5.0 4.0 3.03.0

Figure 1. Nmr spectrum (GO MHz) of a 10% solution of compound A in CDC1S. •£>- CO 2 .0 4.03.0 5.0 PPMlT) 6.0 7.0 1.0 9.0

400 200 too

OFFSET 200 SW 100

a.o 6.0 5.0 PPM 14) 4.0 3.0 2.0 1.0

Figure 2. Nmr spectrum (60 MHz) of a 10% solution of compound B in DMSO - dg. 45

TABLE 1

CHEMICAL SHIFTS AND COUPLING CONSTANTS FOR THE 1-BENZYLIC

PROTON (Hx ) AND 2-METHYL PROTONS OF THE DIASTEREOMERIC

1 -HYDROXY-2-METHYL-2, 3 ,4 ,5-TETRAHYDRO-3-BENZAZEPINES

N-H

Chemical Shift( 6 )a Coupling Constant(Hz) Compound Solvent HX Methyl (R') XM X, OH M,Me

A CDCI3 4.30 1 . 2 2 0 0 6.5

DMSO-dfi 4.50 0.95 0 0 6.5

DMSO-FTFA 4. 75 1.25 0 -0 .5 0 6.5

pyridine d5 4.73 1.25 0 0 6.5

MeOH-d4 4.60 1 . 1 2 0 - 1 0 6.5

B CDCljj 4.38 0.95 5 .6-6 0 6 .5

DMSO-dfl 4.37 1 . 0 6 .5 -7 0 6.5

DMSO+TFA 4.72 1 . 0 6 0 6 .5

pyridine 4.75 1.23 6.5 0 6 .5

cL Chemical shifts are expressed in 6 ^-downfield from internal TMS standard. 46

indicate that compound A has the cis (threo) configuration (62) and compound

B the trans (erythro) configuration (63). Additional evidence supports this

assignment.

Since the chair conformation 62b is favored for the cis isomer 6 £ both

on steric grounds and for intramolecular hydrogen bonding, the steric fac

tors should enhance intramolecular hydrogen bonding. However, steric fac

tors may slightly oppose intramolecular hydrogen bonding in the trans iso

mer (63b), since the 2-methyl group is oriented in the axial position. The

dihedral angle between the nitrogen and OH is small (30-40°) in both isomers,

which should favor intramolecular hydrogen bonding in both isomers. How

ever, the cis isomer 6 j2 would be expected to show a greater intramolecular

to intermolecular hydrogen bonding ratio.

Infrared spectra of A and B at equal but dilute concentrations showed / a slightly larger free OH peak for compound B. Electrochemical oxidation 93 studies have been carried out on diastereomeric pairs of amino alcohols where the more strongly intramolecular hydrogen bonded diastereomer was

known from other methods. Studies on the diastereomeric ephedrine and pseudoephedrine and related dimethylamino ethanols have shown that the

more strongly intramolecular hydrogen bonded diastereomer in each case was the most difficult to oxidize. Since electrochemical oxidation involves the removal of an electron from the nitrogen lone pair, hydrogen bonding may 93 interfere with this process. Electrochemical oxidation experiments on A 47 and B showed compound A to be the most difficult to oxidize. It must however be pointed out that at this time the relationship between intramolecular hydro gen bonding and ease of electrochemical oxidation is still under investigation 93 and other complicating factors may be involved. Nevertheless, these stu dies would seem to support the IR evidence that compound A is intramolecu- larly hydrogen bonded at least to some extent more than B. This supports the nmr data indicating that compound A has the cis (threo) configuration (62) and compound B the trans (erythro) configuration (63) .

The cis isomer 62 could account for a ® Hz only in a boat con formation (62c or 62d) , where Hx and H^- are nearly eclipsed (~ 15°). How ever, in both of these conformations the OH and methyl are also nearly eclipsed. In addition, no intramolecular hydrogen bonding could occur in either of these conformations. Intramolecular hydrogen bonding could occur in a twist boat form where the OH is pseudoaxial and the methyl pseudoequa- torial, but the dihedral angle between Hx and H^. would again approach 90°.

It would therefore seem highly unlikely that the cis isomer could account for

the JHXHM approximately 6 Hz.

Similarly, the trans isomer could account for a JHxH]y[ zero on^ *n the boat conformation 63d. However, since all other possible conformations would give a sizeable coupling constant, the trans isomer would have to reside almost entirely in this conformation, which seems improbable. In addition, no intramolecular hydrogen bonding is possible in this conformation. 48

Further support for the proposed configurational assignments was ob- 94 tained from an nmr study by Buchanan and McCrae on the conformation of

2 ,3-benzocyclohepten-l, 4-diol (64). This cis diol showed strong intramole

O O

cular hydrogen bonding in CCI 4 in the IR, indicating a diaxial arrangement of

the OH groups. The nmr spectra of 6 £in CDClg showed only a doublet (J =

6 Hz) for the benzylic protons, since an equatorial proton will be at ap proximately 90° to one of the adjacent C-H bonds in either the chair or boat conformation. In the diacetate of 64 and in a DMSO solution of 64, the oxygen substituents were found to be equatorial. The diacetate gave a doublet (J =

9 Hz) for the benzylic protons in CDCI 3 solution. The axial C-H bond sub tends an angle of 90° with one of the adjacent C-H bonds only if the ring is in the chair conformation. It was pointed out that in the boat conformation neither of these angles is 90°, but instead 15° and 150°. Therefore, if the diacetate were in the boat form, a multiplet would have been observed. Simi 49 lar results were obtained for the diol in DMSO, where coupling with the hy droxyl proton was also observed. The values for the coupling constants and bond angles are in agreement with those observed for the diastereomeric

tetrahydro- 3 -benzazepines. They also reported that in CD C lg, the aromatic protons of the diol 64 appeared as a clean singlet but in DMSO they appeared

as an A 2 B 2 system. A similar A 2 B 2 pattern was obtained for the diacetate.

This was attributed to the influence of the equatorial oxygen atoms on the adjacent aromatic protons.

A similar effect was observed for compound A and B . In C D C lg, the aromatic protons appeared as a clean singlet, but in DMSO appeared as a broken multiplet. However, the value of Jhv r ,, increased only slightly iVL (Table 1), indicating little change in conformational equilibrium. This has 34 been considered as evidence for strong intramolecular hydrogen bonding.

The effects of solvent change are shown in Table 1. Coupling with the hydroxylic proton was not observed even in DMSO. The proton resonance shifted downfield as expected in pyridine. However, between compound A and B there was very little difference observed in the chemical shifts of the

H^ proton resonances. In changing solvents from CDClg to DMSO, the H^ proton resonance of A shifted downfield slightly, while no change was ob served for B. Assuming A to be the cis isomer 62, a large contribution would be expected from the intramolecular hydrogen bonded conformation 62b. In this conformation, the dihedral angle between the methyl group and the H^ 50 pi'oton is small, and the methyl group may exert a shielding effect on the Hx 34,95 proton. If DMSO decreases the amount of intramolecular hydrogen 96 bonding, a small increase in the population of conformation 62a may occur, 34,95 where the methyl exerts a deshielding effect on Hx , producing a down field shift.

In the trans isom er 6 £the dihedral angle between the methyl group and

HX proton is the same in both conformations 63a and 63b. Therefore, a

change in conformation should produce little change in the effect of the methyl group on the Hx proton. Since the shift was observed only for one isomer it would seem less likely that a solvent effect is responsible, although this possi bility cannot be ruled out entirely.

The upfield shift of the methyl proton resonance of A on changing from

CDClg to DMSO is also consistent with this explanation. The methyl group in conformation 62b is equatorial, but in 62a is axial. An increase in the population of conformation 62a may produce an upfield shift in the methyl pro- 34 ton resonance due to a shielding effect of the aromatic ring.

In the trans isomer the methyl group is shielded by the aromatic ring in 63b and by the hydroxyl group in 63a. Thus a change in population of con formations might be expected to have little effect on the chemical shift of the methyl group. This is consistent with the data observed for compound B.

The vicinal coupling constants for the methyl group are nearly identical with those reported for ephedrine, pseudoephedrine, and the cis and trans isomers

I 34 of 3-methyl-2-phenylmorpholine (Table 1).

The orientation of an electronegative group trans to a vicinal proton that is coupled to a proton on the same carbon atom as the electronegative group has been shown to lower the coupling constant between the two pro- 3 4 ,97,98 tons. The hydroxyl group of the cis isomer is oriented trans to the Cg-proton in conformation 62b, which could help account for the zero coupling constant observed in compound A. This orientation cannot occur in the trans isomer 63^

From this data it would appear that compound A can be assigned the cis (threo) configuration (62) and compound B the trans (erythro) configura tion (63).

The tosylates of A and B, as well as their 3,5-dinitrobenzoate esters, were also examined by nmr (Table 2). The value of Jx m was nearly the same for both tosylates. This value (2.2 Hz) remained essentially the same for the 3 ,5-dinitrobenzoate ester of A tosylate, but doubled (5 Hz) for the

3, 5-dinitrobenzoate ester of B tosylate. However, because of the size of these groups and the decreased contribution of intramolecular hydrogen bond ing expected, no conclusions could be reached from the data on these com pounds. The large steric interactions may well cause an increased confor mational mobility with boat forms or twist boat forms preferred. An inter action between the equatorial methyl group and the N-tosyl group may serious ly decrease the contribution of any conformation where the methyl group is 52

TABLE 2

PROTON RESONANCE CHARACTERISTICS OF SUBSTITUTED

TETRAHYDRO-3-BENZAZEPINES

N-R"

Chemical Shift( 6 )a Coupling Constant(Hz) Compound Solvent HX Methyl (R1) XM M,Me

(A) CDClg 5.13 0.62 2.2 6.5-7.0 R=H

R ’=CH3 R"=Ts

R=3, 5-DNB c d c i3 6.45 0.90 2.2-2.4 6. 5-7.0

R'=CH3 R"=Ts

(B) CDClg 4.51 0.75 3 6.5 R=H

R'=CH3 R"=

R=3, 5-DNB CDClg 6.00 0.92 5.0-5.2 6. 5-7.0

R'=CH3 R1l=Ts b 29 CDClg 4.85 6.5° ------R=H 2.5 R'=H R"=Ts 53

TABLE 2—Continued

Chemical Shift( 6 ja Coupling Constant(Hz) Compound Solvent Hx Methyl (R') XM M ,M e

R=3, 5-DNB CDClg 6.20 ------6.2 R'=H 1.0 R"=Ts 1

25 R=H CDClg 4.56 ------6.2 ------R,=II 1.8 R"=H

Chemical shifts are expressed in _ 6 -downfield from internal TMS standard, b

D 2 O added. c Apparent coupling constant; may be due to higher order system.

equatorial. A similar interaction has been reported for N-benzyl-3,5-di- 99 methylpiperazine (65) and the amide derivative ( 6 6 ). Conformation 65b is

energetically preferred over 65a. The addition of an amide group as in 6 6 produces a severe interaction so that the conformation in which the methyl group adjacent to the amide function is axial is heavily favored energetically. 54

65a 65b

0CH

The relative stereochemistry of ephedrine and pseudoephedrine, 101 and the C-9 configuration of the cinchona alkaloids has been determined

unambiguously by a chemical transformation which should be applicable to 102 the diastereoisomers A and B. Treatment of the quaternary salts of A

and B with potassium t-butoxide should result in the formation of the corres

ponding oxiranes. The ciis (threo) isomer 62_ should give rise to the cis

epoxide and the trans (erythro) isomer should give rise to the trans epoxide. 100,101 For a given pair of isomeric epoxides, Jc-[s is always larger than Jtrans*

Therefore, nmr analysis of the oxiranes should confirm the stereochemistry

of the benzazepine from which it was derived.

The nmr spectra of l-hydroxy-2,3,4,5-tetrahydro-3-benzazepine (25) is shown in Fig. 3. The benzylic proton subtends an angle of approximately 7.0 9.0 4.0 7.0

900 aoo 900 • crstoo too

: 0

• 0 7.0 «.0 s.o PPM ( i ) 4.0 3.0 3.0 1.0

Figure 3. Nmr spectrum (60 MHz) of a 10% solution of 2£in CDClg. d ci 56 I

90° with one of the adjacent protons in either chair conformation. Thus a

doublet might be expected to be observed for the benzylic C-^ proton. How

ever, as seen in Fig. 3, a quartet is observed. The addition of DgO or tri-

fluoroacetic acid resulted in no change in this quartet, therefore coupling with the hydroxylic proton is not responsible for the observed splitting. A

quartet could result if 2£ were in the boat conformation. However, the di hedral angles between the C-^ proton and the adjacent protons would be ap proximately 15° and 150°. A large coupling constant should result from both of these angles, giving a quartet with wide spacings between the inside and outside lines and a narrow spacing between the two inside lines, or possibly even a triplet.

The larger observed splitting is essentially the same as for compound

B (6.2 Hz) (Table 2), which, along with the IR spectra (intramolecular hydro gen bonding) indicate a chair conformation with an axial OH. The absence of the methyl group may be enough to produce a necessary change for coupling 103 with both adjacent protons to occur.

However the situation may be more complex, since the benzylic proton is now the X portion of an ABX system, and a first order interpreta- 89 tion may not be applicable. The observed four line splitting could possibly be the result of virtual coupling. The H-^ proton could be coupled to only one of the AB geminal nuclei and still give rise to more than two lines for the X part of the spectrum. For an ABX system the total distance between the 57

85 strxmg outer lines of the X part of the spectrum is equal to

In DMSO the larger splitting increased to 7.6 Hz, indicating a larger popula-

! tion of the equatorial OH conformation. The smaller splitting remained

about 2 Hz. Addition of trifluoroacetic acid to the DMSO returned the larger

observed J value to about 6 Hz, and the smaller to about 1.4 Hz. Addition of

trifluoroacetic acid to a CDClg solution of 25_ changed the quartet to a doublet

with J = 5. 8 Hz.

Synthesis of Isomeric Cocaines

Allopseudococaine was prepared essentially by the method of 104 105 Findlay, utilizing slight modifications of Sinnema, et al. The com pound 2-carbomethoxytropinone (69) represents a key intermediate in this synthetic scheme. This intermediate ketone was prepared by condensation

of succindialdehyde (67), methylamine, and monomethyl P-ketoglutarate ( 6 8 ), 106 in a manner similar to Robinson's tropinone synthesis. The mono methyl ester was prepared from (3-ketoglutaric acid (70), by way of the correspond ing anhydride 71. 58

c o 2c h 3 c h 2- c h o

I + c h 3 nh2,hci + ch 2 c h 2- c h o I c=o I 67. CH 2 69 c=o I 0-H

p -ketoglutaric acid (70) was cyclized to the corresponding p-ketoglu-

taric anhydride (71) by treatment with a mixture of glacial acetic acid and

acetic anhydride. Treatment of 71 with cold methanol followed by one hour

at room temperature gave a methanol solution of the monomethyl ester 6 8

which was used directly in the preparation of 69.

/ C H 2-C 0 2H CH3C 02H / CH2_C\ MeOH ^ 0=C Tr^-vt > 0=C O ' - • • • > 68 X CH2-C 0 2H (CH3c ° )2 0 X CH2-C ^ o

70 71

Succindialdehyde (67) was prepared by two different methods. One method involved the treatment of pyrrole with hydroxylamine hydrochloride and potassium hydroxide to give succindialdoxime (72). This dioxime was 59

suspended in 1 N sulfuric acid and a solution of sodium nitrite in water added

over a period of three hours. After two more hours under nitrogen the mix ture was neutralized with barium carbonate and filtered through C elite to give a solution of succindialdehyde which was also used directly in the syn thesis of (39.

An alternate and more simplified method utilized commercially availa ble 2 ,5-dimethoxytetrahydrofuran (73), which upon treatment with dilute acid and neutralization afforded the desired solution of succindialdehyde.

NH2 OH* HC1 NaNO

H 72

Although ( 3 -ketoglutaric acid (acetonedicarboxylic acid) is available commercially, large quantities were desired; thus initially it was prepared from citric acid by reaction with fuming sulfuric acid. However, difficulties 60

c o 2h c o 2h

? H 2

c h 2 ? H 2

c o 2h c o 2h

were encountered in purifying the crude product. The crude acid reportedly decomposes after a few hours, but if recrystallized from ethyl acetate and 107 thoroughly dried will remain stable for some time. Attempts at recry- stallizing the crude sulfuric acid contaminated product resulted in extensive decomposition, presumably to acetone and carbon dioxide, since very little product could be recovered. Since crude acetonedicarboxylic acid can be 108 used directly in the synthesis of tropinone, seemingly the crude material could similarly be used in preparing the anhydride. However, treatment of the crude acid with acetic acid and acetic anhydride resulted in an impure product, not the desired anhydride. Further examination of the litera- 109,110 ture revealed that the use of crude sulfuric acid containing acetone dicarboxylic acid results in considerable formation of the carbon acetylated product 74, which can further cyclize to give the 5-carboxy derivative of de- hydroacetic acid 75. .A. glacial H02c-c CH-C02H 3 ^ ^ 70 HoAc (Ac)20 (h2so4) o=c c=o

ch3 ch3 75

109 Kaushal also reported isolating the enolic pyrone shown below. Later

111 reports have indicated structure 76 to be the correct structure of the compound previously reported to be 75. The use of pure acetonedicarboxylic

-CH CH

76 acid gave the pure anhydride 71. ■

Removal of the sulfuric acid from acetonedicarboxylic acid could be accomplished without recrystallization by thorough repeated washings with 112 ethyl acetate. Product purified in this manner was found suitable for an

hydride formation.

A considerable quantity of resinous by-products generally accompanied

the formation of the intermediate ketone 69, due probably to intermolecular

condensations. However, in most cases succindialdoxime was used as the

precursor for succindialdehyde. In a study on the synthesis of tropinone by 108 the Robinson condensation, Schmidt et al. report diethoxytetrahydrofuran

to be "a far superior aldehyde source," which gives better and more repro

ducible results.

The crude brown solid 2-carbomethoxytropinone (69) obtained was best

purified by vacuum sublimation. The ketone could be recrystallized with

some difficulty from several solvents, but generally colored, less pure pro

ducts were obtained.

Reduction of sublimed 2-carbomethoxytropinone (69) with platinum

catalyst in acetic acid and water afforded racemic allopseudoecgonine methyl

ester (76) as a colorless oil which could be purified by forming the acetate

salt. Treatment of the acetate with potassium carbonate solution followed by

ether extraction gave the solid methyl ester which could be recrystallized from petroleum ether. 63

O2 CH3 H 2 Pt ^ HOAc ‘OH

69 76

Allopseudoecgonine methyl ester (76) was benzoylated with benzoyl

chloride in dry pyridine to give allopseudococaine (77) as the hydrochloride.

0COC1 76 > pyridine

CO0 77

The low yields obtained in the benzoylation reaction are presumably due to

the axial.position of the hydroxyl group since much less difficulty is encoun tered in the benzoylation of pseudoecgonine methyl ester, in which the hy- 105 droxyl group is equatorial. Sinnema et al_. have performed nmr studies of the isomeric ecgonines and cocaines.

Pseudoecgonine methyl ester (79) was prepared by refluxing cocaine 113 free base (78) in methanol with sodium methoxide. Benzoylation of 79^ 64 with benzoyl chloride and pyridine afforded pseudococaine (80), purified as the hydrochloride.

CHr V

NaOMe 0COC1 pyridine - C - 0 MeOH

78 79

The isomeric allopseudococaine (77), pseudococaine (80), and cocaine

(78), and their respective ecgonine methyl esters were examined by thin layer chromatography and gas liquid partition chromatography (glpc). Thin layer chromatography (TLC) on Silica Gel GF provides an excellent and facile method of identification of the three cocaines 77, 78^ and 80, each having well separated Rf values. Pseudococaine and pseudoecgonine methyl ester are easily separated on TLC with acetone as developing solvent. Since these results are readily transferred to column chromatography, using

Silica Gel G for columns, this provides an excellent method of separating 65

pseudococaine from pseudoecgonine methyl ester. Unfortunately allopseudo-

cocaine (77) and allopseudoecgonine methyl ester (76) gave nearly identical

Rf values on TLC. The cocaines were easily separated from their respec

tive ecgonine methyl esters by glpc, the benzoylated derivatives possessing

a much longer retention time than their hydroxylic precursors. Although

allopseudococaine (77) and pseudococaine (80) were easily separated by TLC,

their retention times were nearly identical in glpc.

Benzoylecgonine (81) was obtained by partial hydrolysis of cocaine free base (78) by dissolving in boiling water.

C 0 2 CH3

fi H 78 81

Attempts to prepare ecgonine methyl ester by methanolic hydrolysis of cocaine hydrochloride resulted in largely unhydrolyzed starting material. 66

B. BIOLOGICAL

Results. —Cumulative dose-response curves to norepinephrine was

obtained before (control) and after (experimental) incubation of the vas

deferens with the test drug, unless no change in the experimental dose-

response curve occurred. In this case the experimental curve was taken

as the control norepinephrine curve, and either a higher dose of test drug

used next or a different test drug. The cocaine-related drugs tested for

norepinephrine potentiating effects and their relative activities are shown in Table 3.

For pseudococaine and allopseudococaine concentrations were selected that would give equiactive potentiating responses with cocaine. Thus a con- -4 centration of 10 M of pseudococaine and allopseudococaine was found to be about equiactive to 10- M cocaine, or a 100 fold difference in concentration.

A norepinephrine dose-response curve in the presence of cocaine was ob tained for each tissue as a control for that tissue. Log-dose-response curves for cocaine, pseudococaine and allopseudococaine are shown in Figure 4.

Negative log molar ED50 was calculated for each observation, and the averages, standard error of the means, and dose ratios obtained. These values are shown in Table 4 for the isomeric cocaines, tropacocaine and pseudotropacocaine. The number of observations, concentrations, and rela tive activities of the compounds tested for norepinephrine potentiation are shown in Table 5. Log-dose-response curves were not plotted for those com- TABLE 3

RELATIVE ACTIVITIES OF ISOMERIC COCAINES AND RELATED COMPOUNDS

ON RESPONSE OF RAT VAS DEFERENS TO (-)-NOREPINEPHRINE

Compound Structural Formula Compound Structural Formula CH (-)-Cocaine COQCH (±)-Allo-ii-Ecgonine

1 Methyl Ester

> 1 /I 0 0 0 th -C -0

H OH

CH (+M -Cocaine Tropine Benzoate l/lOOth (i|r-tropacocaine) l / 30th

-C -$

(±) -Allo-ij; -Cocaine i(r-Tropine Benzoate l/lOOth (tropacocaine)

l / l 0 0 th i-C-tf O -C -0 TABLE 3—Continued

Compound Structural Formula Compound Structural Formula CH< V Benzoylecgonine Tropine

> 1 /I 0 0 0 th > l / l 0 0 0 th -C -$

iji-Ecgonine i]r-Tropine

Methyl Ester 31111 > l / l 0 0 0 th

> 1 / 1 0 0 Oth

o CO % CONTRACTION 100 120 0 4 20 60 0 6 t (-)-oeiehie eoe n afer ccie ( cocaine r fte a and before -norepinephrine ) - ( ith w ds-epne uv of oeiehie a rpae. rial i ndiat sadr err of o errors standard te ica d in s e lin l ertica V repeated. was norepinephrine f o curve dose-response a th e mean. Each poin t o f the curves represents an average o f f o average an represents curves the f o t poin Each mean. e th oan (0%) Cnetain of h potntatng aet wr add o te ah mn before min. 3 bath the to added were agents g tin tia ten o p the f o Concentrations ). (10“% cocaine I-)- NOREPINEPHRINE NOREPINEPHRINE I-)- WfTH IQ '6 COCAINE '6 IQ WfTH iue uuaie o-oersos cre cntutd n sol ed rt a deferens vas rat d te la o is on constructed curves log-dose-response Cumulative Figure KT'to' (-)-NOREPINEPHRINE IH )4()ALL0- C0CAI E IN A C 0 -C -^ 0 L L (i)-A K)-4 WITH A GNS ( ) (m AGONIST 0 1 %) alpedccie ( allopseudococaine ), "% 8 - 6 ---- observations. § ( -)-NOREPINEPHRINE -)-NOREPINEPHRINE ( WITH I 0 ‘4 ^ COCAINE ^ ‘4 0 I WITH 0 1 % ad pseudo and "%)

TABLE 4

NOREPINEPHRINE POTENTIATING EFFECTS OF ISOMERIC COCARTES

AND TROPACOCAINES ON RAT VAS DEFERENS

-Log Molar ED50 of -- (-)-Norepinephrine with S.E.M. Concentration 3 min. after d a ^ b Treatment (Molar) Control Treatment Difference n Dose Ratio -4 (+) -pseudococaine 1 0 5.25±0.05 5.84±0.05 0.59±0.04 8 3.89

(6 . 37-3.13)C -4 (±)-allops eudo- 1 0 5 .29±0.04 5 .82±0.06 0 .53±0.03 6 3.40 cocaine (4.06-2.85)

- 6 (-)-cocaine (control 1 0 5 .24±0.04 6 . 0 1 ± 0 . 1 0 0 .76±0.09 7 5.75 for pseudococaine) (9.89-3.41)

- 6 (-)-cocaine (control 1 0 5. 28±0.05 5 .93±0.10 0 .65±0.06 6 4.47 for allops eudococaine) (4.83-3.12)

tropine benzoate 3 x 10 " 5 5.06±0.07 5.51±0.10 0.45 9 2.80 (pseudotropacocaine) I rH o pseudotropine ben 4 .99±0. 04 5 .10±0.09 0 . 1 1 1 1 1.26 zoate (tropacocaine)

- 3 © TABLE 4—Continued

a b c Number of observations. Antilog [(-log ED50 of lai’ger value)-(-log ED50 of smaller value)]. Numbers d in parentheses are 95% confidence intervals. In log units. TABLE 5

RELATIVE ACTIVITIES OF ISOMERIC COCAINES AND RELATED COMPOUNDS ON

NOREPINEPHRINE INDUCED CONTRACTIONS OF RAT VAS DEFERENS

Number of Bath Concentration Compound Observations (Molar) Potentiation

(-)-cocaine 19 i c f 6 1

(+) -pseudococaine 8 io ”4 l / l 0 0 th

(±) -allopseudococaine 6 IO- 4 l / l 0 0 th tropacocainea 7-12 io - 4 l/lOOth

pseudotropacocainea 7-12 3 x IO" 5 l/30th

benzoylecgonine 8 3 x IO - 3 > l / l 0 0 0 th

pseudoecgonine methyl ester 2 io"3 > l / l 0 0 0 th -3 (±)-allopseudoecgonine methyl ester 4 3 x 10 > l / l 0 0 0 th 1 rH O tropine 1

1 1 1 W

4 t— O > l / l 0 0 0 th

pseudotropine 1 1 0 " 4

4 1 0 " 3 > l / l 0 0 0 th to TABLE 5—Continued

a J. B. LaPidus, D. Canowitz, C. Ross, and P. N. Patil, unpublished data; cf. ref. 24, p. 1216. 74 pounds showing no significant potentiation. None of the compounds exhibited any visible agonist effects of their own. A typical control dose-response curve and a test curve for which no potentiation occurred is shown in Figure

Although tropine benzoate exhibits norephinephrine potentiating effects, the structurally similar atropine, homatropine, and benztropine show o r -

adrenergic blocking activity (Table 6 ). The pA2 values were obtained graphi

cally by plotting log (dose ratio- 1 ) against log molar concentration of antago- 114 nist according to the method of Arunlakshana and Schild. Increasing con centrations of the antagonist produced a parallel shift of the log-dose-response

curves to the right, as shown in Figure 6 for homatropine hydrobromide. A three minute incubation period was used for all test drugs.

The tetrahydrobenzazepine derivatives, l-hydroxy-2,3,4,5-tetrahydro-

3-benzazepine (25) and the diastereomeric 1-hydroxy-2-methyl-2,3,4,5- tetrahydro-3-benzazepines (46) and (47) were also tested for biological acti vity on the isolated rat vas deferens system.

Compound 25 showed no appreciable direct, potentiating, or blocking -4 -3 activity at 10 M. At relatively high concentrations (10 M) it exhibited a direct response, giving a maximum height of slightly less than one-half that of the norepinephrine control. Pretreatment with cocaine only slightly anta gonized the response. It also showed no appreciable beta activity on isolated guinea pig atria. (-)-NOREPINEPHRINE/* CONTROL / /3 X IO -*

i - 5 * 10” 3 MIN

IO-3 3xiO"7 ^-ECGONINE METHYL ESTER

Figure 5- Cumulative dose-response curves constructed on isolated rat vas deferens with (-)- norepinephrine. Wo potentiation was observed after incubating for 3 min. with pseudoecgonine methyl ester and repeating the norepinephrine dose-response curve.

V* 76

TABLE 6

RELATIVE ACTIVITIES FOR ALPHA-ADRENERGIC

BLOCKADE BY ATROPINE RELATED COMPOUNDS

Compound Structural Formula

Atropine Sulfate

pAg = 5.10

CHoOH -C-CH

I N0 Homatropine HBr

pA^ = 4# 35

OH -CH

CH Benztropine Mesylate

non-competitive

-CH

CH Hyoscine HBr

none CHoOH -C-CH

O % % CONTRACTION 100 120 0 6 0 4 0 6 20 t (-)-oeiehie eoe n afer hmtoie B (05, x 0% 1“ . Concentra ). 10“% 10“%, x 3 (10“5m, HBr homatropine r fte a and before -norepinephrine ) - ( ith w i of oarpn Hr ee de t he ah mn bfr a oersos cre norepine f o curve dose-response a before min. 3 bath e th to added HBr were repeated. was homatropine phrine f o s n tio (-)-NOREPINEPHRINE WITH HOMATROPINE HBr HBr HOMATROPINE WITH Figure Figure I -5M) (IO 6 Cmltv lgds-epne uvs osrce o iol ed rt a deferens vas rat d te la iso on constructed curves log-dose-response Cumulative . (-)-NOREPINEPHRlNE WITH HOMATROPINE HBr HBr HOMATROPINE WITH 3 ( -6 d x GNS ( ) (m AGONIST 5M) '5 0 | (-)-NOREPINEPHRINE WITH HOMATROPINE HBr HBr HOMATROPINE WITH (IO'4M)

Kf4 78

Of the two diastereomeric benzazepines, compound A showed no agonist

activity even at high concentrations, but did appear to have a slight blocking -3 action at 10 M. On the other hand, while compound B also gave no direct -4 agonist response, in 10 M concentration it potentiated the response of nor

epinephrine to almost the same degree as pseudococaine and allopseudococaine

at the same concentration. High concentrations increased its potentiating

effect. The following values were obtained for compound B.

-Log Molar ED50 N o repinephrin e 3 min. after Treatment Control Treatment Difference Dose Ratio n

Compound B 5.31 5.85 0.54 3.47 5

( 1 0 “% )

Discussion. —The optical isomers of cocaine were reportedly studied 115 for local anesthetic activity in 1923. However, very little study has been done on the steric structure-activity relationship of the cocaine molecule for 116 inhibition of norepinephrine uptake. Schmidt et al. investigated central and peripheral effects of (-)-cocaine and (+)-pseudococaine, and found only 79

(-)-cocaine to potentiate epinephrine. This is in contrast to our results using

norepinephrine. However, it has been shown that epinephrine is taken up by 48 the nerve to a lesser extent than norepinephrine, which may account at

least in part for this discrepancy. In addition, the test tissues used were not

the sam e.

Pseudococaine and (±)-allops eudococaine potentiated norepinephrine

response to about the same degree. The 2-carbomethoxy group of each is

oriented in the equatorial position, but the 3-benzoyloxy group is axial in the

alio series and equatorial in cocaine and pseudococaine.

The presence of the 3-benzoyloxy group appears to be more essential

for potentiating activity than does the 2-carbomethoxy group. Pseudoecgonine

methyl ester and (±)-allopseudoecgonine methyl ester'exhibited no significant

potentiating effects, while both tropine and pseudotropine benzoate potentiated

norepinephrine response. Thus while the sampling of compounds is too in

complete to permit any definite conclusions, the presence of a benzoyloxy

group appears to be necessary for potentiating activity. However the pre

sence of a benzoyloxy group does not automatically insure this activity, as

evidenced by the results with benzoylecgonine, the partial hydrolysis product

of cocaine. These compounds differ only in that cocaine is the methyl ester

of benzoylecgonine. Thus the more polar free carboxylic acid group apparent ly prevents the normal action of the methyl ester derivative, probably due to the zwitterionic nature of benzoylecgonine. « 80

A comparison of the racemic threo- and erythro-methylphenidates 117 was made by Buckner eta l., and the threo isomer was found to have a

300-fold greater potency than the erythro isomer in potentiating norepinephrine response on rat vas deferens. A structural comparison was made between the chair form of cocaine and the most stable conformations of threo- and I 1 1 1 erythro-methylphenidate. There is a 60° skew relationship between the nitrogen and the carbomethoxy groups in both cocaine and threo-methylphenidate, whereas in the less active erythro -methylpheni date the nitrogen and the carbomethoxy groups are trans-staggered.

o c h 3

o=c I c h 3

(-)-Cocaine (4) -Pseudococaine (±) -Allops eudoco caine

(±) -Threo-Methylphenidate (±) -E rythro -Methylpheni date 81

It is interesting to note that the equatorial carbomethoxy group of both (+)-

pseudococaine and (±)-allopseudococaine is trans to the nitrogen, and that

at 10“4M concentration, the dose ratio of pseudococaine (3.89) and allopseudo-

cocaine (3.40) is similar to that reported for erythro-methylphenidate (3.7).

No conclusions can be drawn as to absolute steric activity requirements

for the cocaine molecule until all stereochemical and optical isomers are

examined. Nevertheless it becomes apparent from the compounds examined

that, regardless of the mechanism by which cocaine potentiates, a steric

and structural specificity exists, and that the steric isomers of cocaine re

tain potentiating activity.

Atropine, homatropine, benztropine, and hyoscine are all derivatives

of tropine (3-a-tropanol). While neither tropine nor the isomeric pseudo-

tropine (3-(3-tropanol) exhibited potentiating or a-blocking activity, the ben- i zoate esters of both tropine and pseudotropine potentiated norepinephrine

response. However, the tropate ester of tropine (atropine) and the mandelate

ester of tropine (homatropine) both competitively blocked a-adrenergic recep tors. The benzhydryl ether of tropine (benztropine) also displyaed a-blocking activity but appeared to be acting in a non-competitive manner. Hyoscine hydrobromide, structurally identical to atropine except for the presence of a

6 p, 7(3 -epoxy group, possessed neither af-blocking nor norepinephrine poten tiating activity. Thus small structural changes in either the tropane nucleus 82

or substituents can effect a change in biological activity varying from inactive

to a-blockade to potentiation.

The low potency exhibited by l-hydroxy-2,3 ,4 ,5-tetrahydro-3-benz-

azepine may be due partially to the restricted rotation available to the ring

nitrogen. While hydrogen bonding between the hydroxyl and the ring nitrogen

can occur in one chair and one skew boat form, the nitrogen cannot orient

itself trans to the phenyl ring, as in the favored hydrogen bonding conforma

tion of N-methyl phenylethanolamine. The restricted rotation of the benzene

“N-CH3

N-Methyl phenylethanolamine l-Hydroxy-2, 3,4,5-tetrahydro- 3-benzazepine

ring and the testing as racemates may also be factors. These same factors are also present in the diastereomeric l-hydroxy-2-methyl-2,3,4,5-tetrahydro-3-benzazepines. 118 LaPidus et al. proposed that the phenyl, hydroxyl, and amino group of both ephedrine (erythro) and pseudoephedrine (threo) can occupy the same 83

three sites on a receptor surface. Only the configuration of the methyl group

a to the secondary amine differs, and may be responsible for their different

biological responses.

CH HCH HCH3

c h 3

Ephedrine (erythro) Pseudoephedrine (threo)

-H CH3 -H

trans (erythro) -1-Hydroxy-2 - cis (threo)-1-Hydroxy-2- methyl-2,3,4,5-tetrahydro-3- methyl-2,3,4,5-tetrahydro-3- benzazepine benzazepine

As evidenced by the conformational comparison of the ephedrines and

their cyclic analogs, the same configurational difference applies. However,

in the open chain systems, the methyl group on the nitrogen can rotate away

from the OH-N side. But in the tetrahydrobenzazepines, either the four or the five carbon must protrude into this area. This may decrease effective 84

bonding to a biological site, and contribute to the lower potency observed in these compounds. The greater activity of compound B compared to A in po tentiating the effects of norepinephrine is qualitatively analogous to the re

sults reported for the racemic erythro- and threo-3-amino- 2 -phenyl- 2 -buta- 46 nols and racemic ephedrine (erythro) and pseudoephedrine (threo). The

erythro isomers have been the most active, which is consistent with the as

signment of the erythro configuration to compound B. EXPERIMENTAL

A. SYNTHETIC

Melting points were obtained using a Thomas-Hoover melting point apparatus, and are uncorrected. Infrared spectra were obtained with a

Perkin-Elmer Model 257 and/or Model 237 grating spectrophotometer, and a Digilab Model FTS14. The NMR spectra were recorded with a Varian

A-60A NMR spectrometer at 60 MHz using TMS as an internal reference.

Gas chromatographs were taken using an F & M Model 402 gas chromato graph equipped with flame ionization detector and glass columns. Elemental analyses were determined by Alfred Bernhardt Microanalytical Laboratory,

Fritz-Pregl-Strasse 14-16, West Germany.

Phenethylamine tosylate (33). —Standard procedures were used in the preparation of this compound. To a cooled solution (5°) of phenethylamine

(55 ml, 0.437 mol.) in pyridine (200 ml) was added with stirring a solution of p-toluenesulfonyl chloride (83.3 g, 0.437 mol) in pyridine (200 ml). After stirring at room temperature for 28 hours, the red solution was cooled in an

ice bath and acidified with 2 0 per cent hydrochloric acid to afford an orange solid. The solid was dissolved in ether and combined with the ether extracts of the supernatant liquid. The ether solution was washed with dilute hydro-

85 86

chloric acid (3X) and water (3X), then dried (saturated sodium chloride solu

tion, magnesium sulfate) and the ether removed under vacuum to give a light tan solid. Recrystallization from benzene-petroleum ether (30-60°) afforded

91.5 g (76%) of white needle crystals, mp 64-65°; as hard colorless cubes, o 62 o - i 70-71 ; lit. mp 6 6 . ir , cm , (CHClg) 3300 (NH), 1330, 1160 (SO 2 ). 62 N-Phenethyl-N-toluene-p-sulfonylglycine (35) . —Phenethylamine to

sylate (27.5 g, 0.1 mol), ethyl chloroacetate (20 g, 0.164 mol), anhydrous potassium carbonate (26 g, 0.188 mol), and anhydrous acetone (300 ml) were stirred together at reflux for 24 hours. The potassium carbonate was fil tered off and the acetone removed under vacuum. The remaining yellow oil was washed with water, and extracted with chloroform. Removal of the chloroform in vacuum left a white solid which showed no NH peak in the infra red. This ester was dissolved in 125 ml of methylated spirits (ethanol: methanol, 19:1), and 10 per cent sodium hydroxide (250 ml) added. After

refluxing for 1 l/4 hr., the alcohol was distilled off. The remaining aqueous mixture was cooled (15°) and water added to keep the solid in solution. The

mixture was acidified with cold 2 2 % hydrochloric acid and placed in the re frigerator overnight. The white solid precipitate was collected on a buchner and dried over phosphorous pentoxide under vacuum (50°). The dry solid

(32 g, 96%) was recrystallized from benzene to give 27.6 g (83%) of the acid,

mp 146-147°, lit . 6 2 mp 148-149°. ir , cm ”1, (KBr) 3400-2500 (COOH), 1790-

1690 (COOH), 1600 (Ar), 1350, 1160 (SO2 ). 64 3-p-Toluenesulfonyl-2,3,4,5-tetrahydro-3-benzazcpine-l-one (38) . —

A mixture of 6 6 . 6 g (0.2 mol) of N-phenethyl-N-toluene-p-sulfonylglycine

(35), thionyl cliloride (40 g, 0.336 mol) and dry benzene (350 ml) was stirred

for 2 hr. at 50-80°, then left at room temperature overnight, protected from

moisture. The benzene and thionyl chloride were removed under vacuum to

leave the acid chloride as an orange oil. The infrared displayed no absorp

tion in the carboxylic acid carbonyl or OH region, but gave strong absorption

for an acid chloride. A rapidly stirred suspension of aluminum chloride

(80 g, 0 . 6 mol) in dry dichloromethane (400 ml) was cooled with dry ice and

acetone (-78°) and the above acid chloride in dry dichloromethane (200 ml)

added dropwise over a three hour period. Stirring was continued at -78° for o 2 hr. longer, and the mixture then allowed to warm gradually to - 2 0 over a

three hour period. The cooling was removed and the mixture stirred at room temperature for another three hours. The yellow-orange reaction mixture was poured onto an ice-dilute hydrochloric acid mixture and extracted with di chloromethane. The dichloromethane extract was decolorized with charcoal

(2X), dried over magnesium sulfate, and the dichloromethane removed in vacuum. Recrystallization of the resulting solid from ethanol afforded 51.5 g o 64 o -1 (82%) of white rhombic crystals, mp 156-157 , lit. mp 156-157 . ir, cm ,

(CHClg) 1685 (C=0), 1600 (Ar), 1350, 1160 (S02); nmr (CDClg), 6 , 2.35

(singlet, 3H, CH 3 ), 2.95 (triplet, 2H, CHg), 3.65 (triplet, 2H, CH2), 4.2

(singlet, 2H, CH 2 ), 7.25 (multiplet, 8 H, aromatic). 88

l-Hydrox.y-3-p-toluenesulfonyl-2,3 ,4 ,5-tetrahydro-3-benzazepine 64 (39). —The preceding ketone 38J12 g, 0. 038 mol) was suspended in 95% ! ethanol, and sodium borohydride (1.5 g, 0.039 mol) added in portions. The

mixture was warmed to 50° for l/2 h r., then left overnight at room tempera

ture. The reaction mixture was poured onto ice-dilute hydrochloric acid,

extracted with chloroform, and the chloroform washed with dilute sodium

carbonate solution and dried (magnesium sulfate). Removal of the chloroform

in vacuum left a white fluffy solid that recrystallized from benzene-petroleum

C*A ether (30-60°) to afford 10.97 g (92%) of the alcohol, mp 116-117°, lit.

mp 118-119°. ir, cm (CHClg) 3600 (free OH), 3500 (broad, hydrogen

bonded OH); nmr (CDClg), 6 , 2.34 (singlet, 3H, CHg), 2.5-3.5 (multiplet,

7H, methylene, OH), 4.95 (broad quartet, 1H, benzylic H^), 7.15, 7.62

(multiplet, 8 H, aromatic) (see Table 2).

3 ,5-Dinitrobenzoate ester of 39. —To a solution of 3 ,5-dinitrobenzoyl chloride (0.5 g) in pyridine was added the alcohol 39_ (0.1 g). The mixture was warmed for 15 m in., then left at room temperature for 1 hr. The mix ture was poured onto ice, dilute HC1, extracted with ether, and the ether solu tion dried over magnesium sulfate. Removal of the ether left a pale yellow solid that was washed with hot methanol and recrystallized from ethanol: chloroform to give pale yellow needles, mp 200-201.5°. ir, cm *, (CHClg),

1730 (ester C=Q), 1545, 1345 (NOg), 1350, 1160 (SOg); nmr (CDCI 3), 6 , 2.38 89

(singlet, 3H, CHg), 2.7-4.7 (broad multiplet, 6 H, methylenes), 7.23, 7.62

(multiplet, 8 H, aromatic), 9.18 (singlet, 3H, aromatic).

l-Hydroxy-2,3 .4 .5-tetrahydro-3-benzazepine (25). —Sodium/ammonia:

The sulfonamide 39 (2 g, 0.0063 mol) was dissolved in ether (100 ml) by warming. The ether solution was added to liquid ammonia (300 ml) which contained enough sodium to give a blue color. The mixture, cooled in a dry

ice/acetone bath, was stirred for 2 h r., and sodium added periodically to maintain the blue color. Ammonium chloride was added until the mixture was colorless, and the ammonia removed under a stream of nitrogen. Ex cess ether was added and the insoluble salts filtered off and washed with hot benzene. Removal of the combined solvents under reduced pressure left a light yellow solid that recrystallized from benzene to give 0.673 g (65%) of o the free amine, mp 130-131 . 70 Lithium Aluminum Hydride : To a stirred suspension of lithium alumi- 119 num. hydride ( 6 g, 0.158 mol) in dry tetrahydrofuran (100 ml) was added

6 g (0. 019 mol) of the sulfonamide 39 in dry tetrahydrofuran (50 ml). The mixture was stirred at reflux for 36 hours. The excess hydride was carefully decomposed with moist ether and water, and extracted with ether to give 1. 7 g

(55%) of the free amine, identical to that obtained from the sodium/ammonia

cleavage reaction, ir, cm \ (CHCI 3 ) 3600 (free OH), 3500-3200 (hydrogen

bonded OH, NH, peak at 3360). No SO 2 absorbance for tosyl; nmr, see Fig.

3, Table 2. 90

Anal. Calcd. for C 1 0 H1 3 NO: C, 73.62; H, 7.97;N, 8.59. Found:

C, 73.67; H, 7.84; N, 8.67. 64 1-M ethyl-N-p-toluenesulfonyl-(3-phenethylaminoacetic acid (41)

Phenethylamine tosylate (60 g, 0. 21 mol), ethyl-2-bromopropionate (45 ml,

0. 36 mol), anhydrous potassium carbonate (44 g, 0.318 mol) and dry acetone

(750 ml) were stirred at reflux for 133 hours. The acetone solution was de

canted off the potassium carbonate and the acetone removed under reduced

pressure. The resulting oil and the potassium carbonate were treated with

water and extracted with ether. The ether solution was washed with dilute

sodium hydroxide (2X), water (IX), dried (magnesium sulfate) and the ether

removed to give the ester as a yellow oil. The infrared displayed no NH peak.

The ester was dissolved in methanol (200 ml), warmed to 50°, and treated

with 10% sodium hydroxide (200 ml) and water (1 L). After stirring for 2

hours at 50° the mixture was left at room temperature overnight. It was then

acidified (22% HC1) and the gummy precipitate dissolved in dilute sodium bi

carbonate solution and extracted with ether. The aqueous solution was again

acidified and extracted with ether. The ether solution was dried (magnesium

sulfate) and the ether removed to give a yellow viscous oil that solidified to a white mass. Recrystallization from benzene-petroleum ether (30-60°) afforded 64 the pure acid as a white solid. (60.3 g, 82. 7%). mp 99-100°, lit. 99-100°.

2-Methyl-3-p-toluenesulfonyl-2,3,4 ,5-tetrahydro-3-benzazepine-l-one 64 (43) . —A mixture of 20 g (0.057 mol) of the acid 41, thionyl chloride (12 g, 0.1 mol) and dry benzene (100 ml) were heated at 50° for 1 l/2 hours. The

thionyl chloride and benzene were then removed under reduced pressure to

give the acid chloride as a red oil. The acid chloride was dissolved in dry

dichloromethane (70 ml) and added dropwise over a 3 hour period to a rapidly

stirred suspension of aluminum chloride (38 g, 0.28 mol) in dry dichloro

methane (120 ml) at -78°. The temperature was maintained at -60 to -78°

for an additional three hours, and then the mixture stirred at room tempera

ture for 3 hours. The reaction mixture was poured over ice-dilute HC1 and

extracted with dichloromethane. The organic layer was washed with dilute

sodium hydroxide, water, dried (magnesium sulfate), and the dichloromethane

removed under reduced pressure to give a reddish oil which, when treated

with methanol-ether, afforded 6.35 g (33.5%) of pale yellow solid. Recry- o 64 o -1 stallization from methanol gave 4.5 g, mp 132-134 , lit. 132 . ir, cm , I

(CHC13) 1685 (C=0), 1350, 1160 (S02); nmr (CDClg), 6 , 1.46 (doublet, 3H,

methyl), 2.33 (singlet, 3H, methyl), 2.75-4.18 (multiplets, 6 H, m ethylenes),

4.84 (quartet, 1H, methine), 7.20 (multiplet, 8 H, aromatic).

l-Hydroxy-2-methyl-3-p-toluenesulfonyl-2,3 ,4 ,5-tetrah.ydro-3-benz-

azepine (44) and (45). —The tosyl ketone 43_ (5 . 8 g, 0.0176 mol) was dissolved

in alcohol ( 1 0 0 ml) with gentle warming, and sodium borohydride ( 1 . 2 g,

0.032 mol) added in portions. The mixture was stirred at 60-80° for l/2 hr.

then at room temperature for 8 hours. The reaction mixture was then poured over ice-dilute hydrochloric acid and extracted with chloroform, The chloro 92 form solution was washed with dilute sodium carbonate, dried (magnesium sulfate) and the chloroform removed under reduced pressure to give a pale yellow solid. Recrystallization from benzene-petroleum ether afforded

4.72 g (81%) of white crystals, a mixture of diastereoisomers melting over a variable range. The diastereoisomers were separated by column chroma tography on Silica Gel G for columns, using a mixture of ether: Skelly C

(2:1) as eluent. The diastereoisomer first off the column (A) melted at 135-

136°. The second isomer (B) melted at 156.5-157. 5°. ir, cm-1, (CHClg),

3600 (free OH), 3500 (broad, hydrogen bonded OH), 1325, 1160 (SO 2 ); nmr see text for discussion, and Table 2.

3, 5-Dinitrobenzoate ester of compound A and B tosylates.—A solution of 3 ,5-dinitrobenzoyl chloride (0. 5 g) in benzene-pyridine was added to a solu tion of compound B tosylate (0. 050 g) in pyridine (3 ml) and the mixture warmed on a steam bath for 15 min. After sitting for two hours at room tem perature, the mixture was poured over ice, dilute sodium bicarbonate added, and extracted with chloroform. The chloroform solution was dried and the chloroform removed to give a red oil, which solidified on adding ether. The solid was recrystallized from methanol-chloroform to give pale orange o - 1 needles (25 mg), mp 201-202 . ir, cm , (CHCI 3 ), 1730 (ester C=0), 1550,

1345 (NOg), 1345, 1160 (S02); nmr (CDClg), 6 , 0.92 (doublet, 3H, methyl),

2.38 (singlet, 3H, CHg), 2. 6-4. 3 (multiplet, 4H, methylenes), 4.58 (multi plet, 1H, methine), 6.00 (doublet, 1H, benzylic H^), 7.25, 7.68 (multiplets, 93

8 H, aromatic), 9.23 (multiplet, 3H, aromatic). See Table 2 for coupling

constants, text for discussion. Compound A: Prepared by same procedure

as above. Recrystallized from ethanol:methanol: chloroform mixture, mp

171-173°. nmr: See Table 2.

l-Hydroxy-2-methyl-2,3,4.5-tetrahydro-3-benzazepine (46) and (47). —

The two diastereomeric tosylates were cleaved by identical methods. To a

stirred suspension of lithium aluminum hydride (4 g, 0.105 mol) in dry tetra

hydrofuran (80 ml) was added a solution of the sulfonamide (4 g, 0. 012 mol)

A in tetrahydrofuran (20 ml), and the reaction stirred at reflux for 31 hours.

The excess hydride was decomposed cautiously with moist ether and water,

and extracted with ether. The ether solution was washed with dilute sodium hydroxide, dried (magnesium sulfate) and the ether removed under reduced pressure to afford a white solid. Recrystallization from ether gave 0.9 g o (42%) of pure A, mp 151 . Detosylation of B was accomplished in a similar manner, mp 100-101°. ir, cm \ (CHClg), 3610 (free OH), 3500-3200

(hydrogen bonded OH, NH), no sulfonamide absorbance; nmr: See Fig. 1 (A),

Fig. 2 (B), Table 1.

Compound A : Anal. Calcd. for C^-jH-^NO: C, 74.59; H, 8.47; N, 7.90.

Found: C, 74.50; k, 8.34; N, 8.00.

Compound B: Anal. Calcd. for C^-jH^gNO: C, 74.59; H, 8.47; N, 7.90.

Found: C, 74.42; H, 8.54; N, 8.02. 94

82 2-(l-Hydroxy-2-nitroethyl)benzaldehyde lactol (57) . —Nitromethane

(100 nil), o-phthalaldehyde (5 g, 0.0374 mol) and sodium carbonate (10 g) were

stirred at room temperature for 18 l/2 hr. The sodium carbonate was fil

tered off and washed with benzene. The combined solvents were removed

under reduced pressure and the resulting solid recrystallized from chloroform n 82 O to give 4. 7 g (64.4%) of needle crystals, mp 122-123 , lit. 121-123 . 121 Attempted synthesis of l-nitromethyl-3-cyanophthalan from 62 . —

The hemiacetal 62^ (1 g, 0.005 mol) was added to a mixture of liquid hydrogen

cyanide (100 ml) and sodium cyanide (5 g) and stirred at ice bath temperature

for one hour, then at room temperature for another hour. The hydrogen

cyanide was removed and the residue extracted with chloroform. Removal of

the chloroform left a brown amorphous material that decomposed upon purifi

cation attempts. Similar results were obtained using potassium cyanide and

glacial acetic acid. 80 Reaction of o-phthalaldehyde with dimethylsulfonium methylide. —A

dispersion of sodium hydride-oil (4.85 g) was washed under nitrogen with petroleum ether and the ether removed. Dimethyl sulfoxide (60 ml) was added and the mixture stirred at 70° for 1 hr. After cooling to room temperature, tetrahydrofuran (60 ml) was added and the mixture cooled in an ice-salt bath.

A solution of trimethyls ulfonium iodide (20.4 g) in dim ethyls ulfoxide (100 ml) was added dropwise over a five minute period, and the mixture stirred for 2 min. A solution of o-phthalaldehyde (3.35 g) in tetrahydrofuran (25 ml) was 95

added rapidly. The mixture was stirred for 5 min. at ice bath temperature,

then at room temperature for 11/2 hr. The mixture was then cooled, water

added (800 ml) and extracted with ether. After drying the ether solution, the

ether was removed to give a viscous brown oil that could not be purified and

was not characterized. 83 1,4-dihydronaphthalene-l, 4-endoxide (59) .—To a refluxing solution

of 1,2-dimethoxyethane (50 ml) and furan (50 ml) was added a solution of iso-

amylnitrite (20 ml) in dimethoxyethane (20 ml) with the simultaneous addition

of a solution of anthranilic acid (13.7 g) in dimethoxyethane (45 ml). After

the addition was complete (1 hr) refluxing was continued for 10 min. more.

The brown solution was made basic (10% NaOH) and extracted with petroleum

ether (30-60°). The extract was washed thoroughly with water and decolorized with charcoal. Removal of the ether left a pale yellow oil, which solidifed and was recrystallized from petroleum ether (30-60°) to give 5.13 g of white needle crystals, mp 54-55 . lit. ^ 56°.

Ozonolysis of 1,4-dihydronaphthalene-l, 4-endoxide (59). —A 3 per cent ozone in oxygen mixture was bubbled through a solution of 6£ (1 g) in methanol

(40 ml) at -78° until the appearance of a blue color (1 1/2 hr.). The methanol was removed at room temperature to give a glassy colorless syrup, which was treated with 90 per cent formic acid (5 ml) and 30% hydrogen peroxide (3 ml).

Removal of the solvent under reduced pressure left a white solid which was re- 96

o crystallized from ethyl acetate. The resulting crystals melted at 202-203 ,

and appeared identical in all respects to phthalic acid. 107 Acetonedicarboxylie acid (70). —The method described by Adams et al.

was employed. Citric acid (100 g) was added in portions to 20% fuming sul

furic acid (222 ml) cooled to 0°. The addition was carried out over a two hour

period, and the reaction mixture stirred for an additional two hours at 0-5°.

The cooling was then removed and the mixture stirred for eight hours at room

temperature. After cooling to 0°, crushed ice (340 g) was slowly added, and the mixture filtered through a sintered glass funnel. The solid material col

lected was pressed dry, then washed repeatedly by stirring with ethyl acetate, o 107 o filtered off and dried to give 20 g (29%) of 70. mp 134-136 , lit. mp 138 .

3 -ketoglutaric anhydride (71). —p-ketoglutaric anhydride (71) was pre- 104 pared according to the method of Findlay. To a stirred mixture of glacial acetic acid (30 ml) and acetic anhydride (21.5 ml) at 5° was added slowly f3- o ketoglutaric acid (20 g). Stirring was continued at 8-10 for four hours, and the product filtered, washed with benzene and dried over potassium hydroxide o 104 in vacuum to afford 14.5 g of 7L, (83%), mp 135-136. 5 , lit. mp 137. 5-

138.5°.

Succindialdoxime (72). —Suecindialdoxime (72) was prepared by a method 104 described by Findlay. Pulverized hydroxylamine hydrochloride (90.5 g,

1.3 mol) was stirred with alcohol (400 ml) for 30 minutes at room temperature.

A solution of potassium hydroxide (44.8 g, 0.8 mol) in water (50 ml) was 97 I added, followed by freshly distilled pyrrole (34.5 ml, 0.5 mol), and the mix ture stirred at reflux for 23 hours. Alcohol (50 ml) was added, and the mix ture refluxed an additional hour. The mixture was filtered hot with suction and the inorganic residue washed white with hot alcohol. The ethanol was re moved from the filtrate in vacuum to yield a brown solid which was washed with a small amount of ethanol and filtered dry. This was recrystallized from boiling water, collected and dried to give 28.5 g (50%) of light tan solid, mp o 104 o 168-169 , lit. mp 169-170 .

Succindialdeh.yde (67). —This compound was prepared from two sources 104 as described by Findlay.

(a) From succindialdoxime: To 1 N sulfuric acid (150 ml) at 0° was added 12 g of 72_. A solution of sodium nitrite (14 g) in water (100 ml) was added dropwise with stirring over a four hour period. The solution was then stirred at room temperature under nitrogen for three hours, neutralized with barium carbonate, and filtered through celite. The clear yellow solution ob tained was diluted with water to 450 ml and used directly in the synthesis of

69.

(b) From 2 ,5-Dimethoxytetrahydrofuran: To 0.5 N hydrochloric acid

(100 ml) was added 13.2 g (0.1 mol) of 2 ,5-dimethoxytetrahydrofuran and the solution stirred under nitrogen for 30 minutes. This solution was used direct ly in the synthesis of 69.

2-Carbomethoxytropinone (69). —The method of Findlay was employed 104 for the preparation of this compound. (3 -Ketoglutaric anhydride (13.5 g,(

0.1 mol) was dissolved in cold methanol (50 ml) and kept at room tempera ture for one hour to give a methanol solution of monomethyl-|3 -ketoglutarate.

This was added to a solution of methylamine hydrochloride (10 g, 0.148 mol) and sodium hydroxide (4 g, 0.1 mol) in water (150 ml), and a solution of succindialdehyde (450 ml, 0.1 mol) stirred in. The reaction mixture was flushed with nitrogen and stirred for 24 hours at room temperature. The solution was then cooled in an ice bath, 6 N HC1 added to pH 4, and washed with chloro form (35 ml). The aqueous solution was basified with potassium carbonate and extracted with chloroform. The chloroform extract was dried with satu rated sodium chloride solution and magnesium sulfate and removed under vacuum to give a dark brown viscous oil. Upon sitting overnight in the re frigerator this solidified to give 15 g (72%) of crude brown solid. The crude material (. 34 g) was sublimed in vacuum to give . 126 g of pale yellow solid n 1°4 (37%). mp 95-97 , lit. mp 93-100°.

Allopseudoecgonine methyl ester (76). —This compound was prepared 105 by a method similar to the one described by Sinnema et al_. Platinum oxide

(0.75 g) was added to glacial acetic acid (85 ml) and water (10 ml) in a 500 ml hydrogenation bottle and saturated with hydrogen on a Paar shaker. To the saturated catalyst was added a solution of sublimed 2-carbomethoxytropinone in glacial acetic acid (110 ml) and water (20 ml). The maximum pressure was

30 psig, and reduction was continued until the theoretical amount of hydrogen 99 had been consumed. After filtering off the catalyst the colorless solution was

evaporated under vacuum at 40° to yield a yellow oil. The oil was dissolved in water (15 ml), saturated potassium carbonate (50 ml), and extracted with ether. The ether, after drying with saturated sodium chloride solution and magnesium sulfate, was removed under vacuum to give a colorless oil. The oil was taken up in acetone (20 ml) and ether (58 ml), and glacial acetic acid

(1.8 ml) added. After 12 hours a white crystalline solid was filtered off and dried under vacuum over phosphorous pentoxide and potassium hydroxide to

<| a r give 5. 36 g (45.5%) of the hydroacetate, mp 108-111°, lit. mp 107-109°.

The hydroacetate (5.25 g) was dissolved in water (25 ml), saturated po tassium carbonate added (30 ml), and the resulting suspension extracted with ether. The ether was dried (magnesium sulfate) and removed in vacuum to give a colorless oil which solidified on cooling under high vacuum. Recry stallization from petroleum ether yielded 2.75 g (67.8%, 30% from 2-carbomethoxytropinone). mp 72-73. 5°, lit. 72-73°.

Allopseudococaine (77). —This cocaine isomer was prepared according 105 to the procedure given by Sinnema. Allopseudoecgonine methyl ester

(1.23 g) was dissolved in dry pyridine (3 ml), cooled in an ice bath, and ben zoyl chloride (0.31 ml) added. The mixture was stirred for 15 min., then

0.5 ml more of benzoyl chloride added, and the mixture allowed to stand at room temperature overnight. Ether (2.6 ml) and methanol (2.6 ml) were added and the mixture filtered. The solvent was removed from the filtrate under re 100

duced pressure, and the residue recrystallized twice from methanol-ether to

give a white solid, mp 199-200°. This was dissolved in water, basified with

sodium bicarbonate and extracted with ether. After drying the ether solution,

the ether was removed to give a colorless oil which solidified to a white solid.

Recrystallization from petroleum ether (30-60°) afforded colorless rhombic o 105 o crystals, mp 82-83 , lit. 82-84 .

Pseudoecgonine methyl ester (79). —To a solution of cocaine free base

(9.12 g) in methanol (40 ml) was added a solution of sodium metal (0.073 g) in

methanol (10 ml), and the resulting mixture refluxed for 4 hours before stand

ing overnight at room temperature. A mixture of potassium bicarbonate (1 g)

in water (5 ml) and benzene (50 ml) was added and the insoluble salts filtered

off. Excess potassium carbonate was added to the filtrate to remove the

water, then filtered off. Removal of the solvent in vacuum left a pale yellow

oil which was taken up in ether, dried (magnesium s ulfate), and the ether re moved to give a pale yellow oil containing a white solid. A small amount of

ether was added and the white solid filtered off and dried to give 2.2 g (36.8%) of pseudoecgonine methyl ester. Additional product was obtained from the filtrate, mp 114.5-115.5°, lit.113 114-115.5°.

Pseudococaine (80). —Pseudoecgonine methyl ester (1.7 g, 0.0085 mol) was dissolved in dry pyridine (8 ml) and a solution of freshly distilled benzoyl chloride (2.4 g, 0.017 mol) in dry pyridine (5 ml) added dropwise. After stir ring at room temperature for 26 hours, the mixture was poured onto ice and 101 acidified with 10% hydrochloric acid and extracted twice with ether. The pale yellow aqueous solution was made basic with concentrated ammonium hydrox- ! ide, and extracted (vith ether. The ether was dried (saturated sodium chloride solution, magnesium sulfate) and removed under vacuum to give a yellow oil smelling strongly of pyridine. The pyridine was removed under high vacuum, the oil taken up in ether, ether/HCl solution added, and the resulting hydro chloride filtered off. The creme colored hydrochloride was recrystallized from methanol-ether to give a white solid, mp 209-210°. The free base was liberated by dissolving the hydrochloride in water, adding sodium bicarbonate, and extracting with ether. The ether was dried (magnesium sulfate) and re moved in vacuum to yield a colorless oil which crystallized with some diffi culty. Infrared examination revealed no hydroxyl peak and the presence of two carbonyl peaks (1725, 1745 cm *). The presence of the benzoate ester was also confirmed by NMR. The white solid was dissolved in ether, ether/

HC1 solution added, and the resulting precipitate recrystallized from methanol- ether to give white pseudococaine hydrochloride, mp 210-210.5°, lit.120 210°. EXPERIMENTAL

B. BIOLOGICAL

Materials. —Vas deferens were obtained from albino rats weighing

300 to 395 g. Equipment included two isolated tissue baths (jacketed, 10

ml-metro) connected in tandem, thermostated water circulator to maintain

the temperature at 37°C., and isotonic myograph transducers (two) connected

to a "physiograph four" recorder. Both the transducers and the "physiograph

four" are manufactured by the E. & M. Instrument Co. of Houston, Texas.

Chemicals include D(-) -norepinephrine (Regis) which was employed as the

standard agonist; (-)-cocaine hydrochloride (Merck & Co., Inc.); atropine

sulfate (Mallinckrodt Chemical Co.); homatropine hydrobromide, hyoscine hydrobromide, and benztropine mesylate (Merck, Sharpe & Dohme), tropine

(Aldrich), and pseudotropine (Regis). All others were prepared as described in the preceding sections.

Methods.—The technique employed in these studies has been described 30 122 by Patil and by Van Rossum. Rats, weighing 300 to 395 g, were sacri ficed by a sharp blow on the head and either one or both vas deferens removed and placed in physiological solution. Any excess fascia was removed from the vas deferens, and the tissue fastened to a glass support by surgical thread. 103

The tissue was then suspended in the jacketed 10 ml muscle chamber which

contained Tyrode’s solution at 37°C. The composition of the Tyrode's solu

tion was as follows (in g): NaCl 8, KC1 0.20, CaCl2*2H20 0.26, MgCl 2 *

6H20 0.10, NaH2P04'H20 0.05, NaHCOg 1.00, glucose 1.00, EDTA 0.010,

and double distilled demineralized water added to make 1000 ml. The chlo

rides (KC1, MgClg, and CaCl2) were added last from a stock solution to a

dilute solution of the other components to prevent precipitation of insoluble

salts. The physiological solution was continuously aerated with a mixture of

oxygen (95%) and C 02 (5%).

In handling the tissue, care was taken at all times to avoid stretching

the tissue. With one end of the tissue fastened to the muscle holder at the

bottom of the muscle chamber, the other end was attached to the isotonic

myograph transducer (balanced with 300 mg tension) which was connected to the "physiograph four" on which the drug-induced effects were recorded. The tissue was allowed to equilibrate for 15 to 20 minutes before the addition of any drug. ■ 30 Patil et aL reported that the second dose-response curve obtained on the vas deferens has a greater height than the first and that the second and third are the same. In other words, a greater reproducibility is obtained in dose-response curves subsequent to the first one. Thus following the -4 initial equilibrium period each tissue was subjected to a single dose (10 M) of (-)-norepinephrine. After the response had reached a maximum, the

/ 104

tissue was washed four times at intervals of 2, 2, 5, and 5 minutes. Follow

ing the final wash period, exactly 10 ml of physiological solution was added

and the tissue again equilibrated for 15 minutes. A control cumulative dose-

response curve for norepinephrine was then constructed by increasing the

dose of the agonist by a factor of about three while the previous dose was

maintained in the bath. The final bath concentrations were increased in the

order: 3 x l(f 7M, 10_6M, 3 x 10_ 6M, 10_5M, 3 x 10“5M, and 10_4M. After

the conti'action had reached maximal to a given dose and leveled off, the

next higher dose was administered.

The control norepinephrine effect was washed out as before and the tissue equilibrated again. The drug to be tested was introduced into the bath and a 3 minute incubation time allowed before construction of the next norepinephrine cumulative dose-response curve. If either potentiation or inhibition was observed, a new norepinephrine control curve was constructed prior to the next test curve. The absolute contraction values were trans formed into percentage values with maximal response to D(-)-norepinephrine being regarded as 100. ED50's were obtained from graphs in which percent contraction was plotted against log dose. Results were statistically analyzed for the S.E.M . and 95% confidence intervals (C.I.). ED50 values are ex pressed as the negative log molar concentration, and the dose ratio repre sents the antilog of [(-log ED50 of larger value)-(-log ED50 of smaller

value)]. For antagonists, pA 2 values were obtained by plotting log (x-1) 105 against log molar concentration of antagonist, where x is equal to the dose 114 ratio.

Drugs. —Fresh solutions of drugs were made each day and fresh phy siological solution made for each new set of tissues. Dilutions of D(-)-nor epinephrine (free base) were made from a 10_^M refrigerated stock solution prepared in 0. 9 percent saline with 0.05 per cent sodium metabisulfite and a few drops of dilute hydrochloric acid. Other drugs were prepared in 0.9 per cent saline, with a few drops of dilute hydrochloric acid added to those in the free base form. SUMMARY

Substituted tetrahydro-3-benzazepines representing semi-rigid analogs of ephedrine and phenylethanolamine have been prepared as a means of study ing stereochemical and conformational requirements of ephedrine and related agents for adrenergic activity. The use of nmr as a means of assigning the relative configuration of the diastereomeric cis and trans l-hydroxy-2- methyl-2, 3 ,4 ,5-tetrahydro~3-benzazepines was discussed. Vicinal proton coupling constants can be related to the dihedral angle between adjacent pro tons. The use of this relationship, along with a consideration of the confor mational preferences of a system, can provide a means of determining rela tive configurations.

Various synthetic approaches used in the attempted synthesis of 2, 3,4,5- tetrahydro-3-benzazepine-l, 5-endoxide were described.

The ability of cocaine to block the uptake of adrenergic agents such as norepinephrine into the nerve terminal is well known. As a means of studying the stereochemical requirements of cocaine for this action, steric isomers of cocaine were prepared.

These cocaine isomers and related derivatives, as well as the tetrahydro-3-benzazepines, were examined for peripheral adrenergic activity on

106 107

the isolated rat vas deferens. The rat vas deferens is a smooth muscle tis

sue with relatively uniform density of adrenergic innervation, containing

predominantly alpha-reccptors.

Compound B (erythro- 1 -hydroxy-2-methyl-2 ,3 ,4 ,5-tetrahydro-3-

benzazepine) potentiated the effects of norepinephrine, while compound A

showed only a slight blocking activity at high concentrations. This is quali

tatively similar to the results reported for the er.ythro- and threo-3-amino-

2-phenyl-2-butanols and ephedrine (erythro) and pseudoephedrine (threo).

The compound 1-hydroxy-2, 3 ,4 ,5-tetrahydro-3-benzazepine displayed only

slight agonist activity at high concentrations.

Pseudococaine, allopseudococaine, tropacocaine (pseudotropine ben

zoate) , and pseudotropacocaine (tropine benzoate) all potentiated the response

of norepinephrine. The methyl ester derivatives and tropine and pseudotro

pine exhibited no significant potentiating effects. The presence of a benzoyloxy

group appears to be necessary for potentiating activity. A structural compar

ison was made between the isomeric cocaines and the most stable conforma

tions of threo- and erythro-methylphenidates. The erythro-methylphenidate, pseudococaine and allopseudococaine have similar potentiating activities and in each the carbomethoxy group is trans to the nitrogen. In the more active threo-methylphenidate and cocaine there is a 60° skew relationship between the nitrogen and the carbomethoxy group.

Tropine derivatives of atropine were also tested on the rat vas deferens 108 preparation. Atropine and homatropine both competitively blocked alpha- adrcnergic receptors, while benztropine showed a non-competitive blocking activity. I-Iyoscine hydrobromide, structurally identical to atropine except for the presence of a 6(3, 7(3-epoxy group, possessed neither alpha-blocking nor norepinephrine potentiating activity.

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